# Patent

## Title
Dual variable domain immunoglobulins and uses thereof

## Abstract

The present invention relates to engineered multivalent and multispecific binding proteins, methods of making, and specifically to their uses in the prevention, diagnosis, and/or treatment of disease.

## Background
REFERENCE TO RELATED APPLICATIONS
This application is a non-provisional application claiming priority to U.S. Provisional Application Ser. No. 61/125,834, filed Apr. 29, 2008, U.S. Provisional Application Ser. No. 61/134,283, filed Jul. 8, 2008, U.S. Provisional Application Ser. No. 61/197,191, filed Oct. 23, 2008, and U.S. Provisional Application Ser. No. 61/199,009, filed Nov. 12, 2008, the contents of which are hereby incorporated by reference.


FIELD OF THE INVENTION
The present invention relates to multivalent and multispecific binding proteins, methods of making, and specifically to their uses in the, diagnosis, prevention and/or treatment of acute and chronic inflammatory diseases, cancer, and other diseases.
BACKGROUND OF THE INVENTION
Engineered proteins, such as multispecific antibodies capable of binding two or more antigens are known in the art. Such multispecific binding proteins can be generated using cell fusion, chemical conjugation, or recombinant DNA techniques.
Bispecific antibodies have been produced using quadroma technology (see Milstein, C. and A. C. Cuello (1983) Nature 305(5934):537-40) based on the somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies (mAbs) with the desired specificities of the bispecific antibody. Because of the random pairing of two different immunoglobulin (Ig) heavy and light chains within the resulting hybrid-hybridoma (or quadroma) cell line, up to ten different Ig species are generated, of which only one is the functional bispecific antibody. The presence of mis-paired by-products, and significantly reduced production yields, means sophisticated purification procedures are required.
Bispecific antibodies can also be produced by chemical conjugation of two different mAbs (see Staerz, U. D., et al. (1985) Nature 314(6012): 628-31). This approach does not yield homogeneous preparation. Other approaches have used chemical conjugation of two different mAbs or smaller antibody fragments (see Brennan, M., et al. (1985) Science 229(4708): 81-3).
Another method used to produce bispecific antibodies is the coupling of two parental antibodies with a hetero-bifunctional crosslinker, but the resulting bispecific antibodies suffer from significant molecular heterogeneity because reaction of the crosslinker with the parental antibodies is not site-directed. To obtain more homogeneous preparations of bispecific antibodies two different Fab fragments have been chemically crosslinked at their hinge cysteine residues in a site-directed manner (see Glennie, M. J., et al. (1987) J. Immunol. 139(7): 2367-75). But this method results in Fab′2 fragments, not full IgG molecule.
A wide variety of other recombinant bispecific antibody formats have been developed (see Kriangkum, J., et al. (2001) Biomol. Eng. 18(2): 31-40). Amongst them tandem single-chain Fv molecules and diabodies, and various derivatives thereof, are the most widely used. Routinely, construction of these molecules starts from two single-chain Fv (scFv) fragments that recognize different antigens (see Economides, A. N., et al. (2003) Nat. Med. 9(1): 47-52). Tandem scFv molecules (taFv) represent a straightforward format simply connecting the two scFv molecules with an additional peptide linker. The two scFv fragments present in these tandem scFv molecules form separate folding entities. Various linkers can be used to connect the two scFv fragments and linkers with a length of up to 63 residues (see Nakanishi, K., et al. (2001) Ann. Rev. Immunol. 19: 423-74). Although the parental scFv fragments can normally be expressed in soluble form in bacteria, it is, however, often observed that tandem scFv molecules form insoluble aggregates in bacteria. Hence, refolding protocols or the use of mammalian expression systems are routinely applied to produce soluble tandem scFv molecules. In a recent study, in vivo expression by transgenic rabbits and cattle of a tandem scFv directed against CD28 and a melanoma-associated proteoglycan was reported (see Gracie, J. A., et al. (1999) J. Clin. Invest. 104(10): 1393-401). In this construct, the two scFv molecules were connected by a CH1 linker and serum concentrations of up to 100 mg/L of the bispecific antibody were found. Various strategies including variations of the domain order or using middle linkers with varying length or flexibility were employed to allow soluble expression in bacteria. A few studies have now reported expression of soluble tandem scFv molecules in bacteria (see Leung, B. P., et al. (2000) J. Immunol. 164(12): 6495-502; Ito, A., et al. (2003) J. Immunol. 170(9): 4802-9; Karni, A., et al. (2002) J. Neuroimmunol. 125(1-2): 134-40) using either a very short Ala3 linker or long glycine/serine-rich linkers. In another recent study, phage display of a tandem scFv repertoire containing randomized middle linkers with a length of 3 or 6 residues was employed to enrich for those molecules that are produced in soluble and active form in bacteria. This approach resulted in the isolation of a tandem scFv molecule with a 6 amino acid residue linker (see Arndt, M. and J. Krauss (2003) Methods Mol. Biol. 207: 305-21). It is unclear whether this linker sequence represents a general solution to the soluble expression of tandem scFv molecules. Nevertheless, this study demonstrated that phage display of tandem scFv molecules in combination with directed mutagenesis is a powerful tool to enrich for these molecules, which can be expressed in bacteria in an active form.
Bispecific diabodies (Db) utilize the diabody format for expression. Diabodies are produced from scFv fragments by reducing the length of the linker connecting the VH and VL domain to approximately 5 residues (see Peipp, M. and T. Valerius (2002) Biochem. Soc. Trans. 30(4): 507-11). This reduction of linker size facilitates dimerization of two polypeptide chains by crossover pairing of the VH and VL domains. Bispecific diabodies are produced by expressing, two polypeptide chains with, either the structure VHA-VLB and VHB-VLA (VH-VL configuration), or VLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. A large variety of different bispecific diabodies have been produced in the past and most of them are expressed in soluble form in bacteria. However, a recent comparative study demonstrates that the orientation of the variable domains can influence expression and formation of active binding sites (see Mack, M. et al. (1995) Proc. Natl. Acad. Sci. USA 92(15): 7021-5). Nevertheless, soluble expression in bacteria represents an important advantage over tandem scFv molecules. However, since two different polypeptide chains are expressed within a single cell inactive homodimers can be produced together with active heterodimers. This necessitates the implementation of additional purification steps in order to obtain homogenous preparations of bispecific diabodies. One approach to force the generation of bispecific diabodies is the production of knob-into-hole diabodies (see Holliger, P., T. Prospero, and G. Winter (1993) Proc. Natl. Acad. Sci. USA 90(14): 6444-8.18). This approach was demonstrated for a bispecific diabody directed against HER2 and CD3. A large knob was introduced in the VH domain by exchanging Val37 with Phe and Leu45 with Trp and a complementary hole was produced in the VL domain by mutating Phe98 to Met and Tyr87 to Ala, either in the anti-HER2 or the anti-CD3 variable domains. By using this approach the production of bispecific diabodies could be increased from 72% by the parental diabody to over 90% by the knob-into-hole diabody. Importantly, production yields only slightly decrease as a result of these mutations. However, a reduction in antigen-binding activity was observed for several constructs. Thus, this rather elaborate approach requires the analysis of various constructs in order to identify those mutations that produce heterodimeric molecule with unaltered binding activity. In addition, such approach requires mutational modification of the immunoglobulin sequence at the constant region, thus creating non-native and non-natural form of the antibody sequence, which may result in increased immunogenicity, poor in vivo stability, as well as undesirable pharmacokinetics.
Single-chain diabodies (scDb) represent an alternative strategy for improving the formation of bispecific diabody-like molecules (see Holliger, P. and G. Winter (1997) Cancer Immunol. Immunother. 45(3-4): 128-30; Wu, A. M., et al. (1996) Immunotechnology 2(1): p. 21-36). Bispecific single-chain diabodies are produced by connecting the two diabody-forming polypeptide chains with an additional middle linker with a length of approximately 15 amino acid residues. Consequently, all molecules with a molecular weight corresponding to monomeric single-chain diabodies (50-60 kDa) are bispecific. Several studies have demonstrated that bispecific single chain diabodies are expressed in bacteria in soluble and active form with the majority of purified molecules present as monomers (see Holliger, P. and G. Winter (1997) Cancer Immunol. Immunother. 45(3-4): 128-30; Wu, A. M., et al. (1996) Immunotechnol. 2(1): 21-36; Pluckthun, A. and P. Pack (1997) Immunotechnol. 3(2): 83-105; Ridgway, J. B., et al. (1996) Protein Engin. 9(7): 617-21). Thus, single-chain diabodies combine the advantages of tandem scFvs (all monomers are bispecific) and diabodies (soluble expression in bacteria).
More recently diabodies have been fused to Fc to generate more Ig-like molecules, named di-diabodies (see Lu, D., et al. (2004) J. Biol. Chem. 279(4): 2856-65). In addition, multivalent antibody construct comprising two Fab repeats in the heavy chain of an IgG and capable of binding four antigen molecules has been described (see WO 0177342A1, and Miller, K., et al. (2003) J. Immunol. 170(9): 4854-61).
There is a need in the art for improved multivalent binding proteins capable of binding two or more antigens. U.S. patent application Ser. No. 11/507,050 provides a novel family of binding proteins capable of binding two or more antigens with high affinity, which are called dual variable domain immunoglobulins (DVD-IgTM). The present invention provides further novel binding proteins capable of binding two or more antigens.
SUMMARY OF THE INVENTION
This invention pertains to multivalent binding proteins capable of binding two or more antigens. The present invention provides a novel family of binding proteins capable of binding two or more antigens with high affinity.
In one embodiment the invention provides a binding protein comprising a polypeptide chain, wherein the polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 represents an amino acid or polypeptide, X2 represents an Fc region and n is 0 or 1. In an embodiment the VD1 and VD2 in the binding protein are heavy chain variable domains. In another embodiment, the heavy chain variable domain is selected from the group consisting of a murine heavy chain variable domain, a human heavy chain variable domain, a CDR grafted heavy chain variable domain, and a humanized heavy chain variable domain. In yet another, embodiment VD1 and VD2 are capable of binding the same antigen. In another embodiment VD1 and VD2 are capable of binding different antigens. In still another embodiment, C is a heavy chain constant domain. For example, X1 is a linker with the proviso that X1 is not CH1. For example, X1 is a linker selected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G4S)4 (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25); GHEAAAVMQVQYPAS (SEQ ID NO: 26). In an embodiment, X2 is an Fc region. In another embodiment, X2 is a variant Fc region.
In an embodiment the binding protein disclosed herein comprises a polypeptide chain, wherein the polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 is an Fc region.
In an embodiment, VD1 and VD2 in the binding protein are light chain variable domains. In an embodiment, the light chain variable domain is selected from the group consisting of a murine light chain variable domain, a human light chain variable domain, a CDR grafted light chain variable domain, and a humanized light chain variable domain. In one embodiment VD1 and VD2 are capable of binding the same antigen. In another embodiment VD1 and VD2 are capable of binding different antigens. In an embodiment, C is a light chain constant domain. In another embodiment, X1 is a linker with the proviso that X1 is not CL1. In an embodiment, X1 is a linker selected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G4S)4 (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ ID NO: 22) GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25); GHEAAAVMQVQYPAS (SEQ ID NO: 26). In an embodiment, the binding protein does not comprise X2.
In an embodiment, both the variable heavy and variable light chain comprise the same linker. In another embodiment, the variable heavy and variable light chain comprise different linkers. In another embodiment, both the variable heavy and variable light chain comprise a short (about 6 amino acids) linker. In another embodiment, both the variable heavy and variable light chain comprise a long (greater than 6 amino acids) linker. In another embodiment, the variable heavy chain comprises a short linker and the variable light chain comprises a long linker. In another embodiment, the variable heavy chain comprises a long linker and the variable light chain comprises a short linker.
In an embodiment the binding protein disclosed herein comprises a polypeptide chain, wherein said polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 does not comprise an Fc region.
In another embodiment the invention provides a binding protein comprising two polypeptide chains, wherein said first polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 is an Fc region; and said second polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 does not comprise an Fc region. In a particular embodiment, the Dual Variable Domain (DVD) binding protein comprises four polypeptide chains wherein the first two polypeptide chains comprises VD1-(X1)n-VD2-C-(X2)n, respectively wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 is an Fc region; and the second two polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n respectively, wherein VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker with the proviso that it is not CH1, and X2 does not comprise an Fc region. Such a Dual Variable Domain (DVD) protein has four antigen binding sites.
In another embodiment the binding proteins disclosed herein are capable of binding one or more targets. In an embodiment, the target is selected from the group consisting of cytokines, cell surface proteins, enzymes and receptors. In another embodiment, the binding protein is capable of modulating a biological function of one or more targets. In another embodiment, the binding protein is capable of neutralizing one or more targets. The binding protein of the invention is capable of binding cytokines selected from the group consisting of lymphokines, monokines, polypeptide hormones, receptors, or tumor markers. For example, the DVD-Ig of the invention is capable of binding two or more of the following: CD-20, CD-19, CD-80, CD-22, CD-40, CD-3, human epidermal growth factor receptor 2 (HER-2), epidermal growth factor receptor (EGFR), insulin-like growth factor 1,2 (IGF1,2), insulin-like growth factor receptor (IGF1R), macrophage stimulating protein receptor tyrosine kinase (RON), hepatocyte growth factor (HGF), mesenchymal-epithelial transition factor (c-MET), vascular endothelial growth factor (VEGF), Drosophila Delta homologue 4 (DLL4), neuropilin 1 (NRP1), placental growth factor (PLGF), v-erbb2 avian erythroblastic leukemia viral oncogene homolog 3 (ErbB3) (see also Table 2). In a specific embodiment the binding protein is capable of binding pairs of targets selected from the group consisting of CD-20 and CD-19; CD-20 and CD-80; CD-20 and CD-22; CD-20 and CD-40; CD-3 and HER-2; CD-3 and CD-19; EGFR and HER-2; EGFR and CD-3; EGFR and IGF1,2; EGFR and IGF1R; EGFR and RON; EGFR and HGF; EGFR and c-MET; HER-2 and IGF1,2; HER-2 and IGF1R; RON and HGF; VEGF and EGFR; VEGF and HER-2; VEGF and CD-20; VEGF and IGF1,2; VEGF and DLL4; VEGF and HGF; VEGF and RON; VEGF and NRP1; CD-20 and CD3; DLL-4 and PLGF; VEGF and PLGF; ErbB3 and EGFR; ErbB3 and HGF; HER-2 and ErbB3; c-Met and ErB3; PLGF and HER-2; HER-2 and HER-2.
In an embodiment, the binding protein capable of binding CD-20 and CD-19 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 99 and SEQ ID NO. 101; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 100 and SEQ ID NO. 102. In an embodiment, the binding protein capable of binding CD-20 and CD-19 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 99 and a DVD light chain amino acid sequence of SEQ ID NO: 100. In another embodiment, the binding protein capable of binding CD-20 and CD-19 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 101 and a DVD light chain amino acid sequence of SEQ ID NO: 102.
In an embodiment, the binding protein capable of binding CD-20 and CD-3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 103 and SEQ ID NO. 105; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 104 and SEQ ID NO. 106. In an embodiment, the binding protein capable of binding CD-20 and CD-3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 103 and a DVD light chain amino acid sequence of SEQ ID NO: 104. In another embodiment, the binding protein capable of binding CD-20 and CD-3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 105 and a DVD light chain amino acid sequence of SEQ ID NO: 106.
In an embodiment, the binding protein capable of binding CD-20 and CD-80 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 107 and SEQ ID NO. 109; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 108 and SEQ ID NO. 110. In an embodiment, the binding protein capable of binding CD-20 and CD-80 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 107 and a DVD light chain amino acid sequence of SEQ ID NO: 108. In another embodiment, the binding protein capable of binding CD-20 and CD-80 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 109 and a DVD light chain amino acid sequence of SEQ ID NO: 110.
In an embodiment, the binding protein capable of binding CD-20 and CD-22 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 111 and SEQ ID NO. 113; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 112 and SEQ ID NO. 114. In an embodiment, the binding protein capable of binding CD-20 and CD-22 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 111 and a DVD light chain amino acid sequence of SEQ ID NO: 112. In another embodiment, the binding protein capable of binding CD-20 and CD-22 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 113 and a DVD light chain amino acid sequence of SEQ ID NO: 114.
In an embodiment, the binding protein capable of binding CD-20 and CD-40 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 115 and SEQ ID NO. 117; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 116 and SEQ ID NO. 118. In an embodiment, the binding protein capable of binding CD-20 and CD-40 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 115 and a DVD light chain amino acid sequence of SEQ ID NO: 116. In another embodiment, the binding protein capable of binding CD-20 and CD-40 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 117 and a DVD light chain amino acid sequence of SEQ ID NO: 118.
In an embodiment, the binding protein capable of binding CD-3 (seq. 1) and HER-2 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 119 and SEQ ID NO. 121; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 120 and SEQ ID NO. 122. In an embodiment, the binding protein capable of binding CD-3 (seq. 1) and HER-2 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 119 and a DVD light chain amino acid sequence of SEQ ID NO: 120. In another embodiment, the binding protein capable of binding CD-3 (seq. 1) and HER-2 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 121 and a DVD light chain amino acid sequence of SEQ ID NO: 122.
In an embodiment, the binding protein capable of binding CD-3 (seq. 1) and CD-19 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 123 and SEQ ID NO. 125; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 124 and SEQ ID NO. 126. In an embodiment, the binding protein capable of binding CD-3 (seq. 1) and CD-19 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 123 and a DVD light chain amino acid sequence of SEQ ID NO: 124. In another embodiment, the binding protein capable of binding CD-3 (seq. 1) and CD-19 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 125 and a DVD light chain amino acid sequence of SEQ ID NO: 126.
In an embodiment, the binding protein capable of binding EGFR (seq. 2) and HER-2 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 127 and SEQ ID NO. 129; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 128 and SEQ ID NO. 130. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and HER-2 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 127 and a DVD light chain amino acid sequence of SEQ ID NO: 128. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and HER-2 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 129 and a DVD light chain amino acid sequence of SEQ ID NO: 130.
In an embodiment, the binding protein capable of binding EGFR (seq. 2) and CD-3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 131 and SEQ ID NO. 133; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 132 and SEQ ID NO. 136. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and CD-3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 131 and a DVD light chain amino acid sequence of SEQ ID NO: 132. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and CD-3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 133 and a DVD light chain amino acid sequence of SEQ ID NO: 134.
In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1,2 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 135 and SEQ ID NO. 137; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 136 and SEQ ID NO. 138. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1,2 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 135 and a DVD light chain amino acid sequence of SEQ ID NO: 136. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1,2 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 137 and a DVD light chain amino acid sequence of SEQ ID NO: 138.
In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 139 and SEQ ID NO. 140; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 141 and SEQ ID NO. 142. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 139 and a DVD light chain amino acid sequence of SEQ ID NO: 140. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 141 and a DVD light chain amino acid sequence of SEQ ID NO: 142.
In a second embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 143 and SEQ ID NO. 145; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 144 and SEQ ID NO. 146. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 143 and a DVD light chain amino acid sequence of SEQ ID NO: 144. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 145 and a DVD light chain amino acid sequence of SEQ ID NO: 146.
In a third embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 147 and SEQ ID NO. 149; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 148 and SEQ ID NO. 150. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 147 and a DVD light chain amino acid sequence of SEQ ID NO: 148. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 149 and a DVD light chain amino acid sequence of SEQ ID NO: 150.
In a fourth embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 151 and SEQ ID NO. 153; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 152 and SEQ ID NO. 154. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 151 and a DVD light chain amino acid sequence of SEQ ID NO: 152. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 153 and a DVD light chain amino acid sequence of SEQ ID NO: 154.
In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 155 and SEQ ID NO. 157; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 156 and SEQ ID NO. 158. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 155 and a DVD light chain amino acid sequence of SEQ ID NO: 156. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 157 and a DVD light chain amino acid sequence of SEQ ID NO: 158.
In a second embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 159 and SEQ ID NO. 161; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 160 and SEQ ID NO. 162. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 159 and a DVD light chain amino acid sequence of SEQ ID NO: 160. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 161 and a DVD light chain amino acid sequence of SEQ ID NO: 162.
In a third embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 163 and SEQ ID NO. 165; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 164 and SEQ ID NO. 166. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 163 and a DVD light chain amino acid sequence of SEQ ID NO: 164. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 165 and a DVD light chain amino acid sequence of SEQ ID NO: 166.
In a fourth embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 167 and SEQ ID NO. 169; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 168 and SEQ ID NO. 170. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 167 and a DVD light chain amino acid sequence of SEQ ID NO: 168. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 169 and a DVD light chain amino acid sequence of SEQ ID NO: 170.
In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 171 and SEQ ID NO. 173; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 172 and SEQ ID NO. 174. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 171 and a DVD light chain amino acid sequence of SEQ ID NO: 172. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 173 and a DVD light chain amino acid sequence of SEQ ID NO: 174.
In a second embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 175 and SEQ ID NO. 177; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 176 and SEQ ID NO. 178. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 175 and a DVD light chain amino acid sequence of SEQ ID NO: 176. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 177 and a DVD light chain amino acid sequence of SEQ ID NO: 178.
In a third embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 179 and SEQ ID NO. 181; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 180 and SEQ ID NO. 182. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 179 and a DVD light chain amino acid sequence of SEQ ID NO: 180. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 181 and a DVD light chain amino acid sequence of SEQ ID NO: 182.
In a fourth embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 183 and SEQ ID NO. 185; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 184 and SEQ ID NO. 186. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 183 and a DVD light chain amino acid sequence of SEQ ID NO: 184. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and IGF1R (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 185 and a DVD light chain amino acid sequence of SEQ ID NO: 186.
In an embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 187 and SEQ ID NO. 189; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 188 and SEQ ID NO. 190. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 187 and a DVD light chain amino acid sequence of SEQ ID NO: 188. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 189 and a DVD light chain amino acid sequence of SEQ ID NO: 190.
In a second embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 191 and SEQ ID NO. 193; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 192 and SEQ ID NO. 194. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 191 and a DVD light chain amino acid sequence of SEQ ID NO: 192. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 193 and a DVD light chain amino acid sequence of SEQ ID NO: 194.
In a third embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 195 and SEQ ID NO. 197; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 196 and SEQ ID NO. 198. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 195 and a DVD light chain amino acid sequence of SEQ ID NO: 196. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 197 and a DVD light chain amino acid sequence of SEQ ID NO: 198.
In a fourth embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 199 and SEQ ID NO. 201; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 200 and SEQ ID NO. 202. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 199 and a DVD light chain amino acid sequence of SEQ ID NO: 200. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and RON (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 201 and a DVD light chain amino acid sequence of SEQ ID NO: 202.
In an embodiment, the binding protein capable of binding EGFR (seq. 2) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 203 and SEQ ID NO. 205; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 204 and SEQ ID NO. 206. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 203 and a DVD light chain amino acid sequence of SEQ ID NO: 204. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and HGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 205 and a DVD light chain amino acid sequence of SEQ ID NO: 206.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and c-MET comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 207 and SEQ ID NO. 209; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 208 and SEQ ID NO. 210. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and c-MET comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 207 and a DVD light chain amino acid sequence of SEQ ID NO: 208. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and c-MET has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 209 and a DVD light chain amino acid sequence of SEQ ID NO: 210.
In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and IGF1,2 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 211 and SEQ ID NO. 213; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 212 and SEQ ID NO. 214. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and IGF1,2 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 211 and a DVD light chain amino acid sequence of SEQ ID NO: 212. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and IGF1,2 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 213 and a DVD light chain amino acid sequence of SEQ ID NO: 214.
In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and IGF1R comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 215 and SEQ ID NO. 217; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 216 and SEQ ID NO. 218. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and IGF1R comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 215 and a DVD light chain amino acid sequence of SEQ ID NO: 216. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and IGF1R has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 217 and a DVD light chain amino acid sequence of SEQ ID NO: 218.
In an embodiment, the binding protein capable of binding RON (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 219 and SEQ ID NO. 221; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 220 and SEQ ID NO. 222. In an embodiment, the binding protein capable of binding RON (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 219 and a DVD light chain amino acid sequence of SEQ ID NO: 220. In another embodiment, the binding protein capable of binding RON (seq. 1) and HGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 221 and a DVD light chain amino acid sequence of SEQ ID NO: 222.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and EGFR (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 223 and SEQ ID NO. 225; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 224 and SEQ ID NO. 226. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and EGFR (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 223 and a DVD light chain amino acid sequence of SEQ ID NO: 224. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and EGFR (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 225 and a DVD light chain amino acid sequence of SEQ ID NO: 226.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and HER-2 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 227 and SEQ ID NO. 229; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 228 and SEQ ID NO. 230. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and HER-2 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 227 and a DVD light chain amino acid sequence of SEQ ID NO: 228. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and HER-2 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 229 and a DVD light chain amino acid sequence of SEQ ID NO: 230.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and CD-20 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 231 and SEQ ID NO. 233; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 232 and SEQ ID NO. 234. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and CD-20 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 231 and a DVD light chain amino acid sequence of SEQ ID NO: 232. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and CD-20 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 233 and a DVD light chain amino acid sequence of SEQ ID NO: 234.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and IGF1,2 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 235 and SEQ ID NO. 237; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 236 and SEQ ID NO. 238. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and IGF1,2 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 235 and a DVD light chain amino acid sequence of SEQ ID NO: 236. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and IGF1,2 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 237 and a DVD light chain amino acid sequence of SEQ ID NO: 238.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 239 and SEQ ID NO. 241; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 240 and SEQ ID NO. 242. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 239 and a DVD light chain amino acid sequence of SEQ ID NO: 240. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 241 and a DVD light chain amino acid sequence of SEQ ID NO: 242.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 243 and SEQ ID NO. 245; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 244 and SEQ ID NO. 246. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 243 and a DVD light chain amino acid sequence of SEQ ID NO: 244. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 245 and a DVD light chain amino acid sequence of SEQ ID NO: 246.
In a second embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 247 and SEQ ID NO. 249; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 248 and SEQ ID NO. 250. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 247 and a DVD light chain amino acid sequence of SEQ ID NO: 248. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 249 and a DVD light chain amino acid sequence of SEQ ID NO: 250.
In a third embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 251 and SEQ ID NO. 253; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 252 and SEQ ID NO. 254. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 251 and a DVD light chain amino acid sequence of SEQ ID NO: 252. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 253 and a DVD light chain amino acid sequence of SEQ ID NO: 254.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 255 and SEQ ID NO. 257; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 256 and SEQ ID NO. 258. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 255 and a DVD light chain amino acid sequence of SEQ ID NO: 256. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and HGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 257 and a DVD light chain amino acid sequence of SEQ ID NO: 258.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 259 and SEQ ID NO. 261; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 260 and SEQ ID NO. 262. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 259 and a DVD light chain amino acid sequence of SEQ ID NO: 260. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and RON (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 261 and a DVD light chain amino acid sequence of SEQ ID NO: 262.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and NRP1 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 263 and SEQ ID NO. 265; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 264 and SEQ ID NO. 266. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and NRP1 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 263 and a DVD light chain amino acid sequence of SEQ ID NO: 264. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and NRP1 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 265 and a DVD light chain amino acid sequence of SEQ ID NO: 266.
In an embodiment, the binding protein capable of binding RON (seq. 2) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 267 and SEQ ID NO. 269; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 268 and SEQ ID NO. 270. In an embodiment, the binding protein capable of binding RON (seq. 2) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 267 and a DVD light chain amino acid sequence of SEQ ID NO: 268. In another embodiment, the binding protein capable of binding RON (seq. 2) and HGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 269 and a DVD light chain amino acid sequence of SEQ ID NO: 270.
In an embodiment, the binding protein capable of binding RON (seq. 2) and EGFR (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 271 and SEQ ID NO. 273; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 272 and SEQ ID NO. 274. In an embodiment, the binding protein capable of binding RON (seq. 2) and EGFR (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 271 and a DVD light chain amino acid sequence of SEQ ID NO: 272. In another embodiment, the binding protein capable of binding RON (seq. 2) and EGFR (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 273 and a DVD light chain amino acid sequence of SEQ ID NO: 274.
In an embodiment, the binding protein capable of binding RON (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 275 and SEQ ID NO. 277; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 276 and SEQ ID NO. 278. In an embodiment, the binding protein capable of binding RON (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 275 and a DVD light chain amino acid sequence of SEQ ID NO: 276. In another embodiment, the binding protein capable of binding RON (seq. 2) and VEGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 277 and a DVD light chain amino acid sequence of SEQ ID NO: 278.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and HER-2 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 279 and SEQ ID NO. 281; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 280 and SEQ ID NO. 282. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and HER-2 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 279 and a DVD light chain amino acid sequence of SEQ ID NO: 280. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and HER-2 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 281 and a DVD light chain amino acid sequence of SEQ ID NO: 282.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and CD-3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 283 and SEQ ID NO. 285; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 284 and SEQ ID NO. 286. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and CD-3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 283 and a DVD light chain amino acid sequence of SEQ ID NO: 284. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and CD-3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 285 and a DVD light chain amino acid sequence of SEQ ID NO: 286.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and IGF1R comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 287 and SEQ ID NO. 289; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 288 and SEQ ID NO. 290. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and IGF1R comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 287 and a DVD light chain amino acid sequence of SEQ ID NO: 288. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and IGF1R has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 289 and a DVD light chain amino acid sequence of SEQ ID NO: 290.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 291 and SEQ ID NO. 293; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 292 and SEQ ID NO. 294. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and RON (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 291 and a DVD light chain amino acid sequence of SEQ ID NO: 292. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and RON (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 293 and a DVD light chain amino acid sequence of SEQ ID NO: 294.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and RON (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 295 and SEQ ID NO. 297; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 296 and SEQ ID NO. 298. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and RON (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 295 and a DVD light chain amino acid sequence of SEQ ID NO: 296. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and RON (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 297 and a DVD light chain amino acid sequence of SEQ ID NO: 298.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 299 and SEQ ID NO. 301; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 300 and SEQ ID NO. 302. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and HGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 299 and a DVD light chain amino acid sequence of SEQ ID NO: 300. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and HGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 301 and a DVD light chain amino acid sequence of SEQ ID NO: 302.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and c-MET comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 303 and SEQ ID NO. 305; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 304 and SEQ ID NO. 306. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and c-MET comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 303 and a DVD light chain amino acid sequence of SEQ ID NO: 304. In another embodiment, the binding protein capable of binding EGFR (seq. 1 and c-MET has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 305 and a DVD light chain amino acid sequence of SEQ ID NO: 306.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 307 and SEQ ID NO. 309; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 308 and SEQ ID NO. 310. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 307 and a DVD light chain amino acid sequence of SEQ ID NO: 308. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and VEGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 309 and a DVD light chain amino acid sequence of SEQ ID NO: 310.
In an embodiment, the binding protein capable of binding NRP1 (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 311 and SEQ ID NO. 313; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 312 and SEQ ID NO. 314. In an embodiment, the binding protein capable of binding NRP1 (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 311 and a DVD light chain amino acid sequence of SEQ ID NO: 312. In another embodiment, the binding protein capable of binding NRP1 (seq. 2) and VEGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 313 and a DVD light chain amino acid sequence of SEQ ID NO: 314.
In an embodiment, the binding protein capable of binding CD-3 (seq. 2) and CD-20 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 315 and SEQ ID NO. 317; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 316 and SEQ ID NO. 318. In an embodiment, the binding protein capable of binding CD-3 (seq. 2) and CD-20 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 315 and a DVD light chain amino acid sequence of SEQ ID NO: 316. In another embodiment, the binding protein capable of binding CD-3 (seq. 2) and CD-20 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 317 and a DVD light chain amino acid sequence of SEQ ID NO: 318.
In an embodiment, the binding protein capable of binding CD-3 (seq. 2) and HER-2 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 319 and SEQ ID NO. 321; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 320 and SEQ ID NO. 322. In an embodiment, the binding protein capable of binding CD-3 (seq. 2) and HER-2 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 319 and a DVD light chain amino acid sequence of SEQ ID NO: 320. In another embodiment, the binding protein capable of binding CD-3 (seq. 2) and HER-2 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 321 and a DVD light chain amino acid sequence of SEQ ID NO: 322.
In an embodiment, the binding protein capable of binding CD-3 (seq. 2) and CD-19 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 323 and SEQ ID NO. 325; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 324 and SEQ ID NO. 326. In an embodiment, the binding protein capable of binding CD-3 (seq. 2) and CD-19 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 323 and a DVD light chain amino acid sequence of SEQ ID NO: 324. In another embodiment, the binding protein capable of binding CD-3 (seq. 2) and CD-19 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 325 and a DVD light chain amino acid sequence of SEQ ID NO: 326.
In an embodiment, the binding protein capable of binding CD-3 (seq. 2) and EGFR (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 327 and SEQ ID NO. 329; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 328 and SEQ ID NO. 330. In an embodiment, the binding protein capable of binding CD-3 (seq. 2) and EGFR (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 327 and a DVD light chain amino acid sequence of SEQ ID NO: 328. In another embodiment, the binding protein capable of binding CD-3 (seq. 2) and EGFR (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 329 and a DVD light chain amino acid sequence of SEQ ID NO: 330.
In an embodiment, the binding protein capable of binding CD-3 (seq. 2) and EGFR (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 331 and SEQ ID NO. 333; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 332 and SEQ ID NO. 334. In an embodiment, the binding protein capable of binding CD-3 (seq. 2) and EGFR (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 331 and a DVD light chain amino acid sequence of SEQ ID NO: 332. In another embodiment, the binding protein capable of binding CD-3 (seq. 2) and EGFR (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 333 and a DVD light chain amino acid sequence of SEQ ID NO: 334.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and IGF1,2 comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 335 and SEQ ID NO. 337; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 336 and SEQ ID NO. 338. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and IGF1,2 comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 335 and a DVD light chain amino acid sequence of SEQ ID NO: 336. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and IGF1,2 has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 337 and a DVD light chain amino acid sequence of SEQ ID NO: 338.
In an embodiment, the binding protein capable of binding DLL-4 (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 339 and SEQ ID NO. 341; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 340 and SEQ ID NO. 342. In an embodiment, the binding protein capable of binding DLL-4 (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 339 and a DVD light chain amino acid sequence of SEQ ID NO: 340. In another embodiment, the binding protein capable of binding DLL-4 (seq. 1) and PLGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 341 and a DVD light chain amino acid sequence of SEQ ID NO: 342.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 343 and SEQ ID NO. 345; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 344 and SEQ ID NO. 346. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 343 and a DVD light chain amino acid sequence of SEQ ID NO: 344. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 345 and a DVD light chain amino acid sequence of SEQ ID NO: 346.
In a second embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 347 and SEQ ID NO. 349; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 348 and SEQ ID NO. 350. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 347 and a DVD light chain amino acid sequence of SEQ ID NO: 348. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 349 and a DVD light chain amino acid sequence of SEQ ID NO: 350.
In a third embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 351 and SEQ ID NO. 353; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 352 and SEQ ID NO. 354. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 351 and a DVD light chain amino acid sequence of SEQ ID NO: 352. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 353 and a DVD light chain amino acid sequence of SEQ ID NO: 354.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 355 and SEQ ID NO. 357; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 356 and SEQ ID NO. 358. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 355 and a DVD light chain amino acid sequence of SEQ ID NO: 356. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 357 and a DVD light chain amino acid sequence of SEQ ID NO: 358.
In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 359 and SEQ ID NO. 361; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 360 and SEQ ID NO. 362. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 359 and a DVD light chain amino acid sequence of SEQ ID NO: 360. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 361 and a DVD light chain amino acid sequence of SEQ ID NO: 362.
In a second embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 363 and SEQ ID NO. 365; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 364 and SEQ ID NO. 366. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 363 and a DVD light chain amino acid sequence of SEQ ID NO: 364. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 365 and a DVD light chain amino acid sequence of SEQ ID NO: 366.
In a third embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 367 and SEQ ID NO. 369; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 368 and SEQ ID NO. 370. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 367 and a DVD light chain amino acid sequence of SEQ ID NO: 368. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 369 and a DVD light chain amino acid sequence of SEQ ID NO: 370.
In a fourth embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 371 and SEQ ID NO. 373; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 372 and SEQ ID NO. 374. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 371 and a DVD light chain amino acid sequence of SEQ ID NO: 372. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 373 and a DVD light chain amino acid sequence of SEQ ID NO: 374.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 375 and SEQ ID NO. 377; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 376 and SEQ ID NO. 378. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 375 and a DVD light chain amino acid sequence of SEQ ID NO: 376. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and ErbB3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 377 and a DVD light chain amino acid sequence of SEQ ID NO: 378.
In an embodiment, the binding protein capable of binding HGF (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 379 and SEQ ID NO. 381; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 380 and SEQ ID NO. 382. In an embodiment, the binding protein capable of binding HGF (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 379 and a DVD light chain amino acid sequence of SEQ ID NO: 380. In another embodiment, the binding protein capable of binding HGF (seq. 1) and ErbB3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 381 and a DVD light chain amino acid sequence of SEQ ID NO: 382.
In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 383 and SEQ ID NO. 385; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 384 and SEQ ID NO. 386. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 383 and a DVD light chain amino acid sequence of SEQ ID NO: 384. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 385 and a DVD light chain amino acid sequence of SEQ ID NO: 386.
In a second embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 387 and SEQ ID NO. 389; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 388 and SEQ ID NO. 390. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 387 and a DVD light chain amino acid sequence of SEQ ID NO: 388. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 389 and a DVD light chain amino acid sequence of SEQ ID NO: 390.
In a third embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 391 and SEQ ID NO. 393; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 392 and SEQ ID NO. 394. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 391 and a DVD light chain amino acid sequence of SEQ ID NO: 392. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 393 and a DVD light chain amino acid sequence of SEQ ID NO: 394.
In a fourth embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 395 and SEQ ID NO. 397; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 396 and SEQ ID NO. 398. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 395 and a DVD light chain amino acid sequence of SEQ ID NO: 396. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 397 and a DVD light chain amino acid sequence of SEQ ID NO: 398.
In an embodiment, the binding protein capable of binding EGFR (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 399 and SEQ ID NO. 401; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 400 and SEQ ID NO. 402. In an embodiment, the binding protein capable of binding EGFR (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 399 and a DVD light chain amino acid sequence of SEQ ID NO: 400. In another embodiment, the binding protein capable of binding EGFR (seq. 1) and ErbB3 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 401 and a DVD light chain amino acid sequence of SEQ ID NO: 402.
In an embodiment, the binding protein capable of binding HGF (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 403 and SEQ ID NO. 405; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 404 and SEQ ID NO. 406. In an embodiment, the binding protein capable of binding HGF (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 403 and a DVD light chain amino acid sequence of SEQ ID NO: 404. In another embodiment, the binding protein capable of binding HGF (seq. 1) and ErbB3 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 405 and a DVD light chain amino acid sequence of SEQ ID NO: 406.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 407 and SEQ ID NO. 409; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 408 and SEQ ID NO. 410. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 407 and a DVD light chain amino acid sequence of SEQ ID NO: 408. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 409 and a DVD light chain amino acid sequence of SEQ ID NO: 410.
In a second embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 411 and SEQ ID NO. 413; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 412 and SEQ ID NO. 414. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 411 and a DVD light chain amino acid sequence of SEQ ID NO: 412. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 413 and a DVD light chain amino acid sequence of SEQ ID NO: 414.
In a third embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 415 and SEQ ID NO. 417; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 416 and SEQ ID NO. 418. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 415 and a DVD light chain amino acid sequence of SEQ ID NO: 416. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 417 and a DVD light chain amino acid sequence of SEQ ID NO: 418.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 419 and SEQ ID NO. 421; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 420 and SEQ ID NO. 422. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 419 and a DVD light chain amino acid sequence of SEQ ID NO: 420. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 421 and a DVD light chain amino acid sequence of SEQ ID NO: 422.
In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 423 and SEQ ID NO. 425; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 424 and SEQ ID NO. 426. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 423 and a DVD light chain amino acid sequence of SEQ ID NO: 424. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 425 and a DVD light chain amino acid sequence of SEQ ID NO: 426.
In a second embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 427 and SEQ ID NO. 429; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 428 and SEQ ID NO. 430. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 427 and a DVD light chain amino acid sequence of SEQ ID NO: 428. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 429 and a DVD light chain amino acid sequence of SEQ ID NO: 430.
In a third embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 431 and SEQ ID NO. 433; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 432 and SEQ ID NO. 434. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 431 and a DVD light chain amino acid sequence of SEQ ID NO: 432. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 433 and a DVD light chain amino acid sequence of SEQ ID NO: 434.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 435 and SEQ ID NO. 437; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 436 and SEQ ID NO. 438. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 435 and a DVD light chain amino acid sequence of SEQ ID NO: 436. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 437 and a DVD light chain amino acid sequence of SEQ ID NO: 438.
In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 439 and SEQ ID NO. 441; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 440 and SEQ ID NO. 442. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 439 and a DVD light chain amino acid sequence of SEQ ID NO: 440. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 441 and a DVD light chain amino acid sequence of SEQ ID NO: 442.
In a second embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 443 and SEQ ID NO. 445; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 444 and SEQ ID NO. 446. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 443 and a DVD light chain amino acid sequence of SEQ ID NO: 444. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 445 and a DVD light chain amino acid sequence of SEQ ID NO: 446.
In a third embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 447 and SEQ ID NO. 449; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 448 and SEQ ID NO. 450. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 447 and a DVD light chain amino acid sequence of SEQ ID NO: 448. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 449 and a DVD light chain amino acid sequence of SEQ ID NO: 450.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 451 and SEQ ID NO. 453; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 452 and SEQ ID NO. 454. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 451 and a DVD light chain amino acid sequence of SEQ ID NO: 452. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 453 and a DVD light chain amino acid sequence of SEQ ID NO: 454.
In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 455 and SEQ ID NO. 457; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 456 and SEQ ID NO. 458. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 455 and a DVD light chain amino acid sequence of SEQ ID NO: 456. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 457 and a DVD light chain amino acid sequence of SEQ ID NO: 458.
In a second embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 459 and SEQ ID NO. 461; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 460 and SEQ ID NO. 462. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 459 and a DVD light chain amino acid sequence of SEQ ID NO: 460. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 461 and a DVD light chain amino acid sequence of SEQ ID NO: 462.
In a third embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 463 and SEQ ID NO. 465; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 464 and SEQ ID NO. 466. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 463 and a DVD light chain amino acid sequence of SEQ ID NO: 464. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 465 and a DVD light chain amino acid sequence of SEQ ID NO: 466.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 467 and SEQ ID NO. 469; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 468 and SEQ ID NO. 470. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 467 and a DVD light chain amino acid sequence of SEQ ID NO: 468. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 469 and a DVD light chain amino acid sequence of SEQ ID NO: 470.
In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 471 and SEQ ID NO. 473; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 472 and SEQ ID NO. 474. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 471 and a DVD light chain amino acid sequence of SEQ ID NO: 472. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 473 and a DVD light chain amino acid sequence of SEQ ID NO: 474.
In a second embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 475 and SEQ ID NO. 477; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 476 and SEQ ID NO. 478. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 475 and a DVD light chain amino acid sequence of SEQ ID NO: 476. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 477 and a DVD light chain amino acid sequence of SEQ ID NO: 478.
In a third embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 479 and SEQ ID NO. 481; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 480 and SEQ ID NO. 482. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 479 and a DVD light chain amino acid sequence of SEQ ID NO: 480. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 481 and a DVD light chain amino acid sequence of SEQ ID NO: 482.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 483 and SEQ ID NO. 485; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 484 and SEQ ID NO. 486. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 483 and a DVD light chain amino acid sequence of SEQ ID NO: 484. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 485 and a DVD light chain amino acid sequence of SEQ ID NO: 486.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 487 and SEQ ID NO. 489; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 488 and SEQ ID NO. 490. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 487 and a DVD light chain amino acid sequence of SEQ ID NO: 488. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 489 and a DVD light chain amino acid sequence of SEQ ID NO: 490.
In a second embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 491 and SEQ ID NO. 493; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 492 and SEQ ID NO. 494. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 491 and a DVD light chain amino acid sequence of SEQ ID NO: 492. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 493 and a DVD light chain amino acid sequence of SEQ ID NO: 494.
In a third embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 495 and SEQ ID NO. 497; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 496 and SEQ ID NO. 498. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 495 and a DVD light chain amino acid sequence of SEQ ID NO: 496. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 497 and a DVD light chain amino acid sequence of SEQ ID NO: 498.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 499 and SEQ ID NO. 501; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 500 and SEQ ID NO. 502. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 499 and a DVD light chain amino acid sequence of SEQ ID NO: 500. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 501 and a DVD light chain amino acid sequence of SEQ ID NO: 502.
In a second embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 503 and SEQ ID NO. 505; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 504 and SEQ ID NO. 506. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 503 and a DVD light chain amino acid sequence of SEQ ID NO: 504. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 505 and a DVD light chain amino acid sequence of SEQ ID NO: 506.
In a third embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 507 and SEQ ID NO. 509; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 508 and SEQ ID NO. 510. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 507 and a DVD light chain amino acid sequence of SEQ ID NO: 508. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 509 and a DVD light chain amino acid sequence of SEQ ID NO: 510.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 511 and SEQ ID NO. 513; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 512 and SEQ ID NO. 514. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 511 and a DVD light chain amino acid sequence of SEQ ID NO: 512. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 513 and a DVD light chain amino acid sequence of SEQ ID NO: 514.
In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 515 and SEQ ID NO. 517; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 516 and SEQ ID NO. 518. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 515 and a DVD light chain amino acid sequence of SEQ ID NO: 516. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 517 and a DVD light chain amino acid sequence of SEQ ID NO: 518.
In a second embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 519 and SEQ ID NO. 521; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 520 and SEQ ID NO. 522. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 519 and a DVD light chain amino acid sequence of SEQ ID NO: 520. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 521 and a DVD light chain amino acid sequence of SEQ ID NO: 522.
In a third embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 523 and SEQ ID NO. 525; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 524 and SEQ ID NO. 526. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 523 and a DVD light chain amino acid sequence of SEQ ID NO: 524. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 525 and a DVD light chain amino acid sequence of SEQ ID NO: 526.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 527 and SEQ ID NO. 529; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 528 and SEQ ID NO. 530. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 527 and a DVD light chain amino acid sequence of SEQ ID NO: 528. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 529 and a DVD light chain amino acid sequence of SEQ ID NO: 530.
In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 531 and SEQ ID NO. 533; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 532 and SEQ ID NO. 534. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 531 and a DVD light chain amino acid sequence of SEQ ID NO: 532. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 533 and a DVD light chain amino acid sequence of SEQ ID NO: 534.
In a second embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 535 and SEQ ID NO. 537; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 536 and SEQ ID NO. 538. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 535 and a DVD light chain amino acid sequence of SEQ ID NO: 536. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 537 and a DVD light chain amino acid sequence of SEQ ID NO: 538.
In a third embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 539 and SEQ ID NO. 541; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 540 and SEQ ID NO. 542. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 539 and a DVD light chain amino acid sequence of SEQ ID NO: 540. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 541 and a DVD light chain amino acid sequence of SEQ ID NO: 542.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 543 and SEQ ID NO. 545; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 544 and SEQ ID NO. 546. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 543 and a DVD light chain amino acid sequence of SEQ ID NO: 544. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 545 and a DVD light chain amino acid sequence of SEQ ID NO: 546.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 547 and SEQ ID NO. 549; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 548 and SEQ ID NO. 550. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 547 and a DVD light chain amino acid sequence of SEQ ID NO: 548. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 549 and a DVD light chain amino acid sequence of SEQ ID NO: 550.
In a second embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 551 and SEQ ID NO. 553; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 552 and SEQ ID NO. 554. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 551 and a DVD light chain amino acid sequence of SEQ ID NO: 552. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 553 and a DVD light chain amino acid sequence of SEQ ID NO: 554.
In a third embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 555 and SEQ ID NO. 557; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 556 and SEQ ID NO. 558. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 555 and a DVD light chain amino acid sequence of SEQ ID NO: 556. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 557 and a DVD light chain amino acid sequence of SEQ ID NO: 558.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 559 and SEQ ID NO. 561; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 560 and SEQ ID NO. 562. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 559 and a DVD light chain amino acid sequence of SEQ ID NO: 560. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 561 and a DVD light chain amino acid sequence of SEQ ID NO: 562.
In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 563 and SEQ ID NO. 565; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 564 and SEQ ID NO. 566. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 563 and a DVD light chain amino acid sequence of SEQ ID NO: 564. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 565 and a DVD light chain amino acid sequence of SEQ ID NO: 566.
In a second embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 567 and SEQ ID NO. 569; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 568 and SEQ ID NO. 570. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 567 and a DVD light chain amino acid sequence of SEQ ID NO: 568. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 569 and a DVD light chain amino acid sequence of SEQ ID NO: 570.
In a third embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 571 and SEQ ID NO. 573; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 572 and SEQ ID NO. 574. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 571 and a DVD light chain amino acid sequence of SEQ ID NO: 572. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 573 and a DVD light chain amino acid sequence of SEQ ID NO: 574.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 575 and SEQ ID NO. 577; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 576 and SEQ ID NO. 578. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 575 and a DVD light chain amino acid sequence of SEQ ID NO: 576. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 577 and a DVD light chain amino acid sequence of SEQ ID NO: 578.
In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 579 and SEQ ID NO. 581; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 580 and SEQ ID NO. 582. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 579 and a DVD light chain amino acid sequence of SEQ ID NO: 580. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 581 and a DVD light chain amino acid sequence of SEQ ID NO: 582.
In a second embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 583 and SEQ ID NO. 585; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 584 and SEQ ID NO. 586. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 583 and a DVD light chain amino acid sequence of SEQ ID NO: 584. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 585 and a DVD light chain amino acid sequence of SEQ ID NO: 586.
In a third embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 587 and SEQ ID NO. 589; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 588 and SEQ ID NO. 590. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 587 and a DVD light chain amino acid sequence of SEQ ID NO: 588. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 589 and a DVD light chain amino acid sequence of SEQ ID NO: 590.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 591 and SEQ ID NO. 593; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 592 and SEQ ID NO. 594. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 591 and a DVD light chain amino acid sequence of SEQ ID NO: 592. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and DLL-4 (seq. 4) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 593 and a DVD light chain amino acid sequence of SEQ ID NO: 594.
In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 595 and SEQ ID NO. 597; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 596 and SEQ ID NO. 598. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 595 and a DVD light chain amino acid sequence of SEQ ID NO: 596. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 597 and a DVD light chain amino acid sequence of SEQ ID NO: 598.
In a second embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 599 and SEQ ID NO. 601; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 600 and SEQ ID NO. 602. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 599 and a DVD light chain amino acid sequence of SEQ ID NO: 600. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 601 and a DVD light chain amino acid sequence of SEQ ID NO: 602.
In a third embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 603 and SEQ ID NO. 605; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 604 and SEQ ID NO. 606. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 603 and a DVD light chain amino acid sequence of SEQ ID NO: 604. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 605 and a DVD light chain amino acid sequence of SEQ ID NO: 606.
In a fourth embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 607 and SEQ ID NO. 609; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 608 and SEQ ID NO. 610. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 607 and a DVD light chain amino acid sequence of SEQ ID NO: 608. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 609 and a DVD light chain amino acid sequence of SEQ ID NO: 610.
In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 611 and SEQ ID NO. 613; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 612 and SEQ ID NO. 614. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 611 and a DVD light chain amino acid sequence of SEQ ID NO: 612. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 613 and a DVD light chain amino acid sequence of SEQ ID NO: 614.
In a second embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 615 and SEQ ID NO. 617; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 616 and SEQ ID NO. 618. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 615 and a DVD light chain amino acid sequence of SEQ ID NO: 616. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 617 and a DVD light chain amino acid sequence of SEQ ID NO: 618.
In a third embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 619 and SEQ ID NO. 621; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 622 and SEQ ID NO. 624. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 619 and a DVD light chain amino acid sequence of SEQ ID NO: 620. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 621 and a DVD light chain amino acid sequence of SEQ ID NO: 622.
In a fourth embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 623 and SEQ ID NO. 625; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 624 and SEQ ID NO. 626. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 623 and a DVD light chain amino acid sequence of SEQ ID NO: 624. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 625 and a DVD light chain amino acid sequence of SEQ ID NO: 626.
In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 627 and SEQ ID NO. 629; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 628 and SEQ ID NO. 630. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 627 and a DVD light chain amino acid sequence of SEQ ID NO: 628. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 629 and a DVD light chain amino acid sequence of SEQ ID NO: 630.
In a second embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 631 and SEQ ID NO. 633; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 632 and SEQ ID NO. 634. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 631 and a DVD light chain amino acid sequence of SEQ ID NO: 632. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 633 and a DVD light chain amino acid sequence of SEQ ID NO: 634.
In a third embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 635 and SEQ ID NO. 637; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 636 and SEQ ID NO. 638. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 635 and a DVD light chain amino acid sequence of SEQ ID NO: 636. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 637 and a DVD light chain amino acid sequence of SEQ ID NO: 638.
In a fourth embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 639 and SEQ ID NO. 641; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 640 and SEQ ID NO. 642. In an embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 639 and a DVD light chain amino acid sequence of SEQ ID NO: 640. In another embodiment, the binding protein capable of binding EGFR (seq. 2) and ErbB3 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 641 and a DVD light chain amino acid sequence of SEQ ID NO: 642.
In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 643 and SEQ ID NO. 645; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 644 and SEQ ID NO. 646. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 643 and a DVD light chain amino acid sequence of SEQ ID NO: 644. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 645 and a DVD light chain amino acid sequence of SEQ ID NO: 646.
In a second embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 647 and SEQ ID NO. 649; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 648 and SEQ ID NO. 650. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 647 and a DVD light chain amino acid sequence of SEQ ID NO: 648. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 649 and a DVD light chain amino acid sequence of SEQ ID NO: 650.
In a third embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 651 and SEQ ID NO. 653; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 652 and SEQ ID NO. 654. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 651 and a DVD light chain amino acid sequence of SEQ ID NO: 652. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 653 and a DVD light chain amino acid sequence of SEQ ID NO: 654.
In a fourth embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 655 and SEQ ID NO. 657; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 656 and SEQ ID NO. 658. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 655 and a DVD light chain amino acid sequence of SEQ ID NO: 656. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and ErbB3 (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 657 and a DVD light chain amino acid sequence of SEQ ID NO: 658.
In an embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 659 and SEQ ID NO. 661; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 660 and SEQ ID NO. 662. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 659 and a DVD light chain amino acid sequence of SEQ ID NO: 660. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 661 and a DVD light chain amino acid sequence of SEQ ID NO: 662.
In a second embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 663 and SEQ ID NO. 665; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 664 and SEQ ID NO. 666. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 663 and a DVD light chain amino acid sequence of SEQ ID NO: 664. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 665 and a DVD light chain amino acid sequence of SEQ ID NO: 666.
In a third embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 667 and SEQ ID NO. 669; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 668 and SEQ ID NO. 670. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 667 and a DVD light chain amino acid sequence of SEQ ID NO: 668. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 669 and a DVD light chain amino acid sequence of SEQ ID NO: 670.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 671 and SEQ ID NO. 673; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 672 and SEQ ID NO. 674. In an embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 671 and a DVD light chain amino acid sequence of SEQ ID NO: 672. In another embodiment, the binding protein capable of binding VEGF (seq. 1) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 673 and a DVD light chain amino acid sequence of SEQ ID NO: 674.
In an embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 675 and SEQ ID NO. 677; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 676 and SEQ ID NO. 678. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 675 and a DVD light chain amino acid sequence of SEQ ID NO: 676. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 677 and a DVD light chain amino acid sequence of SEQ ID NO: 678.
In a second embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 679 and SEQ ID NO. 681; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 680 and SEQ ID NO. 682. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 679 and a DVD light chain amino acid sequence of SEQ ID NO: 680. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 681 and a DVD light chain amino acid sequence of SEQ ID NO: 682.
In a third embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 683 and SEQ ID NO. 685; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 684 and SEQ ID NO. 686. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 683 and a DVD light chain amino acid sequence of SEQ ID NO: 684. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 685 and a DVD light chain amino acid sequence of SEQ ID NO: 686.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 687 and SEQ ID NO. 689; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 688 and SEQ ID NO. 690. In an embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 687 and a DVD light chain amino acid sequence of SEQ ID NO: 688. In another embodiment, the binding protein capable of binding VEGF (seq. 2) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 689 and a DVD light chain amino acid sequence of SEQ ID NO: 690.
In an embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 691 and SEQ ID NO. 693; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 692 and SEQ ID NO. 694. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 691 and a DVD light chain amino acid sequence of SEQ ID NO: 692. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 693 and a DVD light chain amino acid sequence of SEQ ID NO: 694
In a second embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 695 and SEQ ID NO. 697; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 696 and SEQ ID NO. 698. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 695 and a DVD light chain amino acid sequence of SEQ ID NO: 696. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 697 and a DVD light chain amino acid sequence of SEQ ID NO: 698.
In a third embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 699 and SEQ ID NO. 701; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 700 and SEQ ID NO. 702. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 699 and a DVD light chain amino acid sequence of SEQ ID NO: 700. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 701 and a DVD light chain amino acid sequence of SEQ ID NO: 702.
In a fourth embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 703 and SEQ ID NO. 705; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 704 and SEQ ID NO. 706. In an embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 703 and a DVD light chain amino acid sequence of SEQ ID NO: 704. In another embodiment, the binding protein capable of binding VEGF (seq. 3) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 705 and a DVD light chain amino acid sequence of SEQ ID NO: 706.
In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 707 and SEQ ID NO. 709; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 708 and SEQ ID NO. 710. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 707 and a DVD light chain amino acid sequence of SEQ ID NO: 708. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 709 and a DVD light chain amino acid sequence of SEQ ID NO: 710.
In a second embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 711 and SEQ ID NO. 713; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 712 and SEQ ID NO. 714. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 711 and a DVD light chain amino acid sequence of SEQ ID NO: 712. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 713 and a DVD light chain amino acid sequence of SEQ ID NO: 714.
In a third embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 715 and SEQ ID NO. 717; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 716 and SEQ ID NO. 718. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 715 and a DVD light chain amino acid sequence of SEQ ID NO: 716. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 717 and a DVD light chain amino acid sequence of SEQ ID NO: 718.
In a fourth embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 719 and SEQ ID NO. 721; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 720 and SEQ ID NO. 722. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 719 and a DVD light chain amino acid sequence of SEQ ID NO: 720. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 721 and a DVD light chain amino acid sequence of SEQ ID NO: 722.
In an embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 723 and SEQ ID NO. 725; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 724 and SEQ ID NO. 726. In an embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 723 and a DVD light chain amino acid sequence of SEQ ID NO: 724. In another embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 725 and a DVD light chain amino acid sequence of SEQ ID NO: 726.
In a second embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 727 and SEQ ID NO. 729; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 728 and SEQ ID NO. 730. In an embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 727 and a DVD light chain amino acid sequence of SEQ ID NO: 728. In another embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 729 and a DVD light chain amino acid sequence of SEQ ID NO: 730.
In a third embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 731 and SEQ ID NO. 733; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 732 and SEQ ID NO. 734. In an embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 731 and a DVD light chain amino acid sequence of SEQ ID NO: 732. In another embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 733 and a DVD light chain amino acid sequence of SEQ ID NO: 734.
In a fourth embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 735 and SEQ ID NO. 737; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 736 and SEQ ID NO. 738. In an embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 735 and a DVD light chain amino acid sequence of SEQ ID NO: 736. In another embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 737 and a DVD light chain amino acid sequence of SEQ ID NO: 738.
In an embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 739 and SEQ ID NO. 741; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 740 and SEQ ID NO. 742. In an embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 739 and a DVD light chain amino acid sequence of SEQ ID NO: 740. In another embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 741 and a DVD light chain amino acid sequence of SEQ ID NO: 742.
In a second embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 743 and SEQ ID NO. 745; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 744 and SEQ ID NO. 746. In an embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 743 and a DVD light chain amino acid sequence of SEQ ID NO: 744. In another embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 745 and a DVD light chain amino acid sequence of SEQ ID NO: 746.
In a third embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 747 and SEQ ID NO. 749; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 748 and SEQ ID NO. 750. In an embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 747 and a DVD light chain amino acid sequence of SEQ ID NO: 748. In another embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 749 and a DVD light chain amino acid sequence of SEQ ID NO: 750.
In a fourth embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 751 and SEQ ID NO. 753; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 752 and SEQ ID NO. 754. In an embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 751 and a DVD light chain amino acid sequence of SEQ ID NO: 752. In another embodiment, the binding protein capable of binding PLGF (seq. 1) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 753 and a DVD light chain amino acid sequence of SEQ ID NO: 754.
In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 755 and SEQ ID NO. 757; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 756 and SEQ ID NO. 758. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 755 and a DVD light chain amino acid sequence of SEQ ID NO: 756. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 757 and a DVD light chain amino acid sequence of SEQ ID NO: 758.
In a second embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 759 and SEQ ID NO. 761; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 760 and SEQ ID NO. 762. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 759 and a DVD light chain amino acid sequence of SEQ ID NO: 760. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 761 and a DVD light chain amino acid sequence of SEQ ID NO: 762.
In a third embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 763 and SEQ ID NO. 765; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 764 and SEQ ID NO. 766. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 763 and a DVD light chain amino acid sequence of SEQ ID NO: 764. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 765 and a DVD light chain amino acid sequence of SEQ ID NO: 766.
In a fourth embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 767 and SEQ ID NO. 769; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 768 and SEQ ID NO. 770. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 767 and a DVD light chain amino acid sequence of SEQ ID NO: 768. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and PLGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 769 and a DVD light chain amino acid sequence of SEQ ID NO: 770.
In an embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 771 and SEQ ID NO. 773; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 772 and SEQ ID NO. 774. In an embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 771 and a DVD light chain amino acid sequence of SEQ ID NO: 772. In another embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 773 and a DVD light chain amino acid sequence of SEQ ID NO: 774.
In a second embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 775 and SEQ ID NO. 777; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 776 and SEQ ID NO. 778. In an embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 775 and a DVD light chain amino acid sequence of SEQ ID NO: 776. In another embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 777 and a DVD light chain amino acid sequence of SEQ ID NO: 778.
In a third embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 779 and SEQ ID NO. 781; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 780 and SEQ ID NO. 782. In an embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 779 and a DVD light chain amino acid sequence of SEQ ID NO: 780. In another embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 781 and a DVD light chain amino acid sequence of SEQ ID NO: 782.
In a fourth embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 783 and SEQ ID NO. 785; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 784 and SEQ ID NO. 786. In an embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 783 and a DVD light chain amino acid sequence of SEQ ID NO: 784. In another embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 785 and a DVD light chain amino acid sequence of SEQ ID NO: 786.
In an embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 787 and SEQ ID NO. 789; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 788 and SEQ ID NO. 790. In an embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 787 and a DVD light chain amino acid sequence of SEQ ID NO: 788. In another embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 789 and a DVD light chain amino acid sequence of SEQ ID NO: 790.
In a second embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 791 and SEQ ID NO. 793; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 792 and SEQ ID NO. 794. In an embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 791 and a DVD light chain amino acid sequence of SEQ ID NO: 792. In another embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 793 and a DVD light chain amino acid sequence of SEQ ID NO: 794.
In a third embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 795 and SEQ ID NO. 797; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 796 and SEQ ID NO. 798. In an embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 795 and a DVD light chain amino acid sequence of SEQ ID NO: 796. In another embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 797 and a DVD light chain amino acid sequence of SEQ ID NO: 798.
In a fourth embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 799 and SEQ ID NO. 801; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 800 and SEQ ID NO. 802. In an embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 799 and a DVD light chain amino acid sequence of SEQ ID NO: 800. In another embodiment, the binding protein capable of binding HGF (seq. 1) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 801 and a DVD light chain amino acid sequence of SEQ ID NO: 802.
In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 803 and SEQ ID NO. 805; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 804 and SEQ ID NO. 806. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 803 and a DVD light chain amino acid sequence of SEQ ID NO: 804. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 805 and a DVD light chain amino acid sequence of SEQ ID NO: 806.
In a second embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 807 and SEQ ID NO. 809; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 808 and SEQ ID NO. 810. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 807 and a DVD light chain amino acid sequence of SEQ ID NO: 808. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 809 and a DVD light chain amino acid sequence of SEQ ID NO: 810.
In a third embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 811 and SEQ ID NO. 813; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 812 and SEQ ID NO. 814. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 811 and a DVD light chain amino acid sequence of SEQ ID NO: 812. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 813 and a DVD light chain amino acid sequence of SEQ ID NO: 814.
In a fourth embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 815 and SEQ ID NO. 817; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 816 and SEQ ID NO. 818. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 815 and a DVD light chain amino acid sequence of SEQ ID NO: 816. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 1) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 817 and a DVD light chain amino acid sequence of SEQ ID NO: 818.
In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 819 and SEQ ID NO. 821; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 820 and SEQ ID NO. 822. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 819 and a DVD light chain amino acid sequence of SEQ ID NO: 820. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 821 and a DVD light chain amino acid sequence of SEQ ID NO: 822.
In a second embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 823 and SEQ ID NO. 825; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 824 and SEQ ID NO. 826. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 823 and a DVD light chain amino acid sequence of SEQ ID NO: 824. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 825 and a DVD light chain amino acid sequence of SEQ ID NO: 826.
In a third embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 827 and SEQ ID NO. 829; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 828 and SEQ ID NO. 830. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 827 and a DVD light chain amino acid sequence of SEQ ID NO: 828. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 829 and a DVD light chain amino acid sequence of SEQ ID NO: 830.
In a fourth embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 831 and SEQ ID NO. 833; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 832 and SEQ ID NO. 834. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 831 and a DVD light chain amino acid sequence of SEQ ID NO: 832. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 833 and a DVD light chain amino acid sequence of SEQ ID NO: 834.
In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 835 and SEQ ID NO. 837; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 836 and SEQ ID NO. 838. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 835 and a DVD light chain amino acid sequence of SEQ ID NO: 836. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 837 and a DVD light chain amino acid sequence of SEQ ID NO: 838.
In a second embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 839 and SEQ ID NO. 841; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 840 and SEQ ID NO. 842. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 839 and a DVD light chain amino acid sequence of SEQ ID NO: 840. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 841 and a DVD light chain amino acid sequence of SEQ ID NO: 842.
In a third embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 843 and SEQ ID NO. 845; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 844 and SEQ ID NO. 846. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 843 and a DVD light chain amino acid sequence of SEQ ID NO: 844. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 845 and a DVD light chain amino acid sequence of SEQ ID NO: 846.
In a fourth embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 847 and SEQ ID NO. 849; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 848 and SEQ ID NO. 850. In an embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 847 and a DVD light chain amino acid sequence of SEQ ID NO: 848. In another embodiment, the binding protein capable of binding HGF (seq. 2) and VEGF (seq. 3) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 849 and a DVD light chain amino acid sequence of SEQ ID NO: 850.
In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 851 and SEQ ID NO. 853; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 852 and SEQ ID NO. 854. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 851 and a DVD light chain amino acid sequence of SEQ ID NO: 852. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 853 and a DVD light chain amino acid sequence of SEQ ID NO: 854.
In a second embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 855 and SEQ ID NO. 857; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 856 and SEQ ID NO. 858. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 855 and a DVD light chain amino acid sequence of SEQ ID NO: 856. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 857 and a DVD light chain amino acid sequence of SEQ ID NO: 858.
In a third embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 859 and SEQ ID NO. 861; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 860 and SEQ ID NO. 862. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 859 and a DVD light chain amino acid sequence of SEQ ID NO: 860. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 861 and a DVD light chain amino acid sequence of SEQ ID NO: 862.
In a fourth embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) comprises a DVD heavy chain amino acid sequence selected from the group consisting of SEQ ID NO. 863 and SEQ ID NO. 865; and a DVD light chain amino acid sequence selected from the group consisting of SEQ ID NO. 864 and SEQ ID NO. 866. In an embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 863 and a DVD light chain amino acid sequence of SEQ ID NO: 864. In another embodiment, the binding protein capable of binding HER-2 (seq. 1) and HER-2 (seq. 2) has a reverse orientation and comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 865 and a DVD light chain amino acid sequence of SEQ ID NO: 866.
In another embodiment the invention provides a binding protein comprising a polypeptide chain, wherein said polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein; VD1 is a first heavy chain variable domain obtained from a first parent antibody or antigen binding portion thereof; VD2 is a second heavy chain variable domain obtained from a second parent antibody or antigen binding portion thereof; C is a heavy chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein said (X1)n is either present or absent; and (X2)n is an Fc region, wherein said (X2)n is either present or absent. In an embodiment, the Fc region is absent from the binding protein.
In another embodiment, the invention provides a binding protein comprising a polypeptide chain, wherein said polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first light chain variable domain obtained from a first parent antibody or antigen binding portion thereof; VD2 is a second light chain variable domain obtained from a second parent antibody or antigen binding portion thereof; C is a light chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein said (X1)n is either present or absent; and (X2)n does not comprise an Fc region, wherein said (X2)n is either present or absent. In an embodiment, (X2)n is absent from the binding protein.
In another embodiment the binding protein of the invention comprises first and second polypeptide chains, wherein said first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain variable domain obtained from a first parent antibody or antigen binding portion thereof; VD2 is a second heavy chain variable domain obtained from a second parent antibody or antigen binding portion thereof; C is a heavy chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein said (X1)n is either present or absent; and (X2)n is an Fc region, wherein said (X2)n is either present or absent; and wherein said second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variable domain obtained from a first parent antibody or antigen binding portion thereof; VD2 is a second light chain variable domain obtained from a second parent antibody or antigen binding portion thereof; C is a light chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein said (X1)n is either present or absent; and (X2)n does not comprise an Fc region, wherein said (X2)n is either present or absent. In another embodiment, the binding protein comprises two first polypeptide chains and two second polypeptide chains. In yet another embodiment, (X2)n is absent from the second polypeptide. In still another embodiment, the Fc region, if present in the first polypeptide is selected from the group consisting of native sequence Fc region and a variant sequence Fc region. In still another embodiment, the Fc region is selected from the group consisting of an Fc region from an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
In another embodiment the binding protein of the invention is a DVD-Ig capable of binding two antigens comprising four polypeptide chains, wherein, first and third polypeptide chains comprise VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first heavy chain variable domain obtained from a first parent antibody or antigen binding portion thereof; VD2 is a second heavy chain variable domain obtained from a second parent antibody or antigen binding portion thereof; C is a heavy chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein said (X1)n is either present or absent; and (X2)n is an Fc region, wherein said (X2)n is either present or absent; and wherein second and fourth polypeptide chains comprise VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variable domain obtained from a first parent antibody or antigen binding portion thereof; VD2 is a second light chain variable domain obtained from a second parent antibody or antigen binding portion thereof; C is a light chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein said (X1)n is either present or absent; and (X2)n does not comprise an Fc region, wherein said (X2)n is either present or absent.
The invention provides a method of making a DVD-Ig binding protein by preselecting the parent antibodies. In an embodiment, the method of making a Dual Variable Domain Immunoglobulin capable of binding two antigens comprising the steps of a) obtaining a first parent antibody or antigen binding portion thereof, capable of binding a first antigen; b) obtaining a second parent antibody or antigen binding portion thereof, capable of binding a second antigen; c) constructing first and third polypeptide chains comprising VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first heavy chain variable domain obtained from said first parent antibody or antigen binding portion thereof; VD2 is a second heavy chain variable domain obtained from said second parent antibody or antigen binding portion thereof; C is a heavy chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein said (X1)n is either present or absent; and (X2)n is an Fc region, wherein said (X2)n is either present or absent; d) constructing second and fourth polypeptide chains comprising VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first light chain variable domain obtained from said first parent antibody or antigen binding portion thereof; VD2 is a second light chain variable domain obtained from said second parent antibody or antigen binding thereof; C is a light chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein said (X1)n is either present or absent; and (X2)n does not comprise an Fc region, wherein said (X2)n is either present or absent; e) expressing said first, second, third and fourth polypeptide chains; such that a Dual Variable Domain Immunoglobulin capable of binding said first and said second antigen is generated.
In still another embodiment, the invention provides a method of generating a Dual Variable Domain Immunoglobulin capable of binding two antigens with desired properties comprising the steps of a) obtaining a first parent antibody or antigen binding portion thereof, capable of binding a first antigen and possessing at least one desired property exhibited by the Dual Variable Domain Immunoglobulin; b) obtaining a second parent antibody or antigen binding portion thereof, capable of binding a second antigen and possessing at least one desired property exhibited by the Dual Variable Domain Immunoglobulin; c) constructing first and third polypeptide chains comprising VD1-(X1)n-VD2-C-(X2)n, wherein; VD1 is a first heavy chain variable domain obtained from said first parent antibody or antigen binding portion thereof; VD2 is a second heavy chain variable domain obtained from said second parent antibody or antigen binding portion thereof; C is a heavy chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein said (X1)n is either present or absent; and (X2)n is an Fc region, wherein said (X2)n is either present or absent; d) constructing second and fourth polypeptide chains comprising VD1-(X1)n-VD2-C-(X2)n, wherein; VD1 is a first light chain variable domain obtained from said first parent antibody or antigen binding portion thereof; VD2 is a second light chain variable domain obtained from said second parent antibody or antigen binding portion thereof; C is a light chain constant domain; (X1)n is a linker with the proviso that it is not CH1, wherein said (X1)n is either present or absent; and (X2)n does not comprise an Fc region, wherein said (X2)n is either present or absent; e) expressing said first, second, third and fourth polypeptide chains; such that a Dual Variable Domain Immunoglobulin capable of binding said first and said second antigen with desired properties is generated.
In one embodiment, the VD1 of the first and second polypeptide chains disclosed herein are obtained from the same parent antibody or antigen binding portion thereof. In another embodiment, the VD1 of the first and second polypeptide chains disclosed herein are obtained from different parent antibodies or antigen binding portions thereof. In another embodiment, the VD2 of the first and second polypeptide chains disclosed herein are obtained from the same parent antibody or antigen binding portion thereof. In another embodiment, the VD2 of the first and second polypeptide chains disclosed herein are obtained from different parent antibodies or antigen binding portions thereof.
In one embodiment the first parent antibody or antigen binding portion thereof, and the second parent antibody or antigen binding portion thereof, are the same antibody. In another embodiment the first parent antibody or antigen binding portion thereof, and the second parent antibody or antigen binding portion thereof, are different antibodies.
In one embodiment the first parent antibody or antigen binding portion thereof, binds a first antigen and the second parent antibody or antigen binding portion thereof, binds a second antigen. In a particular embodiment, the first and second antigens are the same antigen. In another embodiment, the parent antibodies bind different epitopes on the same antigen. In another embodiment the first and second antigens are different antigens. In another embodiment, the first parent antibody or antigen binding portion thereof, binds the first antigen with a potency different from the potency with which the second parent antibody or antigen binding portion thereof, binds the second antigen. In yet another embodiment, the first parent antibody or antigen binding portion thereof, binds the first antigen with an affinity different from the affinity with which the second parent antibody or antigen binding portion thereof, binds the second antigen.
In another embodiment the first parent antibody or antigen binding portion thereof, and the second parent antibody or antigen binding portion thereof, are selected from the group consisting of, human antibody, CDR grafted antibody, and humanized antibody. In an embodiment, the antigen binding portions are selected from the group consisting of a Fab fragment, a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementarity determining region (CDR), a single chain antibody, and diabodies.
In another embodiment the binding protein of the invention possesses at least one desired property exhibited by the first parent antibody or antigen binding portion thereof, or the second parent antibody or antigen binding portion thereof. Alternatively, the first parent antibody or antigen binding portion thereof and the second parent antibody or antigen binding portion thereof possess at least one desired property exhibited by the Dual Variable Domain Immunoglobulin. In an embodiment, the desired property is selected from one or more antibody parameters. In another embodiment, the antibody parameters are selected from the group consisting of antigen specificity, affinity to antigen, potency, biological function, epitope recognition, stability, solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross reactivity, and orthologous antigen binding. In an embodiment the binding protein is multivalent. In another embodiment, the binding protein is multispecific. The multivalent and or multispecific binding proteins described herein have desirable properties particularly from a therapeutic standpoint. For instance, the multivalent and or multispecific binding protein may (1) be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind; (2) be an agonist antibody; and/or (3) induce cell death and/or apoptosis of a cell expressing an antigen which the multivalent antibody is capable of binding to. The “parent antibody” which provides at least one antigen binding specificity of the multivalent and or multispecific binding proteins may be one which is internalized (and/or catabolized) by a cell expressing an antigen to which the antibody binds; and/or may be an agonist, cell death-inducing, and/or apoptosis-inducing antibody, and the multivalent and or multispecific binding protein as described herein may display improvement(s) in one or more of these properties. Moreover, the parent antibody may lack any one or more of these properties, but may be endowed with them when constructed as a multivalent binding protein as described herein.
In another embodiment the binding protein of the invention has an on rate constant (Kon) to one or more targets selected from the group consisting of: at least about 102M−1s−1; at least about 103M−1s−1; at least about 104M−1s−1; at least about 105M−1s−1; and at least about 106M−1s−1, as measured by surface plasmon resonance. In an embodiment, the binding protein of the invention has an on rate constant (Kon) to one or more targets between 102M−1s−1 and 103M−1s−1; between 103M−1s−1 and 104M−1s−1; between 104M−1s−1 and 105M−1s−1; or between 105M−1s−1 and 106M−1s−1, as measured by surface plasmon resonance.
In another embodiment the binding protein has an off rate constant (Koff) for one or more targets selected from the group consisting of: at most about 10−3 s−1; at most about 10−4 s−1; at most about 10−5 s−1; and at most about 10−6 s−1, as measured by surface plasmon resonance. In an embodiment, the binding protein of the invention has an off rate constant (Koff) to one or more targets of 10−3 s−1 to 10−4 s−1; of 10−4 s−1 to 10−5 s−1; or of 10−5 s−1 to 10−6 s−1, as measured by surface plasmon resonance.
In another embodiment the binding protein has a dissociation constant (KD) to one or more targets selected from the group consisting of: at most about 10−7 M; at most about 10−8 M; at most about 10−9 M; at most about 10−10 M; at most about 10−11 M; at most about 10−12 M; and at most 10−13M. In an embodiment, the binding protein of the invention has a dissociation constant M; (KD) to its targets of 10−7 M to 10−8 M; of 10−8 M to 10−9 M; of 10−9 M to 10−10 M; of 10−10 to 10−11 M; of 10−11 M to 10−12 M; or of 10−12 M to 10−13M.
In another embodiment, the binding protein described herein is a conjugate further comprising an agent selected from the group consisting of an immunoadhesion molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent. In an embodiment, the imaging agent is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. In another embodiment, the imaging agent is a radiolabel selected from the group consisting of: 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm. In yet another embodiment, the therapeutic or cytotoxic agent is selected from the group consisting of an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.
In another embodiment, the binding protein described herein is a crystallized binding protein and exists as a crystal. In an embodiment, the crystal is a carrier-free pharmaceutical controlled release crystal. In yet another embodiment, the crystallized binding protein has a greater half life in vivo than the soluble counterpart of said binding protein. In still another embodiment, the crystallized binding protein retains biological activity.
In another embodiment, the binding protein described herein is glycosylated. For example, the glycosylation is a human glycosylation pattern.
One aspect of the invention pertains to an isolated nucleic acid encoding any one of the binding proteins disclosed herein. A further embodiment provides a vector comprising the isolated nucleic acid disclosed herein wherein said vector is selected from the group consisting of pcDNA; pTT (Durocher et al., Nucleic Acids Research 2002, Vol 30, No. 2); pTT3 (pTT with additional multiple cloning site; pEFBOS (Mizushima, S, and Nagata, S., (1990) Nucleic acids Research Vol 18, No. 17); pBV; pJV; pcDNA3.1 TOPO, pEF6 TOPO and pBJ. In an embodiment, the vector is a vector disclosed in U.S. Patent Application Ser. No. 61/021,282.
In another aspect a host cell is transformed with the vector disclosed herein. In an embodiment, the host cell is a prokaryotic cell. In another embodiment, the host cell is E. Coli. In a related embodiment the host cell is a eukaryotic cell. In another embodiment, the eukaryotic cell is selected from the group consisting of protist cell, animal cell, plant cell and fungal cell. In yet another embodiment, the host cell is a mammalian cell including, but not limited to, CHO, COS; NS0, SP2, PER.C6 or a fungal cell such as Saccharomyces cerevisiae; or an insect cell such as Sf9.
In an embodiment, two or more DVD-Igs, e.g., with different specificities, are produced in a single recombinant host cell. For example, the expression of a mixture of antibodies has been called OligoclonicsTM, (Merus B. V., The Netherlands) U.S. Pat. Nos. 7,262,028; 7,429,486.
Another aspect of the invention provides a method of producing a binding protein disclosed herein comprising culturing any one of the host cells also disclosed herein in a culture medium under conditions sufficient to produce the binding protein. In an embodiment, 50%-75% of the binding protein produced by this method is a dual specific tetravalent binding protein. In a particular embodiment, 75%-90% of the binding protein produced by this method is a dual specific tetravalent binding protein. In a particular embodiment, 90%-95% of the binding protein produced is a dual specific tetravalent binding protein.
One embodiment provides a composition for the release of a binding protein wherein the composition comprises a formulation that in turn comprises a crystallized binding protein, as disclosed herein, and an ingredient, and at least one polymeric carrier. For example, the polymeric carrier is a polymer selected from one or more of the group consisting of: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof. For example, the ingredient is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol. Another embodiment provides a method for treating a mammal comprising the step of administering to the mammal an effective amount of the composition disclosed herein.
The invention also provides a pharmaceutical composition comprising a binding protein, as disclosed herein and a pharmaceutically acceptable carrier. In a further embodiment the pharmaceutical composition comprises at least one additional therapeutic agent for treating a disorder. For example, the additional agent is selected from the group consisting of: a therapeutic agent, an imaging agent, a cytotoxic agent, an angiogenesis inhibitor (including but not limited to an anti-VEGF antibody or a VEGF-trap), a kinase inhibitor (including but not limited to a KDR and a TIE-2 inhibitor), a co-stimulation molecule blocker (including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig, anti-CD20), an adhesion molecule blocker (including but not limited to an anti-LFA-1 antibody, an anti-E/L selectin antibody, a small molecule inhibitor), an anti-cytokine antibody or functional fragment thereof (including but not limited to an anti-IL-18, an anti-TNF, and an anti-IL-6/cytokine receptor antibody), methotrexate, cyclosporin, rapamycin, FK506, a detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or analog, a cytokine, and a cytokine antagonist.
In another aspect, the invention provides a method for treating a human subject suffering from a disorder in which the target, or targets, capable of being bound by the binding protein disclosed herein is detrimental, comprising administering to the human subject a binding protein disclosed herein such that the activity of the target, or targets in the human subject is inhibited and one of more symptoms is alleviated or treatment is achieved. For example, the disorder is selected from the group comprising arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease, sporadic polyglandular deficiency type I and polyglandular deficiency type II, Schmidt's syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia greata, seronegative arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, chlamydia, yersinia and salmonella associated arthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjögren's disease associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjögren's syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, choleosatatis, idiosyncratic liver disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group B streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 Type and Th1 Type mediated diseases, acute and chronic pain (different forms of pain), and cancers such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti cd3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aortic and peripheral aneuryisms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage transplant rejection, cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chronic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, corpulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated disorders, Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, dermatologic conditions, diabetes, diabetes mellitus, diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's Syndrome in middle age, drug-induced movement disorders induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, epstein-barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hematophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallerrorden-Spatz disease, hashimoto's thyroiditis, hay fever, heart transplant rejection, hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis (A), His bundle arrythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody mediated cytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammation of the aorta, influenza a, ionizing radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, legionella, leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver transplant rejection, lymphederma, malaria, malignant Lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic diseases, migraine headache, mitochondrial multi.system disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple systems degenerations (Mencel Dejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis, mycobacterium avium intracellulare, mycobacterium tuberculosis, myelodyplastic syndrome, myocardial infarction, myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscular atrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of the abdominal aorta and its branches, occlusive arterial disorders, okt3 therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, peripheral atherlosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleo Palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynoud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, Senile Dementia of Lewy body type, seronegative arthropathies, shock, sickle cell anemia, skin allograft rejection, skin changes syndrome, small bowel transplant rejection, solid tumors, specific arrythmias, spinal ataxia, spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, Subacute sclerosing panencephalitis, Syncope, syphilis of the cardiovascular system, systemic anaphalaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type III hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose veins, vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal infections, vital encephalitis/aseptic meningitis, vital-associated hemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or tissue, acute coronary syndromes, acute idiopathic polyneuritis, acute inflammatory demyelinating polyradiculoneuropathy, acute ischemia, adult Still's disease, alopecia greata, anaphylaxis, antiphospholipid antibody syndrome, aplastic anemia, arteriosclerosis, atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmune disorder associated with streptococcus infection, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliferative syndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarian failure, blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular disease, catastrophic antiphospholipid syndrome, celiac disease, cervical spondylosis, chronic ischemia, cicatricial pemphigoid, clinically isolated syndrome (cis) with risk for multiple sclerosis, conjunctivitis, childhood onset psychiatric disorder, chronic obstructive pulmonary disease (COPD), dacryocystitis, dermatomyositis, diabetic retinopathy, diabetes mellitus, disk herniation, disk prolaps, drug induced immune hemolytic anemia, endocarditis, endometriosis, endophthalmitis, episcleritis, erythema multiforme, erythema multiforme major, gestational pemphigoid, Guillain-Barré syndrome (GBS), hay fever, Hughes syndrome, idiopathic Parkinson's disease, idiopathic interstitial pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion body myositis, infectious ocular inflammatory disease, inflammatory demyelinating disease, inflammatory heart disease, inflammatory kidney disease, IPF/UIP, iritis, keratitis, keratojuntivitis sicca, Kussmaul disease or Kussmaul-Meier disease, Landry's paralysis, Langerhan's cell histiocytosis, livedo reticularis, macular degeneration, microscopic polyangiitis, morbus bechterev, motor neuron disorders, mucous membrane pemphigoid, multiple organ failure, myasthenia gravis, myelodysplastic syndrome, myocarditis, nerve root disorders, neuropathy, non-A non-B hepatitis, optic neuritis, osteolysis, ovarian cancer, pauciarticular JRA, peripheral artery occlusive disease (PAOD), peripheral vascular disease (PVD), peripheral artery, disease (PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa), polychondritis, polymyalgia rheumatica, poliosis, polyarticular JRA, polyendocrine deficiency syndrome, polymyositis, polymyalgia rheumatica (PMR), post-pump syndrome, primary Parkinsonism, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), prostatitis, pure red cell aplasia, primary adrenal insufficiency, recurrent neuromyelitis optica, restenosis, rheumatic heart disease, sapho (synovitis, acne, pustulosis, hyperostosis, and osteitis), scleroderma, secondary amyloidosis, shock lung, scleritis, sciatica, secondary adrenal insufficiency, silicone associated connective tissue disease, sneddon-wilkinson dermatosis, spondilitis ankylosans, Stevens-Johnson syndrome (SJS), systemic inflammatory response syndrome, temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumor necrosis factor receptor, type 1 allergic reaction, type II diabetes, urticaria, usual interstitial pneumonia (UIP), vasculitis, vernal conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration, wound healing, yersinia and salmonella associated arthropathy.
In an embodiment, diseases that can be treated or diagnosed with the compositions and methods of the invention include, but are not limited to, primary and metastatic cancers, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract (including cervix, uterus, and ovaries as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as well as Kaposi's sarcoma), tumors of the brain, nerves, eyes, and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, and meningiomas), solid tumors arising from hematopoietic malignancies such as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin's lymphomas).
In an embodiment, the antibodies of the invention or antigen-binding portions thereof, are used to treat cancer or in the prevention of metastases from the tumors described herein either when used alone or in combination with radiotherapy and/or other chemotherapeutic agents.
In another aspect the invention provides a method of treating a patient suffering from a disorder comprising the step of administering any one of the binding proteins disclosed herein before, concurrent, or after the administration of a second agent, as discussed herein. In a particular embodiment the second agent is selected from the group consisting of budenoside, epidermal growth factor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates, 6-mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists, anti-IL-1β mAbs, anti-IL-6 or IL-6 receptor mAbs, growth factors, elastase inhibitors, pyridinyl-imidazole compounds, antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-23, EMAP-II, GM-CSF, FGF, and PDGF, antibodies of CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands, methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen, corticosteroids, prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-1β converting enzyme inhibitors, TNFα converting enzyme inhibitors, T-cell signalling inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor, sIL-1RI, sIL-1RII, sIL-6R, antiinflammatory cytokines, IL-4, IL-10, IL-11, IL-13 and TGFβ.
In a particular embodiment the pharmaceutical compositions disclosed herein are administered to the patient by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.
One aspect of the invention provides at least one anti-idiotype antibody to at least one binding protein of the present invention. The anti-idiotype antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule such as, but not limited to, at least one complementarily determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, that can be incorporated into a binding protein of the present invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a schematic representation of Dual Variable Domain (DVD)-Ig constructs and shows the strategy for generation of a DVD-Ig from two parent antibodies;
FIG. 1B, is a schematic representation of constructs DVD1-Ig, DVD2-Ig, and two chimeric mono-specific antibodies from hybridoma clones 2D13.E3 (anti-IL-1α) and 13F5.G5 (anti-IL-1β).



DETAILED DESCRIPTION OF THE INVENTION
This invention pertains to multivalent and/or multispecific binding proteins capable of binding two or more antigens. Specifically, the invention relates to dual variable domain immunoglobulins (DVD-Ig), and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such DVD-Igs. Methods of using the DVD-Igs of the invention to detect specific antigens, either in vitro or in vivo are also encompassed by the invention.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
That the present invention may be more readily understood, select terms are defined below.
The term “polypeptide” as used herein, refers to any polymeric chain of amino acids. The terms “peptide” and “protein” are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term “polypeptide” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric. Use of “polypeptide” herein is intended to encompass polypeptide and fragments and variants (including fragments of variants) thereof, unless otherwise contradicted by context. For an antigenic polypeptide, a fragment of polypeptide optionally contains at least one contiguous or nonlinear epitope of polypeptide. The precise boundaries of the at least one epitope fragment can be confirmed using ordinary skill in the art. The fragment comprises at least about 5 contiguous amino acids, such as at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, or at least about 20 contiguous amino acids. A variant of polypeptide is as described herein.
The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
The term “recovering” as used herein, refers to the process of rendering a chemical species such as a polypeptide substantially free of naturally associated components by isolation, e.g., using protein purification techniques well known in the art.
“Biological activity” as used herein, refers to any one or more inherent biological properties of a molecule (whether present naturally as found in vivo, or provided or enabled by recombinant means). Biological properties include but are not limited to binding receptor; induction of cell proliferation, inhibiting cell growth, inductions of other cytokines, induction of apoptosis, and enzymatic activity. Biological activity also includes activity of an Ig molecule.
The terms “specific binding” or “specifically binding”, as used herein, in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
The term “antibody”, as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Nonlimiting embodiments of which are discussed below.
In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.
The term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (Winter, et al. U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions e.g., cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRs and complement C1q, respectively. Neonatal Fc receptors (FcRn) are the critical components determining the circulating half-life of antibodies. In still another embodiment at least one amino acid residue is replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered. The dimerization of two identical heavy chains of an immunoglobulin is mediated by the dimerization of CH3 domains and is stabilized by the disulfide bonds within the hinge region (Huber et al. Nature; 264: 415-20; Thies et al 1999 J Mol Biol; 293: 67-79). Mutation of cysteine residues within the hinge regions to prevent heavy chain-heavy chain disulfide bonds will destabilize dimeration of CH3 domains. Residues responsible for CH3 dimerization have been identified (Dall'Acqua 1998 Biochemistry 37: 9266-73). Therefore, it is possible to generate a monovalent half-Ig. Interestingly, these monovalent half Ig molecules have been found in nature for both IgG and IgA subclasses (Seligman 1978 Ann Immunol 129: 855-70; Biewenga et al 1983 Clin Exp Immunol 51: 395-400). The stoichiometry of FcRn:Ig Fc region has been determined to be 2:1 (West et al 0.2000 Biochemistry 39: 9698-708), and half Fc is sufficient for mediating FcRn binding (Kim et al 1994 Eur J Immunol; 24: 542-548). Mutations to disrupt the dimerization of CH3 domain may not have greater adverse effect on its FcRn binding as the residues important for CH3 dimerization are located on the inner interface of CH3 b sheet structure, whereas the region responsible for FcRn binding is located on the outside interface of CH2-CH3 domains. However the half Ig molecule may have certain advantage in tissue penetration due to its smaller size than that of a regular antibody. In one embodiment at least one amino acid residue is replaced in the constant region of the binding protein of the invention, for example the Fc region, such that the dimerization of the heavy chains is disrupted, resulting in half DVD Ig molecules. The anti-inflammatory activity of IgG is completely dependent on sialylation of the N-linked glycan of the IgG Fc fragment. The precise glycan requirements for anti-inflammatory activity has been determined, such that an appropriate IgG1 Fc fragment can be created, thereby generating a recombinant, sialylated IgG1 Fc with greatly enhanced potency (Anthony, R. M., et al. (2008) Science 320:373-376).
The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 90/05144 A1 herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5). In addition single chain antibodies also include “linear antibodies” comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).
The term “multivalent binding protein” is used throughout this specification to denote a binding protein comprising two or more antigen binding sites. In an embodiment, the multivalent binding protein is engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody. The term “multispecific binding protein” refers to a binding protein capable of binding two or more related or unrelated targets. Dual variable domain (DVD) binding proteins of the invention comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. DVDs may be monospecific, i.e., capable of binding one antigen or multispecific, i.e. capable of binding two or more antigens. DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are referred to as DVD-Ig. Each half of a DVD-Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site.
The term “bispecific antibody”, as used herein, refers to full-length antibodies that are generated by quadroma technology (see Milstein, C. and A. C. Cuello, Nature, 1983. 305(5934): p. 537-40), by chemical conjugation of two different monoclonal antibodies (see Staerz, U. D., et al., Nature, 1985. 314(6012): p. 628-31), or by knob-into-hole or similar approaches which introduces mutations in the Fc region (see Holliger, P., T. Prospero, and G. Winter, Proc Natl Acad Sci USA, 1993. 90(14): p. 6444-8.18), resulting in multiple different immunoglobulin species of which only one is the functional bispecific antibody. By molecular function, a bispecific antibody binds one antigen (or epitope) on one of its two binding arms (one pair of HC/LC), and binds a different antigen (or epitope) on its second arm (a different pair of HC/LC). By this definition, a bispecific antibody has two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen it binds to.
The term “dual-specific antibody”, as used herein, refers to full-length antibodies that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC/LC) (see PCT publication WO 02/02773). Accordingly a dual-specific binding protein has two identical antigen binding arms, with identical specificity and identical CDR sequences, and is bivalent for each antigen it binds to.
A “functional antigen binding site” of a binding protein is one that is capable of binding a target antigen. The antigen binding affinity of the antigen binding site is not necessarily as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating antibody binding to an antigen. Moreover, the antigen binding affinity of each of the antigen binding sites of a multivalent antibody herein need not be quantitatively the same.
The term “cytokine” is a generic term for proteins released by one cell population, which act on another cell population as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-alpha; platelet-growth factor; placental growth factor, transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-1 and -11; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, -beta and -gamma colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-18, IL-21, IL-22, IL-23, IL-33; a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
The term “linker” is used to denote polypeptides comprising two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Exemplary linkers include, but are not limited to, AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G4S)4 (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25); GHEAAAVMQVQYPAS (SEQ ID NO: 26).
An “immunoglobulin constant domain” refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.
The term “monoclonal antibody” or “mAb” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method.
The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section II C, below), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H. R. (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E. (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A, and Green L. L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al. (2000) Immunology Today 21:364-370) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
An “affinity matured” antibody is an antibody with one or more alterations in one or more CDRs thereof which result an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. BidlTechnology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); Hawkins et al, J. Mol. BioL 226:889-896 (1992) and selective mutation at selective mutagenesis positions, contact or hypermutation positions with an activity enhancing amino acid residue as described in U.S. Pat. No. 6,914,128B1.
The term “chimeric antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.
The term “CDR-grafted antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
The term “humanized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences. Also “humanized antibody” is an antibody or a variant, derivative, analog or fragment thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term “substantially” in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In an embodiment, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.
The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
As used herein, the term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs.
As used herein, the term “framework” or “framework sequence” refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.
As used herein, the term “germline antibody gene” or “gene fragment” refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et al., Adv Exp Med. Biol. 484:13-30 (2001)). One of the advantages provided by various embodiments of the present invention stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.
As used herein, the term “neutralizing” refers to counteracting the biological activity of an antigen when a binding protein specifically binds the antigen. In an embodiment, the neutralizing binding protein binds the cytokine and reduces its biologically activity by at least about 20%, 40%, 60%, 80%, 85% or more.
The term “activity” includes activities such as the binding specificity and affinity of a DVD-Ig for two or more antigens.
The term “epitope” includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it recognizes its target antigen in a complex mixture of proteins and/or macromolecules. Antibodies are said to “bind to the same epitope” if the antibodies cross-compete (one prevents the binding or modulating effect of the other). In addition structural definitions of epitopes (overlapping, similar, identical) are informative, but functional definitions are often more relevant as they encompass structural (binding) and functional (modulation, competition) parameters.
The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jönsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.
The term “Kon”, as used herein, is intended to refer to the on rate constant for association of a binding protein (e.g., an antibody) to the antigen to form the, e.g., antibody/antigen complex as is known in the art. The “Kon” also is known by the terms “association rate constant”, or “ka”, as used interchangeably herein. This value indicating the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen also is shown by the equation below:

Antibody(“Ab”)+Antigen(“Ag”)→Ab-Ag.

The term “Koff”, as used herein, is intended to refer to the off rate constant for dissociation, or “dissociation rate constant”, of a binding protein (e.g., an antibody) from the, e.g., antibody/antigen complex as is known in the art. This value indicates the dissociation rate of an antibody from its target antigen or separation of Ab-Ag complex over time into free antibody and antigen as shown by the equation below:

Ab+Ag←Ab-Ag.

The term “KD” as used herein, is intended to refer to the “equilibrium dissociation constant”, and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (koff) by the association rate constant (kon). The association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay can be used (e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used.
“Label” and “detectable label” mean a moiety attached to a specific binding partner, such as an antibody or an analyte, e.g., to render the reaction between members of a specific binding pair, such as an antibody and an analyte, detectable, and the specific binding partner, e.g., antibody or analyte, so labeled is referred to as “detectably labeled.” Thus, the term “labeled binding protein” as used herein, refers to a protein with a label incorporated that provides for the identification of the binding protein. In an embodiment, the label is a detectable marker that can produce a signal that is detectable by visual or instrumental means, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates. Representative examples of labels commonly employed for immunoassays include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety. Use of “detectably labeled” is intended to encompass the latter type of detectable labeling.
The term “conjugate” refers to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. In an embodiment, the therapeutic or cytotoxic agents include, but are not limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. When employed in the context of an immunoassay, the conjugate antibody may be a detectably labeled antibody used as the detection antibody.
The terms “crystal” and “crystallized” as used herein, refer to a binding protein (e.g., an antibody), or antigen binding portion thereof, that exists in the form of a crystal. Crystals are one form of the solid state of matter, which is distinct from other forms such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen/antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well-understood in the field. The fundamental unit, or building block, that is repeated in a crystal is called the asymmetric unit. Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined crystallographic symmetry provides the “unit cell” of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999).”
The term “polynucleotide” means a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.
The term “isolated polynucleotide” shall mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, by virtue of its origin, the “isolated polynucleotide” is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.
The term “vector”, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. “Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
“Transformation”, refers to any process by which exogenous DNA enters a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such “transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.
The term “recombinant host cell” (or simply “host cell”), is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. In an embodiment, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. In another embodiment, eukaryotic cells include protist, fungal, plant and animal cells. In another embodiment, host cells include but are not limited to the prokaryotic cell line E. Coli; mammalian cell lines CHO, HEK 293, COS, NS0, SP2 and PER.C6; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae. 
Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
“Transgenic organism”, as known in the art, refers to an organism having cells that contain a transgene, wherein the transgene introduced into the organism (or an ancestor of the organism) expresses a polypeptide not naturally expressed in the organism. A “transgene” is a DNA construct, which is stably and operably integrated into the genome of a cell from which a transgenic organism develops, directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic organism.
The term “regulate” and “modulate” are used interchangeably, and, as used herein, refers to a change or an alteration in the activity of a molecule of interest (e.g., the biological activity of a cytokine). Modulation may be an increase or a decrease in the magnitude of a certain activity or function of the molecule of interest. Exemplary activities and functions of a molecule include, but are not limited to, binding characteristics, enzymatic activity, cell receptor activation, and signal transduction.
Correspondingly, the term “modulator” is a compound capable of changing or altering an activity or function of a molecule of interest (e.g., the biological activity of a cytokine). For example, a modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor, which decreases the magnitude of at least one activity or function of a molecule. Exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, e.g., in WO01/83525.
The term “agonist”, refers to a modulator that, when contacted with a molecule of interest, causes an increase in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the agonist. Particular agonists of interest may include, but are not limited to, polypeptides, nucleic acids, carbohydrates, or any other molecules that bind to the antigen.
The term “antagonist” or “inhibitor”, refer to a modulator that, when contacted with a molecule of interest causes a decrease in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the antagonist. Particular antagonists of interest include those that block or modulate the biological or immunological activity of the antigen. Antagonists and inhibitors of antigens may include, but are not limited to, proteins, nucleic acids, carbohydrates, or any other molecules, which bind to the antigen.
As used herein, the term “effective amount” refers to the amount of a therapy which is sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more symptoms thereof, prevent the advancement of a disorder, cause regression of a disorder, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).
“Patient” and “subject” may be used interchangeably herein to refer to an animal, such as a mammal, including a primate (for example, a human, a monkey, and a chimpanzee), a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a whale), a bird (e.g., a duck or a goose), and a shark. Preferably, the patient or subject is a human, such as a human being treated or assessed for a disease, disorder or condition, a human at risk for a disease, disorder or condition, a human having a disease, disorder or condition, and/or human being treated for a disease, disorder or condition.
The term “sample”, as used herein, is used in its broadest sense. A “biological sample”, as used herein, includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood, (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen.
“Component,” “components,” and “at least one component,” refer generally to a capture antibody, a detection or conjugate antibody, a control, a calibrator, a series of calibrators, a sensitivity panel, a container, a buffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, a detection reagent, a pretreatment reagent/solution, a substrate (e.g., as a solution), a stop solution, and the like that can be included in a kit for assay of a test sample, such as a patient urine, serum or plasma sample, in accordance with the methods described herein and other methods known in the art. Thus, in the context of the present disclosure, “at least one component,” “component,” and “components” can include a polypeptide or other analyte as above, such as a composition comprising an analyte such as polypeptide, which is optionally immobilized on a solid support, such as by binding to an anti-analyte (e.g., anti-polypeptide) antibody. Some components can be in solution or lyophilized for reconstitution for use in an assay.
“Control” refers to a composition known to not analyte (“negative control”) or to contain analyte (“positive control”). A positive control can comprise a known concentration of analyte. “Control,” “positive control,” and “calibrator” may be used interchangeably herein to refer to a composition comprising a known concentration of analyte. A “positive control” can be used to establish assay performance characteristics and is a useful indicator of the integrity of reagents (e.g., analytes).
“Predetermined cutoff” and “predetermined level” refer generally to an assay cutoff value that is used to assess diagnostic/prognostic/therapeutic efficacy results by comparing the assay results against the predetermined cutoff/level, where the predetermined cutoff/level already has been linked or associated with various clinical parameters (e.g., severity of disease, progression/nonprogression/improvement, etc.). While the present disclosure may provide exemplary predetermined levels, it is well-known that cutoff values may vary depending on the nature of the immunoassay (e.g., antibodies employed, etc.). It further is well within the ordinary skill of one in the art to adapt the disclosure herein for other immunoassays to obtain immunoassay-specific cutoff values for those other immunoassays based on this disclosure. Whereas the precise value of the predetermined cutoff/level may vary between assays, correlations as described herein (if any) should be generally applicable.
“Pretreatment reagent,” e.g., lysis, precipitation and/or solubilization reagent, as used in a diagnostic assay as described herein is one that lyses any cells and/or solubilizes any analyte that is/are present in a test sample. Pretreatment is not necessary for all samples, as described further herein. Among other things, solubilizing the analyte (e.g., polypeptide of interest) may entail release of the analyte from any endogenous binding proteins present in the sample. A pretreatment reagent may be homogeneous (not requiring a separation step) or heterogeneous (requiring a separation step). With use of a heterogeneous pretreatment reagent there is removal of any precipitated analyte binding proteins from the test sample prior to proceeding to the next step of the assay.
“Quality control reagents” in the context of immunoassays and kits described herein, include, but are not limited to, calibrators, controls, and sensitivity panels. A “calibrator” or “standard” typically is used (e.g., one or more, such as a plurality) in order to establish calibration (standard) curves for interpolation of the concentration of an analyte, such as an antibody or an analyte. Alternatively, a single calibrator, which is near a predetermined positive/negative cutoff, can be used. Multiple calibrators (i.e., more than one calibrator or a varying amount of calibrator(s)) can be used in conjunction so as to comprise a “sensitivity panel.”
“Risk” refers to the possibility or probability of a particular event occurring either presently or at some point in the future. “Risk stratification” refers to an array of known clinical risk factors that allows physicians to classify patients into a low, moderate, high or highest risk of developing a particular disease, disorder or condition.
“Specific” and “specificity” in the context of an interaction between members of a specific binding pair (e.g., an antigen (or fragment thereof) and an antibody (or antigenically reactive fragment thereof)) refer to the selective reactivity of the interaction. The phrase “specifically binds to” and analogous phrases refer to the ability of antibodies (or antigenically reactive fragments thereof) to bind specifically to analyte (or a fragment thereof) and not bind specifically to other entities.
“Specific binding partner” is a member of a specific binding pair. A specific binding pair comprises two different molecules, which specifically bind to each other through chemical or physical means. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog Immunoreactive specific binding members include antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies as well as complexes, fragments, and variants (including fragments of variants) thereof, whether isolated or recombinantly produced.
“Variant” as used herein means a polypeptide that differs from a given polypeptide (e.g., IL-18, BNP, NGAL or HIV polypeptide or anti-polypeptide antibody) in amino acid sequence by the addition (e.g., insertion), deletion, or conservative substitution of amino acids, but that retains the biological activity of the given polypeptide (e.g., a variant IL-18 can compete with anti-IL-18 antibody for binding to IL-18). A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity and degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (see, e.g., Kyte et al., J. Mol. Biol. 157: 105-132 (1982)). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids also can be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (see, e.g., U.S. Pat. No. 4,554,101, which is incorporated herein by reference). Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. In one aspect, substitutions are performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. “Variant” also can be used to describe a polypeptide or fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, yet retains its biological activity or antigen reactivity, e.g., the ability to bind to IL-18. Use of “variant” herein is intended to encompass fragments of a variant unless otherwise contradicted by context.
I. Generation of DVD Binding Protein
The invention pertains to Dual Variable Domain binding proteins capable of binding one or more targets and methods of making the same. In an embodiment, the binding protein comprises a polypeptide chain, wherein said polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 represents an amino acid or polypeptide, X2 represents an Fc region and n is 0 or 1. The binding protein of the invention can be generated using various techniques. The invention provides expression vectors, host cell and methods of generating the binding protein.
A. Generation of Parent Monoclonal Antibodies
The variable domains of the DVD binding protein can be obtained from parent antibodies, including polyclonal and mAbs capable of binding antigens of interest. These antibodies may be naturally occurring or may be generated by recombinant technology.
MAbs can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, mAbs can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Hybridomas are selected, cloned and further screened for desirable characteristics, including robust hybridoma growth, high antibody production and desirable antibody characteristics, as discussed in Example 1 below. Hybridomas may be cultured and expanded in vivo in syngeneic animals, in animals that lack an immune system, e.g., nude mice, or in cell culture in vitro. Methods of selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art. In a particular embodiment, the hybridomas are mouse hybridomas. In another embodiment, the hybridomas are produced in a non-human, non-mouse species such as rats, sheep, pigs, goats, cattle or horses. In another embodiment, the hybridomas are human hybridomas, in which a human non-secretory myeloma is fused with a human cell expressing an antibody capable of binding a specific antigen.
Recombinant mAbs are also generated from single, isolated lymphocytes using a procedure referred to in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Pat. No. 5,627,052, PCT Publication WO 92/02551 and Babcock, J. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method, single cells secreting antibodies of interest, e.g., lymphocytes derived from an immunized animal, are identified, and, heavy- and light-chain variable region cDNAs are rescued from the cells by reverse transcriptase-PCR and these variable regions can then be expressed, in the context of appropriate immunoglobulin constant regions (e.g., human constant regions), in mammalian host cells, such as COS or CHO cells. The host cells transfected with the amplified immunoglobulin sequences, derived from in vivo selected lymphocytes, can then undergo further analysis and selection in vitro, for example by panning the transfected cells to isolate cells expressing antibodies to the antigen of interest. The amplified immunoglobulin sequences further can be manipulated in vitro, such as by in vitro affinity maturation methods such as those described in PCT Publication WO 97/29131 and PCT Publication WO 00/56772.
Monoclonal antibodies are also produced by immunizing a non-human animal comprising some, or all, of the human immunoglobulin locus with an antigen of interest. In an embodiment, the non-human animal is a XENOMOUSE transgenic mouse, an engineered mouse strain that comprises large fragments of the human immunoglobulin loci and is deficient in mouse antibody production. See, e.g., Green et al. Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also WO 91/10741, published Jul. 25, 1991, WO 94/02602, published Feb. 3, 1994, WO 96/34096 and WO 96/33735, both published Oct. 31, 1996, WO 98/16654, published Apr. 23, 1998, WO 98/24893, published Jun. 11, 1998, WO 98/50433, published Nov. 12, 1998, WO 99/45031, published Sep. 10, 1999, WO 99/53049, published Oct. 21, 1999, WO 00 09560, published Feb. 24, 2000 and WO 00/037504, published Jun. 29, 2000. The XENOMOUSE transgenic mouse produces an adult-like human repertoire of fully human antibodies, and generates antigen-specific human monoclonal antibodies. The XENOMOUSE transgenic mouse contains approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and x light chain loci. See Mendez et al., Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures of which are hereby incorporated by reference.
In vitro methods also can be used to make the parent antibodies, wherein an antibody library is screened to identify an antibody having the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, US patent application publication 20030186374, and PCT Publication No. WO 97/29131, the contents of each of which are incorporated herein by reference.
Parent antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780, 225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
As described in the herein references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies including human antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).
Alternative to screening of recombinant antibody libraries by phage display, other methodologies known in the art for screening large combinatorial libraries can be applied to the identification of parent antibodies. One type of alternative expression system is one in which the recombinant antibody library is expressed as RNA-protein fusions, as described in PCT Publication No. WO 98/31700 by Szostak and Roberts, and in Roberts, R. W. and Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this system, a covalent fusion is created between an mRNA and the peptide or protein that it encodes by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end. Thus, a specific mRNA can be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein, e.g., antibody, or portion thereof, such as binding of the antibody, or portion thereof, to the dual specificity antigen. Nucleic acid sequences encoding antibodies, or portions thereof, recovered from screening of such libraries can be expressed by recombinant means as described herein (e.g., in mammalian host cells) and, moreover, can be subjected to further affinity maturation by either additional rounds of screening of mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence(s), or by other methods for affinity maturation in vitro of recombinant antibodies, as described herein.
In another approach the parent antibodies can also be generated using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of yeast. In particular, such yeast can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Examples of yeast display methods that can be used to make the parent antibodies include those disclosed in Wittrup, et al. U.S. Pat. No. 6,699,658 incorporated herein by reference.
The antibodies described herein can be further modified to generate CDR grafted and humanized parent antibodies. CDR-grafted parent antibodies comprise heavy and light chain variable region sequences from a human antibody wherein one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of murine antibodies capable of binding antigen of interest. A framework sequence from any human antibody may serve as the template for CDR grafting. However, straight chain replacement onto such a framework often leads to some loss of binding affinity to the antigen. The more homologous a human antibody is to the original murine antibody, the less likely the possibility that combining the murine CDRs with the human framework will introduce distortions in the CDRs that could reduce affinity. Therefore, in an embodiment, the human variable framework that is chosen to replace the murine variable framework apart from the CDRs have at least a 65% sequence identity with the murine antibody variable region framework. In an embodiment, the human and murine variable regions apart from the CDRs have at least 70% sequence identify. In a particular embodiment, that the human and murine variable regions apart from the CDRs have at least 75% sequence identity. In another embodiment, the human and murine variable regions apart from the CDRs have at least 80% sequence identity. Methods for producing such antibodies are known in the art (see EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,352); and anti-idiotypic antibodies.
Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Known human Ig sequences are disclosed, e.g., by the U.S. National Library of Medicine National Institutes of Health, the American Type Culture Collection, SciQuest®, Abcam®, the Antibody Resource Page, Iowa State University, Heidelberg University, W.H. Freeman, Oracle® ThinkQuest, the Howard Hughes Medical Institute, the University of Cambridge, Harvard University Department of Molecular and Cellular Biology, the Immunology Link, Washington University in St. Louis School of Medicine, the University of Florida, Pebio, the United States Department of Agriculture National Agricultural Library, Ehime University, Meridian Life Science®, Inc., the International Society for Advancement of Cytometry, the Technische Universitat Braunschweig, the MRC Centre for Protein Engineering, the UNAM Biology Institute, ImMunoGeneTics, the University College London Research Department of Structural and Molecular Biology, the University of Bath's Center for Protein Analysis and Design Web Antibody Modelling, the All India Institute of Medical Sciences Department of Biochemistry, the University of Zurich, Department of Biological Sciences Birkbeck University of London, the Medical Research Counsel of the National Institute for Medical Research, the University of Missouri, IBM, and Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1983), which is entirely incorporated herein by reference. Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art.
Framework residues in the human framework regions may be substituted with the corresponding residue from the CDR donor antibody to alter, e.g., improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Antibodies can be humanized using a variety of techniques known in the art, such as but not limited to those described in Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska, et al., PNAS 91:969-973 (1994); PCT publication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, each entirely incorporated herein by reference, included references cited therein.
B. Criteria for Selecting Parent Monoclonal Antibodies
An embodiment of the invention pertains to selecting parent antibodies with at least one or more properties desired in the DVD-Ig molecule. In an embodiment, the desired property is selected from one or more antibody parameters. In another embodiment, the antibody parameters are selected from the group consisting of antigen specificity, affinity to antigen, potency, biological function, epitope recognition, stability, solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross reactivity, and orthologous antigen binding.
B1. Affinity to Antigen
The desired affinity of a therapeutic mAb may depend upon the nature of the antigen, and the desired therapeutic end-point. In an embodiment, monoclonal antibodies have higher affinities (Kd=0.01-0.50 pM) when blocking a cytokine-cytokine receptor interaction as such interaction are usually high affinity interactions (e.g., <pM-<nM ranges). In such instances, the mAb affinity for its target should be equal to or better than the affinity of the cytokine (ligand) for its receptor. On the other hand, mAb with lesser affinity (>nM range) could be therapeutically effective e.g., in clearing circulating potentially pathogenic proteins e.g., monoclonal antibodies that bind to, sequester, and clear circulating species of A-β amyloid. In other instances, reducing the affinity of an existing high affinity mAb by site-directed mutagenesis or using a mAb with lower affinity for its target could be used to avoid potential side-effects e.g., a high affinity mAb may sequester/neutralize all of its intended target, thereby completely depleting/eliminating the function(s) of the targeted protein. In this scenario, a low affinity mAb may sequester/neutralize a fraction of the target that may be responsible for the disease symptoms (the pathological or over-produced levels), thus allowing a fraction of the target to continue to perform its normal physiological function(s). Therefore, it may be possible to reduce the Kd to adjust dose and/or reduce side-effects. The affinity of the parental mAb might play a role in appropriately targeting cell surface molecules to achieve desired therapeutic out-come. For example, if a target is expressed on cancer cells with high density and on normal cells with low density, a lower affinity mAb will bind a greater number of targets on tumor cells than normal cells, resulting in tumor cell elimination via ADCC or CDC, and therefore might have therapeutically desirable effects. Thus selecting a mAb with desired affinity may be relevant for both soluble and surface targets.
Signaling through a receptor upon interaction with its ligand may depend upon the affinity of the receptor-ligand interaction. Similarly, it is conceivable that the affinity of a mAb for a surface receptor could determine the nature of intracellular signaling and whether the mAb may deliver an agonist or an antagonist signal. The affinity-based nature of mAb-mediated signaling may have an impact of its side-effect profile. Therefore, the desired affinity and desired functions of therapeutic monoclonal antibodies need to be determined carefully by in vitro and in vivo experimentation.
The desired Kd of a binding protein (e.g., an antibody) may be determined experimentally depending on the desired therapeutic outcome. In an embodiment parent antibodies with affinity (Kd) for a particular antigen equal to, or better than, the desired affinity of the DVD-Ig for the same antigen are selected. The antigen binding affinity and kinetics are assessed by Biacore or another similar technique. In one embodiment, each parent antibody has a dissociation constant (Kd) to its antigen selected from the group consisting of: at most about 10−7 M; at most about 10−8 M; at most about 10−9 M; at most about 10−10 M; at most about 10−11 M; at most about 10−12 M; and at most 10−13M. First parent antibody from which VD1 is obtained and second parent antibody from which VD2 is obtained may have similar or different affinity (KD) for the respective antigen. Each parent antibody has an on rate constant (Kon) to the antigen selected from the group consisting of: at least about 102M−1s−1; at least about 103M−1s−1; at least about 104M−1s−1; at least about 105M−1s−1; and at least about 106M−1s−1, as measured by surface plasmon resonance. The first parent antibody from which VD1 is obtained and the second parent antibody from which VD2 is obtained may have similar or different on rate constant (Kon) for the respective antigen. In one embodiment, each parent antibody has an off rate constant (Koff) to the antigen selected from the group consisting of: at most about 10−3 s−1; at most about 10−4 s−1; at most about 10−5 s−1; and at most about 10−6 s−1, as measured by surface plasmon resonance. The first parent antibody from which VD1 is obtained and the second parent antibody from which VD2 is obtained may have similar or different off rate constants (Koff) for the respective antigen.
B2. Potency
The desired affinity/potency of parental monoclonal antibodies will depend on the desired therapeutic outcome. For example, for receptor-ligand (R-L) interactions the affinity (kd) is equal to or better than the R-L kd (pM range). For simple clearance of a pathologic circulating protein, the kd could be in low nM range e.g., clearance of various species of circulating A-β peptide. In addition, the kd will also depend on whether the target expresses multiple copies of the same epitope e.g. a mAb targeting conformational epitope in Aβ oligomers.
Where VD1 and VD2 bind the same antigen, but distinct epitopes, the DVD-Ig will contain 4 binding sites for the same antigen, thus increasing avidity and thereby the apparent kd of the DVD-Ig. In an embodiment, parent antibodies with equal or lower kd than that desired in the DVD-Ig are chosen. The affinity considerations of a parental mAb may also depend upon whether the DVD-Ig contains four or more identical antigen binding sites (i.e.; a DVD-Ig from a single mAb). In this case, the apparent kd would be greater than the mAb due to avidity. Such DVD-Igs can be employed for cross-linking surface receptor, increase neutralization potency, enhance clearance of pathological proteins etc.
In an embodiment parent antibodies with neutralization potency for specific antigen equal to or better than the desired neutralization potential of the DVD-Ig for the same antigen are selected. The neutralization potency can be assessed by a target-dependent bioassay where cells of appropriate type produce a measurable signal (i.e. proliferation or cytokine production) in response to target stimulation, and target neutralization by the mAb can reduce the signal in a dose-dependent manner.
B3. Biological Functions
Monoclonal antibodies can perform potentially several functions. Some of these functions are listed in Table 1. These functions can be assessed by both in vitro assays (e.g., cell-based and biochemical assays) and in vivo animal models.







TABLE 1







Some Potential Applications For Therapeutic Antibodies










Target (Class)
Mechanism of Action (target)







Soluble
Neutralization of activity (e.g., a cytokine)



(cytokines, other)
Enhance clearance (e.g., Aβ oligomers)




Increase half-life (e.g., GLP 1)



Cell Surface
Agonist (e.g., GLP1 R; EPO R; etc.)



(Receptors, other)
Antagonist (e.g., integrins; etc.)




Cytotoxic (CD 20; etc.)



Protein deposits
Enhance clearance/degradation (e.g.,




Aβ plaques, amyloid deposits)










MAbs with distinct functions described in the examples herein in Table 1 can be selected to achieve desired therapeutic outcomes. Two or more selected parent monoclonal antibodies can then be used in DVD-Ig format to achieve two distinct functions in a single DVD-Ig molecule. For example, a DVD-Ig can be generated by selecting a parent mAb that neutralizes function of a specific cytokine, and selecting a parent mAb that enhances clearance of a pathological protein. Similarly, we can select two parent monoclonal antibodies that recognize two different cell surface receptors, one mAb with an agonist function on one receptor and the other mAb with an antagonist function on a different receptor. These two selected monoclonal antibodies each with a distinct function can be used to construct a single DVD-Ig molecule that will possess the two distinct functions (agonist and antagonist) of the selected monoclonal antibodies in a single molecule. Similarly, two antagonistic monoclonal antibodies to cell surface receptors each blocking binding of respective receptor ligands (e.g., EGF and IGF) can be used in a DVD-Ig format. Conversely, an antagonistic anti-receptor mAb (e.g., anti-EGFR) and a neutralizing anti-soluble mediator (e.g., anti-IGF1/2) mAb can be selected to make a DVD-Ig.
B4. Epitope Recognition:
Different regions of proteins may perform different functions. For example specific regions of a cytokine interact with the cytokine receptor to bring about receptor activation whereas other regions of the protein may be required for stabilizing the cytokine. In this instance one may select a mAb that binds specifically to the receptor interacting region(s) on the cytokine and thereby block cytokine-receptor interaction. In some cases, for example certain chemokine receptors that bind multiple ligands, a mAb that binds to the epitope (region on chemokine receptor) that interacts with only one ligand can be selected. In other instances, monoclonal antibodies can bind to epitopes on a target that are not directly responsible for physiological functions of the protein, but binding of a mAb to these regions could either interfere with physiological functions (steric hindrance) or alter the conformation of the protein such that the protein cannot function (mAb to receptors with multiple ligand which alter the receptor conformation such that none of the ligand can bind). Anti-cytokine monoclonal antibodies that do not block binding of the cytokine to its receptor, but block signal transduction have also been identified (e.g., 125-2H, an anti-IL-18 mAb).
Examples of epitopes and mAb functions include, but are not limited to, blocking Receptor-Ligand (R-L) interaction (neutralizing mAb that binds R-interacting site); steric hindrance resulting in diminished or no R-binding. An Ab can bind the target at a site other than a receptor binding site, but still interferes with receptor binding and functions of the target by inducing conformational change and eliminate function (e.g., Xolair), binding to R but block signaling (125-2H).
In an embodiment, the parental mAb needs to target the appropriate epitope for maximum efficacy. Such epitope should be conserved in the DVD-Ig. The binding epitope of a mAb can be determined by several approaches, including co-crystallography, limited proteolysis of mAb-antigen complex plus mass spectrometric peptide mapping (Legros V. et al 2000 Protein Sci. 9:1002-10), phage displayed peptide libraries (O'Connor K H et al 2005 J Immunol Methods. 299:21-35), as well as mutagenesis (Wu C. et al. 2003 J Immunol 170:5571-7).
B5. Physicochemical and Pharmaceutical Properties:
Therapeutic treatment with antibodies often requires administration of high doses, often several mg/kg (due to a low potency on a mass basis as a consequence of a typically large molecular weight). In order to accommodate patient compliance and to adequately address chronic disease therapies and outpatient treatment, subcutaneous (s.c.) or intramuscular (i.m.) administration of therapeutic mAbs is desirable. For example, the maximum desirable volume for s.c. administration is  ̃1.0 mL, and therefore, concentrations of >100 mg/mL are desirable to limit the number of injections per dose. In an embodiment, the therapeutic antibody is administered in one dose. The development of such formulations is constrained, however, by protein-protein interactions (e.g., aggregation, which potentially increases immunogenicity risks) and by limitations during processing and delivery (e.g., viscosity). Consequently, the large quantities required for clinical efficacy and the associated development constraints limit full exploitation of the potential of antibody formulation and s.c. administration in high-dose regimens. It is apparent that the physicochemical and pharmaceutical properties of a protein molecule and the protein solution are of utmost importance, e.g., stability, solubility and viscosity features.
B5.1. Stability:
A “stable” antibody formulation is one in which the antibody therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. Stability can be measured at a selected temperature for a selected time period. In an embodiment the antibody in the formulation is stable at room temperature (about 30° C.) or at 40° C. for at least 1 month and/or stable at about 2-8° C. for at least 1 year for at least 2 years. Furthermore, in an embodiment, the formulation is stable following freezing (to, e.g., −70° C.) and thawing of the formulation, hereinafter referred to as a “freeze/thaw cycle.” In another example, a “stable” formulation may be one wherein less than about 10% and less than about 5% of the protein is present as an aggregate in the formulation.
A DVD-Ig stable in vitro at various temperatures for an extended time period is desirable. One can achieve this by rapid screening of parental mAbs stable in vitro at elevated temperature, e.g., at 40° C. for 2-4 weeks, and then assess stability. During storage at 2-8° C., the protein reveals stability for at least 12 months, e.g., at least 24 months. Stability (% of monomeric, intact molecule) can be assessed using various techniques such as cation exchange chromatography, size exclusion chromatography, SDS-PAGE, as well as bioactivity testing. For a more comprehensive list of analytical techniques that may be employed to analyze covalent and conformational modifications please see Jones, A. J. S. (1993) Analytical methods for the assessment of protein formulations and delivery systems. In: Cleland, J. L.; Langer, R., editors. Formulation and delivery of peptides and proteins, 1st edition, Washington, ACS, pg. 22-45; and Pearlman, R.; Nguyen, T. H. (1990) Analysis of protein drugs. In: Lee, V. H., editor. Peptide and protein drug delivery, 1st edition, New York, Marcel Dekker, Inc., pg. 247-301.
Heterogeneity and aggregate formation: stability of the antibody may be such that the formulation may reveal less than about 10%, and, in an embodiment, less than about 5%, in another embodiment, less than about 2%, or, in an embodiment, within the range of 0.5% to 1.5% or less in the GMP antibody material that is present as aggregate. Size exclusion chromatography is a method that is sensitive, reproducible, and very robust in the detection of protein aggregates.
In addition to low aggregate levels, the antibody must, in an embodiment, be chemically stable. Chemical stability may be determined by ion exchange chromatography (e.g., cation or anion exchange chromatography), hydrophobic interaction chromatography, or other methods such as isoelectric focusing or capillary electrophoresis. For instance, chemical stability of the antibody may be such that after storage of at least 12 months at 2-8° C. the peak representing unmodified antibody in a cation exchange chromatography may increase not more than 20%, in an embodiment, not more than 10%, or, in another embodiment, not more than 5% as compared to the antibody solution prior to storage testing.
In an embodiment, the parent antibodies display structural integrity; correct disulfide bond formation, and correct folding: Chemical instability due to changes in secondary or tertiary structure of an antibody may impact antibody activity. For instance, stability as indicated by activity of the antibody may be such that after storage of at least 12 months at 2-8° C. the activity of the antibody may decrease not more than 50%, in an embodiment not more than 30%, or even not more than 10%, or in an embodiment not more than 5% or 1% as compared to the antibody solution prior to storage testing. Suitable antigen-binding assays can be employed to determine antibody activity.
B5.2. Solubility:
The “solubility” of a mAb correlates with the production of correctly folded, monomeric IgG. The solubility of the IgG may therefore be assessed by HPLC. For example, soluble (monomeric) IgG will give rise to a single peak on the HPLC chromatograph, whereas insoluble (e.g., multimeric and aggregated) will give rise to a plurality of peaks. A person skilled in the art will therefore be able to detect an increase or decrease in solubility of an IgG using routine HPLC techniques. For a more comprehensive list of analytical techniques that may be employed to analyze solubility (see Jones, A. G. Dep. Chem. Biochem. Eng., Univ. Coll. London, London, UK. Editor(s): Shamlou, P. Ayazi. Process. Solid-Liq. Suspensions (1993), 93-117. Publisher: Butterworth-Heinemann, Oxford, UK and Pearlman, Rodney; Nguyen, Tue H, Advances in Parenteral Sciences (1990), 4 (Pept. Protein Drug Delivery), 247-301). Solubility of a therapeutic mAb is critical for formulating to high concentration often required for adequate dosing. As outlined herein, solubilities of >100 mg/mL may be required to accommodate efficient antibody dosing. For instance, antibody solubility may be not less than about 5 mg/mL in early research phase, in an embodiment not less than about 25 mg/mL in advanced process science stages, or in an embodiment not less than about 100 mg/mL, or in an embodiment not less than about 150 mg/mL. It is obvious to a person skilled in the art that the intrinsic properties of a protein molecule are important the physico-chemical properties of the protein solution, e.g., stability, solubility, viscosity. However, a person skilled in the art will appreciate that a broad variety of excipients exist that may be used as additives to beneficially impact the characteristics of the final protein formulation. These excipients may include: (i) liquid solvents, cosolvents (e.g., alcohols such as ethanol); (ii) buffering agents (e.g., phosphate, acetate, citrate, amino acid buffers); (iii) sugars or sugar alcohols (e.g., sucrose, trehalose, fructose, raffinose, mannitol, sorbitol, dextrans); (iv) surfactants (e.g., polysorbate 20, 40, 60, 80, poloxamers); (v) isotonicity modifiers (e.g., salts such as NaCl, sugars, sugar alcohols); and (vi) others (e.g., preservatives, chelating agents, antioxidants, chelating substances (e.g., EDTA), biodegradable polymers, carrier molecules (e.g., HSA, PEGs)
Viscosity is a parameter of high importance with regard to antibody manufacture and antibody processing (e.g., diafiltration/ultrafiltration), fill-finish processes (pumping aspects, filtration aspects) and delivery aspects (syringeability, sophisticated device delivery). Low viscosities enable the liquid solution of the antibody having a higher concentration. This enables the same dose may be administered in smaller volumes. Small injection volumes inhere the advantage of lower pain on injection sensations, and the solutions not necessarily have to be isotonic to reduce pain on injection in the patient. The viscosity of the antibody solution may be such that at shear rates of 100 (1/s) antibody solution viscosity is below 200 mPa s, in an embodiment below 125 mPa s, in another embodiment below 70 mPa s, and in yet another embodiment below 25 mPa s or even below 10 mPa s.
B 5.3. Production Efficiency
The generation of a DVD-Ig that is efficiently expressed in mammalian cells, such as Chinese hamster ovary cells (CHO), will in an embodiment require two parental monoclonal antibodies which are themselves expressed efficiently in mammalian cells. The production yield from a stable mammalian line (i.e. CHO) should be above about 0.5 g/L, in an embodiment above about 1 g/L, and in another embodiment in the range of from about 2-5 g/L or more (Kipriyanov S M, Little M. 1999 Mol. Biotechnol. 12:173-201; Carroll S, Al-Rubeai M. 2004 Expert Opin Biol Ther. 4:1821-9).
Production of antibodies and Ig fusion proteins in mammalian cells is influenced by several factors. Engineering of the expression vector via incorporation of strong promoters, enhancers and selection markers can maximize transcription of the gene of interest from an integrated vector copy. The identification of vector integration sites that are permissive for high levels of gene transcription can augment protein expression from a vector (Wurm et al, 2004, Nature Biotechnology, 2004, Vol/Iss/Pg. 22/11 (1393-1398)). Furthermore, levels of production are affected by the ratio of antibody heavy and light chains and various steps in the process of protein assembly and secretion (Jiang et al. 2006, Biotechnology Progress, January-February 2006, vol. 22, no. 1, p. 313-8).
B 6. Immunogenicity
Administration of a therapeutic mAb may results in certain incidence of an immune response (i.e., the formation of endogenous antibodies directed against the therapeutic mAb). Potential elements that might induce immunogenicity should be analyzed during selection of the parental monoclonal antibodies, and steps to reduce such risk can be taken to optimize the parental monoclonal antibodies prior to DVD-Ig construction. Mouse-derived antibodies have been found to be highly immunogenic in patients. The generation of chimeric antibodies comprised of mouse variable and human constant regions presents a logical next step to reduce the immunogenicity of therapeutic antibodies (Morrison and Schlom, 1990). Alternatively, immunogenicity can be reduced by transferring murine CDR sequences into a human antibody framework (reshaping/CDR grafting/humanization), as described for a therapeutic antibody by Riechmann et al., 1988. Another method is referred to as “resurfacing” or “veneering”, starting with the rodent variable light and heavy domains, only surface-accessible framework amino acids are altered to human ones, while the CDR and buried amino acids remain from the parental rodent antibody (Roguska et al., 1996). In another type of humanization, instead of grafting the entire CDRs, one technique grafts only the “specificity-determining regions” (SDRs), defined as the subset of CDR residues that are involved in binding of the antibody to its target (Kashmiri et al., 2005). This necessitates identification of the SDRs either through analysis of available three-dimensional structures of antibody-target complexes or mutational analysis of the antibody CDR residues to determine which interact with the target. Alternatively, fully human antibodies may have reduced immunogenicity compared to murine, chimeric or humanized antibodies.
Another approach to reduce the immunogenicity of therapeutic antibodies is the elimination of certain specific sequences that are predicted to be immunogenic. In one approach, after a first generation biologic has been tested in humans and found to be unacceptably immunogenic, the B-cell epitopes can be mapped and then altered to avoid immune detection. Another approach uses methods to predict and remove potential T-cell epitopes. Computational methods have been developed to scan and to identify the peptide sequences of biologic therapeutics with the potential to bind to MHC proteins (Desmet et al., 2005). Alternatively a human dendritic cell-based method can be used to identify CD4+ T-cell epitopes in potential protein allergens (Stickler et al., 2005; S. L. Morrison and J. Schlom, Important Adv. Oncol. (1990), pp. 3-18; Riechmann, L., Clark, M., Waldmann, H. and Winter, G. “Reshaping human antibodies for therapy.” Nature (1988) 332: 323-327; Roguska-M-A, Pedersen-J-T, Henry-A-H, Searle-S-M, Roja-C-M, Avery-B, Hoffee-M, Cook-S, Lambert-J-M, Blättler-W-A, Rees-A-R, Guild-B-C. A comparison of two murine mAbs humanized by CDR-grafting and variable domain resurfacing. Protein engineering, {Protein-Eng}, 1996, vol. 9, p. 895-904; Kashmiri-Syed-V-S, De-Pascalis-Roberto, Gonzales-Noreen-R, Schlom-Jeffrey. SDR grafting—a new approach to antibody humanization. Methods (San Diego Calif.), {Methods}, May 2005, vol. 36, no. 1, p. 25-34; Desmet-Johan, Meersseman-Geert, Boutonnet-Nathalie, Pletinckx-Jurgen, De-Clercq-Krista, Debulpaep-Maja, Braeckman-Tessa, Lasters-Ignace. Anchor profiles of HLA-specific peptides: analysis by a novel affinity scoring method and experimental validation. Proteins, 2005, vol. 58, p. 53-69; Stickler-M-M, Estell-D-A, Harding-F-A. CD4+ T-cell epitope determination using unexposed human donor peripheral blood mononuclear cells. Journal of immunotherapy 2000, vol. 23, p. 654-60.)
B 7. In Vivo Efficacy
To generate a DVD-Ig molecule with desired in vivo efficacy, it is important to generate and select mAbs with similarly desired in vivo efficacy when given in combination. However, in some instances the DVD-Ig may exhibit in vivo efficacy that cannot be achieved with the combination of two separate mAbs. For instance, a DVD-Ig may bring two targets in close proximity leading to an activity that cannot be achieved with the combination of two separate mAbs. Additional desirable biological functions are described herein in section B 3. Parent antibodies with characteristics desirable in the DVD-Ig molecule may be selected based on factors such as pharmacokinetic t 1⁄2; tissue distribution; soluble versus cell surface targets; and target concentration-soluble/density—surface.
B 8. In Vivo Tissue Distribution
To generate a DVD-Ig molecule with desired in vivo tissue distribution, in an embodiment parent mAbs with similar desired in vivo tissue distribution profile must be selected. Alternatively, based on the mechanism of the dual-specific targeting strategy, it may at other times not be required to select parent mAbs with the similarly desired in vivo tissue distribution when given in combination. For instance, in the case of a DVD-Ig in which one binding component targets the DVD-Ig to a specific site thereby bringing the second binding component to the same target site. For example, one binding specificity of a DVD-Ig could target pancreas (islet cells) and the other specificity could bring GLP1 to the pancreas to induce insulin.
B 9. Isotype:
To generate a DVD-Ig molecule with desired properties including, but not limited to, Isotype, Effector functions and the circulating half-life, in an embodiment parent mAbs with appropriate Fc-effector functions depending on the therapeutic utility and the desired therapeutic end-point are selected. There are five main heavy-chain classes or isotypes some of which have several sub-types and these determine the effector functions of an antibody molecule. These effector functions reside in the hinge region, CH2 and CH3 domains of the antibody molecule. However, residues in other parts of an antibody molecule may have effects on effector functions as well. The hinge region Fc-effector functions include: (i) antibody-dependent cellular cytotoxicity, (ii) complement (C1q) binding, activation and complement-dependent cytotoxicity (CDC), (iii) phagocytosis/clearance of antigen-antibody complexes, and (iv) cytokine release in some instances. These Fc-effector functions of an antibody molecule are mediated through the interaction of the Fc-region with a set of class-specific cell surface receptors. Antibodies of the IgG1 isotype are most active while IgG2 and IgG4 having minimal or no effector functions. The effector functions of the IgG antibodies are mediated through interactions with three structurally homologous cellular Fc receptor types (and sub-types) (FcgR1, FcgRII and FcgRIII). These effector functions of an IgG1 can be eliminated by mutating specific amino acid residues in the lower hinge region (e.g., L234A, L235A) that are required for FcgR and C1q binding Amino acid residues in the Fc region, in particular the CH2-CH3 domains, also determine the circulating half-life of the antibody molecule. This Fc function is mediated through the binding of the Fc-region to the neonatal Fc receptor (FcRn) which is responsible for recycling of antibody molecules from the acidic lysosomes back to the general circulation.
Whether a mAb should have an active or an inactive isotype will depend on the desired therapeutic end-point for an antibody. Some examples of usage of isotypes and desired therapeutic outcome are listed below:

    
    
        a) If the desired end-point is functional neutralization of a soluble cytokine then an inactive isotype may be used;
        b) If the desired out-come is clearance of a pathological protein an active isotype may be used;
        c) If the desired out-come is clearance of protein aggregates an active isotype may be used;
        d) If the desired outcome is to antagonize a surface receptor an inactive isotype is used (Tysabri, IgG4; OKT3, mutated IgG1);
        e) If the desired outcome is to eliminate target cells an active isotype is used (Herceptin, IgG1 (and with enhanced effector functions); and
        f) If the desired outcome is to clear proteins from circulation without entering the CNS an IgM isotype may be used (e.g., clearing circulating Ab peptide species).

The Fc effector functions of a parental mAb can be determined by various in vitro methods well known in the art.

    
    


As discussed, the selection of isotype, and thereby the effector functions will depend upon the desired therapeutic end-point. In cases where simple neutralization of a circulating target is desired, for example blocking receptor-ligand interactions, the effector functions may not be required. In such instances isotypes or mutations in the Fc-region of an antibody that eliminate effector functions are desirable. In other instances where elimination of target cells is the therapeutic end-point, for example elimination of tumor cells, isotypes or mutations or de-fucosylation in the Fc-region that enhance effector functions are desirable (Presta G L, Adv. Drug Delivery Rev. 58:640-656, 2006; Satoh M., Iida S., Shitara K. Expert Opinion Biol. Ther. 6:1161-1173, 2006). Similarly, depending up on the therapeutic utility, the circulating half-life of an antibody molecule can be reduced/prolonged by modulating antibody-FcRn interactions by introducing specific mutations in the Fc region (Dall'Acqua W F, Kiener P A, Wu H. J. Biol. Chem. 281:23514-23524, 2006; Petkova S B., Akilesh S., Sproule T J. et al. Internat. Immunol. 18:1759-1769, 2006; Vaccaro C., Bawdon R., Wanjie S et al. PNAS 103:18709-18714, 2007).
The published information on the various residues that influence the different effector functions of a normal therapeutic mAb may need to be confirmed for DVD-Ig. It may be possible that in a DVD-Ig format additional (different) Fc-region residues, other than those identified for the modulation of monoclonal antibody effector functions, may be important.
Overall, the decision as to which Fc-effector functions (isotype) will be critical in the final DVD-Ig format will depend up on the disease indication, therapeutic target, desired therapeutic end-point and safety considerations. Listed below are exemplary appropriate heavy chain and light chain constant regions including, but not limited to:

    
    
        IgG1—allotype: G1mz
        IgG1 mutant—A234, A235
        IgG2—allotype: G2m(n−)
        Kappa—Km3
        Lambda
    
    


Fc Receptor and C1q Studies: The possibility of unwanted antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by antibody complexing to any overexpressed target on cell membranes can be abrogated by the (for example, L234A, L235A) hinge-region mutations. These substituted amino acids, present in the IgG1 hinge region of mAb, are expected to result in diminished binding of mAb to human Fc receptors (but not FcRn), as FcgR binding is thought to occur within overlapping sites on the IgG1 hinge region. This feature of mAb may lead to an improved safety profile over antibodies containing a wild-type IgG. Binding of mAb to human Fc receptors can be determined by flow cytometry experiments using cell lines (e.g., THP-1, K562) and an engineered CHO cell line that expresses FcgRIIb (or other FcgRs). Compared to IgG1 control monoclonal antibodies, mAb show reduced binding to FcgRI and FcgRIIa whereas binding to FcgRIIb is unaffected. The binding and activation of C1q by antigen/IgG immune complexes triggers the classical complement cascade with consequent inflammatory and/or immunoregulatory responses. The C1q binding site on IgGs has been localized to residues within the IgG hinge region. C1q binding to increasing concentrations of mAb was assessed by C1q ELISA. The results demonstrate that mAb is unable to bind to C1q, as expected when compared to the binding of a wildtype control IgG1. Overall, the L234A, L235A hinge region mutation abolishes binding of mAb to FcgRI, FcgRIIa and C1q but does not impact the interaction of mAb with FcgRIIb. This data suggests that in vivo, mAb with mutant Fc will interact normally with the inhibitory FcgRIIb but will likely fail to interact with the activating FcgRI and FcgRIIa receptors or C1q.
Human FcRn binding: The neonatal receptor (FcRn) is responsible for transport of IgG across the placenta and to control the catabolic half-life of the IgG molecules. It might be desirable to increase the terminal half-life of an antibody to improve efficacy, to reduce the dose or frequency of administration, or to improve localization to the target. Alternatively, it might be advantageous to do the converse that is, to decrease the terminal half-life of an antibody to reduce whole body exposure or to improve the target-to-non-target binding ratios. Tailoring the interaction between IgG and its salvage receptor, FcRn, offers a way to increase or decrease the terminal half-life of IgG. Proteins in the circulation, including IgG, are taken up in the fluid phase through micropinocytosis by certain cells, such as those of the vascular endothelia. IgG can bind FcRn in endosomes under slightly acidic conditions (pH 6.0-6.5) and can recycle to the cell surface, where it is released under almost neutral conditions (pH 7.0-7.4). Mapping of the Fc-region-binding site on FcRn80, 16, 17 showed that two histidine residues that are conserved across species, His310 and His435, are responsible for the pH dependence of this interaction. Using phage-display technology, a mouse Fc-region mutation that increases binding to FcRn and extends the half-life of mouse IgG was identified (see Victor, G. et al.; Nature Biotechnology (1997), 15(7), 637-640). Fc-region mutations that increase the binding affinity of human IgG for FcRn at pH 6.0, but not at pH 7.4, have also been identified (see Dall'Acqua William F, et al., Journal of Immunology (2002), 169(9), 5171-80). Moreover, in one case, a similar pH-dependent increase in binding (up to 27-fold) was also observed for rhesus FcRn, and this resulted in a twofold increase in serum half-life in rhesus monkeys compared with the parent IgG (see Hinton, Paul R. et al., Journal of Biological Chemistry (2004), 279(8), 6213-6216). These findings indicate that it is feasible to extend the plasma half-life of antibody therapeutics by tailoring the interaction of the Fc region with FcRn. Conversely, Fc-region mutations that attenuate interaction with FcRn can reduce antibody half-life.
B.10 Pharmacokinetics (PK):
To generate a DVD-Ig molecule with desired pharmacokinetic profile, in an embodiment parent mAbs with the similarly desired pharmacokinetic profile are selected. One consideration is that immunogenic response to monoclonal antibodies (i.e., HAHA, human anti-human antibody response; HACA, human anti-chimeric antibody response) further complicates the pharmacokinetics of these therapeutic agents. In an embodiment, monoclonal antibodies with minimal or no immunogenicity are used for constructing DVD-Ig molecules such that the resulting DVD-Igs will also have minimal or no immunogenicity. Some of the factors that determine the PK of a mAb include, but are not limited to, Intrinsic properties of the mAb (VH amino acid sequence); immunogenicity; FcRn binding and Fc functions.
The PK profile of selected parental monoclonal antibodies can be easily determined in rodents as the PK profile in rodents correlates well with (or closely predicts) the PK profile of monoclonal antibodies in cynomolgus monkey and humans. The PK profile is determined as described in Example section 1.2.2.3.A.
After the parental monoclonal antibodies with desired PK characteristics (and other desired functional properties as discussed herein) are selected, the DVD-Ig is constructed. As the DVD-Ig molecules contain two antigen-binding domains from two parental monoclonal antibodies, the PK properties of the DVD-Ig are assessed as well. Therefore, while determining the PK properties of the DVD-Ig, PK assays may be employed that determine the PK profile based on functionality of both antigen-binding domains derived from the 2 parent monoclonal antibodies. The PK profile of a DVD-Ig can be determined as described in Example 1.2.2.3.A. Additional factors that may impact the PK profile of DVD-Ig include the antigen-binding domain (CDR) orientation; Linker size; and Fc/FcRn interactions. PK characteristics of parent antibodies can be evaluated by assessing the following parameters: absorption, distribution, metabolism and excretion.
Absorption: To date, administration of therapeutic monoclonal antibodies is via parenteral routes (e.g., intravenous [IV], subcutaneous [SC], or intramuscular [IM]). Absorption of a mAb into the systemic circulation following either SC or IM administration from the interstitial space is primarily through the lymphatic pathway. Saturable, presystemic, proteolytic degradation may result in variable absolute bioavailability following extravascular administration. Usually, increases in absolute bioavailability with increasing doses of monoclonal antibodies may be observed due to saturated proteolytic capacity at higher doses. The absorption process for a mAb is usually quite slow as the lymph fluid drains slowly into the vascular system, and the duration of absorption may occur over hours to several days. The absolute bioavailability of monoclonal antibodies following SC administration generally ranges from 50% to 100%.
Distribution: Following IV administration, monoclonal antibodies usually follow a biphasic serum (or plasma) concentration-time profile, beginning with a rapid distribution phase, followed by a slow elimination phase. In general, a biexponential pharmacokinetic model best describes this kind of pharmacokinetic profile. The volume of distribution in the central compartment (Vc) for a mAb is usually equal to or slightly larger than the plasma volume (2-3 liters). A distinct biphasic pattern in serum (plasma) concentration versus time profile may not be apparent with other parenteral routes of administration, such as IM or SC, because the distribution phase of the serum (plasma) concentration-time curve is masked by the long absorption portion. Many factors, including physicochemical properties, site-specific and target-oriented receptor mediated uptake, binding capacity of tissue, and mAb dose can influence biodistribution of a mAb. Some of these factors can contribute to nonlinearity in biodistribution for a mAb.
Metabolism and Excretion: Due to the molecular size, intact monoclonal antibodies are not excreted into the urine via kidney. They are primarily inactivated by metabolism (e.g., catabolism). For IgG-based therapeutic monoclonal antibodies, half-lives typically ranges from hours or 1-2 days to over 20 days. The elimination of a mAb can be affected by many factors, including, but not limited to, affinity for the FcRn receptor, immunogenicity of the mAb, the degree of glycosylation of the mAb, the susceptibility for the mAb to proteolysis, and receptor-mediated elimination.
B.11 Tissue Cross-Reactivity Pattern on Human and Tox Species:
Identical staining pattern suggests that potential human toxicity can be evaluated in tox species. Tox species are those animal in which unrelated toxicity is studied.
The individual antibodies are selected to meet two criteria. (1) Tissue staining appropriate for the known expression of the antibody target. (2) Similar staining pattern between human and tox species tissues from the same organ.
Criterion 1: Immunizations and/or antibody selections typically employ recombinant or synthesized antigens (proteins, carbohydrates or other molecules). Binding to the natural counterpart and counterscreen against unrelated antigens are often part of the screening funnel for therapeutic antibodies. However, screening against a multitude of antigens is often unpractical. Therefore tissue cross-reactivity studies with human tissues from all major organs serve to rule out unwanted binding of the antibody to any unrelated antigens.
Criterion 2: Comparative tissue cross reactivity studies with human and tox species tissues (cynomolgus monkey, dog, possibly rodents and others, the same 36 or 37 tissues are being tested as in the human study) help to validate the selection of a tox species. In the typical tissue cross-reactivity studies on frozen tissues sections therapeutic antibodies may demonstrate the expected binding to the known antigen and/or to a lesser degree binding to tissues based either on low level interactions (unspecific binding, low level binding to similar antigens, low level charge based interactions etc.). In any case the most relevant toxicology animal species is the one with the highest degree of coincidence of binding to human and animal tissue.
Tissue cross reactivity studies follow the appropriate regulatory guidelines including EC CPMP Guideline III/5271/94 “Production and quality control of mAbs” and the 1997 US FDA/CBER “Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use”. Cryosections (5 μm) of human tissues obtained at autopsy or biopsy were fixed and dried on object glass. The peroxidase staining of tissue sections was performed, using the avidin-biotin system. FDA's Guidance “Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use”. Relevant references include Clarke J 2004, Boon L. 2002a, Boon L 2002b, Ryan A 1999.
Tissue cross reactivity studies are often done in two stages, with the first stage including cryosections of 32 tissues (typically: Adrenal Gland, Gastrointestinal Tract, Prostate, Bladder, Heart, Skeletal Muscle, Blood Cells, Kidney, Skin, Bone Marrow, Liver, Spinal Cord, Breast, Lung, Spleen, Cerebellum, Lymph Node, Testes, Cerebral Cortex, Ovary, Thymus, Colon, Pancreas, Thyroid, Endothelium, Parathyroid, Ureter, Eye, Pituitary, Uterus, Fallopian Tube and Placenta) from one human donor. In the second phase a full cross reactivity study is performed with up to 38 tissues (including adrenal, blood, blood vessel, bone marrow, cerebellum, cerebrum, cervix, esophagus, eye, heart, kidney, large intestine, liver, lung, lymph node, breast mammary gland, ovary, oviduct, pancreas, parathyroid, peripheral nerve, pituitary, placenta, prostate, salivary gland, skin, small intestine, spinal cord, spleen, stomach, striated muscle, testis, thymus, thyroid, tonsil, ureter, urinary bladder, and uterus) from 3 unrelated adults. Studies are done typically at minimally two dose levels.
The therapeutic antibody (i.e. test article) and isotype matched control antibody may be biotinylated for avidin-biotin complex (ABC) detection; other detection methods may include tertiary antibody detection for a FITC (or otherwise) labeled test article, or precomplexing with a labeled anti-human IgG for an unlabeled test article.
Briefly, cryosections (about 5 μm) of human tissues obtained at autopsy or biopsy are fixed and dried on object glass. The peroxidase staining of tissue sections is performed, using the avidin-biotin system. First (in case of a precomplexing detection system), the test article is incubated with the secondary biotinylated anti-human IgG and developed into immune complex. The immune complex at the final concentrations of 2 and 10 μg/mL of test article is added onto tissue sections on object glass and then the tissue sections were reacted for 30 minutes with a avidin-biotin-peroxidase kit. Subsequently, DAB (3,3′-diaminobenzidine), a substrate for the peroxidase reaction, was applied for 4 minutes for tissue staining. Antigen-Sepharose beads are used as positive control tissue sections.
Any specific staining is judged to be either an expected (e.g., consistent with antigen expression) or unexpected reactivity based upon known expression of the target antigen in question. Any staining judged specific is scored for intensity and frequency. Antigen or serum completion or blocking studies can assist further in determining whether observed staining is specific or nonspecific.
If two selected antibodies are found to meet the selection criteria appropriate tissue staining, matching staining between human and toxicology animal specific tissue they can be selected for DVD-Ig generation.
The tissue cross reactivity study has to be repeated with the final DVD-Ig construct, but while these studies follow the same protocol as outline herein, they are more complex to evaluate because any binding can come from any of the two parent antibodies, and any unexplained binding needs to be confirmed with complex antigen competition studies.
It is readily apparent that the complex undertaking of tissue crossreactivity studies with a multispecific molecule like a DVD-Ig is greatly simplified if the two parental antibodies are selected for (1) lack of unexpected tissue cross reactivity findings and (2) for appropriate similarity of tissue cross reactivity findings between the corresponding human and toxicology animal species tissues.
B.12 Specificity and Selectivity:
To generate a DVD-Ig molecule with desired specificity and selectivity, one needs to generate and select parent mAbs with the similarly desired specificity and selectivity profile.
Binding studies for specificity and selectivity with a DVD-Ig can be complex due to the four or more binding sites, two each for each antigen. Briefly, binding studies using ELISA, BIAcore. KinExA or other interaction studies with a DVD-Ig need to monitor the binding of one, two or more antigens to the DVD-Ig molecule. While BIAcore technology can resolve the sequential, independent binding of multiple antigens, more traditional methods including ELISA or more modern techniques like KinExA cannot. Therefore careful characterization of each parent antibody is critical. After each individual antibody has been characterized for specificity, confirmation of specificity retention of the individual binding sites in the DVD-Ig molecule is greatly simplified.
It is readily apparent that the complex undertaking of determining the specificity of a DVD-Ig is greatly simplified if the two parental antibodies are selected for specificity prior to being combined into a DVD-Ig.
Antigen-antibody interaction studies can take many forms, including many classical protein protein interaction studies, including ELISA (Enzyme linked immunosorbent assay), Mass spectrometry, chemical cross linking, SEC with light scattering, equilibrium dialysis, gel permeation, ultrafiltration, gel chromatography, large-zone analytical SEC, micropreparative ultracentrigugation (sedimentation equilibrium), spectroscopic methods, titration microcalorimetry, sedimentation equilibrium (in analytical ultracentrifuge), sedimentation velocity (in analytical centrifuge), surface plasmon resonance (including BIAcore). Relevant references include “Current Protocols in Protein Science”, John E. Coligan, Ben M. Dunn, David W. Speicher, Paul T, Wingfield (eds.) Volume 3, chapters 19 and 20, published by John Wiley & Sons Inc., and references included therein and “Current Protocols in Immunology”, John E. Coligan, Barbara E. Bierer, David H. Margulies, Ethan M. Shevach, Warren Strober (eds.) published by John Wiley & Sons Inc and relevant references included therein.
Cytokine Release in Whole Blood: The interaction of mAb with human blood cells can be investigated by a cytokine release assay (Wing, M. G. Therapeutic Immunology (1995), 2(4), 183-190; “Current Protocols in Pharmacology”, S. J. Enna, Michael Williams, John W. Ferkany, Terry Kenakin, Paul Moser, (eds.) published by John Wiley & Sons Inc; Madhusudan, S. Clinical Cancer Research (2004), 10(19), 6528-6534; Cox, J. Methods (2006), 38(4), 274-282; Choi, I. European Journal of Immunology (2001), 31(1), 94-106). Briefly, various concentrations of mAb are incubated with human whole blood for 24 hours. The concentration tested should cover a wide range including final concentrations mimicking typical blood levels in patients (including but not limited to 100 ng/ml-100 μg/ml). Following the incubation, supernatants and cell lysates were analyzed for the presence of IL-1Rα, TNF-α, IL-1b, IL-6 and IL-8. Cytokine concentration profiles generated for mAb were compared to profiles produced by a negative human IgG control and a positive LPS or PHA control. The cytokine profile displayed by mAb from both cell supernatants and cell lysates was comparable to control human IgG. In an embodiment, the monoclonal antibody does not interact with human blood cells to spontaneously release inflammatory cytokines.
Cytokine release studies for a DVD-Ig are complex due to the four or more binding sites, two each for each antigen. Briefly, cytokine release studies as described herein measure the effect of the whole DVD-Ig molecule on whole blood or other cell systems, but can resolve which portion of the molecule causes cytokine release. Once cytokine release has been detected, the purity of the DVD-Ig preparation has to be ascertained, because some co-purifying cellular components can cause cytokine release on their own. If purity is not the issue, fragmentation of DVD-Ig (including but not limited to removal of Fc portion, separation of binding sites etc.), binding site mutagenesis or other methods may need to be employed to deconvolute any observations. It is readily apparent that this complex undertaking is greatly simplified if the two parental antibodies are selected for lack of cytokine release prior to being combined into a DVD-Ig.
B.13 Cross Reactivity to Other Species for Toxicological Studies:
In an embodiment, the individual antibodies selected with sufficient cross-reactivity to appropriate tox species, for example, cynomolgus monkey. Parental antibodies need to bind to orthologous species target (i.e. cynomolgus monkey) and elicit appropriate response (modulation, neutralization, activation). In an embodiment, the cross-reactivity (affinity/potency) to orthologous species target should be within 10-fold of the human target. In practice, the parental antibodies are evaluated for multiple species, including mouse, rat, dog, monkey (and other non-human primates), as well as disease model species (i.e. sheep for asthma model). The acceptable cross-reactivity to tox species from the perantal monoclonal antibodies allows future toxicology studies of DVD-Ig-Ig in the same species. For that reason, the two parental monoclonal antibodies should have acceptable cross-reactivity for a common tox species therefore allowing toxicology studies of DVD-Ig in the same species.
Parent mAbs may be selected from various mAbs capable of binding specific targets and well known in the art. These include, but are not limited to anti-TNF antibody (U.S. Pat. No. 6,258,562), anti-IL-12 and/or anti-IL-12p40 antibody (U.S. Pat. No. 6,914,128); anti-IL-18 antibody (US 2005/0147610 A1), anti-05, anti-CBL, anti-CD147, anti-gp120, anti-VLA-4, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti-CD-40 (e.g., see WO2007124299) anti-Id, anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta 2, anti-HGF, anti-cMet, anti DLL-4, anti-NPR1, anti-PLGF, anti-ErbB3, anti-E-selectin, anti-Fact VII, anti-Her2/neu, anti-F gp, anti-CD11/18, anti-CD14, anti-ICAM-3, anti-RON, anti CD-19, anti-CD80 (e.g., see WO2003039486, anti-CD4, anti-CD3, anti-CD23, anti-beta2-integrin, anti-alpha4beta7, anti-CD52, anti-HLA DR, anti-CD22 (e.g., see U.S. Pat. No. 5,789,554), anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR alpha beta, anti-CD2, anti-Hep B, anti-CA 125, anti-EpCAM, anti-gp120, anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25, anti-CD33, anti-HLA, anti-IGF1,2, anti-IGFR, anti-VNRintegrin, anti-IL-1alpha, anti-IL-1 beta, anti-IL-1 receptor, anti-IL-2 receptor, anti-IL-4, anti-IL-4 receptor, anti-IL-5, anti-IL-5 receptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor, anti-IL-17, and anti-IL-23 (see Presta L G., 2005, Selection, design, and engineering of therapeutic antibodies, J. Allergy Clin. Immunol. 116:731-6 .
Parent mAbs may also be selected from various therapeutic antibodies approved for use, in clinical trials, or in development for clinical use. Such therapeutic antibodies include, but are not limited to, rituximab (Rituxan®, IDEC/Genentech/Roche) (see for example U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approved to treat Non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently being developed by Genmab, an anti-CD20 antibody described in U.S. Pat. No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT/US2003/040426, entitled “Immunoglobulin Variants and Uses Thereof”), trastuzumab (Herceptin®, Genentech) (see for example U.S. Pat. No. 5,677,171), a humanized anti-Her2/neu antibody approved to treat breast cancer; pertuzumab (rhuMab-2C4, Omnitarg®), currently being developed by Genentech; an anti-Her2 antibody described in U.S. Pat. No. 4,753,894; cetuximab (Erbitux®, Imclone) (U.S. Pat. No. 4,943,533; PCT WO 96/40210), a chimeric anti-EGFR antibody in clinical trials for a variety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883), currently being developed by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S. Ser. No. 10/172,317), currently being developed by Genmab; 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem. 35(4):315-20; Kettleborough et al., 1991, Protein Eng. 4(7):773-83); ICR62 (Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(1-3):129-46; Modjtahedi et al., 1993, Br J. Cancer. 1993, 67(2):247-53; Modjtahedi et al, 1996, Br J Cancer, 73(2):228-35; Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No. 6,506,883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO 0162931A2); and SC100 (Scancell) (PCT WO 01/88138); alemtuzumab (Campath®, Millenium), a humanized mAb currently approved for treatment of B-cell chronic lymphocytic leukemia; muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), an anti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67 protein) antibody developed by Celltech/Wyeth, alefacept (Amevive®), an anti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro®), developed by Centocor/Lilly, basiliximab (Simulect®), developed by Novartis, palivizumab (Synagis®), developed by Medimmune, infliximab (Remicade®), an anti-TNFalpha antibody developed by Centocor, adalimumab (Humira®), an anti-TNFalpha antibody developed by Abbott, Humicade®, an anti-TNFalpha antibody developed by Celltech, golimumab (CNTO-148), a fully human TNF antibody developed by Centocor, etanercept (Enbrel®), an p75 TNF receptor Fc fusion developed by Immunex/Amgen, lenercept, an p55TNF receptor Fc fusion previously developed by Roche, ABX-CBL, an anti-CD147 antibody being developed by Abgenix, ABX-IL8, an anti-IL8 antibody being developed by Abgenix, ABX-MA1, an anti-MUC18 antibody being developed by Abgenix, Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 in development by Antisoma, Therex (R1550), an anti-MUC1 antibody being developed by Antisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS1407) being developed by Antisoma, Antegren® (natalizumab), an anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, an anti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody being developed by Biogen, CAT-152, an anti-TGF-β2 antibody being developed by Cambridge Antibody Technology, ABT 874 (J695), an anti-IL-12 p40 antibody being developed by Abbott, CAT-192, an anti-TGFβ1 antibody being developed by Cambridge Antibody Technology and Genzyme, CAT-213, an anti-Eotaxin1 antibody being developed by Cambridge Antibody Technology, LymphoStat-B® an anti-Blys antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc., TRAIL-R1 mAb, an anti-TRAIL-R1 antibody being developed by Cambridge Antibody Technology and Human Genome Sciences, Inc., Avastin® bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed by Genentech, an anti-HER receptor family antibody being developed by Genentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibody being developed by Genentech, Xolair® (Omalizumab), an anti-IgE antibody being developed by Genentech, Raptiva® (Efalizumab), an anti-CD11a antibody being developed by Genentech and Xoma, MLN-02 Antibody (formerly LDP-02), being developed by Genentech and Millenium Pharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed by Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab and Amgen, HuMax-Inflam, being developed by Genmab and Medarex, HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma, being developed by Genmab and Amgen, HuMax-TAC, being developed by Genmab, IDEC-131, and anti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody being developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDEC Pharmaceuticals, IDEC-152, an anti-CD23 being developed by IDEC Pharmaceuticals, anti-macrophage migration factor (MIF) antibodies being developed by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibody being developed by Imclone, IMC-1C11, an anti-KDR antibody being developed by Imclone, DC101, an anti-flk-1 antibody being developed by Imclone, anti-VE cadherin antibodies being developed by Imclone, CEA-Cide® (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibody being developed by Immunomedics, LymphoCide® (Epratuzumab), an anti-CD22 antibody being developed by Immunomedics, AFP-Cide, being developed by Immunomedics, MyelomaCide, being developed by Immunomedics, LkoCide, being developed by Immunomedics, ProstaCide, being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by Medarex, MDX-060, an anti-CD30 antibody being developed by Medarex, MDX-070 being developed by Medarex, MDX-018 being developed by Medarex, Osidem® (IDM-1), and anti-Her2 antibody being developed by Medarex and Immuno-Designed Molecules, HuMax®-CD4, an anti-CD4 antibody being developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Medarex and Genmab, CNTO 148, an anti-TNFα antibody being developed by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokine antibody being developed by Centocor/J&J, MOR101 and MOR102, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies being developed by MorphoSys, MOR201, an anti-fibroblast growth factor receptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion® (visilizumab), an anti-CD3 antibody being developed by Protein Design Labs, HuZAF®, an anti-gamma interferon antibody being developed by Protein Design Labs, Anti-α 5β1 Integrin, being developed by Protein Design Labs, anti-IL-12, being developed by Protein Design Labs, ING-1, an anti-Ep-CAM antibody being developed by Xoma, Xolair® (Omalizumab) a humanized anti-IgE antibody developed by Genentech and Novartis, and MLN01, an anti-Beta2 integrin antibody being developed by Xoma, all of the herein-cited references in this paragraph are expressly incorporated herein by reference. In another embodiment, the therapeutics include KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer Research); CNTO 95 (alpha V integrins, Centocor); MEDI-522 (alpha Vβ3 integrin, Medimmune); volociximab (alpha Vβ1 integrin, Biogen/PDL); Human mAb 216 (B cell glycosolated epitope, NCI); BiTE MT103 (bispecific CD19×CD3, Medimmune); 4G7×H22 (Bispecific BcellxFcgammaR1, Medarex/Merck KGa); rM28 (Bispecific CD28×MAPG, US Pat. No. EP1,444,268); MDX447 (EMD 82633) (Bispecific CD64×EGFR, Medarex); Catumaxomab (removab) (Bispecific EpCAM×anti-CD3, Trion/Fres); Ertumaxomab (bispecific HER2/CD3, Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx); Rencarex® (WX G250) (carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507) (CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab (Rituxan) (CD20, Genentech); veltuzumab (hA20) (CD20, Immunomedics); Epratuzumab (CD22, Amgen); lumiliximab (IDEC 152) (CD23, Biogen); muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc receptor, PDL Biopharma); HeFi-1, CD30, NCI); MDX-060 (CD30, Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, Seattle Genentics); SGN-33 (Lintuzumab) (CD33, Seattle Genentics); Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40, Seattle Genentics); Campath1h (Alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38, Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293 (TRC093/D93) (cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab (Ticilimumab, CP-675,2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab) (DR4 TRAIL-R1 agonist, Human Genome Science/Glaxo Smith Kline); AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2 (lexatumumab) (DR5 TRAIL-R2 agonist, HGS); Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8, (EGFR, Imclone); Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANT Immunotherapeutics); adecatumumab (MT201) (Epcam, Merck); edrecolomab (Panorex, 17-1A) (Epcam, Glaxo/Centocor); MORAb-003 (folate receptor a, Morphotech); KW-2871 (ganglioside GD3, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); Trastuzumab (Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); apolizumab (HLA-DR beta chain, PDL Pharma); AMG-479 (IGF-1R, Amgen); anti-IGF-1R R1507 (IGF1-R, Roche); CP 751871 (IGF1-R, Pfizer); IMC-A12 (IGF1-R, Imclone); BIIB022 (IGF-1R, Biogen); Mik-beta-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor); Anti-KIR (1-7F9) (Killer cell Ig-like Receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research); hCBE-11 (LTβR, Biogen); HuHMFG1 (MUC1, Antisoma/NCI); RAV12 (N-linked carbohydrate epitope, Raven); CAL (parathyroid hormone-related protein (PTH-rP), University of California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1, Medarex/Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa, Imclone); bavituximab (phosphatidylserine, Peregrine); huJ591 (PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab (Remicade) (TNFa, Centocor); A27.15 (transferrin receptor, Salk Institute, INSERN WO 2005/111082); E2.3 (transferrin receptor, Salk Institute); Bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab-WO/2000/034337, University of Texas); IMC-18F1 (VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone).
B. Construction of DVD Molecules:
The dual variable domain immunoglobulin (DVD-Ig) molecule is designed such that two different light chain variable domains (VL) from the two different parent monoclonal antibodies are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CH1 and Fc region (FIG. 1A).
The variable domains can be obtained using recombinant DNA techniques from a parent antibody generated by any one of the methods described herein. In an embodiment, the variable domain is a murine heavy or light chain variable domain. In another embodiment, the variable domain is a CDR grafted or a humanized variable heavy or light chain domain. In an embodiment, the variable domain is a human heavy or light chain variable domain.
In one embodiment the first and second variable domains are linked directly to each other using recombinant DNA techniques. In another embodiment the variable domains are linked via a linker sequence. In an embodiment, two variable domains are linked. Three or more variable domains may also be linked directly or via a linker sequence. The variable domains may bind the same antigen or may bind different antigens. DVD molecules of the invention may include one immunoglobulin variable domain and one non-immunoglobulin variable domain such as ligand binding domain of a receptor, active domain of an enzyme. DVD molecules may also comprise 2 or more non-Ig domains.
The linker sequence may be a single amino acid or a polypeptide sequence. In an embodiment, the linker sequences are selected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G4S)4 (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25); GHEAAAVMQVQYPAS (SEQ ID NO: 26). The choice of linker sequences is based on crystal structure analysis of several Fab molecules. There is a natural flexible linkage between the variable domain and the CH1/CL constant domain in Fab or antibody molecular structure. This natural linkage comprises approximately 10-12 amino acid residues, contributed by 4-6 residues from C-terminus of V domain and 4-6 residues from the N-terminus of CL/CH1 domain. DVD Igs of the invention were generated using N-terminal 5-6 amino acid residues, or 11-12 amino acid residues, of CL or CH1 as linker in light chain and heavy chain of DVD-Ig, respectively. The N-terminal residues of CL or CH1 domains, particularly the first 5-6 amino acid residues, adopt a loop conformation without strong secondary structures, therefore can act as flexible linkers between the two variable domains. The N-terminal residues of CL or CH1 domains are natural extension of the variable domains, as they are part of the Ig sequences, therefore minimize to a large extent any immunogenicity potentially arising from the linkers and junctions.
Other linker sequences may include any sequence of any length of CL/CH1 domain but not all residues of CL/CH1 domain; for example the first 5-12 amino acid residues of the CL/CH1 domains; the light chain linkers can be from Cκ or Cλ; and the heavy chain linkers can be derived from CH1 of any isotypes, including Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may also be derived from other proteins such as Ig-like proteins, (e.g. TCR, FcR, KIR); G/S based sequences (e.g. G4S repeats); hinge region-derived sequences; and other natural sequences from other proteins.
In an embodiment a constant domain is linked to the two linked variable domains using recombinant DNA techniques. In an embodiment, sequence comprising linked heavy chain variable domains is linked to a heavy chain constant domain and sequence comprising linked light chain variable domains is linked to a light chain constant domain. In an embodiment, the constant domains are human heavy chain constant domain and human light chain constant domain respectively. In an embodiment, the DVD heavy chain is further linked to an Fc region. The Fc region may be a native sequence Fc region, or a variant Fc region. In another embodiment, the Fc region is a human Fc region. In another embodiment the Fc region includes Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.
In another embodiment two heavy chain DVD polypeptides and two light chain DVD polypeptides are combined to form a DVD-Ig molecule. Table 2 lists amino acid sequences of VH and VL regions of exemplary antibodies for targets useful for treating disease, e.g., for treating cancer. In an embodiment, the invention provides a DVD comprising at least two of the VH and/or VL regions listed in Table 2, in any orientation.








TABLE 2







List of Amino Acid Sequences of VH and VL regions of



Antibodies for Generating DVD-Igs











SEQ






ID
ABT
Protein
Sequence


No.
Unique ID
region
1234567890123456789012345678901234567890














27
AB001VH
VH CD20
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT






PGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAY





MQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA


 


28
AB001VL
VL CD20
QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWFQQKPG





SSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAE





DAATYYCQQWTSNPPTFGGGTKLEIKR


 


29
AB002VH
VH CD3 (seq.
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQR




1)
PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAY





MQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS


 


30
AB002VL
VL CD3 (seq.
QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSG




1)
TSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAE





DAATYYCQQWSSNPLTFGSGTKLEINR


 


31
AB003VH
VH EGFR
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIR




(seq. 1)
QSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQF





SLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS


 


32
AB003VL
VL EGFR
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKP




(seq. 1)
GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP





EDIATYFCQHFDHLPLAFGGGTKVEIKR


 


33
AB004VH
VH HER2
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA




(seq. 1)
PGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY





LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


34
AB004VL
VL HER2
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP




(seq. 1)
GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQP





EDFATYYCQQHYTTPPTFGQGTKVEIKR


 


35
AB005VH
VH RON (seq.
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYAMHWVRQA




1)
PGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLY





LQMNSLRAEDTAVYYCARFSGWPNNYYYYGMDVWGQGTTV





TVSS


 


36
AB005VL
VL RON (seq.
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGFNYVDW




1)
YLQKPGQSPHLLIYFGSYRASGVPDRFSGSGSGTDFTLKI





SRVEAEDVGVYYCMQALQTPPWTFGQGTKVEIRR


 


37
AB006VH
VH CD-19
QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQR





PGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAY





MQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSV





TVSS


 


38
AB006VL
VL CD-19
DILLTQTPASLAVSLGQRATISCKASQSVDYDGDSYLNWY





QQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIH





PVEKVDAATYHCQQSTEDPWTFGGGTKLEIKR


 


39
AB007VH
VH CD-80
QVQLQESGPGLVKPSETLSLTCAVSGGSISGGYGWGWIRQ





PPGKGLEWIGSFYSSSGNTYYNPSLKSQVTISTDTSKNQF





SLKLNSMTAADTAVYYCVRDRLFSVVGMVYNNWFDVWGPG





VLVTVSS


 


40
AB007VL
VL CD-80
ESALTQPPSVSGAPGQKVTISCTGSTSNIGGYDLHWYQQL





PGTAPKLLIYDINKRPSGISDRFSGSKSGTAASLAITGLQ





TEDEADYYCQSYDSSLNAQVFGGGTRLTVLG


 


41
AB008VH
VH CD-22
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQA





PGQGLEWIGYINPRNDYTEYNQNFKDKATITADESTNTAY





MELSSLRSEDTAFYFCARRDITTFYWGQGTTVTVSS


 


42
AB008VL
VL CD-22
DIQLTQSPSSLSASVGDRVTMSCKSSQSVLYSANHKNYLA





WYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFT





ISSLQPEDIATYYCHQYLSSWTFGGGTKLEIKR


 


43
AB009VH
VH CD-40
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQA





PGKGLEWVAVISYEESNRYHADSVKGRFTISRDNSKITLY





LQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTVSS


 


44
AB009VL
VL CD-40
DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDW





YLQKPGQSPQVLISLGSNRASGVPDRFSGSGSGTDFTLKI





SRVEAEDVGVYYCMQARQTPFTFGPGTKVDIRR


 


45
AB010VH
VH IGF1, 2
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQA





TGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAY





MELSSLRSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSS


 


46
AB010VL
VL IGF1, 2
QSVLTQPPSVSAAPGQKVTISCSGSSSNIENNHVSWYQQL





PGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQ





TGDEADYYCETWDTSLSAGRVFGGGTKLTVLG


 


47
AB011VH
VH IGF1R
EVQLLESGGGLVQPGGSLRLSCTASGFTFSSYAMNWVRQA




(seq. 1)
PGKGLEWVSAISGSGGTTFYADSVKGRFTISRDNSRTTLY





LQMNSLRAEDTAVYYCAKDLGWSDSYYYYYGMDVWGQGTT





VTVSS


 


48
AB011VL
VL IGF1R
DIQMTQFPSSLSASVGDRVTITCRASQGIRNDLGWYQQKP




(seq. 1)
GKAPKRLIYAASRLHRGVPSRFSGSGSGTEFTLTISSLQP





EDFATYYCLQHNSYPCSFGQGTKLEIKR


 


49
AB012VH
VH HGF (seq.
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQA




1)
PGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLY





LQMNSLRAEDTAVYYCARDEYNSGWYVLFDYWGQGTLVTV





SS


 


50
AB012VL
VL HGF (seq.
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP




1)
GKAPNLLIYEASSLQSGVPSRFGGSGSGTDFTLTISSLQP





EDFATYYCQQANGFPWTFGQGTKVEIKR


 


51
AB013VH
VH c-MET
QVQLQQSGPELVRPGASVKWSCPASGYTFTSYWLHWVKKQ





RPGQGLEWIGMIDPSNSDTRFNPPNFKDKATLNVDRSSNT





AYNLLSSLTSADSAVYYCATYGSYVSPLDYWGQGTSVYVSS


 


52
AB013VL
VL c-MET
DIMMSQSPSSLTVSVGEKVTVSCKSSQSLLVTSSQKNYLA





WYQQKPQQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLT





ITSVKADDLAVYYCQQYYAYPWTFGDGTKLEIKR


 


53
AB014VH
VH VEGF
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQA




(seq. 1)
PGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAY





LQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVT





VSS


 


54
AB014VL
VL VEGF
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKP




(seq. 1)
GKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQP





EDFATYYCQQYSTVPWTFGQGTKVEIKR


 


55
AB015VH
VH DLL-4
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWISWVRQA




(seq. 1)
PGKGLEWVGYISPNSGFTYYADSVKGRFTISADTSKNTAY





LQMNSLRAEDTAVYYCARDNFGGYFDYWGQGTLVTVSS


 


56
AB015VL
VL DLL-4
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKP




(seq. 1)
GKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQP





EDFATTYYCQQSYTGTVTFGQGTKVEIKR


 


57
AB016VH
VH NRP1
EVQLVESGGGLVQPGGSLRLSCAASGFSFSSEPISWVRQA




(seq. 1)
PGKGLEWVSSITGKNGYTYYADSVKGRFTISADTSKNTAY





LQMNSLRAEDTAVYYCARWGKKVYGMDVWGQGTLVTVSS


 


58
AB016VL
VL NRP1
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKP




(seq. 1)
GKAPKLLIYGASSRASGVPSRFSGSGSGTDFTLTISSLQP





EDFATYYCQQYMSVPITFGQGTKVEIKR


 


59
AB033VH
VH EGFR
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQS




(seq. 2)
PGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFF





KMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


60
AB033VL
VL EGFR
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRT




(seq. 2)
NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVES





EDIADYYCQQNNNWPTTFGAGTKLELKR


 


61
AB034VH
VH RON (seq.
QVQLQESGPGLVKPSEILSLTCTVSGGSISSHYWSWVRQP




2)
PGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSL





NLSSVTAADTAVYYCARIPNYYDRSGYYPGYWYFDLWGRG





TLVTVSS


 


62
AB034VL
VL RON (seq.
QAVLTQPSSLSAPPGASASLTCTLRSGFNVDSYRISWYQQ




2)
KPGSPPQYLLRYKSDSDKQQGSGVPSRFSGSKDASANAGI





LLISGLQSEDEADYYCMIWHSSAWVFGGGTKLTVLR


 


63
AB035VH
VH NRP1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA




(seq. 2)
PGKGLEWVSQISPAGGYTNYADSVKGRFTISADTSKNTAY





LQMNSLRAEDTAVYYCARELPYYRMSKVMDVQGQGTLVTV





SS


 


64
AB035VL
VL NRP1
DIQMTQSPSSLSASVGDRVTITCRASQYFSSYLAWYQQKP




(seq. 2)
GKAPKLLIYGASSRASGVPSRFSGSGSGTDFTLTISSLQP





EDFATYYCQQYLGSPPTFGQGTKVEIKR


 


65
AB039VH
VH CD3 (seq.
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQR




2)
PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAY





MQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS


 


66
AB039VL
VL CD3 (seq.
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSG




2)
TSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAE





DAATYYCQQWSSNPFTFGSGTKLEINR


 


67
AB047VH
VH PLGF
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYINWVKLA




(seq. 1)
PGQGLEWIGWIYPGSGNTKYNEKFKGKATLTIDTSSSTAY





MQLSSLTSEDTAVYFCVRDSPFFDYWGQGTLLTVSS


 


68
AB047VL
VL PLGF
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMRKSFLA




(seq. 1)
WYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLT





ISSVQAEDVAVYYCKQSYHLFTFGSGTKLEIKR


 


69
AB062VH
VH ErbB3
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQP




(seq. 1)
PGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSL





KLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS


 


70
AB062VL
VL ErbB3
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLA




(seq. 1)
WYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLT





ISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKR


 


71
AB063VH
VH ErbB3
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMNWVRQA




(seq. 2)
PGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLY





LQMNSLRDEDTAVYYCARDRGDFDAFDIWGQGTMVTVSS


 


72
AB063VL
VL ErbB3
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKP




(seq. 2)
GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP





EDIATYNCQQCENFPITFGQGTRLEIKR


 


73
AB067VH
VH ErbB3
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMAWVRQA




(seq. 3)
PGKGLEWVSSISSSGGWTLYADSVKGRFTISRDNSKNTLY





LQMNSLRAEDTAVYYCTRGLKMATIFDYWGQGTLVTVSS


 


74
AB067VL
VL ErbB3
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVVSWYQQ




(seq. 3)
HPGKAPKLIIYEVSQRPSGVSNRFSGSKSGNTASLTISGL





QTEDEADYYCCSYAGSSIFVIFGGGTKVTVLG


 


75
AB069VH
VH DLL4
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQA




(seq. 2)
PGQGLEWIGYISSYNGATNYNQKFKGRVTFTTDTSTSTAY





MELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS


 


76
AB069VL
VL DLL4
DIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWF




(seq. 2)
QQKPGQPPKLLIYAASNQGSGVPDRFSGSGSGTDFTLTIS





SLQAEDVAVYYCQQSKEVPWTFGGGTKVEIKR


 


77
AB070VH
VH VEGF
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQA




(seq. 2)
PGKGLEWVAGITPAGGYTYYADSVKGRFTISADTSKNTAY





LQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS


 


78
AB070VL
VL VEGF
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKP




(seq. 2)
GKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQP





EDFATYYCQQSYTTPPTFGQGTKVEIKR


 


79
AB071VH
VH VEGF
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIHWVRQA




(seq. 3)
PGKGLEWVGAIYPYSGYTNYADSVKGRFTISADTSKNTAY





LQMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS


 


80
AB071VL
VL VEGF
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAWYQQKP




(seq. 3)
GKAPKLLIYAASNLASGVPSRFSGSGSGTDFTLTISSLQP





EDFATYYCQQSNTSPLTFGQGTKVEIKR


 


81
AB072VH
VH DLL4
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVINWVKQK




(seq. 3)
PGQGLEWIGLINPYNDGTKYNEKFKVKATLTSDKSSSTAY





MELSSLTSEDSAVYYCASYYYGSRYYFDYWGQGTTLTVSS


 


82
AB072VL
VL DLL4
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKP




(seq. 3)
GNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQT





EDVATYYCQQYWSIPLTFGAGTKLELKR


 


83
AB073VH
VH DLL4
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQS




(seq. 4)
NGKSLEWIGNIDPYFGGTNYNQKFKGKATLTVDKSSSTAY





MQLKSLTSEDSAVYYCARNYDYDGGCFDYWGQGTTLTVSS


 


84
AB073VL
VL DLL4
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQE




(seq. 4)
KPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGA





QTEDEAIYFCALWYSNHWVFGGGTKLTVLG


 


85
AB074VH
VH PLGF
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIHWVRQS




(seq. 2)
PGKGLEWLGVMWSGGDTDYDAAFISRLSISKDNSKSQVFF





KMNSLQANDTGIYYCARYRFYGMDYWGQGTSVTVSS


 


86
AB074VL
VL PLGF
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAWYQQKP




(seq. 2)
GNAPRLLISGAASLEAGVPSRFSGSGSGQDYTLSITSLQT





EDVATYYCQQYWSTPWTFGGGTKLEIKR


 


87
AB075VH
VH IGF1R
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA




(seq. 2)
PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAY





MELSSLRSEDTAVYYCARAPLRFLEWSTQDHYYYYYMDVW





GKGTTVTVSS


 


88
AB075VL
VL IGF1R
SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPG




(seq. 2)
QAPILVIYGENKRPSGIPDRFSGSSSGNTASLTITGAQAE





DEADYYCKSRDGSGQHLVFGGGTKLTVLG


 


89
AB077VH
VH IGF1R
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSFAMHWVRQA




(seq. 3)
PGKGLEWISVIDTRGATYYADSVKGRFTISRDNAKNSLYL





QMNSLRAEDTAVYYCARLGNFYYGMDVWGQGTTVTVSS


 


90
AB077VL
VL IGF1R
EIVLTQSPGTLSVSPGERATLSCRASQSIGSSLHWYQQKP




(seq. 3)
GQAPRLLIKYASQSLSGIPDRFSGSGSGTDFTLTISRLEP





EDFAVYYCHQSSRLPHTFGQGTKVEIKR


 


91
AB079VH
VH HGF (seq.
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMHWVRQM




2)
PGKGLEWMGEINPTNGHTNYNPSFQGQVTISADKSISTAY





LQWSSLKASDTAMYYCARNYVGSIFDYWGQGTLVTVSS


 


92
AB079VL
VL HGF (seq.
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSWYQQKP




2)
GKAPKLLIYGASNRNTGVPSRFSGSGSGTDFTLTISSLQP





EDFATYYCGQSYNYPYTFGQGTKLEIKR


 


93
AB080VH
VH HER2
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQA




(seq. 2)
PGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLY





LQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS


 


94
AB080VL
VL HER2
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKP




(seq. 2)
GKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQP





EDFATYYCQQYYIYPYTFGQGTKVEIKR









Detailed description of specific DVD-Ig molecules capable of binding specific targets, and methods of making the same, is provided in the Examples section below.
C. Production of DVD Proteins
Binding proteins of the present invention may be produced by any of a number of techniques known in the art. For example, expression from host cells, wherein expression vector(s) encoding the DVD heavy and DVD light chains is (are) transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is possible to express the DVD proteins of the invention in either prokaryotic or eukaryotic host cells, DVD proteins are expressed in eukaryotic cells, for example, mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active DVD protein.
Exemplary mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr− CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells, SP2 and PER.C6 cells. When recombinant expression vectors encoding DVD proteins are introduced into mammalian host cells, the DVD proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the DVD proteins in the host cells or secretion of the DVD proteins into the culture medium in which the host cells are grown. DVD proteins can be recovered from the culture medium using standard protein purification methods.
In an exemplary system for recombinant expression of DVD proteins of the invention, a recombinant expression vector encoding both the DVD heavy chain and the DVD light chain is introduced into dhfr− CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the DVD heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the DVD heavy and light chains and intact DVD protein is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the DVD protein from the culture medium. Still further the invention provides a method of synthesizing a DVD protein of the invention by culturing a host cell of the invention in a suitable culture medium until a DVD protein of the invention is synthesized. The method can further comprise isolating the DVD protein from the culture medium.
An important feature of DVD-Ig is that it can be produced and purified in a similar way as a conventional antibody. The production of DVD-Ig results in a homogeneous, single major product with desired dual-specific activity, without any sequence modification of the constant region or chemical modifications of any kind. Other previously described methods to generate “bi-specific”, “multi-specific”, and “multi-specific multivalent” full length binding proteins do not lead to a single primary product but instead lead to the intracellular or secreted production of a mixture of assembled inactive, mono-specific, multi-specific, multivalent, full length binding proteins, and multivalent full length binding proteins with combination of different binding sites. As an example, based on the design described by Miller and Presta (PCT publication WO2001/077342(A1), there are 16 possible combinations of heavy and light chains. Consequently only 6.25% of protein is likely to be in the desired active form, and not as a single major product or single primary product compared to the other 15 possible combinations. Separation of the desired, fully active forms of the protein from inactive and partially active forms of the protein using standard chromatography techniques, typically used in large scale manufacturing, is yet to be demonstrated.
Surprisingly the design of the “dual-specific multivalent full length binding proteins” of the present invention leads to a dual variable domain light chain and a dual variable domain heavy chain which assemble primarily to the desired “dual-specific multivalent full length binding proteins”.
At least 50%, at least 75% and at least 90% of the assembled, and expressed dual variable domain immunoglobulin molecules are the desired dual-specific tetravalent protein. This aspect of the invention particularly enhances the commercial utility of the invention. Therefore, the present invention includes a method to express a dual variable domain light chain and a dual variable domain heavy chain in a single cell leading to a single primary product of a “dual-specific tetravalent full length binding protein”.
The present invention provides a methods of expressing a dual variable domain light chain and a dual variable domain heavy chain in a single cell leading to a “primary product” of a “dual-specific tetravalent full length binding protein”, where the “primary product” is more than 50% of all assembled protein, comprising a dual variable domain light chain and a dual variable domain heavy chain.
The present invention provides methods of expressing a dual variable domain light chain and a dual variable domain heavy chain in a single cell leading to a single “primary product” of a “dual-specific tetravalent full length binding protein”, where the “primary product” is more than 75% of all assembled protein, comprising a dual variable domain light chain and a dual variable domain heavy chain.
The present invention provides methods of expressing a dual variable domain light chain and a dual variable domain heavy chain in a single cell leading to a single “primary product” of a “dual-specific tetravalent full length binding protein”, where the “primary product” is more than 90% of all assembled protein, comprising a dual variable domain light chain and a dual variable domain heavy chain.
II. Derivatized DVD Binding Proteins:
One embodiment provides a labeled binding protein wherein the binding protein of the invention is derivatized or linked to another functional molecule (e.g., another peptide or protein). For example, a labeled binding protein of the invention can be derived by functionally linking an binding protein of the invention (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the binding protein with another molecule (such as a streptavidin core region or a polyhistidine tag).
Useful detectable agents with which a binding protein of the invention may be derivatized include fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and the like. A binding protein may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When a binding protein is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. A binding protein may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
Another embodiment of the invention provides a crystallized binding protein and formulations and compositions comprising such crystals. In one embodiment the crystallized binding protein has a greater half-life in vivo than the soluble counterpart of the binding protein. In another embodiment the binding protein retains biological activity after crystallization.
Crystallized binding protein of the invention may be produced according to methods known in the art and as disclosed in WO 02072636, incorporated herein by reference.
Another embodiment of the invention provides a glycosylated binding protein wherein the antibody or antigen-binding portion thereof comprises one or more carbohydrate residues. Nascent in vivo protein production may undergo further processing, known as post-translational modification. In particular, sugar (glycosyl) residues may be added enzymatically, a process known as glycosylation. The resulting proteins bearing covalently linked oligosaccharide side chains are known as glycosylated proteins or glycoproteins. Antibodies are glycoproteins with one or more carbohydrate residues in the Fc domain, as well as the variable domain. Carbohydrate residues in the Fc domain have important effect on the effector function of the Fc domain, with minimal effect on antigen binding or half-life of the antibody (R. Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the variable domain may have an effect on the antigen binding activity of the antibody. Glycosylation in the variable domain may have a negative effect on antibody binding affinity, likely due to steric hindrance (Co, M. S., et al., Mol. Immunol. (1993) 30:1361-1367), or result in increased affinity for the antigen (Wallick, S. C., et al., Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717 2723).
One aspect of the present invention is directed to generating glycosylation site mutants in which the O- or N-linked glycosylation site of the binding protein has been mutated. One skilled in the art can generate such mutants using standard well-known technologies. Glycosylation site mutants that retain the biological activity but have increased or decreased binding activity are another object of the present invention.
In still another embodiment, the glycosylation of the antibody or antigen-binding portion of the invention is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos. 5,714,350 and 6,350,861, each of which is incorporated herein by reference in its entirety.
Additionally or alternatively, a modified binding protein of the invention can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues (see Kanda, Yutaka et al., Journal of Biotechnology (2007), 130(3), 300-310.) or an antibody having increased bisecting GlcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. See, for example, Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP 1,176,195; PCT Publications WO 03/035835; WO 99/54342 80, each of which is incorporated herein by reference in its entirety.
Protein glycosylation depends on the amino acid sequence of the protein of interest, as well as the host cell in which the protein is expressed. Different organisms may produce different glycosylation enzymes (e.g., glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available. Due to such factors, protein glycosylation pattern, and composition of glycosyl residues, may differ depending on the host system in which the particular protein is expressed. Glycosyl residues useful in the invention may include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. In an embodiment, the glycosylated binding protein comprises glycosyl residues such that the glycosylation pattern is human.
It is known to those skilled in the art that differing protein glycosylation may result in differing protein characteristics. For instance, the efficacy of a therapeutic protein produced in a microorganism host, such as yeast, and glycosylated utilizing the yeast endogenous pathway may be reduced compared to that of the same protein expressed in a mammalian cell, such as a CHO cell line. Such glycoproteins may also be immunogenic in humans and show reduced half-life in vivo after administration. Specific receptors in humans and other animals may recognize specific glycosyl residues and promote the rapid clearance of the protein from the bloodstream. Other adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. Accordingly, a practitioner may choose a therapeutic protein with a specific composition and pattern of glycosylation, for example glycosylation composition and pattern identical, or at least similar, to that produced in human cells or in the species-specific cells of the intended subject animal.
Expressing glycosylated proteins different from that of a host cell may be achieved by genetically modifying the host cell to express heterologous glycosylation enzymes. Using techniques known in the art a practitioner may generate antibodies or antigen-binding portions thereof exhibiting human protein glycosylation. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylation enzymes such that glycosylated proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation identical to that of animal cells, especially human cells (U.S. patent applications 20040018590 and 20020137134 and PCT publication WO2005100584 A2).
In addition to the binding proteins, the present invention is also directed to anti-idiotypic (anti-Id) antibodies specific for such binding proteins of the invention. An anti-Id antibody is an antibody, which recognizes unique determinants generally associated with the antigen-binding region of another antibody. The anti-Id can be prepared by immunizing an animal with the binding protein or a CDR containing region thereof. The immunized animal will recognize, and respond to the idiotypic determinants of the immunizing antibody and produce an anti-Id antibody. It is readily apparent that it may be easier to generate anti-idiotypic antibodies to the two or more parent antibodies incorporated into a DVD-Ig molecule; and confirm binding studies by methods well recognized in the art (e.g., BIAcore, ELISA) to verify that anti-idiotypic antibodies specific for the idiotype of each parent antibody also recognize the idiotype (e.g., antigen binding site) in the context of the DVD-Ig. The anti-idiotypic antibodies specific for each of the two or more antigen binding sites of a DVD-Ig provide ideal reagents to measure DVD-Ig concentrations of a human DVD-Ig in patient serum; DVD-Ig concentration assays can be established using a “sandwich assay ELISA format” with an antibody to a first antigen binding regions coated on the solid phase (e.g., BIAcore chip, ELISA plate etc.), rinsed with rinsing buffer, incubation with the serum sample, another rinsing step and ultimately incubation with another anti-idiotypic antibody to the another antigen binding site, itself labeled with an enzyme for quantitation of the binding reaction. In an embodiment, for a DVD-Ig with more than two different binding sites, anti-idiotypic antibodies to the two outermost binding sites (most distal and proximal from the constant region) will not only help in determining the DVD-Ig concentration in human serum but also document the integrity of the molecule in vivo. Each anti-Id antibody may also be used as an “immunogen” to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
Further, it will be appreciated by one skilled in the art that a protein of interest may be expressed using a library of host cells genetically engineered to express various glycosylation enzymes, such that member host cells of the library produce the protein of interest with variant glycosylation patterns. A practitioner may then select and isolate the protein of interest with particular novel glycosylation patterns. In an embodiment, the protein having a particularly selected novel glycosylation pattern exhibits improved or altered biological properties.
III. Uses of DVD-Ig
Given their ability to bind to two or more antigens the binding proteins of the invention can be used to detect the antigens (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. The DVD-Ig is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm.
In an embodiment, the binding proteins of the invention are capable of neutralizing the activity of the antigens both in vitro and in vivo. Accordingly, such DVD-Igs can be used to inhibit antigen activity, e.g., in a cell culture containing the antigens, in human subjects or in other mammalian subjects having the antigens with which a binding protein of the invention cross-reacts. In another embodiment, the invention provides a method for reducing antigen activity in a subject suffering from a disease or disorder in which the antigen activity is detrimental. A binding protein of the invention can be administered to a human subject for therapeutic purposes.
As used herein, the term “a disorder in which antigen activity is detrimental” is intended to include diseases and other disorders in which the presence of the antigen in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which antigen activity is detrimental is a disorder in which reduction of antigen activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of the antigen in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of antigen in serum, plasma, synovial fluid, etc. of the subject). Non-limiting examples of disorders that can be treated with the binding proteins of the invention include those disorders discussed below and in the section pertaining to pharmaceutical compositions of the antibodies of the invention.
The DVD-Igs of the invention may bind one antigen or multiple antigens. Such antigens include, but are not limited to, therapeutic targets (see, e.g., the Bioinformatics and Drug Design Group of the National University of Singapore), cytokines and cytokine receptors (see, e.g., the Cytokines & Cells Online Pathfinder Encyclopedia), chemokines, chemokine receptors and GPCRs, olfactory receptors (see, e.g., SenseLab at Yale University), receptors (see, e.g., the IUPHAR Committee on Receptor Nomenclature and Drug Classification), cancer targets (see, e.g., the Human Genome Center at the Institute of Medical Science, University of Tokyo), secreted proteins and protein kinases (see, e.g., the Secreted Protein Database from the Center of Bioinformatics, Peking University), and human CD markers (see, e.g., Zola H, 2005, CD molecules 2005: human cell differentiation molecules, Blood, 106:3123-6).
DVD-Igs are useful as therapeutic agents to simultaneously block two different targets to enhance efficacy/safety and/or increase patient coverage. Such targets may include soluble targets (TNF) and cell surface receptor targets (VEGFR and EGFR). It can also be used to induce redirected cytotoxicity between tumor cells and T cells (Her2 and CD3) for cancer therapy, or between autoreactive cell and effector cells for autoimmune disease or transplantation, or between any target cell and effector cell to eliminate disease-causing cells in any given disease.
In addition, DVD-Ig can be used to trigger receptor clustering and activation when it is designed to target two different epitopes on the same receptor. This may have benefit in making agonistic and antagonistic anti-GPCR therapeutics. In this case, DVD-Ig can be used to target two different epitopes (including epitopes on both the loop regions and the extracellular domain) on one cell for clustering/signaling (two cell surface molecules) or signaling (on one molecule). Similarly, a DVD-Ig molecule can be designed to trigger CTLA-4 ligation, and a negative signal by targeting two different epitopes (or 2 copies of the same epitope) of CTLA-4 extracellular domain, leading to down regulation of the immune response. CTLA-4 is a clinically validated target for therapeutic treatment of a number of immunological disorders. CTLA-4/B7 interactions negatively regulate T cell activation by attenuating cell cycle progression, IL-2 production, and proliferation of T cells following activation, and CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by agonistic antibody engagement of CTLA-4 has been unsuccessful since CTLA-4 activation requires ligation. The molecular interaction of CTLA-4/B7 is in “skewed zipper” arrays, as demonstrated by crystal structural analysis (Stamper 2001 Nature 410:608). However none of the currently available CTLA-4 binding reagents have ligation properties, including anti-CTLA-4 mAbs. There have been several attempts to address this issue. In one case, a cell member-bound single chain antibody was generated, and significantly inhibited allogeneic rejection in mice (Hwang 2002 JI 169:633). In a separate case, artificial APC surface-linked single-chain antibody to CTLA-4 was generated and demonstrated to attenuate T cell responses (Griffin 2000 JI 164:4433). In both cases, CTLA-4 ligation was achieved by closely localized member-bound antibodies in artificial systems. While these experiments provide proof-of-concept for immune down-regulation by triggering CTLA-4 negative signaling, the reagents used in these reports are not suitable for therapeutic use. To this end, CTLA-4 ligation may be achieved by using a DVD-Ig molecule, which target two different epitopes (or 2 copies of the same epitope) of CTLA-4 extracellular domain. The rationale is that the distance spanning two binding sites of an IgG, approximately 150-170 Å, is too large for active ligation of CTLA-4 (30-50 Å between 2 CTLA-4 homodimer). However the distance between the two binding sites on DVD-Ig (one arm) is much shorter, also in the range of 30-50 Å, allowing proper ligation of CTLA-4.
Similarly, DVD-Ig can target two different members of a cell surface receptor complex (e.g., IL-12R alpha and beta). Furthermore, DVD-Ig can target CR1 and a soluble protein/pathogen to drive rapid clearance of the target soluble protein/pathogen.
Additionally, DVD-Igs of the invention can be employed for tissue-specific delivery (target a tissue marker and a disease mediator for enhanced local PK thus higher efficacy and/or lower toxicity), including intracellular delivery (targeting an internalizing receptor and a intracellular molecule), delivering to inside brain (targeting transferrin receptor and a CNS disease mediator for crossing the blood-brain barrier). DVD-Ig can also serve as a carrier protein to deliver an antigen to a specific location via binding to a non-neutralizing epitope of that antigen and also to increase the half-life of the antigen. Furthermore, DVD-Ig can be designed to either be physically linked to medical devices implanted into patients or target these medical devices (see Burke, Sandra E.; Kuntz, Richard E.; Schwartz, Lewis B., Zotarolimus eluting stents. Advanced Drug Delivery Reviews (2006), 58(3), 437-446; Surface coatings for biological activation and functionalization of medical devices, Hildebrand, H. F.; Blanchemain, N.; Mayer, G.; Chai, F.; Lefebvre, M.; Boschin, F., Surface and Coatings Technology (2006), 200(22-23), 6318-6324; Drug/device combinations for local drug therapies and infection prophylaxis, Wu, Peng; Grainger, David W., Biomaterials (2006), 27(11), 2450-2467; Mediation of the cytokine network in the implantation of orthopedic devices., Marques, A. P.; Hunt, J. A.; Reis, Rui L., Biodegradable Systems in Tissue Engineering and Regenerative Medicine (2005), 377-397). Briefly, directing appropriate types of cell to the site of medical implant may promote healing and restoring normal tissue function. Alternatively, inhibition of mediators (including but not limited to cytokines), released upon device implantation by a DVD coupled to or target to a device is also provided. For example, Stents have been used for years in interventional cardiology to clear blocked arteries and to improve the flow of blood to the heart muscle. However, traditional bare metal stents have been known to cause restenosis (re-narrowing of the artery in a treated area) in some patients and can lead to blood clots. Recently, an anti-CD34 antibody coated stent has been described which reduced restenosis and prevents blood clots from occurring by capturing endothelial progenitor cells (EPC) circulating throughout the blood. Endothelial cells are cells that line blood vessels, allowing blood to flow smoothly. The EPCs adhere to the hard surface of the stent forming a smooth layer that not only promotes healing but prevents restenosis and blood clots, complications previously associated with the use of stents (Aoji et al. 2005 J Am Coll Cardiol. 45(10):1574-9). In addition to improving outcomes for patients requiring stents, there are also implications for patients requiring cardiovascular bypass surgery. For example, a prosthetic vascular conduit (artificial artery) coated with anti-EPC antibodies would eliminate the need to use arteries from patients legs or arms for bypass surgery grafts. This would reduce surgery and anesthesia times, which in turn will reduce coronary surgery deaths. DVD-Ig are designed in such a way that it binds to a cell surface marker (such as CD34) as well as a protein (or an epitope of any kind, including but not limited to proteins, lipids and polysaccharides) that has been coated on the implanted device to facilitate the cell recruitment. Such approaches can also be applied to other medical implants in general. Alternatively, DVD-Igs can be coated on medical devices and upon implantation and releasing all DVDs from the device (or any other need which may require additional fresh DVD-Ig, including aging and denaturation of the already loaded DVD-Ig) the device could be reloaded by systemic administration of fresh DVD-Ig to the patient, where the DVD-Ig is designed to binds to a target of interest (a cytokine, a cell surface marker (such as CD34) etc.) with one set of binding sites and to a target coated on the device (including a protein, an epitope of any kind, including but not limited to lipids, polysaccharides and polymers) with the other. This technology has the advantage of extending the usefulness of coated implants.
A. Use of DVD-Igs in Various Diseases
DVD-Ig molecules of the invention are also useful as therapeutic molecules to treat various diseases. Such DVD molecules may bind one or more targets involved in a specific disease. Examples of such targets in various diseases are described below.
1. Human Autoimmune and Inflammatory Response
Many proteins have been implicated in general autoimmune and inflammatory responses, including C5, CCL1 (1-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (I-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1 (endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5, IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144). In one aspect, DVD-Igs capable of binding one or more of the targets listed herein are provided.
2. Asthma
Allergic asthma is characterized by the presence of eosinophilia, goblet cell metaplasia, epithelial cell alterations, airway hyperreactivity (AHR), and Th2 and Th1 cytokine expression, as well as elevated serum IgE levels. It is now widely accepted that airway inflammation is the key factor underlying the pathogenesis of asthma, involving a complex interplay of inflammatory cells such as T cells, B cells, eosinophils, mast cells and macrophages, and of their secreted mediators including cytokines and chemokines. Corticosteroids are the most important anti-inflammatory treatment for asthma today, however their mechanism of action is non-specific and safety concerns exist, especially in the juvenile patient population. The development of more specific and targeted therapies is therefore warranted. There is increasing evidence that IL-13 in mice mimics many of the features of asthma, including AHR, mucus hypersecretion and airway fibrosis, independently of eosinophilic inflammation (Finotto et al., International Immunology (2005), 17(8), 993-1007; Padilla et al., Journal of Immunology (2005), 174(12), 8097-8105).
IL-13 has been implicated as having a pivotal role in causing pathological responses associated with asthma. The development of anti-IL-13 mAb therapy to reduce the effects of IL-13 in the lung is an exciting new approach that offers considerable promise as a novel treatment for asthma. However other mediators of differential immunological pathways are also involved in asthma pathogenesis, and blocking these mediators, in addition to IL-13, may offer additional therapeutic benefit. Such target pairs include, but are not limited to, IL-13 and a pro-inflammatory cytokine, such as tumor necrosis factor-α (TNF-α). TNF-α may amplify the inflammatory response in asthma and may be linked to disease severity (McDonnell, et al., Progress in Respiratory Research (2001), 31 (New Drugs for Asthma, Allergy and COPD), 247-250). This suggests that blocking both IL-13 and TNF-α may have beneficial effects, particularly in severe airway disease. In another embodiment the DVD-Ig of the invention binds the targets IL-13 and TNFα and is used for treating asthma.
Animal models such as OVA-induced asthma mouse model, where both inflammation and AHR can be assessed, are known in the art and may be used to determine the ability of various DVD-Ig molecules to treat asthma. Animal models for studying asthma are disclosed in Coffman, et al., Journal of Experimental Medicine (2005), 201(12), 1875-1879; Lloyd, et al., Advances in Immunology (2001), 77, 263-295; Boyce et al., Journal of Experimental Medicine (2005), 201(12), 1869-1873; and Snibson, et al., Journal of the British Society for Allergy and Clinical Immunology (2005), 35(2), 146-52. In addition to routine safety assessments of these target pairs specific tests for the degree of immunosuppression may be warranted and helpful in selecting the best target pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43; Descotes, et al., Developments in biological standardization (1992), 77 99-102; Hart et al., Journal of Allergy and Clinical Immunology (2001), 108(2), 250-257).
Based on the rationale disclosed herein and using the same evaluation model for efficacy and safety other pairs of targets that DVD-Ig molecules can bind and be useful to treat asthma may be determined. In an embodiment, such targets include, but are not limited to, IL-13 and IL-1beta, since IL-1beta is also implicated in inflammatory response in asthma; IL-13 and cytokines and chemokines that are involved in inflammation, such as IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARO; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13 and ADAM8. The present invention also provides DVD-Igs capable of binding one or more targets involved in asthma selected from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG, histamine and histamine receptors, IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R1, TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STATE, TBX21, TGFB1, TNF, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase.
3. Rheumatoid Arthritis
Rheumatoid arthritis (RA), a systemic disease, is characterized by a chronic inflammatory reaction in the synovium of joints and is associated with degeneration of cartilage and erosion of juxta-articular bone. Many pro-inflammatory cytokines including TNF, chemokines, and growth factors are expressed in diseased joints. Systemic administration of anti-TNF antibody or sTNFR fusion protein to mouse models of RA was shown to be anti-inflammatory and joint protective. Clinical investigations in which the activity of TNF in RA patients was blocked with intravenously administered infliximab (Harriman G, Harper L K, Schaible T F. 1999 Summary of clinical trials in rheumatoid arthritis using infliximab, an anti-TNFalpha treatment. Ann Rheum Dis 58 Suppl 1:I61-4), a chimeric anti-TNF mAb, has provided evidence that TNF regulates IL-6, IL-8, MCP-1, and VEGF production, recruitment of immune and inflammatory cells into joints, angiogenesis, and reduction of blood levels of matrix metalloproteinases-1 and -3. A better understanding of the inflammatory pathway in rheumatoid arthritis has led to identification of other therapeutic targets involved in rheumatoid arthritis. Promising treatments such as interleukin-6 antagonists (IL-6 receptor antibody MRA, developed by Chugai, Roche (see Nishimoto, Norihiro et al., Arthritis & Rheumatism (2004), 50(6), 1761-1769), CTLA4Ig (abatacept, Genovese Mc et al 2005 Abatacept for rheumatoid arthritis refractory to tumor necrosis factor alpha inhibition. N Engl J. Med. 353:1114-23), and anti-B cell therapy (rituximab, Okamoto H, Kamatani N. 2004 Rituximab for rheumatoid arthritis. N Engl J Med. 351:1909) have already been tested in randomized controlled trials over the past year. Other cytokines have been identified and have been shown to be of benefit in animal models, including interleukin-15 (therapeutic antibody HuMax-IL—15, AMG 714 see Baslund, Bo et al., Arthritis & Rheumatism (2005), 52(9), 2686-2692), interleukin-17, and interleukin-18, and clinical trials of these agents are currently under way. Dual-specific antibody therapy, combining anti-TNF and another mediator, has great potential in enhancing clinical efficacy and/or patient coverage. For example, blocking both TNF and VEGF can potentially eradicate inflammation and angiogenesis, both of which are involved in pathophysiology of RA. Blocking other pairs of targets involved in RA including, but not limited to, TNF and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1beta; TNF and MIF; TNF and IL-17; TNF and IL-15 with specific DVD Igs is also contemplated. In addition to routine safety assessments of these target pairs, specific tests for the degree of immunosuppression may be warranted and helpful in selecting the best target pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43; Descotes, et al., Developments in biological standardization (1992), 77 99-102; Hart et al., Journal of Allergy and Clinical Immunology (2001), 108(2), 250-257). Whether a DVD Ig molecule will be useful for the treatment of rheumatoid arthritis can be assessed using pre-clinical animal RA models such as the collagen-induced arthritis mouse model. Other useful models are also well known in the art (see Brand D D., Comp Med. (2005) 55(2):114-22). Based on the cross-reactivity of the parental antibodies for human and mouse othologues (e.g., reactivity for human and mouse TNF, human and mouse IL-15 etc.) validation studies in the mouse CIA model may be conducted with “matched surrogate antibody” derived DVD-Ig molecules; briefly, a DVD-Ig based on two (or more) mouse target specific antibodies may be matched to the extent possible to the characteristics of the parental human or humanized antibodies used for human DVD-Ig construction (similar affinity, similar neutralization potency, similar half-life etc.).
4. SLE
The immunopathogenic hallmark of SLE is the polyclonal B cell activation, which leads to hyperglobulinemia, autoantibody production and immune complex formation. The fundamental abnormality appears to be the failure of T cells to suppress the forbidden B cell clones due to generalized T cell dysregulation. In addition, B and T-cell interaction is facilitated by several cytokines such as IL-10 as well as co-stimulatory molecules such as CD40 and CD40L, B7 and CD28 and CTLA-4, which initiate the second signal. These interactions together with impaired phagocytic clearance of immune complexes and apoptotic material, perpetuate the immune response with resultant tissue injury. The following targets may be involved in SLE and can potentially be used for DVD-Ig approach for therapeutic intervention: B cell targeted therapies: CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81, IFNB1, IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA, and NT5E; co-stimulatory signals: CTLA4 or B7.1/B7.2; inhibition of B cell survival: BlyS, BAFF; Complement inactivation: C5; Cytokine modulation: the key principle is that the net biologic response in any tissue is the result of a balance between local levels of proinflammatory or anti-inflammatory cytokines (see Sfikakis P P et al 2005 Curr Opin Rheumatol 17:550-7). SLE is considered to be a Th-2 driven disease with documented elevations in serum IL-4, IL-6, IL-10. DVD Igs capable of binding one or more targets selected from the group consisting of IL-4, IL-6, IL-10, IFN-α, and TNF-α are also contemplated. Combination of targets discussed herein will enhance therapeutic efficacy for SLE which can be tested in a number of lupus preclinical models (see Peng S L (2004) Methods Mol Med.; 102:227-72). Based on the cross-reactivity of the parental antibodies for human and mouse othologues (e.g., reactivity for human and mouse CD20, human and mouse Interferon alpha etc.) validation studies in a mouse lupus model may be conducted with “matched surrogate antibody” derived DVD-Ig molecules; briefly, a DVD-Ig based two (or more) mouse target specific antibodies may be matched to the extent possible to the characteristics of the parental human or humanized antibodies used for human DVD-Ig construction (similar affinity, similar neutralization potency, similar half-life etc.).
5. Multiple Sclerosis
Multiple sclerosis (MS) is a complex human autoimmune-type disease with a predominantly unknown etiology. Immunologic destruction of myelin basic protein (MBP) throughout the nervous system is the major pathology of multiple sclerosis. MS is a disease of complex pathologies, which involves infiltration by CD4+ and CD8+ T cells and of response within the central nervous system. Expression in the CNS of cytokines, reactive nitrogen species and costimulator molecules have all been described in MS. Of major consideration are immunological mechanisms that contribute to the development of autoimmunity. In particular, antigen expression, cytokine and leukocyte interactions, and regulatory T-cells, which help balance/modulate other T-cells such as Th1 and Th2 cells, are important areas for therapeutic target identification.
IL-12 is a proinflammatory cytokine that is produced by APC and promotes differentiation of Th1 effector cells. IL-12 is produced in the developing lesions of patients with MS as well as in EAE-affected animals. Previously it was shown that interference in IL-12 pathways effectively prevents EAE in rodents, and that in vivo neutralization of IL-12p40 using a anti-IL-12 mAb has beneficial effects in the myelin-induced EAE model in common marmosets.
TWEAK is a member of the TNF family, constitutively expressed in the central nervous system (CNS), with pro-inflammatory, proliferative or apoptotic effects depending upon cell types. Its receptor, Fn14, is expressed in CNS by endothelial cells, reactive astrocytes and neurons. TWEAK and Fn14 mRNA expression increased in spinal cord during experimental autoimmune encephalomyelitis (EAE). Anti-TWEAK antibody treatment in myelin oligodendrocyte glycoprotein (MOG) induced EAE in C57BL/6 mice resulted in a reduction of disease severity and leukocyte infiltration when mice were treated after the priming phase.
One aspect of the invention pertains to DVD Ig molecules capable of binding one or more, for example two, targets selected from the group consisting of IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. An embodiment includes a dual-specific anti-IL-12/TWEAK DVD Ig as a therapeutic agent beneficial for the treatment of MS.
Several animal models for assessing the usefulness of the DVD molecules to treat MS are known in the art (see Steinman L, et al., (2005) Trends Immunol. 26(11):565-71; Lublin F D., et al., (1985) Springer Semin Immunopathol. 8(3):197-208; Genain C P, et al., (1997) J Mol Med. 75(3):187-97; Tuohy V K, et al., (1999) J Exp Med. 189(7):1033-42; Owens T, et al., (1995) Neurol Clin. 13(1):51-73; and 't Hart B A, et al., (2005) J Immunol 175(7):4761-8. Based on the cross-reactivity of the parental antibodies for human and animal species othologues (e.g., reactivity for human and mouse IL-12, human and mouse TWEAK etc.) validation studies in the mouse EAE model may be conducted with “matched surrogate antibody” derived DVD-Ig molecules; briefly, a DVD-Ig based on to (or more) mouse target specific antibodies may be matched to the extent possible to the characteristics of the parental human or humanized antibodies used for human DVD-Ig construction (similar affinity, similar neutralization potency, similar half-life etc.). The same concept applies to animal models in other non-rodent species, where a “matched surrogate antibody” derived DVD-Ig would be selected for the anticipated pharmacology and possibly safety studies. In addition to routine safety assessments of these target pairs specific tests for the degree of immunosuppression may be warranted and helpful in selecting the best target pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43; Descotes, et al., Developments in biological standardization (1992), 77 99-102; Jones R. 2000 Rovelizumab (ICOS Corp). IDrugs. 3(4):442-6).
6. Sepsis
The pathophysiology of sepsis is initiated by the outer membrane components of both gram-negative organisms (lipopolysaccharide [LPS], lipid A, endotoxin) and gram-positive organisms (lipoteichoic acid, peptidoglycan). These outer membrane components are able to bind to the CD14 receptor on the surface of monocytes. By virtue of the recently described toll-like receptors, a signal is then transmitted to the cell, leading to the eventual production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1). Overwhelming inflammatory and immune responses are essential features of septic shock and play a central part in the pathogenesis of tissue damage, multiple organ failure, and death induced by sepsis. Cytokines, especially tumor necrosis factor (TNF) and interleukin (IL-1), have been shown to be critical mediators of septic shock. These cytokines have a direct toxic effect on tissues; they also activate phospholipase A2. These and other effects lead to increased concentrations of platelet-activating factor, promotion of nitric oxide synthase activity, promotion of tissue infiltration by neutrophils, and promotion of neutrophil activity.
The treatment of sepsis and septic shock remains a clinical conundrum, and recent prospective trials with biological response modifiers (i.e. anti-TNF, anti-MIF) aimed at the inflammatory response have shown only modest clinical benefit. Recently, interest has shifted toward therapies aimed at reversing the accompanying periods of immune suppression. Studies in experimental animals and critically ill patients have demonstrated that increased apoptosis of lymphoid organs and some parenchymal tissues contribute to this immune suppression, anergy, and organ system dysfunction. During sepsis syndromes, lymphocyte apoptosis can be triggered by the absence of IL-2 or by the release of glucocorticoids, granzymes, or the so-called ‘death’ cytokines: tumor necrosis factor alpha or Fas ligand. Apoptosis proceeds via auto-activation of cytosolic and/or mitochondrial caspases, which can be influenced by the pro- and anti-apoptotic members of the Bcl-2 family. In experimental animals, not only can treatment with inhibitors of apoptosis prevent lymphoid cell apoptosis; it may also improve outcome. Although clinical trials with anti-apoptotic agents remain distant due in large part to technical difficulties associated with their administration and tissue targeting, inhibition of lymphocyte apoptosis represents an attractive therapeutic target for the septic patient. Likewise, a dual-specific agent targeting both inflammatory mediator and a apoptotic mediator, may have added benefit. One aspect of the invention pertains to DVD Igs capable of binding one or more targets involved in sepsis, in an embodiment two targets, selected from the group consisting TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFKB1, PROC, TNFRSF1A, CSF3, CCR3, IL1RN, MIF, NFKB1, PTAFR, TLR2, TLR4, GPR44, HMOX1, midkine, IRAK1, NFKB2, SERPINA1, SERPINE1, and TREM1. The efficacy of such DVD Igs for sepsis can be assessed in preclinical animal models known in the art (see Buras J A, et al., (2005) Nat Rev Drug Discov. 4(10):854-65 and Calandra T, et al., (2000) Nat Med. 6(2):164-70).
7. Neurological Disorders
7.1. Neurodegenerative Diseases
Chronic neurodegenerative diseases are usually age-dependent diseases characterized by progressive loss of neuronal functions (neuronal cell death, demyelination), loss of mobility and loss of memory. Emerging knowledge of the mechanisms underlying chronic neurodegenerative diseases (e.g., Alzheimer's disease) show a complex etiology and a variety of factors have been recognized to contribute to their development and progression e.g., age, glycemic status, amyloid production and multimerization, accumulation of advanced glycation-end products (AGE) which bind to their receptor RAGE (receptor for AGE), increased brain oxidative stress, decreased cerebral blood flow, neuroinflammation including release of inflammatory cytokines and chemokines, neuronal dysfunction and microglial activation. Thus these chronic neurodegenerative diseases represent a complex interaction between multiple cell types and mediators. Treatment strategies for such diseases are limited and mostly constitute either blocking inflammatory processes with non-specific anti-inflammatory agents (e.g., corticosteroids, COX inhibitors) or agents to prevent neuron loss and/or synaptic functions. These treatments fail to stop disease progression. Recent studies suggest that more targeted therapies such as antibodies to soluble A-b peptide (including the A-b oligomeric forms) can not only help stop disease progression but may help maintain memory as well. These preliminary observations suggest that specific therapies targeting more than one disease mediator (e.g., A-b and a pro-inflammatory cytokine such as TNF) may provide even better therapeutic efficacy for chronic neurodegenerative diseases than observed with targeting a single disease mechanism (e.g., soluble A-balone) (see C. E. Shepherd, et al, Neurobiol Aging. 2005 Oct. 24; Nelson R B., Curr Pharm Des. 2005; 11:3335; William L. Klein; Neurochem Int. 2002; 41:345; Michelle C Janelsins, et al., J. Neuroinflammation. 2005; 2:23; Soloman B., Curr Alzheimer Res. 2004; 1:149; Igor Klyubin, et al., Nat. Med. 2005; 11:556-61; Arancio O, et al., EMBO Journal (2004) 1-10; Bornemann K D, et al., Am J. Pathol. 2001; 158:63; Deane R, et al., Nat. Med. 2003; 9:907-13; and Eliezer Masliah, et al., Neuron. 2005; 46:857).
The DVD-Ig molecules of the invention can bind one or more targets involved in Chronic neurodegenerative diseases such as Alzheimers. Such targets include, but are not limited to, any mediator, soluble or cell surface, implicated in AD pathogenesis e.g. AGE (S100 A, amphoterin), pro-inflammatory cytokines (e.g., IL-1), chemokines (e.g., MCP 1), molecules that inhibit nerve regeneration (e.g., Nogo, RGM A), molecules that enhance neurite growth (neurotrophins). The efficacy of DVD-Ig molecules can be validated in pre-clinical animal models such as the transgenic mice that over-express amyloid precursor protein or RAGE and develop Alzheimer's disease-like symptoms. In addition, DVD-Ig molecules can be constructed and tested for efficacy in the animal models and the best therapeutic DVD-Ig can be selected for testing in human patients. DVD-Ig molecules can also be employed for treatment of other neurodegenerative diseases such as Parkinson's disease. Alpha-Synuclein is involved in Parkinson's pathology. A DVD-Ig capable of targeting alpha-synuclein and inflammatory mediators such as TNF, IL-1, MCP-1 can prove effective therapy for Parkinson's disease and are contemplated in the invention.
7.2 Neuronal Regeneration and Spinal Cord Injury
Despite an increase in knowledge of the pathologic mechanisms, spinal cord injury (SCI) is still a devastating condition and represents a medical indication characterized by a high medical need. Most spinal cord injuries are contusion or compression injuries and the primary injury is usually followed by secondary injury mechanisms (inflammatory mediators e.g., cytokines and chemokines) that worsen the initial injury and result in significant enlargement of the lesion area, sometimes more than 10-fold. These primary and secondary mechanisms in SCI are very similar to those in brain injury caused by other means e.g., stroke. No satisfying treatment exists and high dose bolus injection of methylprednisolone (MP) is the only used therapy within a narrow time window of 8 h post injury. This treatment, however, is only intended to prevent secondary injury without causing any significant functional recovery. It is heavily critisized for the lack of unequivocal efficacy and severe adverse effects, like immunosuppression with subsequent infections and severe histopathological muscle alterations. No other drugs, biologics or small molecules, stimulating the endogenous regenerative potential are approved, but promising treatment principles and drug candidates have shown efficacy in animal models of SCI in recent years. To a large extent the lack of functional recovery in human SCI is caused by factors inhibiting neurite growth, at lesion sites, in scar tissue, in myelin as well as on injury-associated cells. Such factors are the myelin-associated proteins NogoA, OMgp and MAG, RGM A, the scar-associated CSPG (Chondroitin Sulfate Proteoglycans) and inhibitory factors on reactive astrocytes (some semaphorins and ephrins). However, at the lesion site not only growth inhibitory molecules are found but also neurite growth stimulating factors like neurotrophins, laminin, L1 and others. This ensemble of neurite growth inhibitory and growth promoting molecules may explain that blocking single factors, like NogoA or RGM A, resulted in significant functional recovery in rodent SCI models, because a reduction of the inhibitory influences could shift the balance from growth inhibition to growth promotion. However, recoveries observed with blocking a single neurite outgrowth inhibitory molecule were not complete. To achieve faster and more pronounced recoveries either blocking two neurite outgrowth inhibitory molecules e.g. Nogo and RGM A, or blocking an neurite outgrowth inhibitory molecule and enhancing functions of a neurite outgrowth enhancing molecule e.g. Nogo and neurotrophins, or blocking a neurite outgrowth inhibitory molecule e.g., Nogo and a pro-inflammatory molecule e.g., TNF, may be desirable (see McGee A W, et al., Trends Neurosci. 2003; 26:193; Marco Domeniconi, et al., J Neurol Sci. 2005; 233:43; Milan Makwanal, et al., FEBS J. 2005; 272:2628; Barry J. Dickson, Science. 2002; 298:1959; Felicia Yu Hsuan Teng, et al., J Neurosci Res. 2005; 79:273; Tara Karnezis, et al., Nature Neuroscience 2004; 7, 736; Gang Xu, et al., J. Neurochem. 2004; 91; 1018).
In one aspect, DVD-Igs capable of binding target pairs such as NgR and RGM A; NogoA and RGM A; MAG and RGM A; OMGp and RGM A; RGM A and RGM B; CSPGs and RGM A; aggrecan, midkine, neurocan, versican, phosphacan, Te38 and TNF-α; Aβ globulomer-specific antibodies combined with antibodies promoting dendrite & axon sprouting are provided. Dendrite pathology is a very early sign of AD and it is known that NOGO A restricts dendrite growth. One can combine such type of ab with any of the SCI-candidate (myelin-proteins) Ab. Other DVD-Ig targets may include any combination of NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG or Omgp. Additionally, targets may also include any mediator, soluble or cell surface, implicated in inhibition of neurite e.g. Nogo, Ompg, MAG, RGM A, semaphorins, ephrins, soluble A-b, pro-inflammatory cytokines (e.g., IL-1), chemokines (e.g., MIP 1a), molecules that inhibit nerve regeneration. The efficacy of anti-nogo/anti-RGM A or similar DVD-Ig molecules can be validated in pre-clinical animal models of spinal cord injury. In addition, these DVD-Ig molecules can be constructed and tested for efficacy in the animal models and the best therapeutic DVD-Ig can be selected for testing in human patients. In addition, DVD-Ig molecules can be constructed that target two distinct ligand binding sites on a single receptor e.g., Nogo receptor which binds three ligand Nogo, Ompg, and MAG and RAGE that binds A-b and S100 A. Furthermore, neurite outgrowth inhibitors e.g., nogo and nogo receptor, also play a role in preventing nerve regeneration in immunological diseases like multiple sclerosis. Inhibition of nogo-nogo receptor interaction has been shown to enhance recovery in animal models of multiple sclerosis. Therefore, DVD-Ig molecules that can block the function of one immune mediator e.g. a cytokine like IL-12 and a neurite outgrowth inhibitor molecule e.g. nogo or RGM may offer faster and greater efficacy than blocking either an immune or an neurite outgrowth inhibitor molecule alone.
8. Oncological Disorders
Monoclonal antibody therapy has emerged as an important therapeutic modality for cancer (von Mehren M, et al 2003 Monoclonal antibody therapy for cancer. Annu Rev Med.; 54:343-69). Antibodies may exert antitumor effects by inducing apoptosis, redirected cytotoxicity, interfering with ligand-receptor interactions, or preventing the expression of proteins that are critical to the neoplastic phenotype. In addition, antibodies can target components of the tumor microenvironment, perturbing vital structures such as the formation of tumor-associated vasculature. Antibodies can also target receptors whose ligands are growth factors, such as the epidermal growth factor receptor. The antibody thus inhibits natural ligands that stimulate cell growth from binding to targeted tumor cells. Alternatively, antibodies may induce an anti-idiotype network, complement-mediated cytotoxicity, or antibody-dependent cellular cytotoxicity (ADCC). The use of dual-specific antibody that targets two separate tumor mediators will likely give additional benefit compared to a mono-specific therapy. DVD Igs capable of binding the following pairs of targets to treat oncological disease are also contemplated: IGF1 and IGF2; IGF1/2 and HER-2; VEGFR and EGFR; CD20 and CD3; CD138 and CD20; CD38 and CD20; CD38 and CD138; CD40 and CD20; CD138 and CD40; CD38 and CD40; CD-20 and CD-19; CD-20 and EGFR; CD-20 and CD-80; CD-20 and CD-22; CD-3 and HER-2; CD-3 and CD-19; EGFR and HER-2; EGFR and CD-3; EGFR and IGF1,2; EGFR and IGF1R; EGFR and RON; EGFR and HGF; EGFR and c-MET; HER-2 and IGF1,2; HER-2 and IGF1R; RON and HGF; VEGF and EGFR; VEGF and HER-2; VEGF and CD-20; VEGF and IGF1,2; VEGF and DLL4; VEGF and HGF; VEGF and RON; VEGF and NRP1; CD20 and CD3; VEGF and PLGF; DLL4 and PLGF; ErbB3 and EGFR; HGF and ErbB3, HER-2 and ErbB3; c-Met and ErbB3; HER-2 and PLGF; HER-2 and HER-2.
In another embodiment, a DVD of the invention is capable of binding VEGF and phosphatidylserine; VEGF and ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1; VEGF and ANPEP; VEGF and c-MET; HER-2 and ERB3; HER-2 and BSG2; HER-2 and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR and CDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20; CD20 and CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 and CD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF and phosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF1,2; c-Met and Her-2; c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20; IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 and VEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and VEGFR2; PDGFRa and PLGF; PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR2; PDGFRb and c-Met; PDGFRb and EGFR; RON and c-Met; RON and MTSP1; RON and MSP; RON and CDCP1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR; VEGFR2 and PLGF; VEGFR2 and NRP1; VEGFR2 and RON; VEGFR2 and DLL4; VEGFR2 and EGFR; VEGFR2 and ROBO4; VEGFR2 and CD55; LPA and S1P; EPHB2 and RON; CTLA4 and VEGF; CD3 and EPCAM; CD40 and IL6; CD40 and IGF; CD40 and CD56; CD40 and CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4; CD40 and APRIL; CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 and CD40; CD80 and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80 and CD3; CD80 and CD19; CD80 and CD4; CD80 and CD52; CD80 and VEGF; CD80 and DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80; CD22 and CD40; CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22 and DR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20; CD30 and CD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30 and CD74; CD30 and CD19; CD30 and DR5; CD30 and DR4; CD30 and VEGFR2; CD30 and CD52; CD30 and CD4; CD138 and RANKL; CD33 and FTL3; CD33 and VEGF; CD33 and VEGFR2; CD33 and CD44; CD33 and DR4; CD33 and DR5; DR4 and CD137; DR4 and IGF1,2; DR4 and IGF1R; DR4 and DR5; DR5 and CD40; DR5 and CD137; DR5 and CD20; DR5 and EGFR; DR5 and IGF1,2; DR5 and IGFR, DR5 and HER-2, EGFR and DLL4. Other target combinations include one or more members of the EGF/erb-2/erb-3 family. Other targets (one or more) involved in oncological diseases that DVD Igs may bind include, but are not limited to those selected from the group consisting of: CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2, BCL2, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1, IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1, IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR113, NR2F6, NR4A3, ESR1, ESR2, NROB1, NROB2, NR1D2, NR1H2, NR1H4, NR112, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1, BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2, IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3, CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164, COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB21P, DES, DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6, ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR, LAMAS, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3, BAI1, COL4A3, IL8, LAMAS, NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4, PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B (p27Kip1), CDKN2A (p161NK4a), COL6A1, CTNNB1 (b-catenin), CTSB (cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3, GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin), SERPINE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A (topoisomerase Ea), TP53, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cipl), CLDN7 (claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin), KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type II keratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Sprl), THBS1, THBS2, THBS4, and TNFAIP2 (B94), RON, c-Met, CD64, DLL4, PLGF, CTLA4, phosphatidylserine, ROBO4, CD80, CD22, CD40, CD23, CD28, CD80, CD55, CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5, RANKL, VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2, EpCAM, PGE2, NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFR alpha, PDGFR beta, ROR1, PSMA, PSCA, SCD1, and CD59.
IV. Pharmaceutical Compositions
The invention also provides pharmaceutical compositions comprising a binding protein, of the invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions comprising binding proteins of the invention are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder, in preventing, treating, managing, or ameliorating of a disorder or one or more symptoms thereof, and/or in research. In a specific embodiment, a composition comprises one or more binding proteins of the invention. In another embodiment, the pharmaceutical composition comprises one or more binding proteins of the invention and one or more prophylactic or therapeutic agents other than binding proteins of the invention for treating a disorder. In an embodiment, the prophylactic or therapeutic agents known to be useful for or having been or currently being used in the prevention, treatment, management, or amelioration of a disorder or one or more symptoms thereof. In accordance with these embodiments, the composition may further comprise of a carrier, diluent or excipient.
The binding proteins of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises a binding protein of the invention and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In some embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, are included in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.
Various delivery systems are known and can be used to administer one or more antibodies of the invention or the combination of one or more antibodies of the invention and a prophylactic agent or therapeutic agent useful for preventing, managing, treating, or ameliorating a disorder or one or more symptoms thereof, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of administering a prophylactic or therapeutic agent of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidurala administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes). In addition, pulmonary administration can be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, each of which is incorporated herein by reference their entireties. In one embodiment, a binding protein of the invention, combination therapy, or a composition of the invention is administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.). In a specific embodiment, prophylactic or therapeutic agents of the invention are administered intramuscularly, intravenously, intratumorally, orally, intranasally, pulmonary, or subcutaneously. The prophylactic or therapeutic agents may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
In an embodiment, specific binding of antibody-coupled carbon nanotubes (CNTs) to tumor cells in vitro, followed by their highly specific ablation with near-infrared (NIR) light can be used to target tumor cells. For example, biotinylated polar lipids can be used to prepare stable, biocompatible, noncytotoxic CNT dispersions that are then attached to one or two different neutralite avidin-derivatized DVD-Igs directed against one or more tumor antigens (e.g., CD22) (Chakravarty, P. et al. (2008) Proc. Natl. Acad. Sci. USA 105:8697-8702.
In a specific embodiment, it may be desirable to administer the prophylactic or therapeutic agents of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous or non-porous material, including membranes and matrices, such as sialastic membranes, polymers, fibrous matrices (e.g., Tissuel®), or collagen matrices. In one embodiment, an effective amount of one or more antibodies of the invention antagonists is administered locally to the affected area to a subject to prevent, treat, manage, and/or ameliorate a disorder or a symptom thereof. In another embodiment, an effective amount of one or more antibodies of the invention is administered locally to the affected area in combination with an effective amount of one or more therapies (e.g., one or more prophylactic or therapeutic agents) other than a binding protein of the invention of a subject to prevent, treat, manage, and/or ameliorate a disorder or one or more symptoms thereof.
In another embodiment, the prophylactic or therapeutic agent can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the invention (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In an embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the invention. See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel,” Radiotherapy &Oncology 39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science &Technology 50:372-397, Cleek et al., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al., 1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in their entireties.
In a specific embodiment, where the composition of the invention is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868). Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection.
If the compositions of the invention are to be administered topically, the compositions can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). In an embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in an embodiment, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.
If the method of the invention comprises intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
If the method of the invention comprises oral administration, compositions can be formulated orally in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, but not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or therapeutic agent(s).
The method of the invention may comprise pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, each of which is incorporated herein by reference their entireties. In a specific embodiment, a binding protein of the invention, combination therapy, and/or composition of the invention is administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).
The method of the invention may comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
The methods of the invention may additionally comprise of administration of compositions formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
The methods of the invention encompass administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the mode of administration is infusion, composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
In particular, the invention also provides that one or more of the prophylactic or therapeutic agents, or pharmaceutical compositions of the invention is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the agent. In one embodiment, one or more of the prophylactic or therapeutic agents, or pharmaceutical compositions of the invention is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the invention is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. The lyophilized prophylactic or therapeutic agents or pharmaceutical compositions of the invention should be stored at between 2° C. and 8° C. in its original container and the prophylactic or therapeutic agents, or pharmaceutical compositions of the invention should be administered within 1 week, e.g., within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In an alternative embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the invention is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the agent. In an embodiment, the liquid form of the administered composition is supplied in a hermetically sealed container at least 0.25 mg/ml, at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid form should be stored at between 2° C. and 8° C. in its original container.
The binding proteins of the invention can be incorporated into a pharmaceutical composition suitable for parenteral administration. In an embodiment, the antibody or antibody-portions will be prepared as an injectable solution containing 0.1-250 mg/ml binding protein. The injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampule or pre-filled syringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents can be included for a lyophilized dosage form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can be used in both liquid and lyophilized dosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking agents include glycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition comprising the binding proteins of the invention prepared as an injectable solution for parenteral administration, can further comprise an agent useful as an adjuvant, such as those used to increase the absorption, or dispersion of a therapeutic protein (e.g., antibody). A particularly useful adjuvant is hyaluronidase, such as Hylenex® (recombinant human hyaluronidase). Addition of hyaluronidase in the injectable solution improves human bioavailability following parenteral administration, particularly subcutaneous administration. It also allows for greater injection site volumes (i.e. greater than 1 ml) with less pain and discomfort, and minimum incidence of injection site reactions. (see WO2004078140, and US2006104968 incorporated herein by reference).
The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form chosen depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The chosen mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In an embodiment, the antibody is administered by intravenous infusion or injection. In another embodiment, the antibody is administered by intramuscular or subcutaneous injection.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including, in the composition, an agent that delays absorption, for example, monostearate salts and gelatin.
The binding proteins of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, in an embodiment, the route/mode of administration is subcutaneous injection, intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
In certain embodiments, a binding protein of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, a binding protein of the invention is coformulated with and/or coadministered with one or more additional therapeutic agents that are useful for treating disorders with binding protein of the invention. For example, a binding protein of the invention may be coformulated and/or coadministered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules). Furthermore, one or more antibodies of the invention may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
In certain embodiments, a binding protein is linked to a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, the Fc domain, polyethylene glycol, and dextran. Such vehicles are described, e.g., in U.S. application Ser. No. 09/428,082 and published PCT Application No. WO 99/25044, which are hereby incorporated by reference for any purpose.
In a specific embodiment, nucleic acid sequences encoding a binding protein of the invention or another prophylactic or therapeutic agent of the invention are administered to treat, prevent, manage, or ameliorate a disorder or one or more symptoms thereof by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded antibody or prophylactic or therapeutic agent of the invention that mediates a prophylactic or therapeutic effect.
Any of the methods for gene therapy available in the art can be used according to the present invention. For general reviews of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215. Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). Detailed description of various methods of gene therapy are disclosed in US20050042664 A1 which is incorporated herein by reference.
The binding proteins of the invention are useful in treating various diseases wherein the targets that are recognized by the binding proteins are detrimental. Such diseases include, but are not limited to, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease, sporadic, polyglandular deficiency type I and polyglandular deficiency type II, Schmidt's syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia greata, seronegative arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, chlamydia, yersinia and salmonella associated arthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjögren's disease associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjögren's syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, choleosatatis, idiosyncratic liver disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group B streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 Type and Th1 Type mediated diseases, acute and chronic pain (different forms of pain), and cancers such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti cd3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aordic and peripheral aneuryisms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage transplant rejection, cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chromic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated disorders, Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, dermatologic conditions, diabetes, diabetes mellitus, diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's Syndrome in middle age, drug-induced movement disorders induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, epstein-barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hematophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallervorden-Spatz disease, hashimoto's thyroiditis, hay fever, heart transplant rejection, hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis (A), His bundle arrythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody mediated cytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammation of the aorta, influenza a, ionizing radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, legionella, leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver transplant rejection, lymphederma, malaria, malignant Lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic, migraine headache, mitochondrial multi.system disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple systems degenerations (Mencel Dejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis, mycobacterium avium intracellulare, mycobacterium tuberculosis, myelodyplastic syndrome, myocardial infarction, myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscular atrophies, neutropenic fever, non-hodgkins lymphoma, occlusion of the abdominal aorta and its branches, occlusive arterial disorders, okt3 therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, peripheral atherlosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleo Palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynoud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, Senile Dementia of Lewy body type, seronegative arthropathies, shock, sickle cell anemia, skin allograft rejection, skin changes syndrome, small bowel transplant rejection, solid tumors, specific arrythmias, spinal ataxia, spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, Subacute sclerosing panencephalitis, Syncope, syphilis of the cardiovascular system, systemic anaphalaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type III hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose veins, vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal infections, vital encephalitis/aseptic meningitis, vital-associated hemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or tissue. (see Peritt et al. PCT publication No. WO2002097048A2, Leonard et al., PCT publication No. WO9524918 A1, and Salfeld et al., PCT publication No. WO00/56772A1).
The binding proteins of the invention can be used to treat humans suffering from autoimmune diseases, in particular those associated with inflammation, including, rheumatoid arthritis, spondylitis, allergy, autoimmune diabetes, autoimmune uveitis. In an embodiment, the binding proteins of the invention or antigen-binding portions thereof, are used to treat rheumatoid arthritis, Crohn's disease, multiple sclerosis, insulin dependent diabetes mellitus and psoriasis.
In an embodiment, diseases that can be treated or diagnosed with the compositions and methods of the invention include, but are not limited to, primary and metastatic cancers, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract (including cervix, uterus, and ovaries as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as well as Kaposi's sarcoma), tumors of the brain, nerves, eyes, and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, and meningiomas), solid tumors arising from hematopoietic malignancies such as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin's lymphomas).
In an embodiment, the antibodies of the invention or antigen-binding portions thereof, are used to treat cancer or in the prevention of metastases from the tumors described herein either when used alone or in combination with radiotherapy and/or other chemotherapeutic agents.
The antibodies of the invention, or antigen binding portions thereof, may be combined with agents that include but are not limited to, antineoplastic agents, radiotherapy, chemotherapy such as DNA alkylating agents, cisplatin, carboplatin, anti-tubulin agents, paclitaxel, docetaxel, taxol, doxorubicin, gemcitabine, gemzar, anthracyclines, adriamycin, topoisomerase I inhibitors, topoisomerase II inhibitors, 5-fluorouracil (5-FU), leucovorin, irinotecan, receptor tyrosine kinase inhibitors (e.g., erlotinib, gefitinib), COX-2 inhibitors (e.g., celecoxib), kinase inhibitors, and siRNAs.
A binding protein of the invention also can be administered with one or more additional therapeutic agents useful in the treatment of various diseases.
A binding protein of the invention can be used alone or in combination to treat such diseases. It should be understood that the binding proteins can be used alone or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent art-recognized as being useful to treat the disease or condition being treated by the antibody of the present invention. The additional agent also can be an agent that imparts a beneficial attribute to the therapeutic composition e.g., an agent which effects the viscosity of the composition.
It should further be understood that the combinations which are to be included within this invention are those combinations useful for their intended purpose. The agents set forth below are illustrative for purposes and not intended to be limited. The combinations, which are part of this invention, can be the antibodies of the present invention and at least one additional agent selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional agents if the combination is such that the formed composition can perform its intended function.
Combinations to treat autoimmune and inflammatory diseases are non-steroidal anti-inflammatory drug(s) also referred to as NSAIDS which include drugs like ibuprofen. Other combinations are corticosteroids including prednisolone; the well known side-effects of steroid use can be reduced or even eliminated by tapering the steroid dose required when treating patients in combination with the DVD Igs of this invention. Non-limiting examples of therapeutic agents for rheumatoid arthritis with which an antibody, or antibody portion, of the invention can be combined include the following: cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-23, interferons, EMAP-II, GM-CSF, FGF, and PDGF. Binding proteins of the invention, or antigen binding portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligands including CD154 (gp39 or CD40L).
Combinations of therapeutic agents may interfere at different points in the autoimmune and subsequent inflammatory cascade; examples include TNF antagonists like chimeric, humanized or human TNF antibodies, ADALIMUMAB, (PCT Publication No. WO 97/29131), CA2 (RemicadeTM), CDP 571, and soluble p55 or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG (EnbrelTM) or p55TNFR1gG (Lenercept), and also TNF ix converting enzyme (TACE) inhibitors; similarly IL-1 inhibitors (Interleukin-1-converting enzyme inhibitors, IL-1RA etc.) may be effective for the same reason. Other combinations include Interleukin 11. Yet another combination include key players of the autoimmune response which may act parallel to, dependent on or in concert with IL-12 function; especially are IL-18 antagonists including IL-18 antibodies or soluble IL-18 receptors, or IL-18 binding proteins. It has been shown that IL-12 and IL-18 have overlapping but distinct functions and a combination of antagonists to both may be most effective. Yet another combination are non-depleting anti-CD4 inhibitors. Yet other combinations include antagonists of the co-stimulatory pathway CD80 (B7.1) or CD86 (B7.2) including antibodies, soluble receptors or antagonistic ligands.
The binding proteins of the invention may also be combined with agents, such as methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine, olsalazine chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate (intramuscular and oral), azathioprine, cochicine, corticosteroids (oral, inhaled and local injection), beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signalling by proinflammatory cytokines such as TNF-α or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFα converting enzyme (TACE) inhibitors, T-cell signalling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNF receptors and the derivatives p75TNFRIgG (EnbrelTM and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen, valdecoxib, sulfasalazine, methylprednisolone, meloxicam, methylprednisolone acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide, propoxyphene napsylate/apap, folate, nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodone bitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol hcl, salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium, prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin, glucosamine sulf/chondroitin, amitriptyline hcl, sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl, misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485, CDC-801, and Mesopram. Combinations include methotrexate or leflunomide and in moderate or severe rheumatoid arthritis cases, cyclosporine.
Nonlimiting additional agents which can also be used in combination with a binding protein to treat rheumatoid arthritis include, but are not limited to, the following: non-steroidal anti-inflammatory drug(s) (NSAIDs); cytokine suppressive anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNFα antibody; Celltech/Bayer); cA2/infliximab (chimeric anti-TNFα antibody; Centocor); 75 kdTNFR-IgG/etanercept (75 kD TNF receptor-IgG fusion protein; Immunex; see e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody; IDEC/SmithKline; see e.g., Arthritis & Rheumatism (1995) Vol. 38, S185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; see e.g., Arthritis & Rheumatism (1993) Vol. 36, 1223); Anti-Tac (humanized anti-IL-2Ra; Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA (IL-1 receptor antagonist; Synergen/Amgen); anakinra (Kineret®/Amgen); TNF-bp/s-TNF (soluble TNF binding protein; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), 5284; Amer. J. Physiol.—Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42); R973401 (phosphodiesterase Type IV inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); MK-966 (COX-2 Inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S81); Iloprost (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S82); methotrexate; thalidomide (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), 5282) and thalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatory and cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), 5131; Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid (inhibitor of plasminogen activation; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284); T-614 (cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); prostaglandin E1 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); Tenidap (non-steroidal anti-inflammatory drug; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S280); Naproxen (non-steroidal anti-inflammatory drug; see e.g., Neuro Report (1996) Vol. 7, pp. 1209-1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S281); Azathioprine (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S281); ICE inhibitor (inhibitor of the enzyme interleukin-1β converting enzyme); zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinase zap-70 or lck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitors of vascular endothelial cell growth factor or vascular endothelial cell growth factor receptor; inhibitors of angiogenesis); corticosteroid anti-inflammatory drugs (e.g., SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies; anti-IL-18 antibodies; interleukin-11 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S296); interleukin-13 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S308); interleukin-17 inhibitors (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S120); gold; penicillamine; chloroquine; chlorambucil; hydroxychloroquine; cyclosporine; cyclophosphamide; total lymphoid irradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins; orally-administered peptides and collagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligo-deoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycan polysulphate; minocycline; anti-IL2R antibodies; marine and botanical lipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21:759-777); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immune globulin; zileuton; azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (therafectin); cladribine (2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see Bruncko, Milan et al., Journal of Medicinal Chemistry (2007), 50(4), 641-662); antivirals and immune modulating agents.
In one embodiment, the binding protein or antigen-binding portion thereof, is administered in combination with one of the following agents for the treatment of rheumatoid arthritis: small molecule inhibitor of KDR, small molecule inhibitor of Tie-2; methotrexate; prednisone; celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib; etanercept; infliximab; leflunomide; naproxen; valdecoxib; sulfasalazine; methylprednisolone; ibuprofen; meloxicam; methylprednisolone acetate; gold sodium thiomalate; aspirin; azathioprine; triamcinolone acetonide; propoxyphene napsylate/apap; folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium; oxaprozin; oxycodone hcl; hydrocodone bitartrate/apap; diclofenac sodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol hcl; salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen; alendronate sodium; prednisolone; morphine sulfate; lidocaine hydrochloride; indomethacin; glucosamine sulfate/chondroitin; cyclosporine; amitriptyline hcl; sulfadiazine; oxycodone hcl/acetaminophen; olopatadine hcl; misoprostol; naproxen sodium; omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab; IL-1 TRAP; MRA; CTLA4-IG; IL-18 BP; IL-12/23; anti-IL 18; anti-IL 15; BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485; CDC-801; and mesopram.
Non-limiting examples of therapeutic agents for inflammatory bowel disease with which a binding protein of the invention can be combined include the following: budenoside; epidermal growth factor; corticosteroids; cyclosporin, sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1β mAbs; anti-IL-6 mAbs; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention, or antigen binding portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands. The antibodies of the invention, or antigen binding portions thereof, may also be combined with agents, such as methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signalling by proinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFα converting enzyme inhibitors, T-cell signalling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R) and antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ) and bcl-2 inhibitors.
Examples of therapeutic agents for Crohn's disease in which a binding protein can be combined include the following: TNF antagonists, for example, anti-TNF antibodies, ADALIMUMAB (PCT Publication No. WO 97/29131; HUMIRA), CA2 (REMICADE), CDP 571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT)) inhibitors and PDE4 inhibitors. Antibodies of the invention, or antigen binding portions thereof, can be combined with corticosteroids, for example, budenoside and dexamethasone. Binding proteins of the invention or antigen binding portions thereof, may also be combined with agents such as sulfasalazine, 5-aminosalicylic acid and olsalazine, and agents which interfere with synthesis or action of proinflammatory cytokines such as IL-1, for example, IL-1β converting enzyme inhibitors and IL-1ra. Antibodies of the invention or antigen binding portion thereof may also be used with T cell signaling inhibitors, for example, tyrosine kinase inhibitors 6-mercaptopurines. Binding proteins of the invention, or antigen binding portions thereof, can be combined with IL-11. Binding proteins of the invention, or antigen binding portions thereof, can be combined with mesalamine, prednisone, azathioprine, mercaptopurine, infliximab, methylprednisolone sodium succinate, diphenoxylate/atrop sulfate, loperamide hydrochloride, methotrexate, omeprazole, folate, ciprofloxacin/dextrose-water, hydrocodone bitartrate/apap, tetracycline hydrochloride, fluocinonide, metronidazole, thimerosal/boric acid, cholestyramine/sucrose, ciprofloxacin hydrochloride, hyoscyamine sulfate, meperidine hydrochloride, midazolam hydrochloride, oxycodone hcl/acetaminophen, promethazine hydrochloride, sodium phosphate, sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphene napsylate, hydrocortisone, multivitamins, balsalazide disodium, codeine phosphate/apap, colesevelam hcl, cyanocobalamin, folic acid, levofloxacin, methylprednisolone, natalizumab and interferon-gamma
Non-limiting examples of therapeutic agents for multiple sclerosis with which binding proteins of the invention can be combined include the following: corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-β1a (AVONEX; Biogen); interferon-β1b (BETASERON; Chiron/Berlex); interferon α-n3) (Interferon Sciences/Fujimoto), interferon-α (Alfa Wassermann/J&J), interferon β1A-IF (Serono/Inhale Therapeutics), Peginterferon α 2b (Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; clabribine; antibodies to or antagonists of other human cytokines or growth factors and their receptors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Binding proteins of the invention can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. Binding proteins of the invention, may also be combined with agents, such as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signalling by proinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TACE inhibitors, T-cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines (e.g., IL-4, IL-10, IL-13 and TGFβ) and bcl-2 inhibitors.
Examples of therapeutic agents for multiple sclerosis in which binding proteins of the invention can be combined to include interferon-β, for example, IFNβ1a and IFNβ1b; copaxone, corticosteroids, caspase inhibitors, for example inhibitors of caspase-1, IL-1 inhibitors, TNF inhibitors, and antibodies to CD40 ligand and CD80.
The binding proteins of the invention, may also be combined with agents, such as alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone, xaliproden hydrochloride, fampridine, glatiramer acetate, natalizumab, sinnabidol, a-immunokine NNSO3, ABR-215062, AnergiX.MS, chemokine receptor antagonists, BBR-2778, calagualine, CPI-1189, LEM (liposome encapsulated mitoxantrone), THC.CBD (cannabinoid agonist) MBP-8298, mesopram (PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody, neurovax, pirfenidone allotrap 1258 (RDP-1258), sTNF-R1, talampanel, teriflunomide, TGF-beta2, tiplimotide, VLA-4 antagonists (for example, TR-14035, VLA4 Ultrahaler, Antegran-ELAN/Biogen), interferon gamma antagonists, IL-4 agonists.
Non-limiting examples of therapeutic agents for Angina with which binding proteins of the invention can be combined include the following: aspirin, nitroglycerin, isosorbide mononitrate, metoprolol succinate, atenolol, metoprolol tartrate, amlodipine besylate, diltiazem hydrochloride, isosorbide dinitrate, clopidogrel bisulfate, nifedipine, atorvastatin calcium, potassium chloride, furosemide, simvastatin, verapamil hcl, digoxin, propranolol hydrochloride, carvedilol, lisinopril, spironolactone, hydrochlorothiazide, enalapril maleate, nadolol, ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalol hydrochloride, fenofibrate, ezetimibe, bumetanide, losartan potassium, lisinopril/hydrochlorothiazide, felodipine, captopril, bisoprolol fumarate.
Non-limiting examples of therapeutic agents for Ankylosing Spondylitis with which binding proteins of the invention can be combined include the following: ibuprofen, diclofenac and misoprostol, naproxen, meloxicam, indomethacin, diclofenac, celecoxib, rofecoxib, Sulfasalazine, Methotrexate, azathioprine, minocyclin, prednisone, etanercept, infliximab.
Non-limiting examples of therapeutic agents for Asthma with which binding proteins of the invention can be combined include the following: albuterol, salmeterol/fluticasone, montelukast sodium, fluticasone propionate, budesonide, prednisone, salmeterol xinafoate, levalbuterol hcl, albuterol sulfate/ipratropium, prednisolone sodium phosphate, triamcinolone acetonide, beclomethasone dipropionate, ipratropium bromide, azithromycin, pirbuterol acetate, prednisolone, theophylline anhydrous, methylprednisolone sodium succinate, clarithromycin, zafirlukast, formoterol fumarate, influenza virus vaccine, methylprednisolone, amoxicillin trihydrate, flunisolide, allergy injection, cromolyn sodium, fexofenadine hydrochloride, flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhaler assist device, guaifenesin, dexamethasone sodium phosphate, moxifloxacin hcl, doxycycline hyclate, guaifenesin/d-methorphan, p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride, mometasone furoate, salmeterol xinafoate, benzonatate, cephalexin, pe/hydrocodone/chlorphenir, cetirizine hcl/pseudoephed, phenylephrine/cod/promethazine, codeine/promethazine, cefprozil, dexamethasone, guaifenesin/pseudoephedrine, chlorpheniramine/hydrocodone, nedocromil sodium, terbutaline sulfate, epinephrine, methylprednisolone, metaproterenol sulfate.
Non-limiting examples of therapeutic agents for COPD with which binding proteins of the invention can be combined include the following: albuterol sulfate/ipratropium, ipratropium bromide, salmeterol/fluticasone, albuterol, salmeterol xinafoate, fluticasone propionate, prednisone, theophylline anhydrous, methylprednisolone sodium succinate, montelukast sodium, budesonide, formoterol fumarate, triamcinolone acetonide, levofloxacin, guaifenesin, azithromycin, beclomethasone dipropionate, levalbuterol hcl, flunisolide, ceftriaxone sodium, amoxicillin trihydrate, gatifloxacin, zafirlukast, amoxicillin/clavulanate, flunisolide/menthol, chlorpheniramine/hydrocodone, metaproterenol sulfate, methylprednisolone, mometasone furoate, p-ephedrine/cod/chlorphenir, pirbuterol acetate, p-ephedrine/loratadine, terbutaline sulfate, tiotropium bromide, (R,R)-formoterol, TgAAT, Cilomilast, Roflumilast.
Non-limiting examples of therapeutic agents for HCV with which binding proteins of the invention can be combined include the following: Interferon-alpha-2a, Interferon-alpha-2b, Interferon-alpha con1, Interferon-alpha-n1, Pegylated interferon-alpha-2a, Pegylated interferon-alpha-2b, ribavirin, Peginterferon alfa-2b+ribavirin, Ursodeoxycholic Acid, Glycyrrhizic Acid, Thymalfasin, Maxamine, VX-497 and any compounds that are used to treat HCV through intervention with the following targets: HCV polymerase, HCV protease, HCV helicase, HCV IRES (internal ribosome entry site).
Non-limiting examples of therapeutic agents for Idiopathic Pulmonary Fibrosis with which binding proteins of the invention can be combined include the following: prednisone, azathioprine, albuterol, colchicine, albuterol sulfate, digoxin, gamma interferon, methylprednisolone sod succ, lorazepam, furosemide, lisinopril, nitroglycerin, spironolactone, cyclophosphamide, ipratropium bromide, actinomycin d, alteplase, fluticasone propionate, levofloxacin, metaproterenol sulfate, morphine sulfate, oxycodone hcl, potassium chloride, triamcinolone acetonide, tacrolimus anhydrous, calcium, interferon-alpha, methotrexate, mycophenolate mofetil, Interferon-gamma-1β.
Non-limiting examples of therapeutic agents for Myocardial Infarction with which binding proteins of the invention can be combined include the following: aspirin, nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin sodium, clopidogrel bisulfate, carvedilol, atenolol, morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril, isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril, tenecteplase, enalapril maleate, torsemide, retavase, losartan potassium, quinapril hcl/mag carb, bumetanide, alteplase, enalaprilat, amiodarone hydrochloride, tirofiban hcl m-hydrate, diltiazem hydrochloride, captopril, irbesartan, valsartan, propranolol hydrochloride, fosinopril sodium, lidocaine hydrochloride, eptifibatide, cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone, interferon, sotalol hydrochloride, potassium chloride, docusate sodium, dobutamine hcl, alprazolam, pravastatin sodium, atorvastatin calcium, midazolam hydrochloride, meperidine hydrochloride, isosorbide dinitrate, epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin, ezetimibe/simvastatin, avasimibe, cariporide.
Non-limiting examples of therapeutic agents for Psoriasis with which binding proteins of the invention can be combined include the following: small molecule inhibitor of KDR, small molecule inhibitor of Tie-2, calcipotriene, clobetasol propionate, triamcinolone acetonide, halobetasol propionate, tazarotene, methotrexate, fluocinonide, betamethasone diprop augmented, fluocinolone acetonide, acitretin, tar shampoo, betamethasone valerate, mometasone furoate, ketoconazole, pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide, urea, betamethasone, clobetasol propionate/emoll, fluticasone propionate, azithromycin, hydrocortisone, moisturizing formula, folic acid, desonide, pimecrolimus, coal tar, diflorasone diacetate, etanercept folate, lactic acid, methoxsalen, hc/bismuth subgal/znox/resor, methylprednisolone acetate, prednisone, sunscreen, halcinonide, salicylic acid, anthralin, clocortolone pivalate, coal extract, coal tar/salicylic acid, coal tar/salicylic acid/sulfur, desoximetasone, diazepam, emollient, fluocinonide/emollient, mineral oil/castor oil/na lact, mineral oil/peanut oil, petroleum/isopropyl myristate, psoralen, salicylic acid, soap/tribromsalan, thimerosal/boric acid, celecoxib, infliximab, cyclosporine, alefacept, efalizumab, tacrolimus, pimecrolimus, PUVA, UVB, sulfasalazine.
Non-limiting examples of therapeutic agents for Psoriatic Arthritis with which binding proteins of the invention can be combined include the following: methotrexate, etanercept, rofecoxib, celecoxib, folic acid, sulfasalazine, naproxen, leflunomide, methylprednisolone acetate, indomethacin, hydroxychloroquine sulfate, prednisone, sulindac, betamethasone diprop augmented, infliximab, methotrexate, folate, triamcinolone acetonide, diclofenac, dimethylsulfoxide, piroxicam, diclofenac sodium, ketoprofen, meloxicam, methylprednisolone, nabumetone, tolmetin sodium, calcipotriene, cyclosporine, diclofenac sodium/misoprostol, fluocinonide, glucosamine sulfate, gold sodium thiomalate, hydrocodone bitartrate/apap, ibuprofen, risedronate sodium, sulfadiazine, thioguanine, valdecoxib, alefacept, efalizumab and bcl-2 inhibitors.
Non-limiting examples of therapeutic agents for Restenosis with which binding proteins of the invention can be combined include the following: sirolimus, paclitaxel, everolimus, tacrolimus, Zotarolimus, acetaminophen.
Non-limiting examples of therapeutic agents for Sciatica with which binding proteins of the invention can be combined include the following: hydrocodone bitartrate/apap, rofecoxib, cyclobenzaprine hcl, methylprednisolone, naproxen, ibuprofen, oxycodone hcl/acetaminophen, celecoxib, valdecoxib, methylprednisolone acetate, prednisone, codeine phosphate/apap, tramadol hcl/acetaminophen, metaxalone, meloxicam, methocarbamol, lidocaine hydrochloride, diclofenac sodium, gabapentin, dexamethasone, carisoprodol, ketorolac tromethamine, indomethacin, acetaminophen, diazepam, nabumetone, oxycodone hcl, tizanidine hcl, diclofenac sodium/misoprostol, propoxyphene napsylate/apap, asa/oxycod/oxycodone ter, ibuprofen/hydrocodone bit, tramadol hcl, etodolac, propoxyphene hcl, amitriptyline hcl, carisoprodol/codeine phos/asa, morphine sulfate, multivitamins, naproxen sodium, orphenadrine citrate, temazepam.
Examples of therapeutic agents for SLE (Lupus) in which binding proteins of the invention can be combined include the following: NSAIDS, for example, diclofenac, naproxen, ibuprofen, piroxicam, indomethacin; COX2 inhibitors, for example, Celecoxib, rofecoxib, valdecoxib; anti-malarials, for example, hydroxychloroquine; Steroids, for example, prednisone, prednisolone, budenoside, dexamethasone; Cytotoxics, for example, azathioprine, cyclophosphamide, mycophenolate mofetil, methotrexate; inhibitors of PDE4 or purine synthesis inhibitor, for example Cellcept. Binding proteins of the invention, may also be combined with agents such as sulfasalazine, 5-aminosalicylic acid, olsalazine, Imuran and agents which interfere with synthesis, production or action of proinflammatory cytokines such as IL-1, for example, caspase inhibitors like IL-1β converting enzyme inhibitors and IL-1ra. Binding proteins of the invention may also be used with T cell signaling inhibitors, for example, tyrosine kinase inhibitors; or molecules that target T cell activation molecules, for example, CTLA-4-IgG or anti-B7 family antibodies, anti-PD-1 family antibodies. Binding proteins of the invention, can be combined with IL-11 or anti-cytokine antibodies, for example, fonotolizumab (anti-IFNg antibody), or anti-receptor receptor antibodies, for example, anti-IL-6 receptor antibody and antibodies to B-cell surface molecules. Antibodies of the invention or antigen binding portion thereof may also be used with LJP 394 (abetimus), agents that deplete or inactivate B-cells, for example, Rituximab (anti-CD20 antibody), lymphostat-B (anti-BlyS antibody), TNF antagonists, for example, anti-TNF antibodies, Adalimumab (PCT Publication No. WO 97/29131; HUMIRA), CA2 (REMICADE), CDP 571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT)) and bcl-2 inhibitors, because bcl-2 overexpression in transgenic mice has been demonstrated to cause a lupus like phenotype (see Marquina, Regina et al., Journal of Immunology (2004), 172(11), 7177-7185), therefore inhibition is expected to have therapeutic effects.
The pharmaceutical compositions of the invention may include a “therapeutically effective amount” or a “prophylactically effective amount” of a binding protein of the invention. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the binding protein may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the binding protein to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody, or antibody portion, are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a binding protein of the invention is 0.1-20 mg/kg, for example, 1-10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope of the invention or the embodiments disclosed herein. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting of the invention.
V. Diagnostics
The disclosure herein also provides diagnostic applications. This is further elucidated below.
I. Method of Assay
The present disclosure also provides a method for determining the presence, amount or concentration of an analyte (or a fragment thereof) in a test sample using at least one DVD-Ig as described herein. Any suitable assay as is known in the art can be used in the method. Examples include, but are not limited to, immunoassay, such as sandwich immunoassay (e.g., monoclonal, polyclonal and/or DVD-Ig sandwich immunoassays or any variation thereof (e.g., monoclonal/DVD-Ig, DVD-Ig/polyclonal, etc.), including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.))), competitive inhibition immunoassay (e.g., forward and reverse), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescent assay, etc. In a SELDI-based immunoassay, a capture reagent that specifically binds an analyte (or a fragment thereof) of interest is attached to the surface of a mass spectrometry probe, such as a pre-activated protein chip array. The analyte (or a fragment thereof) is then specifically captured on the biochip, and the captured analyte (or a fragment thereof) is detected by mass spectrometry. Alternatively, the analyte (or a fragment thereof) can be eluted from the capture reagent and detected by traditional MALDI (matrix-assisted laser desorption/ionization) or by SELDI. A chemiluminescent microparticle immunoassay, in particular one employing the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park, Ill.), is an example of a preferred immunoassay.
Methods well-known in the art for collecting, handling and processing urine, blood, serum and plasma, and other body fluids, are used in the practice of the present disclosure, for instance, when a DVD-Ig as described herein is employed as an immunodiagnostic reagent and/or in an analyte immunoassay kit. The test sample can comprise further moieties in addition to the analyte of interest, such as antibodies, antigens, haptens, hormones, drugs, enzymes, receptors, proteins, peptides, polypeptides, oligonucleotides and/or polynucleotides. For example, the sample can be a whole blood sample obtained from a subject. It can be necessary or desired that a test sample, particularly whole blood, be treated prior to immunoassay as described herein, e.g., with a pretreatment reagent. Even in cases where pretreatment is not necessary (e.g., most urine samples), pretreatment optionally can be done (e.g., as part of a regimen on a commercial platform).
The pretreatment reagent can be any reagent appropriate for use with the immunoassay and kits of the invention. The pretreatment optionally comprises: (a) one or more solvents (e.g., methanol and ethylene glycol) and optionally, salt, (b) one or more solvents and salt, and optionally, detergent, (c) detergent, or (d) detergent and salt. Pretreatment reagents are known in the art, and such pretreatment can be employed, e.g., as used for assays on Abbott TDx, AxSYM®, and ARCHITECT® analyzers (Abbott Laboratories, Abbott Park, Ill.), as described in the literature (see, e.g., Yatscoff et al., Abbott TDx Monoclonal Antibody Assay Evaluated for Measuring Cyclosporine in Whole Blood, Clin. Chem. 36: 1969-1973 (1990), and Wallemacq et al., Evaluation of the New AxSYM Cyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and EMIT Cyclosporine Assays, Clin. Chem. 45: 432-435 (1999)), and/or as commercially available. Additionally, pretreatment can be done as described in Abbott's U.S. Pat. No. 5,135,875, European Pat. Pub. No. 0 471 293, U.S. Provisional Pat. App. 60/878,017, filed Dec. 29, 2006, and U.S. Pat. App. Pub. No. 2008/0020401 (incorporated by reference in its entirety for its teachings regarding pretreatment). The pretreatment reagent can be a heterogeneous agent or a homogeneous agent.
With use of a heterogeneous pretreatment reagent, the pretreatment reagent precipitates analyte binding protein (e.g., protein that can bind to an analyte or a fragment thereof) present in the sample. Such a pretreatment step comprises removing any analyte binding protein by separating from the precipitated analyte binding protein the supernatant of the mixture formed by addition of the pretreatment agent to sample. In such an assay, the supernatant of the mixture absent any binding protein is used in the assay, proceeding directly to the antibody capture step.
With use of a homogeneous pretreatment reagent there is no such separation step. The entire mixture of test sample and pretreatment reagent are contacted with a labeled specific binding partner for analyte (or a fragment thereof), such as a labeled anti-analyte antibody (or an antigenically reactive fragment thereof). The pretreatment reagent employed for such an assay typically is diluted in the pretreated test sample mixture, either before or during capture by the first specific binding partner. Despite such dilution, a certain amount of the pretreatment reagent is still present (or remains) in the test sample mixture during capture. According to the invention, the labeled specific binding partner can be a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof).
In a heterogeneous format, after the test sample is obtained from a subject, a first mixture is prepared. The mixture contains the test sample being assessed for an analyte (or a fragment thereof) and a first specific binding partner, wherein the first specific binding partner and any analyte contained in the test sample form a first specific binding partner-analyte complex. Preferably, the first specific binding partner is an anti-analyte antibody or a fragment thereof. The first specific binding partner can be a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) as described herein. The order in which the test sample and the first specific binding partner are added to form the mixture is not critical. Preferably, the first specific binding partner is immobilized on a solid phase. The solid phase used in the immunoassay (for the first specific binding partner and, optionally, the second specific binding partner) can be any solid phase known in the art, such as, but not limited to, a magnetic particle, a bead, a test tube, a microtiter plate, a cuvette, a membrane, a scaffolding molecule, a film, a filter paper, a disc and a chip.
After the mixture containing the first specific binding partner-analyte complex is formed, any unbound analyte is removed from the complex using any technique known in the art. For example, the unbound analyte can be removed by washing. Desirably, however, the first specific binding partner is present in excess of any analyte present in the test sample, such that all analyte that is present in the test sample is bound by the first specific binding partner.
After any unbound analyte is removed, a second specific binding partner is added to the mixture to form a first specific binding partner-analyte-second specific binding partner complex. The second specific binding partner is preferably an anti-analyte antibody that binds to an epitope on analyte that differs from the epitope on analyte bound by the first specific binding partner. Moreover, also preferably, the second specific binding partner is labeled with or contains a detectable label as described above. The second specific binding partner can be a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) as described herein.
Any suitable detectable label as is known in the art can be used. For example, the detectable label can be a radioactive label (such as 3H, 125I, 35S, 14C, 32P, and 33P), an enzymatic label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), a thermometric label, or an immuno-polymerase chain reaction label. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), which is a combined handbook and catalogue published by Molecular Probes, Inc., Eugene, Oreg. A fluorescent label can be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated by reference in their entireties). An acridinium compound can be used as a detectable label in a homogeneous or heterogeneous chemiluminescent assay (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem. Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett. 5: 3779-3782 (2003)).
A preferred acridinium compound is an acridinium-9-carboxamide. Methods for preparing acridinium 9-carboxamides are described in Mattingly, J. Biolumin. Chemilumin. 6: 107-114 (1991); Adamczyk et al., J. Org. Chem. 63: 5636-5639 (1998); Adamczyk et al., Tetrahedron 55: 10899-10914 (1999); Adamczyk et al., Org. Lett. 1: 779-781 (1999); Adamczyk et al., Bioconjugate Chem. 11: 714-724 (2000); Mattingly et al., In Luminescence Biotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5: 3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524 and 5,783,699 (each of which is incorporated herein by reference in its entirety for its teachings regarding same). Another preferred acridinium compound is an acridinium-9-carboxylate aryl ester. An example of an acridinium-9-carboxylate aryl ester is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing acridinium 9-carboxylate aryl esters are described in McCapra et al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al., Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15: 239-244 (2000); and U.S. Pat. No. 5,241,070 (each of which is incorporated herein by reference in its entirety for its teachings regarding same). Further details regarding acridinium-9-carboxylate aryl ester and its use are set forth in US 2008-0248493.
Chemiluminescent assays (e.g., using acridinium as described above or other chemiluminescent agents) can be performed in accordance with the methods described in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67 (2006). While any suitable assay format can be used, a microplate chemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, Oak Ridge, Tenn.) enables the assay of multiple samples of small volumes rapidly.
The order in which the test sample and the specific binding partner(s) are added to form the mixture for chemiluminescent assay is not critical. If the first specific binding partner is detectably labeled with a chemiluminescent agent such as an acridinium compound, detectably labeled first specific binding partner-analyte complexes form. Alternatively, if a second specific binding partner is used and the second specific binding partner is detectably labeled with a chemiluminescent agent such as an acridinium compound, detectably labeled first specific binding partner-analyte-second specific binding partner complexes form. Any unbound specific binding partner, whether labeled or unlabeled, can be removed from the mixture using any technique known in the art, such as washing.
Hydrogen peroxide can be generated in situ in the mixture or provided or supplied to the mixture (e.g., the source of the hydrogen peroxide being one or more buffers or other solutions that are known to contain hydrogen peroxide) before, simultaneously with, or after the addition of an above-described acridinium compound. Hydrogen peroxide can be generated in situ in a number of ways such as would be apparent to one skilled in the art.
Upon the simultaneous or subsequent addition of at least one basic solution to the sample, a detectable signal, namely, a chemiluminescent signal, indicative of the presence of analyte is generated. The basic solution contains at least one base and has a pH greater than or equal to 10, preferably, greater than or equal to 12. Examples of basic solutions include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and calcium bicarbonate. The amount of basic solution added to the sample depends on the concentration of the basic solution. Based on the concentration of the basic solution used, one skilled in the art can easily determine the amount of basic solution to add to the sample.
The chemiluminescent signal that is generated can be detected using routine techniques known to those skilled in the art. Based on the intensity of the signal generated, the amount of analyte in the sample can be quantified. Specifically, the amount of analyte in the sample is proportional to the intensity of the signal generated. The amount of analyte present can be quantified by comparing the amount of light generated to a standard curve for analyte or by comparison to a reference standard. The standard curve can be generated using serial dilutions or solutions of known concentrations of analyte by mass spectroscopy, gravimetric methods, and other techniques known in the art. While the above is described with emphasis on use of an acridinium compound as the chemiluminescent agent, one of ordinary skill in the art can readily adapt this description for use of other chemiluminescent agents.
Analyte immunoassays generally can be conducted using any format known in the art, such as, but not limited to, a sandwich format. Specifically, in one immunoassay format, at least two antibodies are employed to separate and quantify analyte, such as human analyte, or a fragment thereof in a sample. More specifically, the at least two antibodies bind to different epitopes on an analyte (or a fragment thereof) forming an immune complex, which is referred to as a “sandwich.” Generally, in the immunoassays one or more antibodies can be used to capture the analyte (or a fragment thereof) in the test sample (these antibodies are frequently referred to as a “capture” antibody or “capture” antibodies) and one or more antibodies can be used to bind a detectable (namely, quantifiable) label to the sandwich (these antibodies are frequently referred to as the “detection antibody,” the “detection antibodies,” the “conjugate,” or the “conjugates”). Thus, in the context of a sandwich immunoassay format, a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) as described herein can be used as a capture antibody, a detection antibody, or both. For example, one DVD-Ig having a domain that can bind a first epitope on an analyte (or a fragment thereof) can be used as a capture antibody and/or another DVD-Ig having a domain that can bind a second epitope on an analyte (or a fragment thereof) can be used as a detection antibody. In this regard, a DVD-Ig having a first domain that can bind a first epitope on an analyte (or a fragment thereof) and a second domain that can bind a second epitope on an analyte (or a fragment thereof) can be used as a capture antibody and/or a detection antibody. Alternatively, one DVD-Ig having a first domain that can bind an epitope on a first analyte (or a fragment thereof) and a second domain that can bind an epitope on a second analyte (or a fragment thereof) can be used as a capture antibody and/or a detection antibody to detect, and optionally quantify, two or more analytes. In the event that an analyte can be present in a sample in more than one form, such as a monomeric form and a dimeric/multimeric form, which can be homomeric or heteromeric, one DVD-Ig having a domain that can bind an epitope that is only exposed on the monomeric form and another DVD-Ig having a domain that can bind an epitope on a different part of a dimeric/multimeric form can be used as capture antibodies and/or detection antibodies, thereby enabling the detection, and optional quantification, of different forms of a given analyte. Furthermore, employing DVD-Igs with differential affinities within a single DVD-Ig and/or between DVD-Igs can provide an avidity advantage. In the context of immunoassays as described herein, it generally may be helpful or desired to incorporate one or more linkers within the structure of a DVD-Ig. When present, optimally the linker should be of sufficient length and structural flexibility to enable binding of an epitope by the inner domains as well as binding of another epitope by the outer domains. In this regard, if a DVD-Ig can bind two different analytes and one analyte is larger than the other, desirably the larger analyte is bound by the outer domains.
Generally speaking, a sample being tested for (for example, suspected of containing) analyte (or a fragment thereof) can be contacted with at least one capture antibody (or antibodies) and at least one detection antibody (which can be a second detection antibody or a third detection antibody or even a successively numbered antibody, e.g., as where the capture and/or detection antibody comprise multiple antibodies) either simultaneously or sequentially and in any order. For example, the test sample can be first contacted with at least one capture antibody and then (sequentially) with at least one detection antibody. Alternatively, the test sample can be first contacted with at least one detection antibody and then (sequentially) with at least one capture antibody. In yet another alternative, the test sample can be contacted simultaneously with a capture antibody and a detection antibody.
In the sandwich assay format, a sample suspected of containing analyte (or a fragment thereof) is first brought into contact with at least one first capture antibody under conditions that allow the formation of a first antibody/analyte complex. If more than one capture antibody is used, a first capture antibody/analyte complex comprising two or more capture antibodies is formed. In a sandwich assay, the antibodies, i.e., preferably, the at least one capture antibody, are used in molar excess amounts of the maximum amount of analyte (or a fragment thereof) expected in the test sample. For example, from about 5 μg to about 1 mg of antibody per mL of buffer (e.g., microparticle coating buffer) can be used.
Competitive inhibition immunoassays, which are often used to measure small analytes because binding by only one antibody is required, comprise sequential and classic formats. In a sequential competitive inhibition immunoassay a capture antibody to an analyte of interest is coated onto a well of a microtiter plate or other solid support. When the sample containing the analyte of interest is added to the well, the analyte of interest binds to the capture antibody. After washing, a known amount of labeled (e.g., biotin or horseradish peroxidase (HRP)) analyte is added to the well. A substrate for an enzymatic label is necessary to generate a signal. An example of a suitable substrate for HRP is 3,3′,5,5′-tetramethylbenzidine (TMB). After washing, the signal generated by the labeled analyte is measured and is inversely proportional to the amount of analyte in the sample. In a classic competitive inhibition immunoassay an antibody to an analyte of interest is coated onto a solid support (e.g., a well of a microtiter plate). However, unlike the sequential competitive inhibition immunoassay, the sample and the labeled analyte are added to the well at the same time. Any analyte in the sample competes with labeled analyte for binding to the capture antibody. After washing, the signal generated by the labeled analyte is measured and is inversely proportional to the amount of analyte in the sample.
Optionally, prior to contacting the test sample with the at least one capture antibody (for example, the first capture antibody), the at least one capture antibody can be bound to a solid support, which facilitates the separation of the first antibody/analyte (or a fragment thereof) complex from the test sample. The substrate to which the capture antibody is bound can be any suitable solid support or solid phase that facilitates separation of the capture antibody-analyte complex from the sample.
Examples include a well of a plate, such as a microtiter plate, a test tube, a porous gel (e.g., silica gel, agarose, dextran, or gelatin), a polymeric film (e.g., polyacrylamide), beads (e.g., polystyrene beads or magnetic beads), a strip of a filter/membrane (e.g., nitrocellulose or nylon), microparticles (e.g., latex particles, magnetizable microparticles (e.g., microparticles having ferric oxide or chromium oxide cores and homo- or hetero-polymeric coats and radii of about 1-10 microns). The substrate can comprise a suitable porous material with a suitable surface affinity to bind antigens and sufficient porosity to allow access by detection antibodies. A microporous material is generally preferred, although a gelatinous material in a hydrated state can be used. Such porous substrates are preferably in the form of sheets having a thickness of about 0.01 to about 0.5 mm, preferably about 0.1 mm. While the pore size may vary quite a bit, preferably the pore size is from about 0.025 to about 15 microns, more preferably from about 0.15 to about 15 microns. The surface of such substrates can be activated by chemical processes that cause covalent linkage of an antibody to the substrate. Irreversible binding, generally by adsorption through hydrophobic forces, of the antigen or the antibody to the substrate results; alternatively, a chemical coupling agent or other means can be used to bind covalently the antibody to the substrate, provided that such binding does not interfere with the ability of the antibody to bind to analyte. Alternatively, the antibody can be bound with microparticles, which have been previously coated with streptavidin (e.g., DYNAL® Magnetic Beads, Invitrogen, Carlsbad, Calif.) or biotin (e.g., using Power-BindTM-SA-MP streptavidin-coated microparticles (Seradyn, Indianapolis, Ind.)) or anti-species-specific monoclonal antibodies. If necessary, the substrate can be derivatized to allow reactivity with various functional groups on the antibody. Such derivatization requires the use of certain coupling agents, examples of which include, but are not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. If desired, one or more capture reagents, such as antibodies (or fragments thereof), each of which is specific for analyte(s) can be attached to solid phases in different physical or addressable locations (e.g., such as in a biochip configuration (see, e.g., U.S. Pat. No. 6,225,047; Int'l Pat. App. Pub. No. WO 99/51773; U.S. Pat. No. 6,329,209; Int'l Pat. App. Pub. No. WO 00/56934, and U.S. Pat. No. 5,242,828). If the capture reagent is attached to a mass spectrometry probe as the solid support, the amount of analyte bound to the probe can be detected by laser desorption ionization mass spectrometry. Alternatively, a single column can be packed with different beads, which are derivatized with the one or more capture reagents, thereby capturing the analyte in a single place (see, antibody-derivatized, bead-based technologies, e.g., the xMAP technology of Luminex (Austin, Tex.)).
After the test sample being assayed for analyte (or a fragment thereof) is brought into contact with the at least one capture antibody (for example, the first capture antibody), the mixture is incubated in order to allow for the formation of a first antibody (or multiple antibody)-analyte (or a fragment thereof) complex. The incubation can be carried out at a pH of from about 4.5 to about 10.0, at a temperature of from about 2° C. to about 45° C., and for a period from at least about one (1) minute to about eighteen (18) hours, preferably from about 1 to about 24 minutes, most preferably for about 4 to about 18 minutes. The immunoassay described herein can be conducted in one step (meaning the test sample, at least one capture antibody and at least one detection antibody are all added sequentially or simultaneously to a reaction vessel) or in more than one step, such as two steps, three steps, etc.
After formation of the (first or multiple) capture antibody/analyte (or a fragment thereof) complex, the complex is then contacted with at least one detection antibody under conditions which allow for the formation of a (first or multiple) capture antibody/analyte (or a fragment thereof)/second detection antibody complex). While captioned for clarity as the “second” antibody (e.g., second detection antibody), in fact, where multiple antibodies are used for capture and/or detection, the at least one detection antibody can be the second, third, fourth, etc. antibodies used in the immunoassay. If the capture antibody/analyte (or a fragment thereof) complex is contacted with more than one detection antibody, then a (first or multiple) capture antibody/analyte (or a fragment thereof)/(multiple) detection antibody complex is formed. As with the capture antibody (e.g., the first capture antibody), when the at least one (e.g., second and any subsequent) detection antibody is brought into contact with the capture antibody/analyte (or a fragment thereof) complex, a period of incubation under conditions similar to those described above is required for the formation of the (first or multiple) capture antibody/analyte (or a fragment thereof)/(second or multiple) detection antibody complex. Preferably, at least one detection antibody contains a detectable label. The detectable label can be bound to the at least one detection antibody (e.g., the second detection antibody) prior to, simultaneously with, or after the formation of the (first or multiple) capture antibody/analyte (or a fragment thereof)/(second or multiple) detection antibody complex. Any detectable label known in the art can be used (see discussion above, including of the Polak and Van Noorden (1997) and Haugland (1996) references).
The detectable label can be bound to the antibodies either directly or through a coupling agent. An example of a coupling agent that can be used is EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, hydrochloride), which is commercially available from Sigma-Aldrich, St. Louis, Mo. Other coupling agents that can be used are known in the art. Methods for binding a detectable label to an antibody are known in the art. Additionally, many detectable labels can be purchased or synthesized that already contain end groups that facilitate the coupling of the detectable label to the antibody, such as CPSP-Acridinium Ester (i.e., 9-[N-tosyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridinium carboxamide) or SPSP-Acridinium Ester (i.e., N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).
The (first or multiple) capture antibody/analyte/(second or multiple) detection antibody complex can be, but does not have to be, separated from the remainder of the test sample prior to quantification of the label. For example, if the at least one capture antibody (e.g., the first capture antibody) is bound to a solid support, such as a well or a bead, separation can be accomplished by removing the fluid (of the test sample) from contact with the solid support. Alternatively, if the at least first capture antibody is bound to a solid support, it can be simultaneously contacted with the analyte-containing sample and the at least one second detection antibody to form a first (multiple) antibody/analyte/second (multiple) antibody complex, followed by removal of the fluid (test sample) from contact with the solid support. If the at least one first capture antibody is not bound to a solid support, then the (first or multiple) capture antibody/analyte/(second or multiple) detection antibody complex does not have to be removed from the test sample for quantification of the amount of the label.
After formation of the labeled capture antibody/analyte/detection antibody complex (e.g., the first capture antibody/analyte/second detection antibody complex), the amount of label in the complex is quantified using techniques known in the art. For example, if an enzymatic label is used, the labeled complex is reacted with a substrate for the label that gives a quantifiable reaction such as the development of color. If the label is a radioactive label, the label is quantified using appropriate means, such as a scintillation counter. If the label is a fluorescent label, the label is quantified by stimulating the label with a light of one color (which is known as the “excitation wavelength”) and detecting another color (which is known as the “emission wavelength”) that is emitted by the label in response to the stimulation. If the label is a chemiluminescent label, the label is quantified by detecting the light emitted either visually or by using luminometers, x-ray film, high speed photographic film, a CCD camera, etc. Once the amount of the label in the complex has been quantified, the concentration of analyte or a fragment thereof in the test sample is determined by appropriate means, such as by use of a standard curve that has been generated using serial dilutions of analyte or a fragment thereof of known concentration. Other than using serial dilutions of analyte or a fragment thereof, the standard curve can be generated gravimetrically, by mass spectroscopy and by other techniques known in the art.
In a chemiluminescent microparticle assay employing the ARCHITECT® analyzer, the conjugate diluent pH should be about 6.0+/−0.2, the microparticle coating buffer should be maintained at about room temperature (i.e., at from about 17 to about 27° C.), the microparticle coating buffer pH should be about 6.5+/−0.2, and the microparticle diluent pH should be about 7.8+/−0.2. Solids preferably are less than about 0.2%, such as less than about 0.15%, less than about 0.14%, less than about 0.13%, less than about 0.12%, or less than about 0.11%, such as about 0.10%.
FPIAs are based on competitive binding immunoassay principles. A fluorescently labeled compound, when excited by a linearly polarized light, will emit fluorescence having a degree of polarization inversely proportional to its rate of rotation. When a fluorescently labeled tracer-antibody complex is excited by a linearly polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time light is absorbed and the time light is emitted. When a “free” tracer compound (i.e., a compound that is not bound to an antibody) is excited by linearly polarized light, its rotation is much faster than the corresponding tracer-antibody conjugate produced in a competitive binding immunoassay. FPIAs are advantageous over RIAs inasmuch as there are no radioactive substances requiring special handling and disposal. In addition, FPIAs are homogeneous assays that can be easily and rapidly performed.
In view of the above, a method of determining the presence, amount, or concentration of analyte (or a fragment thereof) in a test sample is provided. The method comprises assaying the test sample for an analyte (or a fragment thereof) by an assay (i) employing (i′) at least one of an antibody, a fragment of an antibody that can bind to an analyte, a variant of an antibody that can bind to an analyte, a fragment of a variant of an antibody that can bind to an analyte, and a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) that can bind to an analyte, and (ii′) at least one detectable label and (ii) comprising comparing a signal generated by the detectable label as a direct or indirect indication of the presence, amount or concentration of analyte (or a fragment thereof) in the test sample to a signal generated as a direct or indirect indication of the presence, amount or concentration of analyte (or a fragment thereof) in a control or calibrator. The calibrator is optionally part of a series of calibrators, in which each of the calibrators differs from the other calibrators by the concentration of analyte.
The method can comprise (i) contacting the test sample with at least one first specific binding partner for analyte (or a fragment thereof) selected from the group consisting of an antibody, a fragment of an antibody that can bind to an analyte, a variant of an antibody that can bind to an analyte, a fragment of a variant of an antibody that can bind to an analyte, and a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) that can bind to an analyte so as to form a first specific binding partner/analyte (or fragment thereof) complex, (ii) contacting the first specific binding partner/analyte (or fragment thereof) complex with at least one second specific binding partner for analyte (or fragment thereof) selected from the group consisting of a detectably labeled anti-analyte antibody, a detectably labeled fragment of an anti-analyte antibody that can bind to analyte, a detectably labeled variant of an anti-analyte antibody that can bind to analyte, a detectably labeled fragment of a variant of an anti-analyte antibody that can bind to analyte, and a detectably labeled DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) so as to form a first specific binding partner/analyte (or fragment thereof)/second specific binding partner complex, and (iii) determining the presence, amount or concentration of analyte in the test sample by detecting or measuring the signal generated by the detectable label in the first specific binding partner/analyte (or fragment thereof)/second specific binding partner complex formed in (ii). A method in which at least one first specific binding partner for analyte (or a fragment thereof) and/or at least one second specific binding partner for analyte (or a fragment thereof) is a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) as described herein can be preferred.
Alternatively, the method can comprise contacting the test sample with at least one first specific binding partner for analyte (or a fragment thereof) selected from the group consisting of an antibody, a fragment of an antibody that can bind to an analyte, a variant of an antibody that can bind to an analyte, a fragment of a variant of an antibody that can bind to an analyte, and a DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof) and simultaneously or sequentially, in either order, contacting the test sample with at least one second specific binding partner, which can compete with analyte (or a fragment thereof) for binding to the at least one first specific binding partner and which is selected from the group consisting of a detectably labeled analyte, a detectably labeled fragment of analyte that can bind to the first specific binding partner, a detectably labeled variant of analyte that can bind to the first specific binding partner, and a detectably labeled fragment of a variant of analyte that can bind to the first specific binding partner. Any analyte (or a fragment thereof) present in the test sample and the at least one second specific binding partner compete with each other to form a first specific binding partner/analyte (or fragment thereof) complex and a first specific binding partner/second specific binding partner complex, respectively. The method further comprises determining the presence, amount or concentration of analyte in the test sample by detecting or measuring the signal generated by the detectable label in the first specific binding partner/second specific binding partner complex formed in (ii), wherein the signal generated by the detectable label in the first specific binding partner/second specific binding partner complex is inversely proportional to the amount or concentration of analyte in the test sample.
The above methods can further comprise diagnosing, prognosticating, or assessing the efficacy of a therapeutic/prophylactic treatment of a patient from whom the test sample was obtained. If the method further comprises assessing the efficacy of a therapeutic/prophylactic treatment of the patient from whom the test sample was obtained, the method optionally further comprises modifying the therapeutic/prophylactic treatment of the patient as needed to improve efficacy. The method can be adapted for use in an automated system or a semi-automated system.
With regard to the methods of assay (and kit therefor), it may be possible to employ commercially available anti-analyte antibodies or methods for production of anti-analyte as described in the literature. Commercial supplies of various antibodies include, but are not limited to, Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.), GenWay Biotech, Inc. (San Diego, Calif.), and R&D Systems (RDS; Minneapolis, Minn.).
Generally, a predetermined level can be employed as a benchmark against which to assess results obtained upon assaying a test sample for analyte or a fragment thereof, e.g., for detecting disease or risk of disease. Generally, in making such a comparison, the predetermined level is obtained by running a particular assay a sufficient number of times and under appropriate conditions such that a linkage or association of analyte presence, amount or concentration with a particular stage or endpoint of a disease, disorder or condition or with particular clinical indicia can be made. Typically, the predetermined level is obtained with assays of reference subjects (or populations of subjects). The analyte measured can include fragments thereof, degradation products thereof, and/or enzymatic cleavage products thereof.
In particular, with respect to a predetermined level as employed for monitoring disease progression and/or treatment, the amount or concentration of analyte or a fragment thereof may be “unchanged,” “favorable” (or “favorably altered”), or “unfavorable” (or “unfavorably altered”). “Elevated” or “increased” refers to an amount or a concentration in a test sample that is higher than a typical or normal level or range (e.g., predetermined level), or is higher than another reference level or range (e.g., earlier or baseline sample). The term “lowered” or “reduced” refers to an amount or a concentration in a test sample that is lower than a typical or normal level or range (e.g., predetermined level), or is lower than another reference level or range (e.g., earlier or baseline sample). The term “altered” refers to an amount or a concentration in a sample that is altered (increased or decreased) over a typical or normal level or range (e.g., predetermined level), or over another reference level or range (e.g., earlier or baseline sample).
The typical or normal level or range for analyte is defined in accordance with standard practice. Because the levels of analyte in some instances will be very low, a so-called altered level or alteration can be considered to have occurred when there is any net change as compared to the typical or normal level or range, or reference level or range, that cannot be explained by experimental error or sample variation. Thus, the level measured in a particular sample will be compared with the level or range of levels determined in similar samples from a so-called normal subject. In this context, a “normal subject” is an individual with no detectable disease, for example, and a “normal” (sometimes termed “control”) patient or population is/are one(s) that exhibit(s) no detectable disease, respectively, for example. Furthermore, given that analyte is not routinely found at a high level in the majority of the human population, a “normal subject” can be considered an individual with no substantial detectable increased or elevated amount or concentration of analyte, and a “normal” (sometimes termed “control”) patient or population is/are one(s) that exhibit(s) no substantial detectable increased or elevated amount or concentration of analyte. An “apparently normal subject” is one in which analyte has not yet been or currently is being assessed. The level of an analyte is said to be “elevated” when the analyte is normally undetectable (e.g., the normal level is zero, or within a range of from about 25 to about 75 percentiles of normal populations), but is detected in a test sample, as well as when the analyte is present in the test sample at a higher than normal level. Thus, inter alia, the disclosure provides a method of screening for a subject having, or at risk of having, a particular disease, disorder, or condition. The method of assay can also involve the assay of other markers and the like.
Accordingly, the methods described herein also can be used to determine whether or not a subject has or is at risk of developing a given disease, disorder or condition. Specifically, such a method can comprise the steps of:
(a) determining the concentration or amount in a test sample from a subject of analyte (or a fragment thereof) (e.g., using the methods described herein, or methods known in the art); and
(b) comparing the concentration or amount of analyte (or a fragment thereof) determined in step (a) with a predetermined level, wherein, if the concentration or amount of analyte determined in step (a) is favorable with respect to a predetermined level, then the subject is determined not to have or be at risk for a given disease, disorder or condition. However, if the concentration or amount of analyte determined in step (a) is unfavorable with respect to the predetermined level, then the subject is determined to have or be at risk for a given disease, disorder or condition.
Additionally, provided herein is method of monitoring the progression of disease in a subject. Optimally the method comprising the steps of:
(a) determining the concentration or amount in a test sample from a subject of analyte;
(b) determining the concentration or amount in a later test sample from the subject of analyte; and
(c) comparing the concentration or amount of analyte as determined in step (b) with the concentration or amount of analyte determined in step (a), wherein if the concentration or amount determined in step (b) is unchanged or is unfavorable when compared to the concentration or amount of analyte determined in step (a), then the disease in the subject is determined to have continued, progressed or worsened. By comparison, if the concentration or amount of analyte as determined in step (b) is favorable when compared to the concentration or amount of analyte as determined in step (a), then the disease in the subject is determined to have discontinued, regressed or improved.
Optionally, the method further comprises comparing the concentration or amount of analyte as determined in step (b), for example, with a predetermined level. Further, optionally the method comprises treating the subject with one or more pharmaceutical compositions for a period of time if the comparison shows that the concentration or amount of analyte as determined in step (b), for example, is unfavorably altered with respect to the predetermined level.
Still further, the methods can be used to monitor treatment in a subject receiving treatment with one or more pharmaceutical compositions. Specifically, such methods involve providing a first test sample from a subject before the subject has been administered one or more pharmaceutical compositions. Next, the concentration or amount in a first test sample from a subject of analyte is determined (e.g., using the methods described herein or as known in the art). After the concentration or amount of analyte is determined, optionally the concentration or amount of analyte is then compared with a predetermined level. If the concentration or amount of analyte as determined in the first test sample is lower than the predetermined level, then the subject is not treated with one or more pharmaceutical compositions. However, if the concentration or amount of analyte as determined in the first test sample is higher than the predetermined level, then the subject is treated with one or more pharmaceutical compositions for a period of time. The period of time that the subject is treated with the one or more pharmaceutical compositions can be determined by one skilled in the art (for example, the period of time can be from about seven (7) days to about two years, preferably from about fourteen (14) days to about one (1) year).
During the course of treatment with the one or more pharmaceutical compositions, second and subsequent test samples are then obtained from the subject. The number of test samples and the time in which said test samples are obtained from the subject are not critical. For example, a second test sample could be obtained seven (7) days after the subject is first administered the one or more pharmaceutical compositions, a third test sample could be obtained two (2) weeks after the subject is first administered the one or more pharmaceutical compositions, a fourth test sample could be obtained three (3) weeks after the subject is first administered the one or more pharmaceutical compositions, a fifth test sample could be obtained four (4) weeks after the subject is first administered the one or more pharmaceutical compositions, etc.
After each second or subsequent test sample is obtained from the subject, the concentration or amount of analyte is determined in the second or subsequent test sample is determined (e.g., using the methods described herein or as known in the art). The concentration or amount of analyte as determined in each of the second and subsequent test samples is then compared with the concentration or amount of analyte as determined in the first test sample (e.g., the test sample that was originally optionally compared to the predetermined level). If the concentration or amount of analyte as determined in step (c) is favorable when compared to the concentration or amount of analyte as determined in step (a), then the disease in the subject is determined to have discontinued, regressed or improved, and the subject should continue to be administered the one or pharmaceutical compositions of step (b). However, if the concentration or amount determined in step (c) is unchanged or is unfavorable when compared to the concentration or amount of analyte as determined in step (a), then the disease in the subject is determined to have continued, progressed or worsened, and the subject should be treated with a higher concentration of the one or more pharmaceutical compositions administered to the subject in step (b) or the subject should be treated with one or more pharmaceutical compositions that are different from the one or more pharmaceutical compositions administered to the subject in step (b). Specifically, the subject can be treated with one or more pharmaceutical compositions that are different from the one or more pharmaceutical compositions that the subject had previously received to decrease or lower said subject's analyte level.
Generally, for assays in which repeat testing may be done (e.g., monitoring disease progression and/or response to treatment), a second or subsequent test sample is obtained at a period in time after the first test sample has been obtained from the subject. Specifically, a second test sample from the subject can be obtained minutes, hours, days, weeks or years after the first test sample has been obtained from the subject. For example, the second test sample can be obtained from the subject at a time period of about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0 years, about 5.5 years, about 6.0 years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0 years after the first test sample from the subject is obtained.
When used to monitor disease progression, the above assay can be used to monitor the progression of disease in subjects suffering from acute conditions. Acute conditions, also known as critical care conditions, refer to acute, life-threatening diseases or other critical medical conditions involving, for example, the cardiovascular system or excretory system. Typically, critical care conditions refer to those conditions requiring acute medical intervention in a hospital-based setting (including, but not limited to, the emergency room, intensive care unit, trauma center, or other emergent care setting) or administration by a paramedic or other field-based medical personnel. For critical care conditions, repeat monitoring is generally done within a shorter time frame, namely, minutes, hours or days (e.g., about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days or about 7 days), and the initial assay likewise is generally done within a shorter timeframe, e.g., about minutes, hours or days of the onset of the disease or condition.
The assays also can be used to monitor the progression of disease in subjects suffering from chronic or non-acute conditions. Non-critical care or, non-acute conditions, refers to conditions other than acute, life-threatening disease or other critical medical conditions involving, for example, the cardiovascular system and/or excretory system. Typically, non-acute conditions include those of longer-term or chronic duration. For non-acute conditions, repeat monitoring generally is done with a longer timeframe, e.g., hours, days, weeks, months or years (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0 years, about 5.5 years, about 6.0 years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0 years), and the initial assay likewise generally is done within a longer time frame, e.g., about hours, days, months or years of the onset of the disease or condition.
Furthermore, the above assays can be performed using a first test sample obtained from a subject where the first test sample is obtained from one source, such as urine, serum or plasma. Optionally, the above assays can then be repeated using a second test sample obtained from the subject where the second test sample is obtained from another source. For example, if the first test sample was obtained from urine, the second test sample can be obtained from serum or plasma. The results obtained from the assays using the first test sample and the second test sample can be compared. The comparison can be used to assess the status of a disease or condition in the subject.
Moreover, the present disclosure also relates to methods of determining whether a subject predisposed to or suffering from a given disease, disorder or condition will benefit from treatment. In particular, the disclosure relates to analyte companion diagnostic methods and products. Thus, the method of “monitoring the treatment of disease in a subject” as described herein further optimally also can encompass selecting or identifying candidates for therapy.
Thus, in particular embodiments, the disclosure also provides a method of determining whether a subject having, or at risk for, a given disease, disorder or condition is a candidate for therapy. Generally, the subject is one who has experienced some symptom of a given disease, disorder or condition or who has actually been diagnosed as having, or being at risk for, a given disease, disorder or condition, and/or who demonstrates an unfavorable concentration or amount of analyte or a fragment thereof, as described herein.
The method optionally comprises an assay as described herein, where analyte is assessed before and following treatment of a subject with one or more pharmaceutical compositions (e.g., particularly with a pharmaceutical related to a mechanism of action involving analyte), with immunosuppressive therapy, or by immunoabsorption therapy, or where analyte is assessed following such treatment and the concentration or the amount of analyte is compared against a predetermined level. An unfavorable concentration of amount of analyte observed following treatment confirms that the subject will not benefit from receiving further or continued treatment, whereas a favorable concentration or amount of analyte observed following treatment confirms that the subject will benefit from receiving further or continued treatment. This confirmation assists with management of clinical studies, and provision of improved patient care.
It goes without saying that, while certain embodiments herein are advantageous when employed to assess a given disease, disorder or condition as discussed herein, the assays and kits can be employed to assess analyte in other diseases, disorders and conditions. The method of assay can also involve the assay of other markers and the like.
The method of assay also can be used to identify a compound that ameliorates a given disease, disorder or condition. For example, a cell that expresses analyte can be contacted with a candidate compound. The level of expression of analyte in the cell contacted with the compound can be compared to that in a control cell using the method of assay described herein.
II. Kit
A kit for assaying a test sample for the presence, amount or concentration of an analyte (or a fragment thereof) in a test sample is also provided. The kit comprises at least one component for assaying the test sample for the analyte (or a fragment thereof) and instructions for assaying the test sample for the analyte (or a fragment thereof). The at least one component for assaying the test sample for the analyte (or a fragment thereof) can include a composition comprising an anti-analyte DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof), which is optionally immobilized on a solid phase.
The kit can comprise at least one component for assaying the test sample for an analyte by immunoassay, e.g., chemiluminescent microparticle immunoassay, and instructions for assaying the test sample for an analyte by immunoassay, e.g., chemiluminescent microparticle immunoassay. For example, the kit can comprise at least one specific binding partner for an analyte, such as an anti-analyte, monoclonal/polyclonal antibody (or a fragment thereof that can bind to the analyte, a variant thereof that can bind to the analyte, or a fragment of a variant that can bind to the analyte) or an anti-analyte DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof), either of which can be detectably labeled. Alternatively or additionally, the kit can comprise detectably labeled analyte (or a fragment thereof that can bind to an anti-analyte, monoclonal/polyclonal antibody or an anti-analyte DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof)), which can compete with any analyte in a test sample for binding to an anti-analyte, monoclonal/polyclonal antibody (or a fragment thereof that can bind to the analyte, a variant thereof that can bind to the analyte, or a fragment of a variant that can bind to the analyte) or an anti-analyte DVD-Ig (or a fragment, a variant, or a fragment of a variant thereof), either of which can be immobilized on a solid support. The kit can comprise a calibrator or control, e.g., isolated or purified analyte. The kit can comprise at least one container (e.g., tube, microtiter plates or strips, which can be already coated with a first specific binding partner, for example) for conducting the assay, and/or a buffer, such as an assay buffer or a wash buffer, either one of which can be provided as a concentrated solution, a substrate solution for the detectable label (e.g., an enzymatic label), or a stop solution. Preferably, the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary to perform the assay. The instructions can be in paper form or computer-readable form, such as a disk, CD, DVD, or the like.
Any antibodies, such as an anti-analyte antibody or an anti-analyte DVD-Ig, or tracer can incorporate a detectable label as described herein, such as a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, or the like, or the kit can include reagents for carrying out detectable labeling. The antibodies, calibrators and/or controls can be provided in separate containers or pre-dispensed into an appropriate assay format, for example, into microtiter plates.
Optionally, the kit includes quality control components (for example, sensitivity panels, calibrators, and positive controls). Preparation of quality control reagents is well-known in the art and is described on insert sheets for a variety of immunodiagnostic products. Sensitivity panel members optionally are used to establish assay performance characteristics, and further optionally are useful indicators of the integrity of the immunoassay kit reagents, and the standardization of assays.
The kit can also optionally include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, enzyme substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit. The kit can additionally include one or more other controls. One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components.
The various components of the kit optionally are provided in suitable containers as necessary, e.g., a microtiter plate. The kit can further include containers for holding or storing a sample (e.g., a container or cartridge for a urine sample). Where appropriate, the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit can also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.
If the detectable label is at least one acridinium compound, the kit can comprise at least one acridinium-9-carboxamide, at least one acridinium-9-carboxylate aryl ester, or any combination thereof. If the detectable label is at least one acridinium compound, the kit also can comprise a source of hydrogen peroxide, such as a buffer, a solution, and/or at least one basic solution. If desired, the kit can contain a solid phase, such as a magnetic particle, bead, test tube, microtiter plate, cuvette, membrane, scaffolding molecule, film, filter paper, disc or chip.
III. Adaptation of Kit and Method
The kit (or components thereof), as well as the method of determining the presence, amount or concentration of an analyte in a test sample by an assay, such as an immunoassay as described herein, can be adapted for use in a variety of automated and semi-automated systems (including those wherein the solid phase comprises a microparticle), as described, e.g., in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as commercially marketed, e.g., by Abbott Laboratories (Abbott Park, Ill.) as ARCHITECT®.
Some of the differences between an automated or semi-automated system as compared to a non-automated system (e.g., ELISA) include the substrate to which the first specific binding partner (e.g., an anti-analyte, monoclonal/polyclonal antibody (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) or an anti-analyte DVD-Ig (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) is attached; either way, sandwich formation and analyte reactivity can be impacted), and the length and timing of the capture, detection and/or any optional wash steps. Whereas a non-automated format, such as an ELISA, may require a relatively longer incubation time with sample and capture reagent (e.g., about 2 hours), an automated or semi-automated format (e.g., ARCHITECT®, Abbott Laboratories) may have a relatively shorter incubation time (e.g., approximately 18 minutes for ARCHITECT®). Similarly, whereas a non-automated format, such as an ELISA, may incubate a detection antibody, such as the conjugate reagent, for a relatively longer incubation time (e.g., about 2 hours), an automated or semi-automated format (e.g., ARCHITECT®) may have a relatively shorter incubation time (e.g., approximately 4 minutes for the ARCHITECT®).
Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404, which is hereby incorporated by reference in its entirety), PRISM®, EIA (bead), and QuantumTM II, as well as other platforms. Additionally, the assays, kits and kit components can be employed in other formats, for example, on electrochemical or other hand-held or point-of-care assay systems. The present disclosure is, for example, applicable to the commercial Abbott Point of Care (i-STAT®, Abbott Laboratories) electrochemical immunoassay system that performs sandwich immunoassays Immunosensors and their methods of manufacture and operation in single-use test devices are described, for example in, U.S. Pat. No. 5,063,081, U.S. Pat. App. Pub. No. 2003/0170881, U.S. Pat. App. Pub. No. 2004/0018577, U.S. Pat. App. Pub. No. 2005/0054078, and U.S. Pat. App. Pub. No. 2006/0160164, which are incorporated in their entireties by reference for their teachings regarding same.
In particular, with regard to the adaptation of an analyte assay to the I-STAT® system, the following configuration is preferred. A microfabricated silicon chip is manufactured with a pair of gold amperometric working electrodes and a silver-silver chloride reference electrode. On one of the working electrodes, polystyrene beads (0.2 mm diameter) with immobilized anti-analyte, monoclonal/polyclonal antibody (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) or anti-analyte DVD-Ig (or a fragment thereof, a variant thereof, or a fragment of a variant thereof), are adhered to a polymer coating of patterned polyvinyl alcohol over the electrode. This chip is assembled into an I-STAT® cartridge with a fluidics format suitable for immunoassay. On a portion of the wall of the sample-holding chamber of the cartridge there is a layer comprising a specific binding partner for an analyte, such as an anti-analyte, monoclonal/polyclonal antibody (or a fragment thereof, a variant thereof, or a fragment of a variant thereof that can bind the analyte) or an anti-analyte DVD-Ig (or a fragment thereof, a variant thereof, or a fragment of a variant thereof that can bind the analyte), either of which can be detectably labeled. Within the fluid pouch of the cartridge is an aqueous reagent that includes p-aminophenol phosphate.
In operation, a sample suspected of containing an analyte is added to the holding chamber of the test cartridge, and the cartridge is inserted into the I-STAT® reader. After the specific binding partner for an analyte has dissolved into the sample, a pump element within the cartridge forces the sample into a conduit containing the chip. Here it is oscillated to promote formation of the sandwich. In the penultimate step of the assay, fluid is forced out of the pouch and into the conduit to wash the sample off the chip and into a waste chamber. In the final step of the assay, the alkaline phosphatase label reacts with p-aminophenol phosphate to cleave the phosphate group and permit the liberated p-aminophenol to be electrochemically oxidized at the working electrode. Based on the measured current, the reader is able to calculate the amount of analyte in the sample by means of an embedded algorithm and factory-determined calibration curve.
It further goes without saying that the methods and kits as described herein necessarily encompass other reagents and methods for carrying out the immunoassay. For instance, encompassed are various buffers such as are known in the art and/or which can be readily prepared or optimized to be employed, e.g., for washing, as a conjugate diluent, microparticle diluent, and/or as a calibrator diluent. An exemplary conjugate diluent is ARCHITECT® conjugate diluent employed in certain kits (Abbott Laboratories, Abbott Park, Ill.) and containing 2-(N-morpholino)ethanesulfonic acid (MES), a salt, a protein blocker, an antimicrobial agent, and a detergent. An exemplary calibrator diluent is ARCHITECT® human calibrator diluent employed in certain kits (Abbott Laboratories, Abbott Park, Ill.), which comprises a buffer containing MES, other salt, a protein blocker, and an antimicrobial agent. Additionally, as described in U.S. Patent Application No. 61/142,048 filed Dec. 31, 2008, improved signal generation may be obtained, e.g., in an I-Stat cartridge format, using a nucleic acid sequence linked to the signal antibody as a signal amplifier.
EXEMPLIFICATION
Example 1
Design, Construction, and Analysis of a DVD-Ig
Example 1.1
Assays Used to Identify and Characterize Parent Antibodies and DVD-Ig
The following assays were used throughout the Examples to identify and characterize parent antibodies and DVD-Ig, unless otherwise stated.
Example 1.1.1
Assays Used to Determine Binding and Affinity of Parent Antibodies and DVD-Ig for Their Target Antigen(s)
Example 1.1.1A
Direct Bind ELISA
Enzyme Linked Immunosorbent Assays to screen for antibodies that bind a desired target antigen were performed as follows. High bind ELISA plates (Corning Costar #3369, Acton, Mass.) were coated with 100 μL/well of 10 μg/ml of desired target antigen (R&D Systems, Minneapolis, Minn.) or desired target antigen extra-cellular domain/FC fusion protein (R&D Systems, Minneapolis, Minn.) or monoclonal mouse anti-polyHistidine antibody (R&D Systems # MAB050, Minneapolis, Minn.) in phosphate buffered saline (10×PBS, Abbott Bioresearch Center, Media Prep# MPS-073, Worcester, Mass.) overnight at 4° C. Plates were washed four times with PBS containing 0.02% Tween 20. Plates were blocked by the addition of 300 μL/well blocking solution (non-fat dry milk powder, various retail suppliers, diluted to 2% in PBS) for 1⁄2 hour at room temperature. Plates were washed four times after blocking with PBS containing 0.02% Tween 20.
Alternatively, one hundred microliters per well of 10 μg/ml of Histidine (His) tagged desired target antigen (R&D Systems, Minneapolis, Minn.) was added to ELISA plates coated with monoclonal mouse anti-polyHistidine antibody as described above and incubated for 1 hour at room temperature. Wells were washed four times with PBS containing 0.02% Tween 20.
One hundred microliters of antibody or DVD-Ig preparations diluted in blocking solution as described above was added to the desired target antigen plate or desired target antigen/FC fusion plate or the anti-polyHistidine antibody/His tagged desired target antigen plate prepared as described above and incubated for 1 hour at room temperature. Wells are washed four times with PBS containing 0.02% Tween 20.
One hundred microliters of 10 ng/mL goat anti-human IgG FC specific HRP conjugated antibody (Southern Biotech #2040-05, Birmingham, Ala.) was added to each well of the desired target antigen plate or anti-polyHistidine antibody/Histidine tagged desired target antigen plate. Alternatively, one hundred microliters of 10 ng/mL goat anti-human IgG kappa light chain specific HRP conjugated antibody (Southern Biotech #2060-05 Birmingham, Ala.) was added to each well of the desired target antigen/FC fusion plate and incubated for 1 hour at room temperature. Plates were washed 4 times with PBS containing 0.02% Tween 20.
One hundred microliters of enhanced TMB solution (Neogen Corp. #308177, K Blue, Lexington, Ky.) was added to each well and incubated for 10 minutes at room temperature. The reaction was stopped by the addition of 50 μL 1N sulphuric acid. Plates were read spectrophotometrically at a wavelength of 450 nm.
Table 3 contains a list of the antigens used in the Direct Bind Assay.
Table 4 contains the binding data expressed as EC50 in nM for those antibodies and DVD-Ig constructs tested in the Direct Bind ELISA assay.
In the Direct Bind ELISA, binding was sometimes not observed, probably because the antibody binding site on the target antigen was either “masked” or the antigen is “distorted” when coated to the plastic surface. The inability of a DVD-Ig to bind its target may also be due to steric limitation imposed on DVD-Ig by the Direct Bind ELISA format. The parent antibodies and DVD-Igs that did not bind in the Direct Bind ELISA format bound to target antigen in other ELISA formats, such as FACS, Biacore or bioassay. Non-binding of a DVD-Ig was also restored by adjusting the linker length between the two variable domains of the DVD-Ig, as shown previously.







TABLE 3







Antigens Used in Direct Bind ELISA











Assay
Antigen
Vendor Designation
Vendor
Catalog #





CD-22
CD22/FC
Siglec-2 ECD/FC
R&D
1968-SL-050




chimera


CD-40
CD40/FC
CD40 ECD/FC
R&D
1493-CD-050




chimera-His tag


CD-80
CD80/FC
B7-1 ECD/FC chimera
R&D
140-B1-100


DLL4
DLL4
DLL4 ECD/His tag
R&D
1506-D4-050


EGFR
EGFR/FC
EGFR ECD/FC chimera
R&D
344-ER-050


HER-2
HER-2/FC
ErbB2 ECD/FC
R&D
1129-ER-050




chimera-His tag


HGF
HGF
HGF-His tag
R&D
294-HG-025


IGF1
IGF1
IGF-I
R&D
291-G1-050


IGF2
IGF2
IGF-2
R&D
292-G2-050


IGF1R
IGF1R
IGF1R ECD
R&D
391-GR-050


NRP1
NRP1
Neuropilin-1 Npn-1-
R&D
3870-N1-025




His tag


PlGF
PlGF
Placental GF
R&D
264-PG-050


RON
RON
MSP Receptor ECD-
R&D
1947-MS-050




His tag


VEGF
VEGF
VEGF 1-165 aa
R&D
293-VE-010


ErbB3
ErbB3
ErbB3 ECD/FC
R&D
348-RB-050




Chimera-His tag





ECD = Extracellular Domain


/FC chimera = antigen/IgG FC domain fusion protein













TABLE 4







Direct Bind ELISA Of Parent Antibodies And DVD-Ig Constructs











Parent
N-terminal
C-terminal
Direct Bind
Direct Bind


Antibody
Variable
Variable
ELISA N-
ELISA C-


or DVD-
Domain
Domain
terminal VD
terminal VD


Ig ID
(VD)
(VD)
EC50 (nM)
EC50 (nM)












AB006
CD-19
0.04


AB001
CD-20












DVD001
CD-20
CD-19

12.93


DVD002
CD-19
CD-20
0.07









AB007
CD-80
0.77


AB001
CD-20












DVD005
CD-20
CD-80

9.77


DVD006
CD-80
CD-20
1.09









AB008
CD-22
3.44


AB001
CD-20












DVD007
CD-20
CD-22

30.54


DVD008
CD-22
CD-20
4.79









AB009
CD-40
0.99


AB001
CD-20












DVD009
CD-20
CD-40

3.55


DVD010
CD-40
CD-20
0.82









AB002
CD-3



AB004
HER-2
0.15











DVD011
CD-3
HER-2

6.63


DVD012
HER-2
CD-3
1.24









AB002
CD-3



AB006
CD-19
0.14











DVD013
CD-3
CD-19

7.29


DVD014
CD-19
CD-3
0.12









AB033
EGFR
0.66


AB004
HER-2
1.9











DVD015
EGFR
HER-2
0.41
148.48


DVD016
HER-2
EGFR
1.25
5.23









AB002
CD-3



AB033
EGFR
0.28











DVD017
EGFR
CD-3
0.3



DVD018
CD-3
EGFR

1.93









AB033
EGFR
0.27


AB011
IGF1R
0.28











DVD021
EGFR
IGF1R
0.33
134.95


DVD022
IGF1R
EGFR
0.26
0.74









AB005
RON
0.26


AB033
EGFR
0.28











DVD023
EGFR
RON
0.23
40.18


DVD024
RON
EGFR
0.54
1.87









AB033
EGFR
0.27


AB012
HGF
0.22











DVD025
EGFR
HGF
0.31
251.45


DVD026
HGF
EGFR
0.33
7.89









AB004
HER-2
0.44


AB010
IGF1, 2
4.77/10.02











DVD029
HER-2
IGF1/IGF2
1226.04
229.33/206.12


DVD030
IGF1/IGF2
HER-2
1.75/5.49 
10.65









AB004
HER-2
0.15


AB011
IGF1R
0.28











DVD031
HER-2
IGF1R
0.28
171.04


DVD032
IGF1R
HER-2
0.16
11.03









AB005
RON
0.21


AB012
HGF
0.19











DVD033
RON
HGF
0.22
11.95


DVD034
HGF
RON
0.27
116.46









AB014
VEGF
0.87


AB033
EGFR
0.28











DVD035
VEGF
EGFR
0.74
1.24


DVD036
EGFR
VEGF
0.18
336.13









AB014
VEGF
2.71


AB004
HER-2
0.15











DVD037
VEGF
HER-2
1.86
1.44


DVD038
HER-2
VEGF
0.66
106.77









AB001
CD-20



AB014
VEGF
2.71











DVD039
VEGF
CD-20
1.27



DVD040
CD-20
VEGF

71.18









AB014
VEGF
0.87


AB010
IGF1, 2
4.77/18.13











DVD041
VEGF
IGF1/IGF2
0.68
 381.18/7335.24


DVD042
IGF1/IGF2
VEGF
2.54/17.62
42.3









AB015
DLL-4
0.31


AB014
VEGF
2.71











DVD043
VEGF
DLL4
28.25
2.12


DVD044
DLL4
VEGF
0.53
10.88









AB014
VEGF
2.95


AB012
HGF
0.19











DVD045
VEGF
HGF
2.87
184.53


DVD046
HGF
VEGF
0.5
46.3









AB005
RON
0.26


AB014
VEGF
2.95











DVD047
VEGF
RON
19.2
94.82


DVD048
RON
VEGF
0.46
42.95









AB014
VEGF
2.95


AB016
NRP1
0.58











DVD049
VEGF
NRP1
1.71
0.5


DVD050
NRP1
VEGF
0.46
219.02









AB015
DLL-4
0.31


AB047
PlGF
0.2











DVD257
DLL4
PlGF
0.28
2


DVD258
PlGF
DLL4
0.08
2.3









AB014
VEGF
1.09


AB047
PlGF
0.2











DVD259
VEGF
PlGF
0.79
1.3


DVD260
PlGF
VEGF
0.12
19.06









AB062
ErbB3
1.93


AB033
EGFR
0.68











DVD299
ErbB3
EGFR
1.96
17.38


DVD300
EGFR
ErbB3
0.49
7633.3









AB063
ErbB3
0.28


AB033
EGFR
0.68











DVD305
ErbB3
EGFR
0.44
24.39


DVD306
EGFR
ErbB3
0.36
150.92





Binding of all DVD-Ig constructs was maintained and comparable to that of parent antibodies. All N-terminal variable domains bound with a similar high affinity as the parent antibody as well as the C-terminal variable domains of DVD-Ig constructs DVD009, DVD016, DVD018, DVD022, DVD024, DVD035, DVD37, DVD043, DVD044, DVD49, DVD257, DVD258 and DVD259.






Tables 5 and 6 contain VEGF Direct Bind ELISA data for three VEGF parent antibodies and 96 DVD-Ig constructs with either C-terminal (C-term.) or N-terminal (N-term.) variable domains derived from the variable domains of the parent VEGF Reference Antibodies (Ref Ab.) AB014-VEGF (seq. 1), AB071-VEGF (seq. 2) and AB070-VEGF (seq. 3). These variable domains are paired with four DLL-4 variable domains derived from four DLL-4 Ref. Ab. (AB015-DLL-4 (seq. 1), AB069-DLL-4 (seq. 2), AB073-DLL-4 (seq. 3), and AB072-DLL-4 (seq. 4). The DVD-Ig variable domains are connected by 2 linker lengths (Short and Long in either the heavy chain (HC linker) and/or light chain (LC linker), resulting in four possible linker combinations: Short-Short, Long-Long, Short-Long and Long-Short. The combination of these 5 factors (3 VEGF sequences×2 Orientations×2 HC Linkers×2 LC Linkers×4 DLL-4 sequences) results in the full factorial experiment of 96 DVDs.







TABLE 5







Direct Bind ELISA Of 96 DVD Constructs With Various VEGF Sequences,


Orientations And Linker Length Combinations To VEGF


















HC
LC
Other DVD
DVD VEGF
Ref.
RefAb VEGF


DVD ID
Sequence ID
Position
Linker
Linker
Domain
EC50 (nM)
Ab. ID
EC50 (nM)


















DVD043
VEGF (Seq. 1)
N-term.
Short
Short
DLL4 (seq. 1)
1.25
AB014
2.36


DVD044
VEGF (Seq. 1)
C-term.
Short
Short
DLL4 (seq. 1)
407.52
AB014
2.36


DVD469
VEGF (Seq. 1)
C-term.
Long
Long
DLL4 (seq. 1)
1262.59
AB014
2.36


DVD470
VEGF (Seq. 1)
N-term.
Long
Long
DLL4 (seq. 1)
1.87
AB014
2.36


DVD475
VEGF (Seq. 1)
C-term.
Long
Short
DLL4 (seq. 1)
19.17
AB014
1.51


DVD476
VEGF (Seq. 1)
N-term.
Long
Short
DLL4 (seq. 1)
0.55
AB014
1.51


DVD481
VEGF (Seq. 1)
C-term.
Short
Long
DLL4 (seq. 1)
150.24
AB014
1.51


DVD482
VEGF (Seq. 1)
N-term.
Short
Long
DLL4 (seq. 1)
1.30
AB014
1.51


DVD467
VEGF (Seq. 2)
C-term.
Short
Short
DLL4 (seq. 1)
1805.23
AB071
60.82


DVD468
VEGF (Seq. 2)
N-term.
Short
Short
DLL4 (seq. 1)
25.55
AB071
60.82


DVD473
VEGF (Seq. 2)
C-term.
Long
Long
DLL4 (seq. 1)
388.01
AB071
60.82


DVD474
VEGF (Seq. 2)
N-term.
Long
Long
DLL4 (seq. 1)
17.93
AB071
60.82


DVD479
VEGF (Seq. 2)
C-term.
Long
Short
DLL4 (seq. 1)
455.50
AB071
68.55


DVD480
VEGF (Seq. 2)
N-term.
Long
Short
DLL4 (seq. 1)
22.98
AB071
68.55


DVD485
VEGF (Seq. 2)
C-term.
Short
Long
DLL4 (seq. 1)
121.21
AB071
68.55


DVD486
VEGF (Seq. 2)
N-term.
Short
Long
DLL4 (seq. 1)
27.96
AB071
68.55


DVD465
VEGF (Seq. 3)
C-term.
Short
Short
DLL4 (seq. 1)
396.45
AB070
1.82


DVD466
VEGF (Seq. 3)
N-term.
Short
Short
DLL4 (seq. 1)
2.61
AB070
1.82


DVD471
VEGF (Seq. 3)
C-term.
Long
Long
DLL4 (seq. 1)
30.05
AB070
1.82


DVD472
VEGF (Seq. 3)
N-term.
Long
Long
DLL4 (seq. 1)
9.22
AB070
1.82


DVD477
VEGF (Seq. 3)
C-term.
Long
Short
DLL4 (seq. 1)
41.66
AB070
3.59


DVD478
VEGF (Seq. 3)
N-term.
Long
Short
DLL4 (seq. 1)
2.72
AB070
3.59


DVD483
VEGF (Seq. 3)
C-term.
Short
Long
DLL4 (seq. 1)
46.09
AB070
3.59


DVD484
VEGF (Seq. 3)
N-term.
Short
Long
DLL4 (seq. 1)
4.84
AB070
3.59


DVD441
VEGF (Seq. 1)
C-term.
Short
Short
DLL4 (seq. 2)
247.99
AB014
1.52


DVD442
VEGF (Seq. 1)
N-term.
Short
Short
DLL4 (seq. 2)
0.59
AB014
1.52


DVD447
VEGF (Seq. 1)
C-term.
Long
Long
DLL4 (seq. 2)
5.05
AB014
1.52


DVD448
VEGF (Seq. 1)
N-term.
Long
Long
DLL4 (seq. 2)
1.59
AB014
1.52


DVD453
VEGF (Seq. 1)
C-term.
Long
Short
DLL4 (seq. 2)
35.28
AB014
1.50


DVD454
VEGF (Seq. 1)
N-term.
Long
Short
DLL4 (seq. 2)
1.43
AB014
1.50


DVD459
VEGF (Seq. 1)
C-term.
Short
Long
DLL4 (seq. 2)
6.17
AB014
1.50


DVD460
VEGF (Seq. 1)
N-term.
Short
Long
DLL4 (seq. 2)
0.94
AB014
1.50


DVD445
VEGF (Seq. 2)
C-term.
Short
Short
DLL4 (seq. 2)
14482.02
AB071
48.07


DVD446
VEGF (Seq. 2)
N-term.
Short
Short
DLL4 (seq. 2)
14.38
AB071
48.07


DVD451
VEGF (Seq. 2)
C-term.
Long
Long
DLL4 (seq. 2)
2404.95
AB071
48.07


DVD452
VEGF (Seq. 2)
N-term.
Long
Long
DLL4 (seq. 2)
17.75
AB071
48.07


DVD457
VEGF (Seq. 2)
C-term.
Long
Short
DLL4 (seq. 2)
1475.74
AB071
40.24


DVD458
VEGF (Seq. 2)
N-term.
Long
Short
DLL4 (seq. 2)
21.51
AB071
40.24


DVD463
VEGF (Seq. 2)
C-term.
Short
Long
DLL4 (seq. 2)
1730.97
AB071
40.24


DVD464
VEGF (Seq. 2)
N-term.
Short
Long
DLL4 (seq. 2)
19.31
AB071
40.24


DVD443
VEGF (Seq. 3)
C-term.
Short
Short
DLL4 (seq. 2)
610.53
AB070
1.05


DVD444
VEGF (Seq. 3)
N-term.
Short
Short
DLL4 (seq. 2)
2.44
AB070
1.05


DVD449
VEGF (Seq. 3)
C-term.
Long
Long
DLL4 (seq. 2)
15.61
AB070
1.05


DVD450
VEGF (Seq. 3)
N-term.
Long
Long
DLL4 (seq. 2)
12.06
AB070
1.05


DVD455
VEGF (Seq. 3)
C-term.
Long
Short
DLL4 (seq. 2)
26.71
AB070
1.11


DVD456
VEGF (Seq. 3)
N-term.
Long
Short
DLL4 (seq. 2)
1.63
AB070
1.11


DVD461
VEGF (Seq. 3)
C-term.
Short
Long
DLL4 (seq. 2)
10.44
AB070
1.11


DVD462
VEGF (Seq. 3)
N-term.
Short
Long
DLL4 (seq. 2)
8.00
AB070
1.11


DVD511
VEGF (Seq. 1)
C-term.
Short
Short
DLL4 (seq. 3)
35.11
AB014
0.75


DVD512
VEGF (Seq. 1)
N-term.
Short
Short
DLL4 (seq. 3)
0.71
AB014
0.75


DVD517
VEGF (Seq. 1)
C-term.
Long
Long
DLL4 (seq. 3)
9.78
AB014
0.75


DVD518
VEGF (Seq. 1)
N-term.
Long
Long
DLL4 (seq. 3)
162.44
AB014
0.75


DVD523
VEGF (Seq. 1)
C-term.
Long
Short
DLL4 (seq. 3)
9.67
AB014
0.49


DVD524
VEGF (Seq. 1)
N-term.
Long
Short
DLL4 (seq. 3)
0.30
AB014
0.49


DVD529
VEGF (Seq. 1)
C-term.
Short
Long
DLL4 (seq. 3)
2.64
AB014
0.49


DVD530
VEGF (Seq. 1)
N-term.
Short
Long
DLL4 (seq. 3)
0.79
AB014
0.49


DVD515
VEGF (Seq. 2)
C-term.
Short
Short
DLL4 (seq. 3)
3015.10
AB071
35.19


DVD516
VEGF (Seq. 2)
N-term.
Short
Short
DLL4 (seq. 3)
24.65
AB071
35.19


DVD521
VEGF (Seq. 2)
C-term.
Long
Long
DLL4 (seq. 3)
654.85
AB071
35.19


DVD522
VEGF (Seq. 2)
N-term.
Long
Long
DLL4 (seq. 3)
23.53
AB071
35.19


DVD527
VEGF (Seq. 2)
C-term.
Long
Short
DLL4 (seq. 3)
2380.25
AB071
52.97


DVD528
VEGF (Seq. 2)
N-term.
Long
Short
DLL4 (seq. 3)
27.69
AB071
52.97


DVD533
VEGF (Seq. 2)
C-term.
Short
Long
DLL4 (seq. 3)
1260.61
AB071
52.97


DVD534
VEGF (Seq. 2)
N-term.
Short
Long
DLL4 (seq. 3)
23.61
AB071
52.97


DVD513
VEGF (Seq. 3)
C-term.
Short
Short
DLL4 (seq. 3)
58.54
AB070
0.97


DVD514
VEGF (Seq. 3)
N-term.
Short
Short
DLL4 (seq. 3)
67.41
AB070
0.97


DVD519
VEGF (Seq. 3)
C-term.
Long
Long
DLL4 (seq. 3)
77.17
AB070
0.97


DVD520
VEGF (Seq. 3)
N-term.
Long
Long
DLL4 (seq. 3)
247.74
AB070
0.97


DVD525
VEGF (Seq. 3)
C-term.
Long
Short
DLL4 (seq. 3)
21.79
AB070
1.42


DVD526
VEGF (Seq. 3)
N-term.
Long
Short
DLL4 (seq. 3)
96.22
AB070
1.42


DVD531
VEGF (Seq. 3)
C-term.
Short
Long
DLL4 (seq. 3)
60.39
AB070
1.42


DVD532
VEGF (Seq. 3)
N-term.
Short
Long
DLL4 (seq. 3)
28.91
AB070
1.42


DVD487
VEGF (Seq. 1)
C-term.
Short
Short
DLL4 (seq. 4)
39.78
AB014
1.61


DVD488
VEGF (Seq. 1)
N-term.
Short
Short
DLL4 (seq. 4)
4.98
AB014
1.61


DVD493
VEGF (Seq. 1)
C-term.
Long
Long
DLL4 (seq. 4)
2.26
AB014
1.61


DVD494
VEGF (Seq. 1)
N-term.
Long
Long
DLL4 (seq. 4)
712.21
AB014
1.61


DVD499
VEGF (Seq. 1)
C-term.
Long
Short
DLL4 (seq. 4)
5.07
AB014
1.41


DVD500
VEGF (Seq. 1)
N-term.
Long
Short
DLL4 (seq. 4)
5.28
AB014
1.41


DVD505
VEGF (Seq. 1)
C-term.
Short
Long
DLL4 (seq. 4)
1.27
AB014
1.41


DVD506
VEGF (Seq. 1)
N-term.
Short
Long
DLL4 (seq. 4)
1.84
AB014
1.41


DVD491
VEGF (Seq. 2)
C-term.
Short
Short
DLL4 (seq. 4)
1249.97
AB071
45.67


DVD492
VEGF (Seq. 2)
N-term.
Short
Short
DLL4 (seq. 4)
8.42
AB071
45.67


DVD497
VEGF (Seq. 2)
C-term.
Long
Long
DLL4 (seq. 4)
858.21
AB071
45.67


DVD498
VEGF (Seq. 2)
N-term.
Long
Long
DLL4 (seq. 4)
30.61
AB071
45.67


DVD503
VEGF (Seq. 2)
C-term.
Long
Short
DLL4 (seq. 4)
429.96
AB071
37.84


DVD504
VEGF (Seq. 2)
N-term.
Long
Short
DLL4 (seq. 4)
16.74
AB071
37.84


DVD509
VEGF (Seq. 2)
C-term.
Short
Long
DLL4 (seq. 4)
121.28
AB071
37.84


DVD510
VEGF (Seq. 2)
N-term.
Short
Long
DLL4 (seq. 4)
23.89
AB071
37.84


DVD489
VEGF (Seq. 3)
C-term.
Short
Short
DLL4 (seq. 4)
180.95
AB070
2.80


DVD490
VEGF (Seq. 3)
N-term.
Short
Short
DLL4 (seq. 4)
6.70
AB070
2.80


DVD495
VEGF (Seq. 3)
C-term.
Long
Long
DLL4 (seq. 4)
69.95
AB070
2.80


DVD496
VEGF (Seq. 3)
N-term.
Long
Long
DLL4 (seq. 4)
107.87
AB070
2.80


DVD501
VEGF (Seq. 3)
C-term.
Long
Short
DLL4 (seq. 4)
45.16
AB070
2.44


DVD502
VEGF (Seq. 3)
N-term.
Long
Short
DLL4 (seq. 4)
3.25
AB070
2.44


DVD507
VEGF (Seq. 3)
C-term.
Short
Long
DLL4 (seq. 4)
34.85
AB070
2.44


DVD508
VEGF (Seq. 3)
N-term.
Short
Long
DLL4 (seq. 4)
7.88
AB070
2.44









Binding of all DVD-Ig constructs to VEGF was maintained and comparable to that of parent antibodies. All N-terminal variable domains bound with a similar high affinity as the parent antibody. Some specific combinations of linker length in the heavy chain and light chain improved the binding affinity of the C-terminal domains comparable to the parent antibody. Specifically there is a statistically significant (p=0.019) improvement in affinity of the C-terminal domain with a long linker rather than a short linker in the light chain.







TABLE 6







Direct Bind ELISA Of 96 DVD-Ig Constructs With Various DLL4


Sequences, Orientations And Linker Length Combinations To DLL




















Other DVD-








HC
LC
Ig Variable
DVD-Ig DLL4
Ref.
Ref. Ab. DLL4


DVD-Ig ID
Sequence ID
Orientation
linker
linker
Domain
EC50 (nM)
Ab. ID
EC50 (nM)


















DVD043
DLL4 (Seq. 1)
C-term.
Short
Short
VEGF (Seq. 1)
0.67
AB015
1.39


DVD044
DLL4 (Seq. 1)
N-term.
Short
Short
VEGF (Seq. 1)
0.25
AB015
1.39


DVD469
DLL4 (Seq. 1)
N-term.
Long
Long
VEGF (Seq. 1)
2.78
AB015
1.39


DVD470
DLL4 (Seq. 1)
C-term.
Long
Long
VEGF (Seq. 1)
0.19
AB015
1.39


DVD475
DLL4 (Seq. 1)
N-term.
Long
Short
VEGF (Seq. 1)
0.23
AB015
1.23


DVD476
DLL4 (Seq. 1)
C-term.
Long
Short
VEGF (Seq. 1)
0.30
AB015
1.23


DVD481
DLL4 (Seq. 1)
N-term.
Short
Long
VEGF (Seq. 1)
3.30
AB015
1.23


DVD482
DLL4 (Seq. 1)
C-term.
Short
Long
VEGF (Seq. 1)
0.36
AB015
1.23


DVD441
DLL4 (Seq. 4)
N-term.
Short
Short
VEGF (Seq. 1)
0.04
AB069
0.10


DVD442
DLL4 (Seq. 4)
C-term.
Short
Short
VEGF (Seq. 1)
10.55
AB069
0.10


DVD447
DLL4 (Seq. 4)
N-term.
Long
Long
VEGF (Seq. 1)
0.12
AB069
0.10


DVD448
DLL4 (Seq. 4)
C-term.
Long
Long
VEGF (Seq. 1)
0.41
AB069
0.10


DVD453
DLL4 (Seq. 4)
N-term.
Long
Short
VEGF (Seq. 1)
0.09
AB069
0.04


DVD454
DLL4 (Seq. 4)
C-term.
Long
Short
VEGF (Seq. 1)
1.01
AB069
0.04


DVD459
DLL4 (Seq. 4)
N-term.
Short
Long
VEGF (Seq. 1)
0.26
AB069
0.04


DVD460
DLL4 (Seq. 4)
C-term.
Short
Long
VEGF (Seq. 1)
0.64
AB069
0.04


DVD511
DLL4 (Seq. 2)
N-term.
Short
Short
VEGF (Seq. 1)
0.10
AB073
0.08


DVD512
DLL4 (Seq. 2)
C-term.
Short
Short
VEGF (Seq. 1)
1.71
AB073
0.08


DVD517
DLL4 (Seq. 2)
N-term.
Long
Long
VEGF (Seq. 1)
0.10
AB073
0.08


DVD518
DLL4 (Seq. 2)
C-term.
Long
Long
VEGF (Seq. 1)
5.90
AB073
0.08


DVD523
DLL4 (Seq. 2)
N-term.
Long
Short
VEGF (Seq. 1)
0.09
AB073
0.03


DVD524
DLL4 (Seq. 2)
C-term.
Long
Short
VEGF (Seq. 1)
0.22
AB073
0.03


DVD529
DLL4 (Seq. 2)
N-term.
Short
Long
VEGF (Seq. 1)
0.10
AB073
0.03


DVD530
DLL4 (Seq. 2)
C-term.
Short
Long
VEGF (Seq. 1)
0.22
AB073
0.03


DVD487
DLL4 (Seq. 3)
N-term.
Short
Short
VEGF (Seq. 1)
0.14
AB072
1.29


DVD488
DLL4 (Seq. 3)
C-term.
Short
Short
VEGF (Seq. 1)
1.84
AB072
1.29


DVD493
DLL4 (Seq. 3)
N-term.
Long
Long
VEGF (Seq. 1)
0.06
AB072
1.29


DVD494
DLL4 (Seq. 3)
C-term.
Long
Long
VEGF (Seq. 1)
39.48
AB072
1.29


DVD499
DLL4 (Seq. 3)
N-term.
Long
Short
VEGF (Seq. 1)
0.09
AB072
0.70


DVD500
DLL4 (Seq. 3)
C-term.
Long
Short
VEGF (Seq. 1)
0.57
AB072
0.70


DVD505
DLL4 (Seq. 3)
N-term.
Short
Long
VEGF (Seq. 1)
0.09
AB072
0.70


DVD506
DLL4 (Seq. 3)
C-term.
Short
Long
VEGF (Seq. 1)
0.32
AB072
0.70


DVD467
DLL4 (Seq. 1)
N-term.
Short
Short
VEGF (Seq. 2)
0.54
AB015
0.30


DVD468
DLL4 (Seq. 1)
C-term.
Short
Short
VEGF (Seq. 2)
2.28
AB015
0.30


DVD473
DLL4 (Seq. 1)
N-term.
Long
Long
VEGF (Seq. 2)
1.80
AB015
0.30


DVD474
DLL4 (Seq. 1)
C-term.
Long
Long
VEGF (Seq. 2)
0.57
AB015
0.30


DVD479
DLL4 (Seq. 1)
N-term.
Long
Short
VEGF (Seq. 2)
1.69
AB015
0.28


DVD480
DLL4 (Seq. 1)
C-term.
Long
Short
VEGF (Seq. 2)
0.60
AB015
0.28


DVD485
DLL4 (Seq. 1)
N-term.
Short
Long
VEGF (Seq. 2)
1.41
AB015
0.28


DVD486
DLL4 (Seq. 1)
C-term.
Short
Long
VEGF (Seq. 2)
0.59
AB015
0.28


DVD445
DLL4 (Seq. 4)
N-term.
Short
Short
VEGF (Seq. 2)
0.17
AB069
0.20


DVD446
DLL4 (Seq. 4)
C-term.
Short
Short
VEGF (Seq. 2)
13.92
AB069
0.20


DVD451
DLL4 (Seq. 4)
N-term.
Long
Long
VEGF (Seq. 2)
0.40
AB069
0.20


DVD452
DLL4 (Seq. 4)
C-term.
Long
Long
VEGF (Seq. 2)
0.34
AB069
0.20


DVD457
DLL4 (Seq. 4)
N-term.
Long
Short
VEGF (Seq. 2)
0.13
AB069
0.21


DVD458
DLL4 (Seq. 4)
C-term.
Long
Short
VEGF (Seq. 2)
1.54
AB069
0.21


DVD463
DLL4 (Seq. 4)
N-term.
Short
Long
VEGF (Seq. 2)
0.50
AB069
0.21


DVD464
DLL4 (Seq. 4)
C-term.
Short
Long
VEGF (Seq. 2)
0.80
AB069
0.21


DVD515
DLL4 (Seq. 2)
N-term.
Short
Short
VEGF (Seq. 2)
0.20
AB073
0.11


DVD516
DLL4 (Seq. 2)
C-term.
Short
Short
VEGF (Seq. 2)
4.33
AB073
0.11


DVD521
DLL4 (Seq. 2)
N-term.
Long
Long
VEGF (Seq. 2)
0.26
AB073
0.11


DVD522
DLL4 (Seq. 2)
C-term.
Long
Long
VEGF (Seq. 2)
0.48
AB073
0.11


DVD527
DLL4 (Seq. 2)
N-term.
Long
Short
VEGF (Seq. 2)
0.20
AB073
0.11


DVD528
DLL4 (Seq. 2)
C-term.
Long
Short
VEGF (Seq. 2)
0.81
AB073
0.11


DVD533
DLL4 (Seq. 2)
N-term.
Short
Long
VEGF (Seq. 2)
0.25
AB073
0.11


DVD534
DLL4 (Seq. 2)
C-term.
Short
Long
VEGF (Seq. 2)
0.47
AB073
0.11


DVD491
DLL4 (Seq. 3)
N-term.
Short
Short
VEGF (Seq. 2)
0.39
AB072
1.99


DVD492
DLL4 (Seq. 3)
C-term.
Short
Short
VEGF (Seq. 2)
1.77
AB072
1.99


DVD497
DLL4 (Seq. 3)
N-term.
Long
Long
VEGF (Seq. 2)
0.53
AB072
1.99


DVD498
DLL4 (Seq. 3)
C-term.
Long
Long
VEGF (Seq. 2)
0.45
AB072
1.99


DVD503
DLL4 (Seq. 3)
N-term.
Long
Short
VEGF (Seq. 2)
0.33
AB072
1.85


DVD504
DLL4 (Seq. 3)
C-term.
Long
Short
VEGF (Seq. 2)
0.53
AB072
1.85


DVD509
DLL4 (Seq. 3)
N-term.
Short
Long
VEGF (Seq. 2)
0.21
AB072
1.85


DVD510
DLL4 (Seq. 3)
C-term.
Short
Long
VEGF (Seq. 2)
0.42
AB072
1.85


DVD465
DLL4 (Seq. 1)
N-term.
Short
Short
VEGF (Seq. 3)
0.57
AB015
0.60


DVD466
DLL4 (Seq. 1)
C-term.
Short
Short
VEGF (Seq. 3)
19.84
AB015
0.60


DVD471
DLL4 (Seq. 1)
N-term.
Long
Long
VEGF (Seq. 3)
0.89
AB015
0.60


DVD472
DLL4 (Seq. 1)
C-term.
Long
Long
VEGF (Seq. 3)
2.78
AB015
0.60


DVD477
DLL4 (Seq. 1)
N-term.
Long
Short
VEGF (Seq. 3)
0.69
AB015
0.58


DVD478
DLL4 (Seq. 1)
C-term.
Long
Short
VEGF (Seq. 3)
2.53
AB015
0.58


DVD483
DLL4 (Seq. 1)
N-term.
Short
Long
VEGF (Seq. 3)
0.63
AB015
0.58


DVD484
DLL4 (Seq. 1)
C-term.
Short
Long
VEGF (Seq. 3)
1.75
AB015
0.58


DVD443
DLL4 (Seq. 4)
N-term.
Short
Short
VEGF (Seq. 3)
0.20
AB069
0.23


DVD444
DLL4 (Seq. 4)
C-term.
Short
Short
VEGF (Seq. 3)
30.54
AB069
0.23


DVD449
DLL4 (Seq. 4)
N-term.
Long
Long
VEGF (Seq. 3)
0.36
AB069
0.23


DVD450
DLL4 (Seq. 4)
C-term.
Long
Long
VEGF (Seq. 3)
1.99
AB069
0.23


DVD455
DLL4 (Seq. 4)
N-term.
Long
Short
VEGF (Seq. 3)
0.24
AB069
0.24


DVD456
DLL4 (Seq. 4)
C-term.
Long
Short
VEGF (Seq. 3)
2.48
AB069
0.24


DVD461
DLL4 (Seq. 4)
N-term.
Short
Long
VEGF (Seq. 3)
0.52
AB069
0.24


DVD462
DLL4 (Seq. 4)
C-term.
Short
Long
VEGF (Seq. 3)
7.99
AB069
0.24


DVD513
DLL4 (Seq. 2)
N-term.
Short
Short
VEGF (Seq. 3)
0.66
AB073
0.22


DVD514
DLL4 (Seq. 2)
C-term.
Short
Short
VEGF (Seq. 3)
21.05
AB073
0.22


DVD519
DLL4 (Seq. 2)
N-term.
Long
Long
VEGF (Seq. 3)
1.53
AB073
0.22


DVD520
DLL4 (Seq. 2)
C-term.
Long
Long
VEGF (Seq. 3)
17.34
AB073
0.22


DVD525
DLL4 (Seq. 2)
N-term.
Long
Short
VEGF (Seq. 3)
1.00
AB073
0.21


DVD526
DLL4 (Seq. 2)
C-term.
Long
Short
VEGF (Seq. 3)
1.57
AB073
0.21


DVD531
DLL4 (Seq. 2)
N-term.
Short
Long
VEGF (Seq. 3)
0.57
AB073
0.21


DVD532
DLL4 (Seq. 2)
C-term.
Short
Long
VEGF (Seq. 3)
1.43
AB073
0.21


DVD489
DLL4 (Seq. 3)
N-term.
Short
Short
VEGF (Seq. 3)
0.62
AB072
63.43


DVD490
DLL4 (Seq. 3)
C-term.
Short
Short
VEGF (Seq. 3)
9.27
AB072
63.43


DVD495
DLL4 (Seq. 3)
N-term.
Long
Long
VEGF (Seq. 3)
0.33
AB072
63.43


DVD496
DLL4 (Seq. 3)
C-term.
Long
Long
VEGF (Seq. 3)
5.59
AB072
63.43


DVD501
DLL4 (Seq. 3)
N-term.
Long
Short
VEGF (Seq. 3)
0.18
AB072
63.24


DVD502
DLL4 (Seq. 3)
C-term.
Long
Short
VEGF (Seq. 3)
3.17
AB072
63.24


DVD507
DLL4 (Seq. 3)
N-term.
Short
Long
VEGF (Seq. 3)
0.71
AB072
63.24


DVD508
DLL4 (Seq. 3)
C-term.
Short
Long
VEGF (Seq. 3)
0.61
AB072
63.24









Binding of all DVD-Ig constructs to DLL4 was maintained and comparable to that of parent antibodies. All N-terminal variable domains bound with a similar high affinity as the parent antibody. Some specific combinations of linker length in the heavy chain and light chain improved the binding affinity of the C-terminal domains comparable to the parent antibody. Specifically there is a trend for improvement in the affinity of the C-terminal domain with a long linker in the light chain and/or heavy chain rather than a short linker in both the light and heavy chain.
Example 1.1.1.B
Capture ELISA-VEGF
ELISA plates (Nunc, MaxiSorp, Rochester, N.Y.) were incubated overnight at 4° C. with anti-human Fc antibody (5 μg/ml in PBS, Jackson Immunoresearch, West Grove, Pa.). Plates were washed three times in washing buffer (PBS containing 0.05% Tween 20), and blocked for 1 hour at 25° C. in blocking buffer (PBS containing 1% BSA). Wells were washed three times, and serial dilutions of each antibody or DVD-Ig in PBS containing 0.1% BSA were added to the wells and incubated at 25° C. for 1 hour. The wells were washed three times, and biotinylated VEGF (2 nM) was added to the plates and incubated for 1 hour at 25° C. The wells were three times, and then incubated for 1 hour at 25° C. with streptavidin-HRP (KPL #474-3000, Gaithersburg, Md.). The wells were washed three times, and 100 μl of ULTRA-TMB ELISA (Pierce, Rockford, Ill.) were added per well. Following color development the reaction was stopped with 1N HCL and absorbance at 450 nM was measured. VEGF capture ELISA data is shown in Table 7.
Example 1.1.1.C
IgG-Fc Capture ELISA-RON
96-well Nunc-Immuno plates were coated with 2 μg/mL goat-anti-human IgG Fc specific antibody (Jackson Immunoresearch #109-055-098, West Grove, Pa., 50 μL/well) in PBS (Gibco #10010-023 from Invitrogen, Grand Island, N.Y.), and incubated overnight at 4° C. Plates were washed three times with washing buffer (PBS, 0.05% Tween 20) and subsequently blocked with 100 uL/well of blocking buffer (PBS, 2% BSA) for one hour at room temperature. Plates were washed three times and incubated with 50 μL/well of a 1 μg/mL solution of the appropriate antibody or DVD-Ig for one hour at room temperature. After the one hour incubation, the plates were washed three times and incubated with 50 μL/well of his-tagged, recombinant RON protein (R&D Systems #1947-MS, Minneapolis, Minn., 1000 nM to 0 nM final dose range) for one hour at room temperature. Plates were washed three times, and 50 μL/well of a rabbit-anti-His tag-HRP antibody (Abeam ab1187, Cambridge, Mass., diluted at 1:10,000 in 2% BSA/PBS solution) was added and plates were incubated at room temperature for one hour. After the final wash, 50 μl/well of TMB substrate (Pierce #34028, Rockford, Ill.) was added, and the reaction was terminated after five minutes using 50 μl/well of 2N H2SO4. The absorbance was read at 450 nm (Spectra Max Plus plate reader, Molecular Devices, Sunnyvale, Calif.). EC50s were calculated in GraphPad Prism 4.03. RON capture ELISA data is shown in Table 7.
Example 1.1.1.D
Capture ELISA-IGF1,2
96-well Nunc-Immuno plates were coated with 5 μg/ml antibody against human IgG (Fcγ fragment specific, Jackson ImmunoResearch, West Grove, Pa., #109-005-098, 100 μl/well) in D-PBS (Gibco #14190, San Diego, Calif.) and incubated overnight at 4° C. ELISA plates were washed three times in wash buffer (PBS, 0.05% Tween 20) and then blocked with 200 μl/well blocking buffer (D-PBS, 1% BSA) for 1 hour at 25° C. Plates were then washed and incubated with 100 μl/well antibodies or DVD-Igs (0.01 μg/mL-100 μg/mL in blocking buffer) for 1 hour at 37° C. Plates were then washed three times and incubated with biotin-labeled human IGF1 or IGF2 (0.02 nM-100 nM dose range in blocking buffer, 100 μl/well) for 1 hour at 37° C. Plates were washed three times and incubated with streptavidin conjugated with HRP (KPL #474-3000, Gaithersburg, Md., 1:10,000 dilution in blocking buffer, 100 μl/well) for 1 hour at 25° C. After the final wash, plates were incubated with 100 μl/well ELISA substrate (1-Step Ultra TMB-ELISA, Pierce #340280, Rockford, Ill.). The reaction was stopped after 5 minutes at 25° C. with 100 μl/well 2N H2SO4 and the absorbance was read at 450 nm. IGF1,2 capture ELISA data is shown in Table 7.
Example 1.1.1.E
Capture ELISA DLL4
96-well Nunc-Immuno plates (#439454, Rochester, N.Y.) were coated with 5 μg/ml antibody against human IgG (Fc fragment specific, Jackson ImmunoResearch, West Grove, Pa. #109-005-098, 100 μl/well) in D-PBS (Gibco #14190, Grand Island, N.Y.) and incubated overnight at 4° C. ELISA plates were washed three times with wash buffer (PBS, 0.05% Tween 20) and then blocked with 200 μl/well blocking buffer (D-PBS, 1% BSA, 1 mM CaCl2, 0.05% Tween 20) for 1 hour at 25° C. Plates were washed three times and incubated with 100 μl/well DLL4 antibodies (0.0001-100 nM, 10-fold serial dilution in blocking buffer) for 1 hour at 25° C., and then washed again three times. Plates containing captured DLL4 Ab were incubated with biotin-labeled human DLL4 extracellular domain (10 nM in blocking buffer, 100 μl/well) for 1 hour at 25° C., washed three times, and incubated with streptavidin conjugated with HRP (KPL #474-3000C, washed three times, and incubated with streptavidin conjugated with HRP (KPL #474-3000, Gaithersburg, Md., 1:10,000 dilution in blocking buffer, 100 μl/well) for 1 hour at 25° C. After the final wash, plates were incubated with 100 μl/well ELISA substrate (1-Step Ultra TMB-ELISA, Pierce #340280, Rockford, Ill.). The reaction was stopped after 2 minutes at 25° C. with 100 μl/well 2N H2SO4 and the absorbance was read at 450 nm. DLL4 capture ELISA data is shown in Table 7.
Example 1.1.1.F
Capture ELISA ErbB3 or EGFR
96-well ELISA plates were coated with goat anti-human IgG Fc (Jackson Immunoresearch, Pa.) at a concentration of 33 nM, and incubated overnight at 4° C. The plates were washed with PBS containing 0.05% Tween 20 three times and blocked with 200 μl/well of 1% BSA/PBS for 1 hour at room temperature. Fifty μl of 5 nM DVD-Ig or antibody were added to each well and incubated for 1 hour at room temperature. The plates were washed, and then incubated with 50 μl/well of biotinylated ErbB3 or biotinylated EGFR at various concentrations for 1 hour at room temperature. The plates were washed again and then incubated with 50 μl/well of streptavidin-conjugated HRP (KPL #474-3000, Protein Research Products, Md.) and incubated for 1 hour at room temperature. The wells were washed three times, and 100 μl of ULTRA-TMB ELISA (Pierce, Rockford, Ill.) were added per well. Following color development, the reaction was stopped with 1N HCL and absorbance at 450 nM was measured.
Table 7 contains the affinity, expressed as EC50 in nM, of parent antibodies and DVD-Ig constructs in the VEGF, RON, EGFR, IGFR, IGF-1,2, HER-2, DLL4, and ErbB3.







TABLE 7







VEGF, RON, EGFR, ErbB3, IGFR, IGF-1, 2, DLL4 and HER-2


Antigen Capture ELISA of Parent Antibodies and DVD-Ig


Constructs














N-terminal
C-terminal


Parent
N-terminal
C-terminal
Ag binding in
Ag binding in


Antibody or
Variable
Variable
Capture ELISA
Capture ELISA


DVD-Ig ID
Domain
Domain
(EC50, nM)
(EC50, nM)












AB004
HER-2
0.4


AB010
IGF1, 2
4.8, 10











DVD029
HER-2
IGF1, 2

229, 206


DVD030
IGF1, 2
HER-2
1.8, 5.5
10.7









AB033
EGFR
0.3


AB011
IGF1R
0.3











DVD021
EGFR
IGF1R
0.3
135


DVD022
IGF1R
EGFR
0.3
0.7









AB005
RON
1.6











DVD024
RON
EGFR
1.96



DVD023
EGFR
RON

>100









AB004
HER-2
0.2


AB011
IGF1R
0.3











DVD031
HER-2
IGF1R
0.3
171


DVD032
IGF1R
HER-2
0.2
11









AB005
RON
0.7


AB012
HGF











DVD033
RON
HGF
2.5



DVD034
HGF
RON









AB014
VEGF
0.24











DVD035
VEGF
EGFR
0.24










AB014
VEGF
0.24










DVD038
HER-2
VEGF
2.00









AB014
VEGF
0.9


AB010
IGF1, 2
4.8, 18.1











DVD041
VEGF
IGF1, 2
0.7
381, 7335


DVD042
IGF1, 2
VEGF
2.5, 17.6
42.3









AB015
DLL-4
0.03


AB014
VEGF
0.24











DVD044
DLL4
VEGF
0.06
4.20


DVD043
VEGF
DLL4
0.20
0.50









AB014
VEGF
0.24


AB012
HGF











DVD045
VEGF
HGF
0.29
>100









AB005
RON
1.3


AB014
VEGF
0.24











DVD048
RON
VEGF
2.7
3.60


DVD047
VEGF
RON
0.36
>100









AB014
VEGF
0.24











DVD049
VEGF
NRP1
4.80



DVD050
NRP1
VEGF

2.30









AB014
VEGF
0.53










DVD260
P1GF
VEGF
1.70









AB062
ErbB3
0.26











DVD299
ErbB3
EGFR
0.32
>10.0









AB063
ErbB3
0.67


AB033
EGFR











DVD305
ErbB3
EGFR
1.16



DVD306
EGFR
ErbB3

>10.0









Binding of all DVD-Ig constructs to soluble antigen was maintained and comparable to parent antibodies. All N-terminal variable domains bound with a similar high affinity as to the parent antibody as well as the C-terminal variable domains of DVD022, DVD038, DVD043, DVD048, DVD50 and DVD260.
Example 1.1.1.G
Affinity Determination Using BIACORE Technology







TABLE 8







Reagent Used in Biacore Analyses











Assay
Antigen
Vendor Designation
Vendor
Catalog #





DLL4
DLL4
DLL ECD His tag
R&D
1506-D4-050


EGFR
EGFR
EGFR ECD
R&D
1095-ER


HER-2
HER-2/FC
ErbB2/FC chimera-
R&D
1129-ER-050




His tag


HGF
HGF
HGF
R&D
294-HG-025


IGF1
IGF1
IGF-I
R&D
291-G1-050


IGF2
IGF2
IGF-2
R&D
292-G2-050


IGF1R
IGF1R
IGF1R ECD
R&D
391-GR-050


NRP1
NRP1
Neuropilin-1 Npn-1-
R&D
3870-N1-025




His tag


PlGF
PlGF
Placental GF
R&D
264-PG-050


RON
RON
MSP Receptor ECD-
R&D
1947-MS-050




His tag


VEGF
VEGF
VEGF
R&D
293-VE-010


ErbB3
ErbB3
ErbB3/FC Chimera-
R&D
348-RB




His tag





ECD = Extracellular Domain


/FC = antigen/IgG FC domain fusion protein






BIACORE Methods:

The BIACORE assay (Biacore, Inc, Piscataway, N.J.) determines the affinity of antibodies or DVD-Ig with kinetic measurements of on-rate and off-rate constants. Binding of antibodies or DVD-Ig to a target antigen (for example, a purified recombinant target antigen) is determined by surface plasmon resonance-based measurements with a Biacore® 1000 or 3000 instrument (Biacore® AB, Uppsala, Sweden) using running HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25° C. All chemicals are obtained from Biacore® AB (Uppsala, Sweden) or otherwise from a different source as described in the text. For example, approximately 5000 RU of goat anti-mouse IgG, (Fcγ), fragment specific polyclonal antibody (Pierce Biotechnology Inc, Rockford, Ill.) diluted in 10 mM sodium acetate (pH 4.5) is directly immobilized across a CM5 research grade biosensor chip using a standard amine coupling kit according to manufacturer's instructions and procedures at 25 μg/ml. Unreacted moieties on the biosensor surface are blocked with ethanolamine. Modified carboxymethyl dextran surface in flowcell 2 and 4 is used as a reaction surface. Unmodified carboxymethyl dextran without goat anti-mouse IgG in flow cell 1 and 3 is used as the reference surface. For kinetic analysis, rate equations derived from the 1:1 Langmuir binding model are fitted simultaneously to association and dissociation phases of all eight injections (using global fit analysis) with the use of Biaevaluation 4.0.1 software. Purified antibodies or DVD-Ig are diluted in HEPES-buffered saline for capture across goat anti-mouse IgG specific reaction surfaces. Antibodies or DVD-Ig to be captured as a ligand (25 μg/ml) are injected over reaction matrices at a flow rate of 5 μl/min. The association and dissociation rate constants, kon (M−1s−1) and koff (s−1) are determined under a continuous flow rate of 25 μl/min. Rate constants are derived by making kinetic binding measurements at different antigen concentrations ranging from 10-200 nM. The equilibrium dissociation constant (M) of the reaction between antibodies or DVD-Igs and the target antigen is then calculated from the kinetic rate constants by the following formula: KD=koff/kon. Binding is recorded as a function of time and kinetic rate constants are calculated. In this assay, on-rates as fast as 106 M−1s−1 and off-rates as slow as 10−6 s−1 can be measured.







TABLE 9





BIACORE Analysis of Parental Antibodies and DVD Constructs























kon
koff
KD


Parent Antibody
N-Terminal
C-Terminal
(M−1s−1)
(s−1)
(M)














AB011
IGF1R
7.32E+04
6.21E−05
8.48E−10












DVD021
EGFR

1.22E+06
1.80E−03
1.47E−9 


DVD021

IGF1R


DVD022
IGF1R

1.06E+05
8.25E−05
7.78E−10


DVD022

EGFR
8.03E+04
2.26E−04
2.81E−9 











AB004
HER-2
3.22E+05
1.28E−05
3.97E−11


AB011
IGF1R
7.32E+04
6.21E−05
8.48E−10












DVD031
HER-2

3.30E+05
7.02E−06
2.12E−11


DVD031

IGF1R


DVD032
IGF1R

5.10E+04
4.23E−05
8.29E−10


DVD032

HER-2
1.84E+04
9.31E−06
5.05E−10











AB004
HER-2
3.22E+05
1.28E−05
3.97E−11


AB010
IGF1
3.17E+06
1.23E−03
3.88E−10


AB010
IGF2
6.47E+06
2.74E−06
4.23E−13












DVD029
HER-2

2.42E+05
9.64E−06
3.98E−11


DVD029

IGF1
6.65E+04
4.23E−04
6.36E−9 


DVD029

IGF2
1.04E+05
3.15E−05
3.02E−10


DVD030
IGF1

3.96E+06
1.16E−03
2.92E−10


DVD030
IGF2

8.46E+06
1.98E−05
2.34E−12


DVD030

HER-2
1.54E+05
7.36E−04
4.77E−9 











AB033
EGFR
7.93E+04
1.39E−03
1.75E−8 


AB004
HER-2
3.22E+05
1.28E−05
3.97E−11












DVD015
EGFR

1.79E+06
1.96E−03
1.09E−9 


DVD015

HER-2


DVD016
HER-2

2.44E+05
4.36E−06
1.78E−11


DVD016

EGFR
2.36E+04
4.53E−04
1.92E−8 











AB014
VEGF
1.47E+05
3.03E−05
2.07E−10


AB010
IGF1


AB010
IGF2
3.35E+06
<1.00E−06
<2.99E−13  












DVD041
VEGF

5.26E+05
2.39E−06
4.55E−12


DVD041

IGF1


DVD041

IGF2
8.09E+04
<1.00E−06
<1.24E−11  


DVD042
IGF1


DVD042
IGF2

5.20E+06
5.18E−06
9.98E−13


DVD042

VEGF
3.45E+04
1.46E−05
4.21E−10











AB005
RON
3.66E+04
7.39E−04
2.02E−8 


AB033
EGFR
7.93E+04
1.39E−03
1.75E−8 












DVD024
RON

4.56E+04
8.52E−04
1.86E−8 


DVD024

EGFR


DVD023
EGFR

2.08E+06
2.22E−03
1.06E−9 


DVD023

RON











AB005
RON
3.66E+04
7.39E−04
2.02E−8 


AB012
HGF
>1.00E+7
2.84E−04
<2.84E−11  












DVD033
RON

1.56E+05
1.18E−03
7.60E−09


DVD033

HGF
2.97E+05
9.20E−05
3.10E−10


DVD034
HGF

3.05E+06
6.55E−05
2.15E−11


DVD034

RON











AB005
RON
3.66E+04
7.39E−04
2.01E−8 


AB014
VEGF
3.06E+05
3.99E−06
1.30E−11








DVD048
RON
















N-Terminal
C-Terminal






Variable
Variable


Parent Antibody
Domain
Domain
kon
koff
KD


or DVD-Ig ID
(VD)
(VD)
(M−1s−1)
(s−1)
(M)





DVD048

VEGF
3.85E+04
4.50E−05
1.17E−9 


DVD047
VEGF

3.46E+05
2.79E−05
8.06E−11


DVD047

RON











AB015
DLL-4
4.00E+05
1.66E−04
4.14E−10


AB014
VEGF
1.47E+05
3.03E−05
2.07E−10












DVD044
DLL4

5.90E+05
2.05E−04
3.47E−10


DVD044

VEGF
3.67E+04
1.13E−05
3.08E−10


DVD043
VEGF

4.94E+05
8.26E−06
1.67E−11


DVD043

DLL4
9.37E+04
2.17E−04
2.32E−09











AB015
DLL-4





AB047
P1GF
3.80E+06
1.08E−04
2.84E−11












DVD257
DLL4

8.35E+05
1.96E−04
2.34E−10


DVD257

P1GF
3.02E+05
1.11E−04
3.67E−10


DVD258
P1GF

>1.00E+07
2.54E−03
<2.54E−10  


DVD258

DLL4











AB014
VEGF
1.47E+05
3.03E−05
2.07E−10


AB033
EGFR
8.85E+05
1.23E−03
1.39E−09












DVD035
VEGF

2.25E+05
1.73E−05
8.23E−11


DVD035

EGFR


DVD036
EGFR

1.21E+06
1.57E−03
1.30E−09


DVD036

VEGF
4.80E+04
3.19E−04
6.65E−09











AB014
VEGF
3.07E+05
3.39E−05
1.06E−11


AB004
HER-2
3.02E+05
1.03E−05
3.40E−11












DVD037
VEGF

4.45E+05
<1.00E−06
<2.25E−12  


DVD037

HER-2
7.61E+03
2.83E−06
3.72E−10


DVD038
HER-2

1.95E+05
4.81E−06
2.47E−11


DVD038

VEGF
3.67E+04
<1.00E−06
<2.27E−11  











AB014
VEGF
1.47E+05
3.03E−05
2.07E−10


AB012
HGF
>1.00E+7
2.84E−04
<2.84E−11  












DVD045
VEGF

2.01E+05
3.05E−06
1.52E−11


DVD045

HGF
1.84E+05
1.66E−04
9.01E−10


DVD046
HGF

>1.00E+7
2.36E−04
<2.36E−11  


DVD046

VEGF











AB014
VEGF
3.06E+05
3.99E−06
1.30E−11


AB016
NRP1
1.47E+05
3.90E−04
2.67E−9 












DVD049
VEGF






DVD049

NRP1
1.42E+05
1.81E−04
1.28E−9 


DVD050
NRP1

1.82E+05
2.61E−04
1.440E−9 


DVD050

VEGF
1.82E+05
2.61E−04
1.440E−9 











AB014
VEGF
3.07E+05
3.39E−05
1.06E−10


AB047
P1GF
3.80E+06
1.08E−04
2.85E−11












DVD259
VEGF

5.02E+05
2.06E−05
4.12E−11


DVD259

P1GF
2.99E+05
9.87E−05
3.30E−10


DVD260
P1GF

4.27E+06
5.14E−05
1.20E−11


DVD260

VEGF
4.99E+04
2.05E−05
4.11E−10











AB033
EGFR
8.85E+05
1.23E−03
1.39E−09


AB012
HGF
>1.00E+7
2.84E−04
<2.84E−11  












DVD025
EGFR

7.31E+05
1.44E−03
1.98E−09


DVD025

HGF
3.05E+05
2.28E−04
7.46E−10


DVD026
HGF

4.19E+06
6.67E−05
1.59E−11


DVD026

EGFR











AB062
ErbB3
8.66E+04
1.17E−04
1.360E−9 


AB033
EGFR
7.93E+04
1.39E−03
1.75E−8 












DVD299
ErbB3

1.95E+05
1.60E−04
8.20E−10


DVD299

EGFR
3.26E+04
4.48E−04
1.37E−8 


DVD300
EGFR

1.52E+06
1.69E−03
1.11E−9 


DVD300

ErbB3











AB063
ErbB3
1.17E+06
1.75E−04
1.50E−10


AB033
EGFR
7.93E+04
1.39E−03
1.75E−8 












DVD305
ErbB3

2.27E+06
1.37E−04
6.03E−11


DVD305

EGFR
3.99E+04
3.17E−04
7.940E−9 


DVD306
EGFR

1.82E+06
1.86E−03
1.020E−9 


DVD306

ErbB3









Binding of all DVD-Ig constructs characterized by Biacore technology was maintained and comparable to that of parent antibodies. All N-terminal variable domains bound with a similar high affinity as the parent antibody as well as the C-terminal variable domains of DVD-Ig constructs DVD022, DVD016, DVD042, DVD 044, DVD043, DVD038, DVD049, DVD260, DVD299, and DVD305.
Example 1.1.2
Assays Used to Determine the Functional Activity of Parent Antibodies and DVD-Ig
Example 1.1.2.A
Cytokine Bioassay
The ability of an anti-cytokine or an anti-growth factor parent antibody or DVD-Ig containing anti-cytokine or anti-growth factor sequences to inhibit or neutralize a target cytokine or growth factor bioactivity is analyzed by determining the inhibitory potential of the antibody or DVD-Ig. For example, the ability of an anti-IL-4 antibody to inhibit IL-4 mediated IgE production may be used. For example, human naive B cells are isolated from peripheral blood, respectively, buffy coats by Ficoll-paque density centrifugation, followed by magnetic separation with MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) specific for human sIgD FITC labeled goat F(ab)2 antibodies followed by anti-FITC MACS beads. Magnetically sorted naive B cells are adjusted to 3×105 cells per ml in XV15 and plated out in 100 μl per well of 96-well plates in a 6×6 array in the center of the plate, surrounded by PBS filled wells during the 10 days of culture at 37° C. in the presence of 5% CO2. One plate each is prepared per antibody to be tested, consisting of 3 wells each of un-induced and induced controls and quintuplicate repeats of antibody titrations starting at 7 μg/ml and running in 3-fold dilution down to 29 ng/ml final concentrations added in 50 μl four times concentrated pre-dilution. To induce IgE production, rhIL-4 at 20 ng/ml plus anti-CD40 monoclonal antibody (Novartis, Basel, Switzerland) at 0.5 μg/ml final concentrations in 50 μl each are added to each well, and IgE concentrations are determined at the end of the culture period by a standard sandwich ELISA method.
Example 1.1.2.B
Cytokine Release Assay
The ability of a parent antibody or DVD-Ig to cause cytokine release is analyzed. Peripheral blood is withdrawn from three healthy donors by venipuncture into heparized vacutainer tubes. Whole blood is diluted 1:5 with RPMI-1640 medium and placed in 24-well tissue culture plates at 0.5 mL per well. The anti-cytokine antibodies (e.g., anti-IL-4) are diluted into RPMI-1640 and placed in the plates at 0.5 mL/well to give final concentrations of 200, 100, 50, 10, and 1 μg/mL. The final dilution of whole blood in the culture plates is 1:10. LPS and PHA are added to separate wells at 2 μg/mL and 5 μg/mL final concentration as a positive control for cytokine release. Polyclonal human IgG is used as negative control antibody. The experiment is performed in duplicate. Plates are incubated at 37° C. at 5% CO2. Twenty-four hours later the contents of the wells are transferred into test tubes and spun for 5 minutes at 1200 rpm. Cell-free supernatants are collected and frozen for cytokine assays. Cells left over on the plates and in the tubes are lysed with 0.5 mL of lysis solution, and placed at −20° C. and thawed. 0.5 mL of medium is added (to bring the volume to the same level as the cell-free supernatant samples) and the cell preparations are collected and frozen for cytokine assays. Cell-free supernatants and cell lysates are assayed for cytokine levels by ELISA, for example, for levels of IL-8, IL-6, IL-1β, IL-1RA, or TNF-α.
Example 1.1.2.C
Cytokine Cross-Reactivity Study
The ability of an anti-cytokine parent antibody or DVD-Ig directed to a cytokine(s) of interest to cross react with other cytokines is analyzed. Parent antibodies or DVD-Ig are immobilized on a Biacore biosensor matrix. An anti-human Fc mAb is covalently linked via free amine groups to the dextran matrix by first activating carboxyl groups on the matrix with 100 mM N-hydroxysuccinimide (NHS) and 400 mM N-Ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). Approximately 50 μL of each antibody or DVD-Ig preparation at a concentration of 25 μg/mL, diluted in sodium acetate, pH 4.5, is injected across the activated biosensor and free amines on the protein are bound directly to the activated carboxyl groups. Typically, 5000 Resonance Units (RU's) are immobilized. Unreacted matrix EDC-esters are deactivated by an injection of 1 M ethanolamine. A second flow cell is prepared as a reference standard by immobilizing human IgG1/K using the standard amine coupling kit. SPR measurements are performed using the CM biosensor chip. All antigens to be analyzed on the biosensor surface are diluted in HBS-EP running buffer containing 0.01% P20.
To examine the cytokine binding specificity, excess cytokine of interest (100 nM, e.g., soluble recombinant human) is injected across the anti-cytokine parent antibody or DVD-Ig immobilized biosensor surface (5 minute contact time). Before injection of the cytokine of interest and immediately afterward, HBS-EP buffer alone flows through each flow cell. The net difference in the signals between the baseline and the point corresponding to approximately 30 seconds after completion of cytokine injection are taken to represent the final binding value. Again, the response is measured in Resonance Units. Biosensor matrices are regenerated using 10 mM HCl before injection of the next sample where a binding event is observed, otherwise running buffer was injected over the matrices. Human cytokines (e.g., IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-22, IL-23, IL-27, TNF-α, TNF-β, and IFN-γ, for example) are also simultaneously injected over the immobilized mouse IgG1/K reference surface to record any nonspecific binding background. By preparing a reference and reaction surface, Biacore can automatically subtract the reference surface data from the reaction surface data in order to eliminate the majority of the refractive index change and injection noise. Thus, it is possible to ascertain the true binding response attributed to an anti-cytokine antibody or DVD-Ig binding reaction.
When a cytokine of interest is injected across immobilized anti-cytokine antibody, significant binding is observed. 10 mM HCl regeneration completely removes all non-covalently associated proteins. Examination of the sensorgram shows that immobilized anti-cytokine antibody or DVD-Ig binding to soluble cytokine is strong and robust. After confirming the expected result with the cytokine of interest, the panel of remaining recombinant human cytokines is tested, for each antibody or DVD-Ig separately. The amount of anti-cytokine antibody or DVD-Ig bound or unbound cytokine for each injection cycle is recorded. The results from three independent experiments are used to determine the specificity profile of each antibody or DVD-Ig. Antibodies or DVD-Ig with the expected binding to the cytokine of interest and no binding to any other cytokine are selected.
Example 1.1.2.D
Tissue Cross Reactivity
Tissue cross reactivity studies are done in three stages, with the first stage including cryosections of 32 tissues, second stage including up to 38 tissues, and the 3rd stage including additional tissues from 3 unrelated adults as described below. Studies are done typically at two dose levels.
Stage 1: Cryosections (about 5 μm) of human tissues (32 tissues (typically: Adrenal Gland, Gastrointestinal Tract, Prostate, Bladder, Heart, Skeletal Muscle, Blood Cells, Kidney, Skin, Bone Marrow, Liver, Spinal Cord, Breast, Lung, Spleen, Cerebellum, Lymph Node, Testes, Cerebral Cortex, Ovary, Thymus, Colon, Pancreas, Thyroid, Endothelium, Parathyroid, Ureter, Eye, Pituitary, Uterus, Fallopian Tube and Placenta) from one human donor obtained at autopsy or biopsy) are fixed and dried on object glass. The peroxidase staining of tissue sections is performed, using the avidin-biotin system.
Stage 2: Cryosections (about 5 μm) of human tissues 38 tissues (including adrenal, blood, blood vessel, bone marrow, cerebellum, cerebrum, cervix, esophagus, eye, heart, kidney, large intestine, liver, lung, lymph node, breast mammary gland, ovary, oviduct, pancreas, parathyroid, peripheral nerve, pituitary, placenta, prostate, salivary gland, skin, small intestine, spinal cord, spleen, stomach, striated muscle, testis, thymus, thyroid, tonsil, ureter, urinary bladder, and uterus) from 3 unrelated adults obtained at autopsy or biopsy) are fixed and dried on object glass. The peroxidase staining of tissue sections is performed, using the avidin-biotin system.
Stage 3: Cryosections (about 5 μm) of cynomolgus monkey tissues (38 tissues (including adrenal, blood, blood vessel, bone marrow, cerebellum, cerebrum, cervix, esophagus, eye, heart, kidney, large intestine, liver, lung, lymph node, breast mammary gland, ovary, oviduct, pancreas, parathyroid, peripheral nerve, pituitary, placenta, prostate, salivary gland, skin, small intestine, spinal cord, spleen, stomach, striated muscle, testis, thymus, thyroid, tonsil, ureter, urinary bladder, and uterus) from 3 unrelated adult monkeys obtained at autopsy or biopsy) are fixed and dried on object glass. The peroxidase staining of tissue sections is performed, using the avidin-biotin system.
The antibody or DVD-Ig is incubated with the secondary biotinylated anti-human IgG and developed into immune complex. The immune complex at the final concentrations of 2 and 10 μg/mL of antibody or DVD-Ig is added onto tissue sections on object glass and then the tissue sections are reacted for 30 minutes with a avidin-biotin-peroxidase kit. Subsequently, DAB (3,3′-diaminobenzidine), a substrate for the peroxidase reaction, is applied for 4 minutes for tissue staining. Antigen-Sepharose beads are used as positive control tissue sections. Target antigen and human serum blocking studies serve as additional controls. The immune complex at the final concentrations of 2 and 10 μg/mL of antibody or DVD-Ig is pre-incubated with target antigen (final concentration of 100 μg/ml) or human serum (final concentration 10%) for 30 minutes, and then added onto the tissue sections on object glass and then the tissue sections are reacted for 30 minutes with a avidin-biotin-peroxidase kit. Subsequently, DAB (3,3′-diaminobenzidine), a substrate for the peroxidase reaction, is applied for 4 minutes for tissue staining.
Any specific staining is judged to be either an expected (e.g., consistent with antigen expression) or unexpected reactivity based upon known expression of the target antigen in question. Any staining judged specific is scored for intensity and frequency. The tissue staining between stage 2 (human tissue) and stage 3 (cynomolgus monkey tissue) is either judged to be similar or different.
Example 1.1.2.E
Inhibition of HUVEC Proliferation/Survival by VEGF Parent Antibody and DVD-Ig Constructs
Prior to plating for the assay, normal human umbilical vascular endothelial cells or HUVEC (passage 2-6) were maintained in EBM-2 (Lonza-Clonetics, Walkersville, Md.) supplemented with EGM-2 SingleQuots (Lonza-Clonetics, Walkersville, Md., #CC-4176). HUVEC cells were plated at 10,000 cells/well on collagen coated black 96-well plates in (100 μl) EMB-2 with 0.1% FBS in the absence of growth factors. The following day the media was replaced with 0.1% FBS in the absence of growth factors. The following day the media was replaced with 100 μl of EMB-2 (without growth factors or serum) and incubated for four hours prior to the addition of VEGF and antibodies/DVD-Igs. Anti-VEGF monoclonal antibodies or DVD-Igs (at final concentrations of 67 nM, 6.7 nM and 0.67 nM) were diluted in EMB-2 with 0.1% BSA and were pre-incubated with recombinant human VEGF165 (50 ng/ml) for 1 hour at 25° C. in 504 Antibody/DVD-Ig and VEGF mixtures were then added to the cells (50 μl), and the plates were incubated at 37° C. in a humidified, 5% C02 atmosphere for 72 hours. Cell survival/proliferation was measured indirectly by assessing ATP levels using an ATPlite kit (Perkin Elmer, Waltham, Mass.) according to the manufacturer's instructions. Table 10 provides the data showing inhibition of HUVEC proliferation/survival.







TABLE 10







Inhibition Of HUVEC Proliferation/Survival By VEGF Parent Antibody And


DVD-Ig Constructs














N-terminal VD
C-terminal VD





inhibition of
inhibition of





HUVEC
HUVEC



N-terminal
C-terminal
proliferation
proliferation


Parent
Variable
Variable
(% inhibition at
(% inhibition at


Antibody
Domain
Domain
67, 6.7 and
67, 6.7 and


or DVD-Ig ID
(VD)
(VD)
0.67 nM Ab/DVD-Ig)
0.67 nM Ab/DVD-Ig)












AB014
VEGF
67 nM = 98%, 6.7 nM = 91%, 0.67 nM = 12%










DVD038
HER-2
VEGF
67 nM = 45%,





6.7 nM = −18%,





0.67 nM = −22%









AB014
VEGF
67 nM = 98%, 6.7 nM = 91%, 0.67 nM = 12%











DVD044
DLL4
VEGF

67 nM = 99%,






6.7 nM = 19%,






0.67 nM = 6%


DVD043
VEGF
DLL4
67 nM = 89%,





6.7 nM = 51%,





0.67 nM = −22%









AB014
VEGF
67 nM = 97%, 6.7 nM = 91%, 0.67 nM = 62%











DVD048
RON
VEGF

67 nM = 91%,






6.7 nM = 88%,






0.67 nM = 62%


DVD047
VEGF
RON
67 nM = 65%,





6.7 nM = 31%,





0.67 nM = −5%









AB014
VEGF
67 nM = 97%, 6.7 nM = 91%, 0.67 nM = 62%











DVD049
VEGF
NRP1
67 nM = 108%,






6.7 nM = 86%,





0.67 nM = 43%


DVD050
NRP1
VEGF

67 nM = 103%,






6.7 nM = 74%,






0.67 nM = 15%









AB014
VEGF
67 nM = 98%, 6.7 nM = 91%, 0.67 nM = 12%










DVD260
P1GF
VEGF
67 nM = 51%,





6.7 nM = 7%,





0.67 nM = −16%









All DVD-Igs containing VDs from AB014 inhibited HUVEC cell proliferation caused by VEGF. DVD044, DVD048, DVD040 DVD050 inhibited HUVEC cell proliferation by >90% at 67 nM concentration of DVD-Igs.
Example 1.1.2.F
Tumor Cell Growth Inhibitory Effect of IGF1,2 Monoclonal Antibodies or DVD-Igs In Vitro
IGF1,2 monoclonal antibodies or DVD-Igs diluted in D-PBS-BSA (Dulbecco's phosphate buffered saline with 0.1% BSA) 20 μL were added to human tumor cells at final concentrations of 0.01 μg/mL-100 μg/mL in 200 μL. The plates were incubated at 37° C. in a humidified, 5% CO2 atmosphere for three days. The number of live cells in each well was quantified using MTS reagents according to the manufacturer's instructions (Promega, Madison, Wis.) to determine the percent of tumor growth inhibition. Wells without antibody treatment were used as controls of 0% inhibition whereas wells without cells were considered to show 100% inhibition.







TABLE 11







H929, IGFR Line Proliferation Inhibition Assay With IGF1R and IGF1,


2 Parent Antibodies and DVD-Ig Constructs














N-terminal VD
C-terminal VD



N-terminal
C-terminal
Proliferation
Proliferation



Variable
Variable
Inhibition Cellular
Inhibition Cellular


Parent Antibody
Domain
Domain
Assay IC50 nM
Assay IC50 nM


or DVD-Ig ID
(VD)
(VD)
(H929, IGF1R line)
(H929, IGF1R line)













AB033
EGFR
>67
(5% max)


AB004
HER-2
>67
(10% max)













DVD015
EGFR
HER-2
>50
(20% max)




DVD016
HER-2
EGFR


>50
(15% max)










AB033
EGFR
>67
(5% max)


AB011
IGF1R
0.1
(65% max)













DVD021
EGFR
IGF1R


>50
(10% max)


DVD022
IGF1R
EGFR
0.05
(70% max)










AB004
HER-2
>67
(10% max)


AB010
IGF1, 2
1.6
(70% max)













DVD029
HER-2
IGF1, 2


6.8
(40% max)


DVD030
IGF1, 2
HER-2
0.8
(70% max)










AB004
HER-2
>67
(10%)


AB011
IGF1R
0.1
(65% max)













DVD031
HER-2
IGF1R


>50
(10% max)


DVD032
IGF1R
HER-2
0.1
(60% max)









All DVD-Igs containing VDs from AB033, AB004, AB011, or AB010 in either the N-terminal or C-terminal position showed inhibition in the A431 cell proliferation assay.
Example 1.1.2.G
Growth Inhibitory Effect of an EGFR Monoclonal Antibody or DVD-Igs In Vitro
EGFR monoclonal antibodies or DVD-Igs diluted in D-PBS-BSA (Dulbecco's phosphate buffered saline with 0.1% BSA) 20 μL were added to human tumor cells at final concentrations of 0.01 μg/mL-100 μg/mL in 180 uL. The plates were incubated at 37° C. in a humidified, 5% CO2 atmosphere for 3 days. The number of live cells in each well was quantified using MTS reagents according to the manufacturer's instructions (Promega, Madison, Wis.) to determine the percent of tumor growth inhibition. Wells without antibody treatment were used as controls of 0% inhibition whereas wells without cells were considered to show 100% inhibition.







TABLE 12







A431, EGFR Cell Line Proliferation Inhibition Assay With EGFR Parent Antibodies


And DVD-Ig Constructs












N-terminal
C-terminal
N-terminal VD
C-terminal VD


Parent
Variable
Variable
Proliferation Inhibition
Proliferation Inhibition


Antibody or
Domain
Domain
Cellular Assay IC50 nM
Cellular Assay IC50 nM


DVD-Ig ID
(VD)
(VD)
(A431, EGFR line)
(A431 EGFR line)












AB033
EGFR
 1.0 (65% max)


AB004
HER-2
>67 (10% max)











DVD015
EGFR
HER-2
1.7 (65% max)



DVD016
HER-2
EGFR

5.4 (80% max)









AB033
EGFR
 1.0 (65% max)


AB011
IGF1R
>67 (5% max) 











DVD021
EGFR
IGF1R
1.5 (50% max)



DVD022
IGF1R
EGFR

3.4 (65% max)









AB004
HER-2
>67 (10% max)


AB010
IGF1, 2
>67 (10% max)










DVD029
HER-2
IGF1, 2
>50 (10% max)


DVD030
IGF1, 2
HER-2
>50 (10% max)









AB004
HER-2
>67 (10% max)


AB011
IGF1R
>67 (5% max) 










DVD031
HER-2
IGF1R
>50 (10% max)


DVD032
IGF1R
HER-2
>50 (10% max)









AB014
VEGF
>67 (0%)


AB010
IGF1, 2
>67 (10% max)










DVD041
VEGF
IGF1, 2
>50 (0%)









All DVD-Igs containing VDs from AB033, AB004, AB011, AB010, AB014 in either N-terminal or C-terminal position showed inhibition of A431 cell proliferation assay.







TABLE 13







GEO, EGFR/IGF1R Cell Line Proliferation Inhibition Assay With EGFR,


IGF1R And IGF1, 2 Parent Antibodies And DVD-Ig Constructs












N-
C-
N-terminal
C-terminal


Parent
terminal
terminal
Proliferation
Proliferation


Antibody
Variable
Variable
Inhibition Cellular
Inhibition Cellular


or DVD-Ig
Domain
Domain
Assay IC50 nM
Assay IC50 nM (GEO,


ID
(VD)
(VD)
(GEO, EGFR/IGF1R line)
EGFR/IGF1R line)












AB033
EGFR
 0.2 (60% max)


AB004
HER-2
>67 (15% max)











DVD015
EGFR
HER-2
  13 (60% max)



DVD016
HER-2
EGFR

  10 (40% max)









AB033
EGFR
 0.2 (60% max)


AB011
IGF1R
>67 (15% max)











DVD021
EGFR
IGF1R
0.32 (60% max) 
0.32 (60% max) 


DVD022
IGF1R
EGFR
 3.0 (40% max)
 3.0 (40% max)









AB004
HER-2
>67 (15% max)


AB010
IGF1, 2
20% at 67 nM











DVD029
HER-2
IGF1, 2
>50 (10% max)
>50 (10% max)


DVD030
IGF1, 2
HER-2
>50 (10% max)
>50 (10% max)









AB004
HER-2
>67 (15% max)


AB011
IGF1R
>67 (15% max)











DVD031
HER-2
IGF1R
>50 (10% max)
>50 (10% max)


DVD032
IGF1R
HER-2
>50 (10% max)
>50 (10% max)









All DVD-Igs containing VDs from AB033, AB004, AB011, AB010, AB014 in either N-terminal or C-terminal position showed inhibition of GEO cell proliferation assay.
Example 1.1.2.H
Tumor Cell Growth Inhibitory Effect of HER2 Parent Antibody or DVD-Ig Constructs In Vitro
HER2 monoclonal antibody or DVD-Igs diluted in D-PBS-BSA (Dulbecco's phosphate buffered saline with 0.1% BSA) 204, were added to HER2-expressing human tumor cells (BT474) at final concentrations of 0.01 μg/mL-100 μg/mL (180 μL). The plates were incubated at 37° C. in a humidified, 5% CO2 atmosphere for three days. The number of live cells in each well was quantified using MTS reagents according to the manufacturer's instructions (Promega, Madison, Wis.) to determine the percent of tumor growth inhibition. Wells without antibody treatment were used as controls of 0% inhibition whereas wells without cells were considered to show 100% inhibition.







TABLE 14







BT474, Erb2(Her-2) Cell Line Proliferation Inhibition Assay


With Anti-Her-2 Parent Antibodies And DVD-Ig Constructs














N-terminal VD
C-terminal VD





Proliferation
Proliferation





Inhibition
Inhibition



N-terminal
C-terminal
Cellular Assay
Cellular


Parent
Variable
Variable
IC50 nM
Assay IC50 nM


Antibody or
Domain
Domain
(BT474, ErbB2
(BT474, ErbB2


DVD-Ig ID
(VD)
(VD)
line)
line)












AB004
HER-2
0.9











DVD015
EGFR
HER-2

>50


DVD016
HER-2
EGFR
1.7









AB004
HER-2
0.9











DVD029
HER-2
IGF1, 2
2.7



DVD030
IGF1, 2
HER-2

>50









AB004
HER-2
0.9











DVD031
HER-2
IGF1R
2.2



DVD032
IGF1R
HER-2

>50









All DVD-Igs containing VD from AB004 in either N-terminal or C-terminal position showed inhibition in the BT474 cell proliferation assay.
Example 1.1.2.I
Inhibition of Recombinant DLL4-Dependent Increase of Svegfr1 (Sflt1) in Eahy.926 Cells by DLL4 Parent Antibody and DVD-12 Constructs
96-well tissue culture plates were coated with 100 μl/well human DLL4 extracellular domain at 5 μg/ml in D-PBS (Gibco #14190, Grand Island, N.Y.) and incubated overnight at 4° C. Plates were washed once with D-PBS and 4000 EA.hy926 cells/well were seeded in the absence or presence of antibodies or DVD-Igs. Cell proliferation was measured four days later using the CyQUANT Cell Proliferation Assay Kit (Invitrogen, #C35007, Eugene, Oreg.). sVEGFR1 expression in the conditioned media was detected by an ELISA kit per the manufacturer's recommendations (R&D Systems #DVR100B, Minneapolis, Minn.). Levels of sVEGFR1 were normalized to the RFU determined by CyQUANT assay to account for differences in cell proliferation.







TABLE 15







Inhibition Of DLL4-Dependent Increase Of Svegfr1 (Sflt1) In


Eahy.926 Cells By DLL4 Parent Antibodies Or DVD-Ig Constructs














N-terminal VD
C-terminal VD





Soluble Flt1 Assay
Soluble Flt1 Assay


Parent
N-terminal
C-terminal
(% of
(% of


Antibody or
Variable
Variable
Neutralization @
Neutralization @


DVD-Ig ID
Domain (VD)
Domain (VD)
nM Ab)
nM Ab)












AB015
DLL-4
75.2% @9.2 nM, 78.7% @27.7 nM,




86.6% @83.3 nM











DVD044
DLL4
VEGF
39.8% @9.2 nM,






76.2% @27.7 nM,





79.9% @83.3 nM


DVD043
VEGF
DLL4

 1.1% @9.2 nM,






50.3% @27.7 nM,






57.9% @83.3 nM









DVD-Igs containing VD from AB015 in either N-terminal or C-terminal position showed dose-dependent inhibition of sFLT release induced by DLL4 from Eahy.926 cells.
Example 1.1.2.J
Tumoricidal Effect of a Parent or DVD-Ig Antibody In Vitro
Parent antibodies or DVD-Ig that bind to target antigens on tumor cells may be analyzed for tumoricidal activity. Briefly, parent antibodies or DVD-Ig are diluted in D-PBS-BSA (Dulbecco's phosphate buffered saline with 0.1% BSA) and added to human tumor cells at final concentrations of 0.01 μg/mL to 100 μg/mL 200 μL. The plates are incubated at 37° C. in a humidified, 5% CO2 atmosphere for 3 days. The number of live cells in each well is quantified using MTS reagents according to the manufacturer's instructions (Promega, Madison, Wis.) to determine the percent of tumor growth inhibition. Wells without antibody treatment are used as controls of 0% inhibition whereas wells without cells were considered to show 100% inhibition.
For assessment of apoptosis, caspase-3 activation was determined by the following protocol: antibody-treated cells in 96 well plates are lysed in 120 μl of 1× lysis buffer (1.67 mM Hepes, pH 7.4, 7 mM KCl, 0.83 mM MgCl2, 0.11 mM EDTA, 0.11 mM EGTA, 0.57% CHAPS, 1 mM DTT, 1× protease inhibitor cocktail tablet; EDTA-free; Roche Pharmaceuticals, Nutley, N.J.) at room temperature with shaking for 20 minutes. After cell lysis, 80 μl of a caspase-3 reaction buffer (48 mM Hepes, pH 7.5, 252 mM sucrose, 0.1% CHAPS, 4 mM DTT, and 20 μM Ac-DEVD-AMC substrate; Biomol Research Labs, Inc., Plymouth Meeting, Pa.) is added and the plates are incubated for 2 hours at 37° C. The plates are read on a 1420 VICTOR Multilabel Counter (Perkin Elmer Life Sciences, Downers Grove, Ill.) using the following settings: excitation=360/40, emission=460/40. An increase of fluorescence units from antibody-treated cells relative to the isotype antibody control-treated cells is indicative of apoptosis.
Example 1.1.2.K
Inhibition of U87-MG Tumor Cell Proliferation by HGF Parent Antibody and DVD-Ig Constructs
U87-MG human glioma tumor cells were plated at 2,000 cells/well in 100 μl in 96-well dishes in RPMI medium supplemented with 5% fetal bovine serum, and incubated at 37° C., 5% CO2 overnight. The following day the cells were treated with serial dilutions of antibody or DVD-Igs (0.013 nM to 133 nM dose range), and incubated at 37° C. in a humidified, 5% CO2 atmosphere for 5 days. Cell survival/proliferation was measured indirectly by assessing ATP levels using an ATPlite kit (Perkin Elmer, Waltham, Mass.) according to the manufacturer's instructions.







TABLE 16







U87-MG Tumor Proliferation Inhibition by anti-HGF Parent


Antibody and DVD-Ig Constructs














N-terminal
C-terminal





VD
VD




C-terminal
inhibition of
inhibition of


Parent
N-terminal
Variable
U87MG
U87MG


Antibody or
Variable
Domain
proliferation
proliferation


DVD-Ig ID
Domain (VD)
(VD)
(EC50)
(EC50)












AB012
HGF
12










DVD025
EGFR
HGF
>200









AB012
HGF
12











DVD033
RON
HGF

>200


DVD034
HGF
RON
136









AB012
HGF
12










DVD045
VEGF
HGF
>200









AB008
CD22
>200









DVD-Igs containing a VD from AB012 in the C-terminal position or N-terminal position inhibited U87-MG tumor cell proliferation.
Example 1.1.2.L
Inhibition of RON Interaction with MSP1 by RON Parent Antibody and DVD-Ig Constructs In Vitro
96-well plates were coated with 50 μl/well of an anti-MSPα chain antibody (R&D Systems #MAB352, Minneapolis, Minn., 2 μg/mL), and plates were incubated overnight at 4° C. Plates were washed three times in wash buffer (PBS containing 0.05% Tween 20), and subsequently blocked with 100 μl/well of blocking buffer (PBS containing 2% BSA) for one hour at 25° C. Plates were then washed three times, and incubated with 50 μl/well of a 10 nM solution of recombinant human MSP1 (R&D Systems #352-MS, Minneapolis, Minn.) for one hour at 25° C. During plate incubation, serial 10-fold dilutions of the antibodies to be tested (0 nM to 1000 nM dose range) were pre-incubated with 10 nM recombinant His-RON partial ECD (R&D Systems #1947-MS, Minneapolis, Minn.) at 25° C. for one hour. Plates incubated with recombinant human MSP1 were washed three times, and 50 μl/well of the antibody/His-RON complexes were then added in triplicate. Following a one hour incubation at 25° C., the plates were then washed, and 50 μl/well of a TMB substrate (Pierce #34028, Rockford, Ill.) were added and incubated for five minutes at 25° C. The reaction was terminated after five minutes using 50 μl/well of 2N H2SO4. The absorbance was read at 450 nm (Spectra Max Plus plate reader, Molecular Devices, Sunnyvale, Calif.). EC50s were calculated in GraphPad Prism 4.03. The results are provided in Table 17 below.
Example 1.1.2.M
VEGF Parent Antibody and DVD-Ig Constructs Prevent VEGF165 Interaction with VEGFR1
ELISA plates (Nunc, MaxiSorp, Rochester, N.Y.) were incubated overnight at 4° C. with 100 μl PBS containing recombinant VEGFR1 extra-cellular domain-Fc fusion protein (5 μg/ml, R&D systems, Minneapolis, Minn.). Plates were washed three times in washing buffer (PBS containing 0.05% Tween 20), and blocked for 1 hour at 25° C. in blocking buffer (PBS containing 1% BSA). Serial dilutions of each antibody/DVD-Ig in PBS containing 0.1% BSA were incubated with 50 μl of 2 nM biotinylated VEGF for 1 hour at 25° C. The antibody/DVD-Ig-biotinylated VEGF mixtures (100 μl) were then added to the VEGFR1-Fc coated wells and incubated at 25° C. for 10 minutes. The wells were washed three times, and then incubated for 1 hour at 25° C. with 100 μl of streptavidin-HRP (KPL #474-3000, Gaithersburg, Md.). The wells were washed three times, and 100 μl of ULTRA-TMB ELISA (Pierce, Rockford, Ill.) were added per well. Following color development the reaction was stopped with 1N HCL and absorbance at 450 nM was measured. The results are provided in Table 17 below.
Example 1.1.2.N
Inhibition of DLL4 Interaction with Notch-1 by DLL4 Parent Antibody and DVD-Ig Constructs In Vitro
96-well Nunc-Immuno plates (Nunc, #439-454, Rochester, N.Y.) were coated with 16 nM human Notch-1 (R&D Systems #3647-TK, Minneapolis, Minn., 100 μl/well in D-PBS) and incubated overnight at 4° C. Plates were then washed once with wash buffer (PBS, 0.05% Tween 20) and blocked with 200 μl/well blocking buffer (D-PBS, 1% BSA, 1 mM CaCl2, 0.05% Tween 20) for 1 hour at 25° C. While blocking, 300 μl biotin labeled human DLL4 extracellular domain (14 nM) was mixed with antibody or DVD-Ig (3.4 pM-66 nM, 3-fold serial dilution in blocking buffer) for 1 hour at 25° C. Assay plates were washed after blocking, and incubated with DLL4 antibody or DVD-Ig mixtures (100 μl/well, 1 hour at 25° C.). Plates were washed again and 100 μl/well streptavidin conjugated with HRP (KPL #474-3000, Gaithersburg, Md., diluted 1:10,000 in blocking buffer) was added for 1 hour at 25° C. After a final wash, plates were developed using 100 μl/well substrate (1-Step Ultra TMB-ELISA, Pierce #340280, Rockford, Ill.), and the reaction was stopped after a 10-20 minute incubation at 25° C. using 100 μl/well 2N H2SO4, and the absorbance was read at 450 nm. The results are provided in Table 17 below.
Example 1.1.2.O
Inhibition of HGF Interaction with c-Met by HGF Parent Antibody and DVD-Ig Constructs
ELISA plates (Nunc, MaxiSorp) were coated with 100 μl/well of recombinant human HGF (2 μg/ml of HGF in PBS, R&D systems) overnight at 4° C. Serial dilutions of each antibody/DVD-Ig and 2 nM soluble c-Met Fc fusion (R&D systems) (50 μl), were co-incubated and added to HGF coated wells. c-Met binding was detected with biotinylated anti-c-Met (BAF358, R&D Systems) and 100 μl of streptavidin-HRP (KPL). The wells were washed three times in PBST (PBS containing 0.05% Tween 20), and 100 μl of ULTRA-TMB ELISA (Pierce) were added per well. Following color development the reaction was stopped with 1N HCL and absorbance at 450 nM was measured. The data were evaluated by calculating the percentage inhibition compared with the maximal signal (control antibody or no antibody added) and the IC50 values were calculated.
The following table contains the affinity data, expressed as IC50 in nM, of parent antibodies and DVD-Ig constructs in the ligand-receptor binding competition ELISA assays for RON, VEGF, DLL4, and HGF as described above.







TABLE 17







Inhibition Of Ligand-Receptor Interaction With RON. VEGF


DLL4, And HGF Parent Antibodies And DVD-Ig Constructs In Vitro














N-terminal
C-terminal





VD
VD





Ligand-
Ligand-





Receptor
Receptor



N-terminal
C-terminal
Binding
Binding



Variable
Variable
Competition
Competition


Parent Antibody
Domain
Domain
Assay (IC50,
Assay (IC50,


or DVD-Ig ID
(VD)
(VD)
nM)
nM)












AB005
RON
8.1











DVD024
RON
EGFR
9.6



DVD023
EGFR
RON









AB005
RON
0.32











DVD033
RON
HGF
**0.0000097



DVD034
HGF
RON









AB014
VEGF
21.40











DVD035
VEGF
EGFR
23.90










AB014
VEGF
21.40










DVD038
HER-2
VEGF
73.00









AB015
DLL-4
0.37


AB014
VEGF
21.4











DVD044
DLL4
VEGF
0.50
143


DVD043
VEGF
DLL4
24.1
13.10









AB005
RON
0.1


AB014
VEGF
21.4











DVD048
RON
VEGF
0.1127
114


DVD047
VEGF
RON
30.5
21.28









AB014
VEGF
21.40










DVD050
NRP1
VEGF
79.00









AB015
DLL-4
0.4











DVD257
DLL4
P1GF
0.38



DVD258
P1GF
DLL4

17.47









AB014
VEGF
21.40










DVD260
P1GF
VEGF
13.00









AB012
HGF
0.18










DVD025
EGFR
HGF
>100









AB012
HGF
0.18










DVD045
VEGF
HGF
>100









AB012
HGF
0.18











DVD033
RON
HGF

8.7


DVD034
HGF
RON
0.34









All DVD-Igs containing VD from AB005, AB015, AB015, AB012, in either N-terminal or C-terminal positions showed inhibition of ligand to their respective receptors. The N-terminal domain of DVD-Ig blocked ligand-receptor interaction as well as parent antibody.
Example 1.1.2.P
Inhibition of IGF-Induced IGFR Phosphorylation by Parent Antibodies or DVD-Ig Constructs In Vitro
Human carcinoma cells were plated in 96-well plates at 40,000 cells/well in 180 μl serum-free medium (DMEM+0.1% BSA), and incubated overnight at 37° C., 5% CO2. Costar EIA plates (Lowell, Mass.) were coated with 100 μl/well of IGFR capture Ab (R&D Systems cat #MAB391, Minneapolis, Minn., 4 μg/ml final concentration), and incubated overnight at room temperature while shaking. The following day, IGFR antibody-coated ELISA plates were washed (three times with PBST=0.05% Tween 20 in PBS, pH 7.2-7.4), and 200 μl blocking solution was added (1% BSA, 0.05% NaN3 in PBS, pH 7.2-7.4) to block for 2 hours at room temperature on a rocker. Human tumor cells were co-incubated with antibodies or DVD-Igs and IGF ligand. IGF1,2 monoclonal antibodies or DVD-Igs diluted in D-PBS-BSA (Dulbecco's phosphate buffered saline with 0.1% BSA) were added to human carcinoma cells at final concentrations of 0.01 μg/mL-100 μg/mL. Growth factors (IGF1 and IGF2) were simultaneously added to the cells at concentrations of 1-100 ng/mL (200 μL), and cells were incubated at 37° C. in a humidified, 5% CO2 atmosphere for 1 hour. Cells were lysed in 120 μl/well of cold cell extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, 10% Glycerol, 0.1% SDS, and protease inhibitor cocktail), and incubated at 4° C. for 20 minutes with shaking. Cell lysates (1000 were added to the ELISA plate, and incubated overnight at 4° C. with gentle shaking. The following day, ELISA plates were washed, and 100 μl/well of pTyr-HRP detection Ab was added (p-IGF1R ELISA kit, R&D System # DYC1770, Minneapolis, Minn.), and plates were incubated for 2 hours at 25° C. in the dark. Plates were developed to determine phospho-IGF1R per the manufacturer's instructions. Results are shown in Tables 18 and 19.







TABLE 18







Inhibition Of IGF1R Phosphorylation By IGF1 With


Anti-IGF1, 2 Parent Antibodies Or DVD-Ig Constructs















C-terminal VD



N-terminal
C-terminal
N-terminal VD
pIGF1R Cellular



Variable
Variable
pIGF1R Cellular
Assay (hIGF1


Parent Antibody
Domain
Domain
Assay (hIGF1
Neutralization IC50


or DVD-Ig ID
(VD)
(VD)
Neutralization IC50 nM)
nM)












AB010
IGF1, 2
1.7











DVD029
HER-2
IGF1, 2

14.22 (40% max)


DVD030
IGF1, 2
HER-2
1.9









AB010
IGF1, 2
1.7











DVD041
VEGF
IGF1, 2

 16.8 (40% max)


DVD042
IGF1, 2
VEGF
1.8









DVD-Igs containing the VD from AB010 in either the N-terminal or the C-terminal position showed inhibition of IGF1-induced IGF1R receptor phosphorylation. The VD of AB010 in N-terminal position of DVD-Ig blocked receptor phosphorylation as well as parent antibody AB010.







TABLE 19







Inhibition Of IGF1R Phosphorylation By IGF2 With Anti-IGF1, 2 Parent


Antibodies Or DVD-Ig Constructs












N-terminal
C-terminal
N-terminal VD
C-terminal VD



Variable
Variable
pIGFR1 Cellular
pIGFR1 Cellular


Parent Antibody
Domain
Domain
Assay (IGF2 induced
Assay (IGF2 induced


or DVD-Ig ID
(VD)
(VD)
IC50 nM)
IC50 nM












AB010
IGF1, 2
0.3











DVD029
HER-2
IGF1, 2
ND
1.9


DVD030
IGF1, 2
HER-2
0.4
ND









AB010
IGF1, 2
0.3











DVD041
VEGF
IGF1, 2
ND
1.5


DVD042
IGF1, 2
VEGF
0.3
ND









DVD-Igs containing the VD from AB010 in either the N-terminal or the C-terminal position showed inhibition of IGF2-induced IGF1R receptor phosphorylation. The VD of AB010 in the N-terminal position of DVD-Ig blocked receptor phosphorylation as well as parent antibody AB010.
Example 1.1.2.Q
Inhibition of HGF-Mediated Phosphorylation of Akt by HGF Parent Antibody and DVD-12 Constructs
H1299 non-small cell lung tumor cells were plated at 20,000 cells/well (100 μl total volume) in 96-well plates and serum starved for 18 hours at 37° C., 5% CO2. Anti-HGF monoclonal antibodies or DVD-Igs (final concentrations of 67 nM, 6.7 nM, and 0.67 nM) were diluted in Dulbecco's Minimal Essential Media containing 0.1% BSA and were pre-incubated with recombinant human HGF (50 ng/ml) in 50 μl for 1 hour at 25° C. These antibody/DVD-Ig and HGF mixtures (50 μl) were then added to the cells, and the plates were incubated at 37° C. in a humidified, 5% CO2 atmosphere for approximately 15 minutes. The cells were then fixed by adding an equal volume of 7.6% formaldehyde to each well and the plates were incubated for 15 minutes at 25° C. Following fixation, the cells were washed five times in PBS containing 0.1% Triton X-100. The cells were then treated with 150 μl of LI-COR Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, Nebr.) per well, and incubated for 90 minutes at room temperature with moderate shaking. The blocking buffer was removed and the cells were incubated at 4° C. overnight with primary antibody diluted in blocking buffer (1:300 dilution Phospho Ser473-Akt, Cell Signaling Technology #4060, Boston, Mass.). The wells were then washed five times with PBS containing 0.1% Tween 20, and then incubated with secondary antibody (1:400 dilution of an anti-rabbit IRDyeTM 680CW LI-COR (Li-Cor Biosciences, Lincoln, Nebr.) in 1×PBS with 0.2% Tween 20) for 1 hour at 25° C. The cells were washed five times with PBS containing 0.1% Tween 20, and imaged using an Odyssey Infrared Imaging System.







TABLE 20







Inhibition Of HGF-Mediated Phosphorylation Of Akt By HGF


Parent Antibody And DVD-Ig Constructs














N-Terminal VD
C-Terminal VD





Inhibition Of Akt
Inhibition Of Akt


Parent


Phosphorylation
Phosphorylation


Antibody
N-terminal
C-terminal
(% Inhibition At
(% Inhibition At 67,


or DVD-Ig
Variable
Variable
67, 6.7 And
6.7 And 0.67 nm


ID
Domain (VD)
Domain (VD)
0.67 nM Ab/DVD)
Ab/DVD)












AB012
HGF
67 nM = 106%, 6.7 nM = 110%,




0.67 nM = 79%











DVD033
RON
HGF

  67 nM = 95%,






 6.7 nM = 91%,






0.67 nM = 24%


DVD034
HGF
RON
  67 nM = 111%,





 6.7 nM = 105%,





0.67 nM = 99%









AB012
HGF
67 nM = 106%, 6.7 nM = 110%,




0.67 nM = 79%











DVD045
VEGF
HGF

  67 nM = 64%,






 6.7 nM = −2%,






0.67 nM = −15%









AB008
CD22
67 nM = 8%, 6.7 nM = −13%









DVD-Igs containing the VD from AB012 in either the N-terminal or the C-terminal position showed good inhibition of HGF-induced Akt phosphorylation. The VD of AB012 in the N-terminal position of DVD-Ig blocked Akt phosphorylation as well as parent antibody AB012.
Example 1.1.2.R
Inhibition of VEGFR2 (KDR) Phosphorylation by VEGF Parent Antibody and DVD-Ig Constructs
NIH3T3 cells expressing human VEGFR2 (KDR) were plated at 20,000 cells/well (100 μl) in 96-well plates in DMEM supplemented with 10% FBS. The following day, the cells were washed twice with DMEM and serum-starved for three hours in DMEM without FBS. Anti-VEGF parent antibody or DVD-Igs (at final concentrations of 67 nM, 6.7 nM and 0.67 nM) diluted in DMEM with 0.1% BSA were pre-incubated with recombinant human VEGF165 (50 ng/ml) for 1 hour at 25° C. These antibody/DVD-Ig and VEGF mixtures were then added to the cells, and the plates were incubated at 37° C. in a humidified, 5% CO2 atmosphere for 10 minutes. Cells were washed twice with ice cold PBS and lysed by addition of 100 μl/well of Cell Lysis Buffer (Cell Signaling, Boston, Mass.) supplemented with 0.1% NP40. Duplicate samples were pooled and 170 μl was added to wells of ELISA plates previously coated with anti-VEGFR2 antibody (R&D systems, AF357, Minneapolis, Minn.) and incubated at 25° C. with gentle shaking for two hours. The wells were washed five times with washing buffer (PBS containing 0.05% Tween 20), and incubated with 50 μl of 1:2000 dilution of biotinylated anti-phosphotyrosine antibody (4G10; Millipore, Billerica, Mass.) for 1 hour at 25° C. The wells were washed five times with PBS containing 0.05% Tween 20, and then incubated for 1 hour at 25° C. with streptavidin-HRP (KPL #474-3000, Gaithersburg, Md.). The wells were washed three times with streptavidin-HRP (KPL #474-3000, Gaithersburg, Md.)). The wells were washed three times with PBS containing 0.05% Tween 20, and 100 μl of ULTRA-TMB ELISA (Pierce, Rockford, Ill.) were added per well. Following color development the reaction was stopped with 1N HCL and absorbance at 450 nM was measured. The results are shown in Table 21.







TABLE 21







Inhibition Of VEGFR2 (KDR) Phosphorylation By VEGF Parent Antibodies Or


DVD-Ig Constructs













N-terminal





C-terminal
Variable
N-Terminal VD
C-Terminal VD


Parent Antibody
Variable
Domain
Inhibition Of
Inhibition Of Pkdr


or DVD-Ig ID
Domain (VD)
(VD)
Pkdr (EC50)
(EC50)












AB014
VEGF
0.45











DVD044
DLL4
VEGF

10.40


DVD043
VEGF
DLL4
0.54









AB014
VEGF
0.45











DVD048
RON
VEGF

31.80


DVD047
VEGF
RON
0.47









DVD-Igs containing the VD from AB014 in either N-terminal or the C-terminal position showed good inhibition of VEGF-induced KDR phosphorylation. The VD of AB014 in the N-terminal position of DVD-Ig blocked KDR phosphorylation as well as parent antibody AB014.
Example 1.1.2.S
Inhibition of EGF-Induced EGFR Phosphorylation by EGFR Parent Antibody or DVD-Ig Constructs In Vitro
EGFR monoclonal antibodies or DVD-Igs diluted in D-PBS-BSA (Dulbecco's phosphate buffered saline with 0.1% BSA) were added to human carcinoma cells at final concentrations of 0.01 μg/mL-100 μg/mL (180 μL). The plates were incubated at 37° C. in a humidified, 5% CO2 atmosphere for 1 hour. Growth factors (EGF) at a final concentration of 1-100 ng/mL (20 μL) were added to the cells for 5-15 minutes to stimulate receptor (EGFR) autophosphorylation. Wells without antibody treatment were used as controls of 0% inhibition whereas wells without growth factor stimulation were considered to show 100% inhibition. Cell lysates were made by incubation with cell extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, 10% Glycerol, 0.1% SDS, and protease inhibitor cocktail). Phospho-EGFR in these cell lysates was determined using the p-EGFR ELISA kit from R&D Systems (#DYC1095, Minneapolis, Minn.) according to the manufacturer's instructions. Results are shown in Tables 22 and 23.







TABLE 22







Inhibition Of EGF-Induced EGFR Phosphorylation By Anti-EGFR Parent


Antibodies And DVD-Ig Constructs














N-terminal VD
C-terminal VD


Parent
N-terminal
C-terminal
pEGFR Cellular
pEGFR Cellular


Antibody or
Variable
Variable
Assay (EGF
Assay (EGF


DVD-Ig ID
Domain (VD)
Domain (VD)
induced IC50 nM)
induced IC50 nM))












AB033
EGFR
7.3











DVD015
EGFR
HER-2
7.9



DVD016
HER-2
EGFR

>50 (10% max)









AB033
EGFR
7.3











DVD021
EGFR
IGF1R
7.7



DVD022
IGF1R
EGFR

>50 (20% max)









DVD-Igs containing the VD from AB033 in either the N-terminal or the C-terminal position showed good inhibition of EGF-induced EGFR phosphorylation. The VD of AB033 in the N-terminal position of DVD-Ig (DVD015, DVD021) blocked EGFR phosphorylation as well as parent antibody AB033.







TABLE 23







Inhibition Of EGF-Induced EGFR Phosphorylation By


Anti-EGFR Parent Antibodies And DVD-Ig Constructs














N-Terminal
C-Terminal



N-

Cellular Assay,
Cellular Assay,



terminal
C-terminal
Inhibition Of
Inhibition Of


Parent
Variable
Variable
EGF-Induced
EGF-Induced


Antibody or
Domain
Domain
phEGFR
phEGFR (IC50,


DVD-Ig ID
(VD)
(VD)
(IC50, nM)
nM)












AB033
EGFR
0.35











DVD024
RON
EGFR
ND
7.143


DVD023
EGFR
RON
0.54
ND









DVD-Igs containing the VD from AB033 in either the N-terminal or the C-terminal position showed good inhibition of EGF-induced EGFR phosphorylation. The VD of AB033 in the N-terminal position of DVD-Ig (DVD023) blocked EGFR phosphorylation as well as parent antibody AB033.
Example 1.1.2.T
Inhibition of MSP1-Induced ERK1/2 and AKT Phosphorylation In Vitro by Parent RON Antibody and DVD-Ig Constructs
Sub-confluent DU145 colon tumor cells grown in 10% FBS/Minimal Essential Medium were trypsinized and seeded into 6-well tissue culture plates (0.25×106 cells/2 ml final volume), and incubated at 37° C., 5% CO2 for 18-24 hours. After incubation, the cells were washed twice with 1×D-PBS and starved overnight in serum-free medium. The next day, cells were incubated with 900 μl of serum-free media containing 1 μM of monoclonal antibodies or DVD-Igs for 1 hour at 37° C. Following antibody incubation, cells were then treated with 13 nM MSP1 (37° C., 30 minutes). Cells were washed twice with ice-cold D-PBS and harvested in 150 μl Cell Extraction Buffer (Biosource International, Carlsbad, Calif.) containing 100 μl of HALT® phosphatase inhibitor cocktail (Thermo Scientific, Rockford, Ill.), one Complete® EDTA-free protease inhibitor tablet (1 tablet/10 ml, Roche Diagnostic, Mannheim, Germany), and PMSF. Cell lysates were incubated on ice for 30 minutes with intermittent vortexing, pre-cleared by centrifugation (10 min, 14,000 RPM, 4° C.), and processed for Western blot analysis. A total of 15 μg of cell lysate was resolved by SDS-PAGE on a 4-12% NuPage Bis-Tris Gel with 1×MOPS running buffer (Invitrogen, Carlsbad, Calif.). Proteins were transferred onto a nitrocellulose membrane (Invitrogen, Carlsbad, Calif.), and blocked in 5% nonfat milk in TBS-T (1×TBS/0.1% Tween 20) for 1 hour at room temperature. After blocking, membranes were incubated overnight at 4° C. with a 1:1000 dilution of either a rabbit polyclonal phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cell Signaling, Danvers, Mass.) or a rabbit monoclonal phospho-AKT (Ser473) antibody (Cell Signaling, Danvers, Mass.). Blots were washed three times (1×TBS-T, 15 minutes), and incubated for 1 hour at room temperature with a 1:5000 dilution of anti-rabbit IgG, HRP-conjugated secondary goat antibody (Jackson Immunoresearch, West Grove, Pa.) in a 5% nonfat milk/TBS-T solution. The blots were washed three times (1×TBS-T, 15 min), and peroxidase conjugated secondary antibody was activated by a 5 min. incubation with SuperSignal West Dura Luminol/Enhancer Solution® (Millipore, Temecula, Calif.), and chemiluminescence was detected and quantified with a Luminescent Image Analyzer LAS-3000 (FUJIFilm, Tokyo, Japan). Band density was determined using MultiGauge Software (FUJIFilm, Tokyo, Japan) and the total chemiluminescent signal was quantified for each band. To determine total ERK1/2 and AKT levels, membranes were stripped for 15 minute with 1× Reblot Plus Strong Solution® (Thermo Scientific, Rockford, Ill.) and reprobed with a 1:1000 dilution of either a rabbit polyclonal p44/42 MAPK antibody (Cell Signaling, Danvers, Mass.) or a rabbit monoclonal AKT antibody (Cell Signaling, Danvers, Mass.), and processed as described above for phospho-antibodies. Ph-ERK and ph-Akt levels were standardized to total ERK or total Akt, respectively.







TABLE 24







Inhibition Of MSP1-Induced ERK1/2 Phosphorylation


By RON Parent Antibodies Or DVD-Ig Constructs














N-Terminal VD
C-Terminal VD





Cellular Assay,
Cellular Assay,


Parent


Inhibition Of
Inhibition Of


Antibody
N-terminal
C-terminal
RON-Induced
RON-Induced


or
Variable
Variable
phERK1/2 (% of
phERK1/2 (% of


DVD-Ig
Domain
Domain
neutralization @
neutralization @


ID
(VD)
(VD)
nM Ab)
nM Ab)












AB005
RON
80% at 1000 nM











DVD024
RON
EGFR
97% at 1000 nM










AB005
RON
80% at 1000 nM











DVD033
RON
HGF
60% at 1000 nM










DVD-Igs containing the VD from AB005 in the N-terminal position showed excellent inhibition of MSP1-induced ERK1/2 phosphorylation. The inhibition profile of DVD024 and DVD033 was similar to the inhibition profile of parent antibody AB005.
Example 1.1.2.U
Efficacy of a DVD-Ig on the Growth of Human Carcinoma Subcutaneous Flank Xenografts
A-431 human epidermoid carcinoma cells were grown in vitro to 99% viability, 85% confluence in tissue culture flasks. SCID female mice (Charles Rivers Labs, Wilmington, Mass.) at 19-25 grams were injected subcutaneously into the right flank with 1×106 human tumor cells (1:1 matrigel) on study day 0. Administration (IP, QD, 3×/week) of human IgG control or DVD-Ig was initiated after mice were size matched into groups of mice with mean tumor volumes of approximately 200 to 320 mm3. The tumors were measured twice a week starting on approximately day 10 post tumor cell injection
Reduction in tumor volume was seen in animals administered EGFR+IGF1/2 DVD Ig relative to tumors in animals that received only control IgG. For two different EGFR+IGF1/2 DVD Ig constructs, % TGIs were 69 and 64 as measured four days after the end of the 3 week dosing phase.
Example 1.1.2.V
Binding of Monoclonal Antibodies to the Surface of Human Tumor Cell Lines as Assessed by Flow Cytometry
Stable cell lines overexpressing a cell-surface antigen of interest or human tumor cell lines were harvested from tissue culture flasks and resuspended in phosphate buffered saline (PBS) containing 5% fetal bovine serum (PBS/FBS). Prior to staining, human tumor cells were incubated on ice with (100 μl) human IgG at 5 μg/ml in PBS/FCS. 1-5×105 cells were incubated with antibody or DVD-Ig (2 μg/mL) in PBS/FBS for 30-60 minutes on ice. Cells were washed twice and 100 μl of F(ab′)2 goat anti human IgG, Fcγ-phycoerythrin (1:200 dilution in PBS) (Jackson ImmunoResearch, West Grove, Pa., Cat. #109-116-170) was added. After 30 minutes incubation on ice, cells were washed twice and resuspended in PBS/FBS. Fluorescence was measured using a Becton Dickinson FACSCalibur (Becton Dickinson, San Jose, Calif.).
Table 25 shows the FACS data for the DVD-Ig constructs. The geometric mean is the n root of the multiplication product of n fluorescent signals (a1×a2×a3 . . . an). With log-transformed data the geometric mean is used to normalize the weighting of the data distribution. The following table contains the FACS geometric mean of parent antibodies and DVD-Ig constructs.







TABLE 25







Fluorescent Activated Cell Sorting of DVD-Ig Constructs












N-terminal
C-terminal
FACS
FACS



Variable
Variable
Geometric
Geometric


Parent Antibody or
Domain
Domain
Mean
Mean


DVD-Ig ID
(VD)
(VD)
N-terminal
C-terminal












AB006
CD-19
979


AB001
CD-20











DVD001
CD-20
CD-19

161


DVD002
CD-19
CD20
1102









AB001
CD-20
95.8


AB002
CD-3
461











DVD003
CD-20
CD-3
45.9
5.7


DVD004
CD-3
CD-20
673
3.59









AB007
CD-80
25.4


AB001
CD-20











DVD005
CD-20
CD-80

6


DVD006
CD-80
CD-20
70.7









AB002
CD-3
897


AB004
HER-2
27.7











DVD011
CD-3
HER-2
1096
3.39


DVD012
HER-2
CD-3
7.14
41.6









AB002
CD-3
461/897


AB006
CD-19
661/989











DVD013
CD-3
CD-19
417
103


DVD014
CD-19
CD-3
1090
23.3









AB033
EGFR
955.4


AB004
HER-2
24.77











DVD015
EGFR
HER-2
1006.4
200.07


DVD016
HER-2
EGFR
75.67
384.4









AB033
EGFR
955.4


AB011
IGF1R
1684.5











DVD021
EGFR
IGF1R
888.4
733.5


DVD022
IGF1R
EGFR
2016.5
411.4









AB005
RON
54.46


AB033
EGFR
955.4











DVD024
RON
EGFR
0.38
309.4


DVD023
EGFR
RON
1001.4
164.96









AB033
EGFR
955.4


AB012
HGF











DVD025
EGFR
HGF
1045.4



DVD026
HGF
EGFR

143.4









AB004
HER-2
24.77


AB010
IGF1, 2











DVD029
HER-2
IGF1, 2
22.37



DVD030
IGF1, 2
HER-2

0.23









AB004
HER-2
24.77


AB011
IGF1R
1684.5











DVD031
HER-2
IGF1R
4.99
24.7


DVD032
IGF1R
HER-2
34.6
4.9









AB005
RON
54.46


AB012
HGF











DVD033
RON
HGF
6.46



DVD034
HGF
RON

0.39









AB014
VEGF



AB033
EGFR
955.4











DVD035
VEGF
EGFR

162.4


DVD036
EGFR
VEGF
1081.4









AB014
VEGF



AB004
HER-2
24.77











DVD037
VEGF
HER-2

1.03


DVD038
HER-2
VEGF
7.47









AB015
DLL-4
308.27


AB014
VEGF











DVD044
DLL4
VEGF
297.27



DVD043
VEGF
DLL4

68.37









AB014
VEGF



AB012
HGF









DVD045
VEGF
HGF


DVD046
HGF
VEGF









AB005
RON
54.46


AB014
VEGF











DVD048
RON
VEGF
29.86



DVD047
VEGF
RON

0.51









AB014
VEGF



AB016
NRP1
338.4











DVD049
VEGF
NRP1

352.4


DVD050
NRP1
VEGF
336.4









AB015
DLL-4
308.27


AB047
P1GF











DVD257
DLL4
P1GF
274.27



DVD258
P1GF
DLL4

5.57









All DVDs showed binding to their cell surface targets. The N-terminal domains of DVDs bound their targets on the cell surface as well as or better than the parent antibody. Binding can be restored or improved by adjusting linker length.
Example 1.1.2.W
Binding of Parent EGFR Antibody and DVD-Ig Constructs to the Surface of Human Tumor Cell Lines as Assessed by Flow Cytometry
Stable cell lines overexpressing cell-surface EGFR or human tumor cell lines were harvested from tissue culture flasks and resuspended in Dulbecco's phosphate buffered saline (DPBS) containing 1% fetal calf serum (DPBS/FCS). 1-5×105 cells were incubated with 100 μL antibodies or DVD-Igs (10 ug/mL) in DPBS/FCS for 30-60 minutes on ice. Cells were washed twice and 50 μl of goat anti-human IgG-phycoerythrin (1:50 dilution in DPBS/BSA) (Southern Biotech Associates, Birmingham, Ala. cat#2040-09) was added. After 30-45 minutes incubation on ice, cells were washed twice and resuspended in 125 uL/well 1% formaldehyde in DPBS/FCS. Fluorescence was measured using a Becton Dickinson LSRII (Becton Dickinson, San Jose, Calif.).







TABLE 26







Binding Affinity of anti-EGFR Parent Antibodies and


DVD-Ig Constructs to A431, EGFR Cell Line by FACS














N-terminal
C-terminal



N-terminal
C-terminal
VD Ag
VD Ag


Parent
Variable
Variable
binding A431
binding A431


Antibody or
Domain
Domain
FACS (EC50,
FACS (EC50,


DVD-Ig ID
(VD)
(VD)
nM)
nM)












AB005
RON



AB033
EGFR
4.2











DVD024
RON
EGFR
ND
25.43


DVD023
EGFR
RON
5.38
ND









All DVDs bound to their cell surface targets. The N-terminal domains of DVDs bound their targets on the cell surface as well as the parent antibody.







TABLE 27







Binding Affinity of anti-EGFR Parent Antibodies and DVD-Ig


Constructs to BAFvar3 Cell Line by FACS











Parent
N-
C-




Antibody
terminal
terminal
N-terminal VD
C-terminalVD


or
Variable
Variable
Ag binding
Ag binding


DVD-Ig
Domain
Domain
BAFvar3 FACS
BAFvar3 FACS


ID
(VD)
(VD)
(EC50, nM)
(EC50, nM)












AB005
RON



AB033
EGFR
0.5











DVD024
RON
EGFR
ND
17.8


DVD023
EGFR
RON
0.6
ND









All DVDs bound to their cell surface targets. The N-terminal domains of DVDs bound their targets on the cell surface as well as the parent antibody.
Example 1.2
Generation of Parent Monoclonal Antibodies to a Human Antigen of Interest
Parent mouse mAbs able to bind to and neutralize a human antigen of interest and a variant thereof are obtained as follows:
Example 1.2.A
Immunization of Mice With a Human Antigen of Interest
Twenty micrograms of recombinant purified human antigen (e.g., IGF1,2) mixed with complete Freund's adjuvant or Immunoeasy adjuvant (Qiagen, Valencia, Calif.) is injected subcutaneously into five 6-8 week-old Balb/C, five C57B/6 mice, and five AJ mice on Day 1. On days 24, 38, and 49, twenty micrograms of recombinant purified human antigen variant mixed with incomplete Freund's adjuvant or Immunoeasy adjuvant is injected subcutaneously into the same mice. On day 84 or day 112 or day 144, mice are injected intravenously with 1 μg recombinant purified human antigen of interest.
Example 1.2.B
Generation of a Hybridoma
Splenocytes obtained from the immunized mice described in Example 1.2.A are fused with SP2/O-Ag-14 cells at a ratio of 5:1 according to the established method described in Kohler, G. and Milstein (1975) Nature, 256:495 to generate hybridomas. Fusion products are plated in selection media containing azaserine and hypoxanthine in 96-well plates at a density of 2.5×106 spleen cells per well. Seven to ten days post fusion, macroscopic hybridoma colonies are observed. Supernatant from each well containing hybridoma colonies is tested by ELISA for the presence of antibody to the antigen of interest (as described in Example 1.1.1.A). Supernatants displaying antigen-specific activity are then tested for activity (as described in the assays of Example 1.1.2), for example, the ability to neutralize the antigen of interest in a bioassay such as that described in Example 1.1.2.I).
Example 1.2.C
Identification and Characterization of Parent Monoclonal Antibodies to a Human Target Antigen of Interest
Example 1.2.C.1
Analyzing Parent Monoclonal Antibody Neutralizing Activity
Hybridoma supernatants are assayed for the presence of parent antibodies that bind an antigen of interest, generated according to Examples 1.2.A and 1.2.B, and are also capable of binding a variant of the antigen of interest (“antigen variant”). Supernatants with antibodies positive in both assays are then tested for their antigen neutralization potency, for example, in the cytokine bioassay of Example 1.1.2.I. The hybridomas producing antibodies with IC50 values in the bioassay less than 1000 pM, in an embodiment, less than 100 pM are scaled up and cloned by limiting dilution. Hybridoma cells are expanded into media containing 10% low IgG fetal bovine serum (Hyclone #SH30151, Logan, Utah). On average, 250 mL of each hybridoma supernatant (derived from a clonal population) is harvested, concentrated and purified by protein A affinity chromatography, as described in Harlow, E. and Lane, D. 1988 “Antibodies: A Laboratory Manual”. The ability of purified mAbs to inhibit the activity of its target antigen is determined, for example, using the cytokine bioassay as described in Example 1.1.2.1.
Example 1.2.C.2
Analyzing Parent Monoclonal Antibody Cross-Reactivity to Cynomolgus Target Antigen of Interest
To determine whether the selected mAbs described herein recognize cynomolgus antigen of interest, BIACORE analysis is conducted as described herein (Example 1.1.1.G) using recombinant cynomolgus target antigen. In addition, neutralization potencies of mAbs against recombinant cynomolgus antigen of interest may also be measured in the cytokine bioassay (Example 1.1.2.I). MAbs with good cyno cross-reactivity (in an embodiment, within 5-fold of reactivity for human antigen) are selected for future characterization.
Example 1.2.D
Determination of the Amino Acid Sequence of the Variable Region for Each Murine Anti-Human Monoclonal Antibody
Isolation of the cDNAs, expression and characterization of the recombinant anti-human mouse mAbs is conducted as follows. For each amino acid sequence determination, approximately 1×106 hybridoma cells are isolated by centrifugation and processed to isolate total RNA with Trizol (Gibco BRL/Invitrogen, Carlsbad, Calif.) following manufacturer's instructions. Total RNA is subjected to first strand DNA synthesis using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, Calif.) per the manufacturer's instructions. Oligo(dT) is used to prime first-strand synthesis to select for poly(A)+ RNA. The first-strand cDNA product is then amplified by PCR with primers designed for amplification of murine immunoglobulin variable regions (Ig-Primer Sets, Novagen, Madison, Wis.). PCR products are resolved on an agarose gel, excised, purified, and then subcloned with the TOPO Cloning kit into pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) and transformed into TOP10 chemically competent E. coli (Invitrogen, Carlsbad, Calif.). Colony PCR is performed on the transformants to identify clones containing insert. Plasmid DNA is isolated from clones containing insert using a QIAprep Miniprep kit (Qiagen, Valencia, Calif.). Inserts in the plasmids are sequenced on both strands to determine the variable heavy or variable light chain DNA sequences using M13 forward and M13 reverse primers (Fermentas Life Sciences, Hanover Md.). Variable heavy and variable light chain sequences of the mAbs are identified. In an embodiment, the selection criteria for a panel of lead mAbs for next step development (humanization) includes the following:

    
    
        The antibody does not contain any N-linked glycosylation sites (NXS), except from the standard one in CH2
        The antibody does not contain any extra cysteines in addition to the normal cysteines in every antibody
        The antibody sequence is aligned with the closest human germline sequences for VH and VL and any unusual amino acids should be checked for occurrence in other natural human antibodies
        N-terminal Glutamine (Q) is changed to Glutamic acid (E) if it does not affect the activity of the antibody. This will reduce heterogeneity due to cyclization of Q
        Efficient signal sequence cleavage is confirmed by Mass Spectrophotometry. This can be done with COS cell or 293 cell material
        The protein sequence is checked for the risk of deamidation of Asn that could result in loss of activity
        The antibody has a low level of aggregation
        The antibody has solubility >5-10 mg/ml (in research phase); >25 mg/ml
        The antibody has a normal size (5-6 nm) by Dynamic Light Scattering (DLS)
        The antibody has a low charge heterogeneity
        The antibody lacks cytokine release (see Example 1.1.2.B)
        The antibody has specificity for the intended cytokine (see Example 1.1.2.C)
        The antibody lacks unexpected tissue cross reactivity (see Example 1.1.2.D)
        The antibody has similarity between human and cynomolgus tissue cross reactivity (see Example 1.1.2.D)
    
    


Example 1.2.2
Recombinant Humanized Parent Antibodies
Example 1.2.2.1
Construction and Expression of Recombinant Chimeric Anti Human Parent Antibodies
The DNA encoding the heavy chain constant region of murine anti-human parent mAbs is replaced by a cDNA fragment encoding the human IgG1 constant region containing 2 hinge-region amino acid mutations by homologous recombination in bacteria. These mutations are a leucine to alanine change at position 234 (EU numbering) and a leucine to alanine change at position 235 (Lund et al., 1991, J. Immunol., 147:2657). The light chain constant region of each of these antibodies is replaced by a human kappa constant region. Full-length chimeric antibodies are transiently expressed in COS cells by co-transfection of chimeric heavy and light chain cDNAs ligated into the pBOS expression plasmid (Mizushima and Nagata, Nucleic Acids Research 1990, Vol 18, pg 5322). Cell supernatants containing recombinant chimeric antibody are purified by Protein A Sepharose chromatography and bound antibody is eluted by addition of acid buffer. Antibodies are neutralized and dialyzed into PBS.
The heavy chain cDNA encoding a chimeric mAb is co-transfected with its chimeric light chain cDNA (both ligated in the pBOS vector) into COS cells. Cell supernatant containing recombinant chimeric antibody is purified by Protein A Sepharose chromatography and bound antibody is eluted by addition of acid buffer. Antibodies are neutralized and dialyzed into PBS.
The purified chimeric anti-human parent mAbs are then tested for their ability to bind (by Biacore) and for functional activity, e.g., to inhibit the cytokine induced production of IgE as described in Examples 1.1.1.G and 1.1.2.B. Chimeric mAbs that maintain the activity of the parent hybridoma mAbs are selected for future development.
Example 1.2.2.2
Construction and Expression of Humanized Anti Human Parent Antibodies
Example 1.2.2.2.A
Selection of Human Antibody Frameworks
Each murine variable heavy and variable light chain gene sequence is separately aligned against 44 human immunoglobulin germline variable heavy chain or 46 germline variable light chain sequences (derived from NCBI Ig Blast website using Vector NTI software.
Humanization is based on amino acid sequence homology, CDR cluster analysis, frequency of use among expressed human antibodies, and available information on the crystal structures of human antibodies. Taking into account possible effects on antibody binding, VH-VL pairing, and other factors, murine residues are mutated to human residues where murine and human framework residues are different, with a few exceptions. Additional humanization strategies are designed based on an analysis of human germline antibody sequences, or a subgroup thereof, that possessed a high degree of homology, i.e., sequence similarity, to the actual amino acid sequence of the murine antibody variable regions.
Homology modeling is used to identify residues unique to the murine antibody sequences that are predicted to be critical to the structure of the antibody combining site, the CDRs. Homology modeling is a computational method whereby approximate three dimensional coordinates are generated for a protein. The source of initial coordinates and guidance for their further refinement is a second protein, the reference protein, for which the three dimensional coordinates are known and the sequence of which is related to the sequence of the first protein. The relationship among the sequences of the two proteins is used to generate a correspondence between the reference protein and the protein for which coordinates are desired, the target protein. The primary sequences of the reference and target proteins are aligned with coordinates of identical portions of the two proteins transferred directly from the reference protein to the target protein. Coordinates for mismatched portions of the two proteins, e.g., from residue mutations, insertions, or deletions, are constructed from generic structural templates and energy refined to insure consistency with the already transferred model coordinates. This computational protein structure may be further refined or employed directly in modeling studies. The quality of the model structure is determined by the accuracy of the contention that the reference and target proteins are related and the precision with which the sequence alignment is constructed.
For the murine mAbs, a combination of BLAST searching and visual inspection is used to identify suitable reference structures. Sequence identity of 25% between the reference and target amino acid sequences is considered the minimum necessary to attempt a homology modeling exercise. Sequence alignments are constructed manually and model coordinates are generated with the program Jackal (see Petrey, D. et al. (2003) Proteins 53 (Suppl. 6): 430-435).
The primary sequences of the murine and human framework regions of the selected antibodies share significant identity. Residue positions that differ are candidates for inclusion of the murine residue in the humanized sequence in order to retain the observed binding potency of the murine antibody. A list of framework residues that differ between the human and murine sequences is constructed manually. Table 28 shows the framework sequences chosen for this study.








TABLE 28







Sequence Of Human IgG Heavy Chain Constant Domain



And Light Chain Constant Domain










SEQ
Sequence


Protein
ID NO
12345678901234567890123456789012345678901













Wild type hIgG1
95
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW



constant region

NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI




CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV




FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG




VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK




VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV




SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF




FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP




GK


 


Mutant hIgG1 constant
96
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW


region

NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI




CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSV




FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG




VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK




VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV




SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF




FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP




GK


 


Ig kappa constant
SEQ ID
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK


region
NO.: 97
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK




VYACEVTHQGLSSPVTKSFNRGEC


 


Ig Lambda
SEQ ID
QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW


constant region
NO.: 98
KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHR




SYSCQVTHEGSTVEKTVAPTECS









The likelihood that a given framework residue would impact the binding properties of the antibody depends on its proximity to the CDR residues. Therefore, using the model structures, the residues that differ between the murine and human sequences are ranked according to their distance from any atom in the CDRs. Those residues that fell within 4.5 Å of any CDR atom are identified as most important and are recommended to be candidates for retention of the murine residue in the humanized antibody (i.e., back mutation).
In silico constructed humanized antibodies are constructed using oligonucleotides. For each variable region cDNA, 6 oligonucleotides of 60-80 nucleotides each are designed to overlap each other by 20 nucleotides at the 5′ and/or 3′ end of each oligonucleotide. In an annealing reaction, all 6 oligonucleotides are combined, boiled, and annealed in the presence of dNTPs. DNA polymerase I, Large (Klenow) fragment (New England Biolabs #M0210, Beverley, Mass.) is added to fill-in the approximately 40 bp gaps between the overlapping oligonucleotides. PCR is performed to amplify the entire variable region gene using two outermost primers containing overhanging sequences complementary to the multiple cloning site in a modified pBOS vector (Mizushima, S, and Nagata, S. (1990) Nucleic Acids Res. 18: 17). The PCR products derived from each cDNA assembly are separated on an agarose gel and the band corresponding to the predicted variable region cDNA size is excised and purified. The variable heavy region is inserted in-frame onto a cDNA fragment encoding the human IgG1 constant region containing 2 hinge-region amino acid mutations by homologous recombination in bacteria. These mutations are a leucine to alanine change at position 234 (EU numbering) and a leucine to alanine change at position 235 (Lund et al. (1991) J. Immunol. 147:2657). The variable light chain region is inserted in-frame with the human kappa constant region by homologous recombination. Bacterial colonies are isolated and plasmid DNA extracted. cDNA inserts are sequenced in their entirety. Correct humanized heavy and light chains corresponding to each antibody are co-transfected into COS cells to transiently produce full-length humanized anti-human antibodies. Cell supernatants containing recombinant chimeric antibody are purified by Protein A Sepharose chromatography and bound antibody is eluted by addition of acid buffer. Antibodies are neutralized and dialyzed into PBS.
Example 1.2.2.3
Characterization of Humanized Antibodies
The ability of purified humanized antibodies to inhibit a functional activity is determined, e.g., using the cytokine bioassay as described in Examples 1.1.2.A. The binding affinities of the humanized antibodies to recombinant human antigen are determined using surface plasmon resonance (Biacore®) measurement as described in Example 1.1.1.B. The IC50 values from the bioassays and the affinity of the humanized antibodies are ranked. The humanized mAbs that fully maintain the activity of the parent hybridoma mAbs are selected as candidates for future development. The top 2-3 most favorable humanized mAbs are further characterized.
Example 1.2.2.3.A
Pharmacokinetic Analysis of Humanized Antibodies
Pharmacokinetic studies are carried out in Sprague-Dawley rats and cynomolgus monkeys. Male and female rats and cynomolgus monkeys are dosed intravenously or subcutaneously with a single dose of 4 mg/kg mAb and samples are analyzed using antigen capture ELISA, and pharmacokinetic parameters are determined by noncompartmental analysis. Briefly, ELISA plates are coated with goat anti-biotin antibody (5 mg/ml, 4° C., overnight), blocked with Superblock (Pierce), and incubated with biotinylated human antigen at 50 ng/ml in 10% Superblock TTBS at room temperature for 2 hours. Serum samples are serially diluted (0.5% serum, 10% Superblock in TTBS) and incubated on the plate for 30 minutes at room temperature. Detection is carried out with HRP-labeled goat anti human antibody and concentrations are determined with the help of standard curves using the four parameter logistic fit. Values for the pharmacokinetic parameters are determined by non-compartmental model using WinNonlin software (Pharsight Corporation, Mountain View, Calif.). Humanized mAbs with good pharmacokinetics profile (T1/2 is 8-13 days or better, with low clearance and excellent bioavailability 50-100%) are selected.
Example 1.2.2.3.B
Physicochemical and In Vitro Stability Analysis of Humanized Monoclonal Antibodies
Size Exclusion Chromatography
Antibodies are diluted to 2.5 mg/mL with water and 20 mL is analyzed on a Shimadzu HPLC system using a TSK gel G3000 SWXL column (Tosoh Bioscience, cat# k5539-05k). Samples are eluted from the column with 211 mM sodium sulfate, 92 mM sodium phosphate, pH 7.0, at a flow rate of 0.3 mL/minutes. The HPLC system operating conditions are the following:
















Mobile phase:
211 mM Na2SO4, 92 mM




Na2HPO4*7H2O, pH 7.0



Gradient:
Isocratic



Flow rate:
0.3 mL/minute



Detector wavelength:
280 nm



Autosampler cooler temp:
4° C.



Column oven temperature:
Ambient



Run time:
50 minutes










Table 29 contains purity data of parent antibodies and DVD-Ig constructs expressed as percent monomer (unaggregated protein of the expected molecular weight) as determined by the above protocol.







TABLE 29







Purity of Parent Antibodies and DVD-Ig Constructs as


Determined by Size Exclusion Chromatography












Parent
N-terminal
C-terminal




Antibody
Variable
Variable
%



or
Domain
Domain
Monomer



DVD-Ig ID
(VD)
(VD)
(purity)















AB033
EGFR
99.75



AB004
HER-2
95.6












DVD015
EGFR
HER-2
96.98



DVD016
HER-2
EGFR
93.41











AB033
EGFR
99.75



AB011
IGF1R
98.23












DVD021
EGFR
IGF1R
99.25



DVD022
IGF1R
EGFR
99.53











AB005
RON
79.34



AB033
EGFR
99.75












DVD024
RON
EGFR
96.86



DVD023
EGFR
RON
97.27











AB033
EGFR
99.75



AB012
HGF
98.97












DVD025
EGFR
HGF
99.21



DVD026
HGF
EGFR
99.48











AB004
HER-2
95.6



AB010
IGF1, 2
93.2












DVD029
HER-2
IGF1, 2
88.2



DVD030
IGF1, 2
HER-2
96.9











AB004
HER-2
95.6



AB011
IGF1R
98.23












DVD031
HER-2
IGF1R
94.53



DVD032
IGF1R
HER-2
98.1











AB005
RON
79.34



AB012
HGF
98.97












DVD033
RON
HGF
100



DVD034
HGF
RON
100











AB014
VEGF
97.81



AB033
EGFR
99.75












DVD035
VEGF
EGFR
97.7



DVD036
EGFR
VEGF
99.36











AB014
VEGF
97.81



AB004
HER-2
95.6












DVD037
VEGF
HER-2
45.4



DVD038
HER-2
VEGF
96.9











AB014
VEGF
97.81



AB010
IGF1, 2
93.2












DVD041
VEGF
IGF1, 2
94.7



DVD042
IGF1, 2
VEGF
92.4











AB015
DLL-4
97.52



AB014
VEGF
97.81












DVD044
DLL4
VEGF
48.66



DVD043
VEGF
DLL4
96.72











AB014
VEGF
97.81



AB012
HGF
98.97












DVD045
VEGF
HGF
97.88



DVD046
HGF
VEGF
98.14











AB005
RON
79.34



AB014
VEGF
97.81












DVD048
RON
VEGF
100



DVD047
VEGF
RON
100











AB014
VEGF
97.81



AB016
NRP1
93.54












DVD049
VEGF
NRP1
97.74



DVD050
NRP1
VEGF
98.59











AB015
DLL-4
97.52



AB047
P1GF
99.47












DVD257
DLL4
P1GF
83.8



DVD258
P1GF
DLL4
100











AB014
VEGF
97.81



AB047
P1GF
99.47












DVD259
VEGF
P1GF
90.61



DVD260
P1GF
VEGF
98.57











AB062
ErbB3
100



AB033
EGFR
99.75












DVD299
ErbB3
EGFR
98.8



DVD300
EGFR
ErbB3
100











AB063
ErbB3
96



AB033
EGFR
99.75












DVD305
ErbB3
EGFR
83.8



DVD306
EGFR
ErbB3
100










DVD-Igs showed an excellent SEC profile with most DVD-Ig showing >90% monomer. This DVD-Ig profile is similar to that observed for parent antibodies.
SDS-PAGE
Antibodies are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under both reducing and non-reducing conditions. Adalimumab lot AFP04C is used as a control. For reducing conditions, the samples are mixed 1:1 with 2× tris glycine SDS-PAGE sample buffer (Invitrogen, cat# LC2676, lot#1323208) with 100 mM DTT, and heated at 60° C. for 30 minutes. For non-reducing conditions, the samples are mixed 1:1 with sample buffer and heated at 100° C. for 5 minutes. The reduced samples (10 mg per lane) are loaded on a 12% pre-cast tris-glycine gel (Invitrogen, cat# EC6005box, lot#6111021), and the non-reduced samples (10 mg per lane) are loaded on an 8%-16% pre-cast tris-glycine gel (Invitrogen, cat# EC6045box, lot#6111021). SeeBlue Plus 2 (Invitrogen, cat#LC5925, lot#1351542) is used as a molecular weight marker. The gels are run in a XCell SureLock mini cell gel box (Invitrogen, cat# EI0001) and the proteins are separated by first applying a voltage of 75 to stack the samples in the gel, followed by a constant voltage of 125 until the dye front reached the bottom of the gel. The running buffer used is 1× tris glycine SDS buffer, prepared from a 10× tris glycine SDS buffer (ABC, MPS-79-080106)). The gels are stained overnight with colloidal blue stain (Invitrogen cat#46-7015, 46-7016) and destained with Milli-Q water until the background is clear. The stained gels are then scanned using an Epson Expression scanner (model 1680, S/N DASX003641).
Sedimentation Velocity Analysis
Antibodies are loaded into the sample chamber of each of three standard two-sector carbon epon centerpieces. These centerpieces have a 1.2 cm optical path length and are built with sapphire windows. PBS is used for a reference buffer and each chamber contained 140 μL. All samples are examined simultaneously using a 4-hole (AN-60Ti) rotor in a Beckman ProteomeLab XL-I analytical ultracentrifuge (serial # PL106C01).
Run conditions are programmed and centrifuge control is performed using ProteomeLab (v5.6). The samples and rotor are allowed to thermally equilibrate for one hour prior to analysis (20.0±0.1° C.). Confirmation of proper cell loading is performed at 3000 rpm and a single scan is recorded for each cell. The sedimentation velocity conditions are the following:
Sample Cell Volume: 420 mL
Reference Cell Volume: 420 mL
Temperature: 20° C.
Rotor Speed: 35,000 rpm
Time: 8:00 hours
UV Wavelength: 280 nm
Radial Step Size: 0.003 cm
Data Collection One data point per step without signal averaging.
Total Number of Scans: 100
LC-MS Molecular Weight Measurement of Intact Antibodies
Molecular weight of intact antibodies are analyzed by LC-MS. Each antibody is diluted to approximately 1 mg/mL with water. An 1100 HPLC (Agilent) system with a protein microtrap (Michrom Bioresources, Inc, cat#004/25109/03) is used to desalt and introduce 5 mg of the sample into an API Qstar pulsar i mass spectrometer (Applied Biosystems). A short gradient is used to elute the samples. The gradient is run with mobile phase A (0.08% FA, 0.02% TFA in HPLC water) and mobile phase B (0.08% FA and 0.02% TFA in acetonitrile) at a flow rate of 50 mL/minute. The mass spectrometer is operated at 4.5 kvolts spray voltage with a scan range from 2000 to 3500 mass to charge ratio.
LC-MS Molecular Weight Measurement of Antibody Light and Heavy Chains
Molecular weight measurement of antibody light chain (LC), heavy chain (HC) and deglycosylated HC are analyzed by LC-MS. An antibody is diluted to 1 mg/mL with water and the sample is reduced to LC and HC with a final concentration of 10 mM DTT for 30 minutes at 37° C. To deglycosylate the antibody, 100 mg of the antibody is incubated with 2 mL of PNGase F, 5 mL of 10% N-octylglucoside in a total volume of 100 mL overnight at 37° C. After deglycosylation the sample is reduced with a final concentration of 10 mM DTT for 30 minutes at 37° C. An Agilent 1100 HPLC system with a C4 column (Vydac, cat#214TP5115, S/N 060206537204069) is used to desalt and introduce the sample (5 mg) into an API Qstar pulsar i mass spectrometer (Applied Biosystems). A short gradient is used to elute the sample. The gradient is run with mobile phase A (0.08% FA, 0.02% TFA in HPLC water) and mobile phase B (0.08% FA and 0.02% TFA in acetonitrile) at a flow rate of 50 mL/minute. The mass spectrometer is operated at 4.5 kvolts spray voltage with a scan range from 800 to 3500 mass to charge ratio.
Peptide Mapping
Antibody is denatured for 15 minutes at room temperature with a final concentration of 6 M guanidine hydrochloride in 75 mM ammonium bicarbonate. The denatured samples are reduced with a final concentration of 10 mM DTT at 37° C. for 60 minutes, followed by alkylation with 50 mM iodoacetic acid (IAA) in the dark at 37° C. for 30 minutes. Following alkylation, the sample is dialyzed overnight against four liters of 10 mM ammonium bicarbonate at 4° C. The dialyzed sample is diluted to 1 mg/mL with 10 mM ammonium bicarbonate, pH 7.8 and 100 mg of antibody is either digested with trypsin (Promega, cat# V5111) or Lys-C (Roche, cat#11 047 825 001) at a 1:20 (w/w) trypsin/Lys-C:antibody ratio at 37° C. for 4 hrs. Digests are quenched with 1 mL of 1 N HCl. For peptide mapping with mass spectrometer detection, 40 mL of the digests are separated by reverse phase high performance liquid chromatography (RPHPLC) on a C18 column (Vydac, cat#218TP51, S/N NE9606 10.3.5) with an Agilent 1100 HPLC system. The peptide separation is run with a gradient using mobile phase A (0.02% TFA and 0.08% FA in HPLC grade water) and mobile phase B (0.02% TFA and 0.08% FA in acetonitrile) at a flow rate of 50 mL/minutes. The API QSTAR Pulsar i mass spectromer is operated in positive mode at 4.5 kvolts spray voltage and a scan range from 800 to 2500 mass to charge ratio.
Disulfide Bond Mapping
To denature the antibody, 100 mL of the antibody is mixed with 300 mL of 8 M guanidine HCl in 100 mM ammonium bicarbonate. The pH is checked to ensure that it is between 7 and 8 and the samples are denatured for 15 minutes at room temperature in a final concentration of 6 M guanidine HCl. A portion of the denatured sample (100 mL) is diluted to 600 mL with Milli-Q water to give a final guanidine-HCl concentration of 1 M. The sample (220 mg) is digested with either trypsin (Promega, cat #V5111, lot#22265901) or Lys-C (Roche, cat#11047825001, lot#12808000) at a 1:50 trypsin or 1:50 Lys-C: antibody (w/w) ratios (4.4 mg enzyme: 220 mg sample) at 37° C. for approximately 16 hours. An additional 5 mg of trypsin or Lys-C is added to the samples and digestion is allowed to proceed for an additional 2 hours at 37° C. Digestions are stopped by adding 1 mL of TFA to each sample. Digested samples are separated by RPHPLC using a C18 column (Vydac, cat#218TP51 S/N NE020630-4-1A) on an Agilent HPLC system. The separation is run with the same gradient used for peptide mapping using mobile phase A (0.02% TFA and 0.08% FA in HPLC grade water) and mobile phase B (0.02% TFA and 0.08% FA in acetonitrile) at a flow rate of 50 mL/minute. The HPLC operating conditions are the same as those used for peptide mapping. The API QSTAR Pulsar i mass spectromer is operated in positive mode at 4.5 kvolts spray voltage and a scan range from 800 to 2500 mass-to-charge ratio. Disulfide bonds are assigned by matching the observed MWs of peptides with the predicted MWs of tryptic or Lys-C peptides linked by disulfide bonds.
Free Sulfhydryl Determination
The method used to quantify free cysteines in an antibody is based on the reaction of Ellman's reagent, 5,5¢-dithio-bis(2-nitrobenzoic acid) (DTNB), with sulfhydryl groups (SH) which gives rise to a characteristic chromophoric product, 5-thio-(2-nitrobenzoic acid) (TNB). The reaction is illustrated in the formula:

DTNB+RSH®RS-TNB+TNB−+H+

The absorbance of the TNB− is measured at 412 nm using a Cary 50 spectrophotometer. An absorbance curve is plotted using dilutions of 2 mercaptoethanol (b-ME) as the free SH standard and the concentrations of the free sulfhydryl groups in the protein are determined from absorbance at 412 nm of the sample.
The b-ME standard stock is prepared by a serial dilution of 14.2 M b-ME with HPLC grade water to a final concentration of 0.142 mM. Then standards in triplicate for each concentration are prepared. Antibody is concentrated to 10 mg/mL using an amicon ultra 10,000 MWCO centrifugal filter (Millipore, cat# UFC801096, lot# L3KN5251) and the buffer is changed to the formulation buffer used for adalimumab (5.57 mM sodium phosphate monobasic, 8.69 mM sodium phosphate dibasic, 106.69 mM NaCl, 1.07 mM sodium citrate, 6.45 mM citric acid, 66.68 mM mannitol, pH 5.2, 0.1% (w/v) Tween). The samples are mixed on a shaker at room temperature for 20 minutes. Then 180 mL of 100 mM Tris buffer, pH 8.1 is added to each sample and standard followed by the addition of 300 mL of 2 mM DTNB in 10 mM phosphate buffer, pH 8.1. After thorough mixing, the samples and standards are measured for absorption at 412 nm on a Cary 50 spectrophotometer. The standard curve is obtained by plotting the amount of free SH and OD412 nm of the b-ME standards. Free SH content of samples are calculated based on this curve after subtraction of the blank.
Weak Cation Exchange Chromatography
Antibody is diluted to 1 mg/mL with 10 mM sodium phosphate, pH 6.0. Charge heterogeneity is analyzed using a Shimadzu HPLC system with a WCX-10 ProPac analytical column (Dionex, cat#054993, S/N 02722). The samples are loaded on the column in 80% mobile phase A (10 mM sodium phosphate, pH 6.0) and 20% mobile phase B (10 mM sodium phosphate, 500 mM NaCl, pH 6.0) and eluted at a flow rate of 1.0 mL/minute.
Oligosaccharide Profiling
Oligosaccharides released after PNGase F treatment of antibody are derivatized with 2-aminobenzamide (2-AB) labeling reagent. The fluorescent-labeled oligosaccharides are separated by normal phase high performance liquid chromatography (NPHPLC) and the different forms of oligosaccharides are characterized based on retention time comparison with known standards.
The antibody is first digested with PNGaseF to cleave N-linked oligosaccharides from the Fc portion of the heavy chain. The antibody (200 mg) is placed in a 500 mL Eppendorf tube along with 2 mL PNGase F and 3 mL of 10% N-octylglucoside. Phosphate buffered saline is added to bring the final volume to 60 mL. The sample is incubated overnight at 37° C. in an Eppendorf thermomixer set at 700 RPM. Adalimumab lot AFP04C is also digested with PNGase F as a control.
After PNGase F treatment, the samples are incubated at 95° C. for 5 minutes in an Eppendorf thermomixer set at 750 RPM to precipitate out the proteins, then the samples are placed in an Eppendorf centrifuge for 2 minutes at 10,000 RPM to spin down the precipitated proteins. The supernatant containing the oligosaccharides are transferred to a 500 mL Eppendorf tube and dried in a speed-vac at 65° C.
The oligosaccharides are labeled with 2AB using a 2AB labeling kit purchased from Prozyme (cat# GKK-404, lot#132026). The labeling reagent is prepared according to the manufacturer's instructions. Acetic acid (150 mL, provided in kit) is added to the DMSO vial (provided in kit) and mixed by pipeting the solution up and down several times. The acetic acid/DMSO mixture (100 mL) is transferred to a vial of 2-AB dye (just prior to use) and mixed until the dye is fully dissolved. The dye solution is then added to a vial of reductant (provided in kit) and mixed well (labeling reagent). The labeling reagent (5 mL) is added to each dried oligosaccharide sample vial, and mixed thoroughly. The reaction vials are placed in an Eppendorf thermomixer set at 65° C. and 700-800 RPM for 2 hours of reaction.
After the labeling reaction, the excess fluorescent dye is removed using GlycoClean S Cartridges from Prozyme (cat# GKI-4726). Prior to adding the samples, the cartridges are washed with 1 mL of milli-Q water followed with 5 ishes of 1 mL 30% acetic acid solution. Just prior to adding the samples, 1 mL of acetonitrile (Burdick and Jackson, cat# AH015-4) is added to the cartridges.
After all of the acetonitrile passed through the cartridge, the sample is spotted onto the center of the freshly washed disc and allowed to adsorb onto the disc for 10 minutes. The disc is washed with 1 mL of acetonitrile followed by five ishes of 1 mL of 96% acetonitrile. The cartridges are placed over a 1.5 mL Eppendorf tube and the 2-AB labeled oligosaccharides are eluted with 3 ishes (400 mL each ish) of milli Q water.
The oligosaccharides are separated using a Glycosep N HPLC (cat# GKI-4728) column connected to a Shimadzu HPLC system. The Shimadzu HPLC system consisted of a system controller, degasser, binary pumps, autosampler with a sample cooler, and a fluorescent detector.
Stability at Elevated Temperatures
The buffer of antibody is either 5.57 mM sodium phosphate monobasic, 8.69 mM sodium phosphate dibasic, 106.69 mM NaCl, 1.07 mM sodium citrate, 6.45 mM citric acid, 66.68 mM mannitol, 0.1% (w/v) Tween, pH 5.2; or 10 mM histidine, 10 mM methionine, 4% mannitol, pH 5.9 using Amicon ultra centrifugal filters. The final concentration of the antibodies is adjusted to 2 mg/mL with the appropriate buffers. The antibody solutions are then filter sterized and 0.25 mL aliquots are prepared under sterile conditions. The aliquots are left at either −80° C., 5° C., 25° C., or 40° C. for 1, 2 or 3 weeks. At the end of the incubation period, the samples are analyzed by size exclusion chromatography and SDS-PAGE.
The stability samples are analyzed by SDS-PAGE under both reducing and non-reducing conditions. The procedure used is the same as described herein. The gels are stained overnight with colloidal blue stain (Invitrogen cat#46-7015, 46-7016) and destained with Milli-Q water until the background is clear. The stained gels are then scanned using an Epson Expression scanner (model 1680, S/N DASX003641). To obtain more sensitivity, the same gels are silver stained using silver staining kit (Owl Scientific) and the recommended procedures given by the manufacturer is used.
Example 1.2.2.3.C
Efficacy of a Humanized Monoclonal Antibody by Itself or in Combination with Chemotherapy on the Growth of Human Carcinoma Xenografts
Human cancer cells are grown in vitro to 99% viability, 85% confluence in tissue culture flasks. SCID female or male mice (Charles Rivers Labs) at 19-25 grams, are ear tagged and shaved. Mice are then inoculated subcutaneously into the right flank with 0.2 ml of 2×106 human tumor cells (1:1 matrigel) on study day 0. Administration (IP, Q3D/week) of vehicle (PBS), humanized antibody, and/or chemotherapy is initiated after mice are size matched into separate cages of mice with mean tumor volumes of approximately 150 to 200 mm3 The tumors are measured by a pair of calipers twice a week starting on approximately day 10 post inoculation and the tumor volumes calculated according to the formula V=L×W2/2 (V: volume, mm3; L: length, mm; W: width, m) Reduction in tumor volume is seen in animals treated with mAb alone or in combination with chemotherapy relative to tumors in animals that received only vehicle or an isotype control mAb.
Example 1.2.2.3.D
FACS Based Redirected Cytotoxicity (rCTL) Assay
Human CD3+ T cells were isolated from previously frozen isolated peripheral blood mononuclear cells (PBMC) by a negative selection enrichment column (R&D Systems, Minneapolis, Minn.; Cat.#HTCC-525). T cells were stimulated for 4 days in flasks (vent cap, Corning, Acton, Mass.) coated with 10 μg/mL anti-CD3 (OKT-3, eBioscience, Inc., San Diego, Calif.) and 2 μg/mL anti-CD28 (CD28.2, eBioscience, Inc., San Diego, Calif.) in D-PBS (Invitrogen, Carlsbad, Calif.) and cultured in 30 U/mL IL-2 (Roche) in complete RPMI 1640 media (Invitrogen, Carlsbad, Calif.) with L-glutamine, 55 mM β-ME, Pen/Strep, 10% FBS). T cells were then rested overnight in 30 U/mL IL-2 before using in assay. DoHH2 or Raji target cells were labeled with PKH26 (Sigma-Aldrich, St. Louis, Mo.) according to manufacturer's instructions. RPMI 1640 media (no phenol, Invitrogen, Carlsbad, Calif.) containing L-glutamine and 10% FBS (Hyclone, Logan, Utah) was used throughout the rCTL assay. (See Dreier et al. (2002) Int J Cancer 100:690).
Effector T cells (E) and targets (T) were plated at a final cell concentration of 105 and 104 cells/well in 96-well plates (Costar #3799, Acton, Mass.), respectively to give an E:T ratio of 10:1. DVD-Ig molecules were diluted to obtain concentration-dependent titration curves. After an overnight incubation cells are pelleted and washed with D-PBS once before resuspending in FACS buffer containing 0.1% BSA (Invitrogen, Carlsbad, Calif.), 0.1% sodium azide and 0.5 μg/mL propidium iodide (BD) in D-PBS. FACS data was collected on a FACS Canto II machine (Becton Dickinson, San Jose, Calif.) and analyzed in Flowjo (Treestar). The percent live targets in the DVD-Ig treated samples divided by the percent total targets (control, no treatment) was calculated to determine percent specific lysis. IC50s were calculated in Prism (Graphpad).
The CD3/CD 19 DVD-Ig (AB sequence IDs, AB002+AB006; Example 2.7) was tested in the redirected toxicity assay for tumor cell killing. This DVD-Ig showed in vitro tumor killing with an IC50=5.0 pM A CD3/CD20 DVD-Ig was also tested for redirected toxicity and showed in vitro tumor killing with an IC50=325 pM. The sequence of this CD3/CD20 DVD-Ig was disclosed in US Patent Application Serial No. 20070071675.
Example 1.4
Generation of a DVD-Ig
DVD-Ig molecules capable of binding two antigens are constructed using two parent monoclonal antibodies, one against human antigen A, and the other against human antigen B, selected as described herein.
Example 1.4.1
Generation of a DVD-Ig Having Two Linker Lengths
A constant region containing μl Fc with mutations at 234, and 235 to eliminate ADCC/CDC effector functions is used. Four different anti-A/B DVD-Ig constructs are generated: 2 with short linker and 2 with long linker, each in two different domain orientations: VA-VB-C and VB-VA-C (see Table 30). The linker sequences, derived from the N-terminal sequence of human Cl/Ck or CH1 domain, are as follows:
For DVDAB constructs:
light chain (if anti-A has λ): Short linker: QPKAAP; Long linker: QPKAAPSVTLFPP
light chain (if anti-A has κ): Short linker: TVAAP; Long linker: TVAAPSVFIFPP
heavy chain (γ1): Short linker: ASTKGP; Long linker: ASTKGPSVFPLAP
For DVDBA constructs:
light chain (if anti-B has λ): Short linker: QPKAAP; Long linker: QPKAAPSVTLFPP
light chain (if anti-B has k): Short linker: TVAAP; Long linker: TVAAPSVFIFPP
heavy chain (γ1): Short linker: ASTKGP; Long linker: ASTKGPSVFPLAP
Heavy and light chain constructs are subcloned into the pBOS expression vector, and expressed in COS cells, followed by purification by Protein A chromatography. The purified materials are subjected to SDS-PAGE and SEC analysis.
Table 30 describes the heavy chain and light chain constructs used to express each anti-A/B DVD-Ig protein.







TABLE 30







Anti-A/B DVD-Ig Constructs









DVD-Ig protein
Heavy chain construct
Light chain construct





DVDABSL
DVDABHC-SL
DVDABLC-SL


DVDABLL
DVDABHC-LL
DVDABLC-LL


DVDBASL
DVDBAHC-SL
DVDBALC-SL


DVDBALL
DVDBAHC-LL
DVDBALC-LL









Example 1.4.2
Molecular Cloning of DNA Constructs for DVDABSL and DVDABLL
To generate heavy chain constructs DVDABHC-LL and DVDABHC-SL, VH domain of A antibody is PCR amplified using specific primers (3′ primers contain short/long liner sequence for SL/LL constructs, respectively); meanwhile VH domain of B antibody is amplified using specific primers (5′ primers contains short/long liner sequence for SL/LL constructs, respectively). Both PCR reactions are performed according to standard PCR techniques and procedures. The two PCR products are gel-purified, and used together as overlapping template for the subsequent overlapping PCR reaction. The overlapping PCR products are subcloned into Srf I and Sal I double digested pBOS-hCγ1,z non-a mammalian expression vector (Abbott) by using standard homologous recombination approach.
To generate light chain constructs DVDABLC-LL and DVDABLC-SL, VL domain of A antibody is PCR amplified using specific primers (3′ primers contain short/long liner sequence for SL/LL constructs, respectively); meanwhile VL domain of B antibody is amplified using specific primers (5′ primers contains short/long liner sequence for SL/LL constructs, respectively). Both PCR reactions are performed according to standard PCR techniques and procedures. The two PCR products are gel-purified, and used together as overlapping template for the subsequent overlapping PCR reaction using standard PCR conditions. The overlapping PCR products are subcloned into Srf I and Not I double digested pBOS-hCk mammalian expression vector (Abbott) by using standard homologous recombination approach. Similar approach has been used to generate DVDBASL and DVDBALL as described below:
Example 1.4.3
Molecular Cloning of DNA Constructs for DVDBASL and DVDBALL
To generate heavy chain constructs DVDBAHC-LL and DVDBAHC-SL, VH domain of antibody B is PCR amplified using specific primers (3′ primers contain short/long liner sequence for SL/LL constructs, respectively); meanwhile VH domain of antibody A is amplified using specific primers (5′ primers contains short/long liner sequence for SL/LL constructs, respectively). Both PCR reactions are performed according to standard PCR techniques and procedures. The two PCR products are gel-purified, and used together as overlapping template for the subsequent overlapping PCR reaction using standard PCR conditions. The overlapping PCR products are subcloned into Srf I and Sal I double digested pBOS-hCγ1,z non-a mammalian expression vector (Abbott) by using standard homologous recombination approach.
To generate light chain constructs DVDBALC-LL and DVDBALC-SL, VL domain of antibody B is PCR amplified using specific primers (3′ primers contain short/long liner sequence for SL/LL constructs, respectively); meanwhile VL domain of antibody A is amplified using specific primers (5′ primers contains short/long liner sequence for SL/LL constructs, respectively). Both PCR reactions are performed according to standard PCR techniques and procedures. The two PCR products are gel-purified, and used together as overlapping template for the subsequent overlapping PCR reaction using standard PCR conditions. The overlapping PCR products are subcloned into Srf I and Not I double digested pBOS-hCk mammalian expression vector (Abbott) by using standard homologous recombination approach.
Example 1.4.4
Construction and Expression of Additional DVD-Ig
Example 1.4.4.1
Preparation of DVD-Ig Vector Constructs
Parent antibody amino acid sequences for specific antibodies, which recognize specific antigens or epitopes thereof, for incorporation into a DVD-Ig can be obtained by preparation of hybridomas as described above or can be obtained by sequencing known antibody proteins or nucleic acids. In addition, known sequences can be obtained from the literature. The sequences can be used to synthesize nucleic acids using standard DNA synthesis or amplification technologies and assembling the desired antibody fragments into expression vectors, using standard recombinant DNA technology, for expression in cells.
For example, nucleic acid codons were determined from amino acids sequences and oligonucleotide DNA was synthesized by Blue Heron Biotechnology, Inc., Bothell, Wash. USA. The oligonucleotides were assembled into 300-2,000 base pair double-stranded DNA fragments, cloned into a plasmid vector and sequence-verified. Cloned fragments were assembled using an enzymatic process to yield the complete gene and subcloned into an expression vector. (See U.S. Pat. Nos. 7,306,914; 7,297,541; 7,279,159; 7,150,969; and U.S. Patent Application Publication Nos. 20080115243; 20080102475; 20080081379; 20080075690; 20080063780; 20080050506; 20080038777; 20080022422; 20070289033; 20070287170; 20070254338; 20070243194; 20070225227; 20070207171; 20070150976; 20070135620; 20070128190; 20070104722; 20070092484; 20070037196; 20070028321; 20060172404; 20060162026; 20060153791; 20030215458; 20030157643).
A group of pHybE vectors (U.S. Patent Application Ser. No. 61/021,282) were used for parental antibody and DVD-Ig cloning. V1, derived from pJP183; pHybE-hCg1,z,non-a V2, was used for cloning of antibody and DVD heavy chains with a wildtype constant region. V2, derived from pJP191; pHybE-hCk V2, was used for cloning of antibody and DVD light chains with a kappa constant region. V3, derived from pJP192; pHybE-hCl V2, was used for cloning of antibody and DVDs light chains with a lambda constant region. V4, built with a lambda signal peptide and a kappa constant region, was used for cloning of DVD light chains with a lambda-kappa hybrid V domain. V5, built with a kappa signal peptide and a lambda constant region, was used for cloning of DVD light chains with a kappa-lambda hybrid V domain. V7, derived from pJP183; pHybE-hCg1,z,non-a V2, was used for cloning of antibody and DVD heavy chains with a (234,235 AA) mutant constant region.
Referring to Table 31, a number of vectors were used in the cloning of the parent antibodies and DVD-Ig VH and VL chains.







TABLE 31







Vectors Used to Clone Parent Antibodies and DVD-Igs












Heavy
Light




chain
chain



ID
vector
vector







AB001
V1
V2



AB002
V1
V2



AB003
V1
V2



AB004
V1
V2



AB005
V1
V2



AB006
V1
V2



AB007
V1
V3



AB008
V1
V2



AB009
V1
V2



AB010
V1
V3



AB011
V1
V2



AB012
V1
V2



AB013
V1
V2



AB014
V1
V2



AB015
V1
V2



AB016
V1
V2



AB033
V1
V2



AB034
V1
V2



AB035
V1
V2



AB039
V1
V2



AB047
V1
V2



AB062
V1
V2



AB063
V1
V2



AB067
V7
V3



AB069
V1
V2



AB070
V1
V2



AB071
V7
V2



AB072
V1
V2



AB073
V1
V3



AB074
V1
V2



AB075
V1
V3



AB077
V1
V2



AB079
V1
V2



AB080
V1
V2



DVD001
V1
V2



DVD002
V1
V2



DVD003
V1
V2



DVD004
V1
V2



DVD005
V1
V5



DVD006
V1
V4



DVD007
V1
V2



DVD008
V1
V2



DVD009
V1
V2



DVD010
V1
V2



DVD011
V1
V2



DVD012
V1
V2



DVD013
V1
V2



DVD014
V1
V2



DVD015
V1
V2



DVD016
V1
V2



DVD017
V1
V2



DVD018
V1
V2



DVD019
V1
V5



DVD020
V1
V4



DVD021
V1
V2



DVD022
V1
V2



DVD023
V1
V2



DVD024
V1
V2



DVD025
V1
V2



DVD026
V1
V2



DVD027
V1
V2



DVD028
V1
V2



DVD029
V1
V5



DVD030
V1
V4



DVD031
V1
V2



DVD032
V1
V2



DVD033
V1
V2



DVD034
V1
V2



DVD035
V1
V2



DVD036
V1
V2



DVD037
V1
V2



DVD038
V1
V2



DVD039
V1
V2



DVD040
V1
V2



DVD041
V1
V5



DVD042
V1
V4



DVD043
V1
V2



DVD044
V1
V2



DVD045
V1
V2



DVD046
V1
V2



DVD047
V1
V2



DVD048
V1
V2



DVD049
V1
V2



DVD050
V1
V2



DVD073
V1
V2



DVD074
V1
V2



DVD075
V1
V2



DVD076
V1
V2



DVD077
V1
V2



DVD078
V1
V2



DVD079
V1
V2



DVD080
V1
V2



DVD081
V1
V2



DVD082
V1
V2



DVD083
V1
V2



DVD084
V1
V2



DVD085
V1
V2



DVD086
V1
V2



DVD087
V1
V2



DVD088
V1
V2



DVD089
V1
V2



DVD090
V1
V2



DVD091
V1
V2



DVD092
V1
V2



DVD093
V1
V2



DVD094
V1
V2



DVD107
V1
V2



DVD108
V1
V2



DVD131
V1
V2



DVD132
V1
V2



DVD135
V1
V2



DVD136
V1
V2



DVD137
V1
V2



DVD138
V1
V2



DVD139
V1
V2



DVD140
V1
V2



DVD141
V1
V2



DVD142
V1
V2



DVD143
V1
V2



DVD144
V1
V2



DVD257
V1
V2



DVD258
V1
V2



DVD259
V1
V2



DVD260
V1
V2



DVD299
V1
V2



DVD300
V1
V2



DVD301
V1
V2



DVD302
V1
V2



DVD303
V1
V2



DVD304
V1
V2



DVD305
V1
V2



DVD306
V1
V2



DVD307
V1
V2



DVD308
V1
V2



DVD309
V1
V2



DVD310
V1
V2



DVD385
V1
V2



DVD386
V1
V2



DVD387
V7
V2



DVD388
V1
V2



DVD389
V1
V2



DVD390
V1
V2



DVD391
V7
V2



DVD392
V1
V2



DVD393
V1
V2



DVD394
V1
V2



DVD395
V1
V2



DVD396
V1
V2



DVD397
V1
V2



DVD398
V1
V2



DVD399
V1
V2



DVD400
V1
V2



DVD401
V1
V2



DVD402
V1
V2



DVD403
V1
V2



DVD404
V1
V2



DVD405
V1
V2



DVD406
V1
V2



DVD407
V1
V2



DVD408
V1
V2



DVD409
V1
V4



DVD410
V1
V5



DVD411
V1
V4



DVD412
V1
V5



DVD413
V1
V4



DVD414
V7
V5



DVD415
V1
V4



DVD416
V1
V5



DVD417
V1
V4



DVD418
V7
V5



DVD419
V1
V4



DVD420
V1
V5



DVD421
V1
V4



DVD422
V1
V5



DVD423
V1
V4



DVD424
V1
V5



DVD441
V1
V2



DVD442
V1
V2



DVD443
V1
V2



DVD444
V1
V2



DVD445
V1
V2



DVD446
V1
V2



DVD447
V1
V2



DVD448
V1
V2



DVD449
V1
V2



DVD450
V1
V2



DVD451
V7
V2



DVD452
V7
V2



DVD453
V1
V2



DVD454
V1
V2



DVD455
V1
V2



DVD456
V1
V2



DVD457
V7
V2



DVD458
V7
V2



DVD459
V1
V2



DVD460
V1
V2



DVD461
V1
V2



DVD462
V1
V2



DVD463
V1
V2



DVD464
V1
V2



DVD465
V1
V2



DVD466
V1
V2



DVD467
V1
V2



DVD468
V1
V2



DVD469
V1
V2



DVD470
V1
V2



DVD471
V7
V2



DVD472
V1
V2



DVD473
V1
V2



DVD474
V1
V2



DVD475
V1
V2



DVD476
V1
V2



DVD477
V7
V2



DVD478
V1
V2



DVD479
V1
V2



DVD480
V1
V2



DVD481
V1
V2



DVD482
V1
V2



DVD483
V1
V2



DVD484
V1
V2



DVD485
V1
V2



DVD486
V1
V2



DVD487
V1
V2



DVD488
V1
V2



DVD489
V7
V2



DVD490
V1
V2



DVD491
V1
V2



DVD492
V1
V2



DVD493
V1
V2



DVD494
V1
V2



DVD495
V1
V2



DVD496
V1
V2



DVD497
V1
V2



DVD498
V1
V2



DVD499
V1
V2



DVD500
V1
V2



DVD501
V1
V2



DVD502
V1
V2



DVD503
V1
V2



DVD504
V1
V2



DVD505
V1
V2



DVD506
V1
V2



DVD507
V7
V2



DVD508
V1
V2



DVD509
V1
V2



DVD510
V1
V2



DVD511
V1
V4



DVD512
V7
V5



DVD513
V1
V4



DVD514
V1
V5



DVD515
V1
V4



DVD516
V1
V5



DVD517
V1
V4



DVD518
V1
V5



DVD519
V1
V4



DVD520
V1
V5



DVD521
V7
V4



DVD522
V7
V5



DVD523
V1
V4



DVD524
V1
V5



DVD525
V1
V4



DVD526
V1
V5



DVD527
V1
V4



DVD528
V1
V5



DVD529
V1
V4



DVD530
V7
V5



DVD531
V1
V4



DVD532
V1
V5



DVD533
V7
V4



DVD534
V7
V5



DVD535
V1
V2



DVD536
V1
V2



DVD537
V1
V2



DVD538
V1
V2



DVD539
V1
V2



DVD540
V1
V2



DVD541
V1
V2



DVD542
V1
V2



DVD543
V1
V2



DVD544
V1
V2



DVD545
V1
V2



DVD546
V1
V2



DVD547
V1
V2



DVD548
V1
V2



DVD549
V1
V2



DVD550
V1
V2



DVD551
V1
V2



DVD552
V1
V2



DVD553
V1
V2



DVD554
V1
V2



DVD555
V1
V2



DVD556
V1
V2



DVD557
V1
V2



DVD558
V1
V2



DVD559
V1
V2



DVD560
V1
V2



DVD561
V1
V2



DVD562
V1
V2



DVD563
V1
V2



DVD564
V1
V2



DVD565
V1
V2



DVD566
V1
V2



DVD567
V1
V2



DVD568
V1
V2



DVD569
V1
V2



DVD570
V1
V2



DVD571
V1
V2



DVD572
V1
V2



DVD573
V1
V2



DVD574
V1
V2



DVD575
V1
V2



DVD576
V1
V2



DVD577
V1
V2



DVD578
V1
V2



DVD579
V1
V2



DVD580
V1
V2



DVD581
V1
V2



DVD582
V1
V2



DVD583
V1
V2



DVD584
V1
V2



DVD585
V1
V2



DVD586
V1
V2



DVD587
V1
V2



DVD588
V1
V2



DVD589
V1
V2



DVD590
V1
V2



DVD591
V1
V2



DVD592
V1
V2



DVD593
V1
V2



DVD594
V1
V2



DVD595
V1
V2



DVD596
V1
V2



DVD597
V1
V2



DVD598
V1
V2



DVD599
V1
V2



DVD600
V1
V2



DVD601
V1
V2



DVD602
V1
V2



DVD603
V1
V4



DVD604
V1
V5



DVD605
V1
V4



DVD606
V1
V5



DVD607
V1
V4



DVD608
V1
V5



DVD609
V1
V4



DVD610
V1
V5



DVD611
V1
V2



DVD612
V1
V2



DVD613
V1
V2



DVD614
V1
V2



DVD615
V1
V2



DVD616
V1
V2



DVD625
V1
V2



DVD626
V1
V2



DVD627
V1
V2



DVD628
V1
V2



DVD629
V1
V2



DVD630
V1
V2



DVD631
V1
V2



DVD632
V1
V2



DVD641
V1
V2



DVD642
V1
V2



DVD643
V1
V2



DVD644
V1
V2



DVD645
V1
V2



DVD646
V1
V2



DVD647
V1
V2



DVD648
V1
V2



DVD649
V1
V2



DVD650
V1
V2



DVD651
V1
V2



DVD652
V1
V2



DVD653
V1
V2



DVD654
V1
V2



DVD655
V1
V2



DVD656
V1
V2



DVD657
V1
V2



DVD658
V1
V2



DVD659
V1
V2



DVD660
V1
V2



DVD661
V1
V2



DVD662
V1
V2



DVD663
V1
V2



DVD664
V1
V2



DVD665
V1
V2



DVD666
V1
V2



DVD667
V1
V2



DVD668
V1
V2



DVD669
V1
V2



DVD670
V1
V2



DVD671
V1
V2



DVD672
V1
V2



DVD673
V1
V2



DVD674
V1
V2



DVD675
V1
V2



DVD676
V1
V2



DVD677
V1
V2



DVD678
V1
V2



DVD679
V1
V2



DVD680
V1
V2



DVD681
V1
V2



DVD682
V1
V2



DVD683
V1
V2



DVD684
V1
V2



DVD685
V1
V2



DVD686
V1
V2



DVD687
V1
V2



DVD688
V1
V2



DVD689
V1
V2



DVD690
V1
V2



DVD691
V1
V2



DVD692
V1
V2



DVD693
V1
V2



DVD694
V1
V2



DVD709
V1
V2



DVD710
V1
V2



DVD711
V1
V2



DVD712
V1
V2










Example 1.4.4.2
Transfection and Expression in 293 Cells
The DVD-Ig vector constructs are transfected into 293 cells for production of DVD-Ig protein. The 293 transient transfection procedure used is a modification of the methods published in Durocher et al. (2002) Nucleic Acids Res. 30(2):E9 and Pham et al. (2005) Biotech. Bioengineering 90(3):332-44. Reagents that were used in the transfection included:

    
    
        HEK 293-6E cells (human embryonic kidney cell line stably expressing EBNA1; obtained from National Research Council Canada) cultured in disposable Erlenmeyer flasks in a humidified incubator set at 130 rpm, 37° C. and 5% CO2.
        Culture medium: FreeStyle 293 Expression Medium (Invitrogen 12338-018) plus 25 μg/mL Geneticin (G418) (Invitrogen 10131-027) and 0.1% Pluronic F-68 (Invitrogen 24040-032).
        Transfection medium: FreeStyle 293 Expression Medium plus 10 mM HEPES (Invitrogen 15630-080).
        Polyethylenimine (PEI) stock: 1 mg/mL sterile stock solution, pH 7.0, prepared with linear 25 kDa PEI (Polysciences) and stored at less than −15° C.
        Tryptone Feed Medium: 5% w/v sterile stock of Tryptone N1 (Organotechnie, 19554) in FreeStyle 293 Expression Medium.

Cell preparation for transfection: Approximately 2-4 hours prior to transfection, HEK 293-6E cells are harvested by centrifugation and resuspended in culture medium at a cell density of approximately 1 million viable cells per mL. For each transfection, 40 mL of the cell suspension is transferred into a disposable 250-mL Erlenmeyer flask and incubated for 2-4 hours.

Transfection: The transfection medium and PEI stock are prewarmed to room temperature (RT). For each transfection, 25 μg of plasmid DNA and 50 μg of polyethylenimine (PEI) are combined in 5 mL of transfection medium and incubated for 15±20 minutes at RT to allow the DNA:PEI complexes to form. For the BR3-Ig transfections, 25 μg of BR3-Ig plasmid is used per transfection. Each 5-mL DNA:PEI complex mixture is added to a 40-mL culture prepared previously and returned to the humidified incubator set at 130 rpm, 37° C. and 5% CO2. After 20-28 hours, 5 mL of Tryptone Feed Medium is added to each transfection and the cultures are continued for six days.

    
    


Table 32 contains the yield data for parent antibodies or DVD-Ig constructs expressed as milligrams per liter in 293 cells.







TABLE 32







Transient Expression in Yields of Parent Antibodies and


DVD-Ig Constructs in 293 Cells












Parent
N-terminal
C-terminal




Antibody
Variable
Variable
Expression



or DVD-
Domain
Domain
Yield



Ig ID
(VD)
(VD)
(mg/L)















AB006
CD-19
9.4



AB001
CD-20
90.2












DVD001
CD-20
CD-19
5.8



DVD002
CD-19
CD-20
2.4











AB001
CD-20
90.2



AB002
CD-3
67.2












DVD003
CD-20
CD-3
4.2



DVD004
CD-3
CD-20
7.4











AB007
CD-80
9.6



AB001
CD-20
90.2












DVD005
CD-20
CD-80
35.4



DVD006
CD-80
CD-20
43.4











AB008
CD-22
50



AB001
CD-20
90.2












DVD007
CD-20
CD-22
0.8



DVD008
CD-22
CD-20
0.22











AB009
CD-40
36.6



AB001
CD-20
90.2












DVD009
CD-20
CD-40
2.6



DVD010
CD-40
CD-20
8











AB002
CD-3
67.2



AB004
HER-2
108.2












DVD011
CD-3
HER-2
30.6



DVD012
HER-2
CD-3
74











AB002
CD-3
67.2



AB006
CD-19
9.4












DVD013
CD-3
CD-19
12.4



DVD014
CD-19
CD-3
7.2











AB033
EGFR
44.4



AB004
HER-2
108.2












DVD015
EGFR
HER-2
42.2



DVD016
HER-2
EGFR
17











AB002
CD-3
67.2



AB033
EGFR
44.4












DVD017
EGFR
CD-3
9.6



DVD018
CD-3
EGFR
4











AB033
EGFR
44.4



AB011
IGF1R
28.5












DVD021
EGFR
IGF1R
10.4



DVD022
IGF1R
EGFR
17.8











AB005
RON
67.4



AB033
EGFR
44.4












DVD024
RON
EGFR
19.2



DVD023
EGFR
RON
17.8











AB033
EGFR
44.4



AB012
HGF
22.8












DVD025
EGFR
HGF
5.8



DVD026
HGF
EGFR
2.2











AB004
HER-2
108.2



AB010
IGF1, 2
38.6












DVD029
HER-2
IGF1, 2
74.4



DVD030
IGF1, 2
HER-2
26.8











AB004
HER-2
108.2



AB011
IGF1R
28.5












DVD031
HER-2
IGF1R
95



DVD032
IGF1R
HER-2
41.4











AB005
RON
67.4



AB012
HGF
22.8












DVD033
RON
HGF
29.4



DVD034
HGF
RON
7.8











AB014
VEGF
52.4



AB033
EGFR
44.4












DVD035
VEGF
EGFR
6.4



DVD036
EGFR
VEGF
5.4











AB014
VEGF
52.4



AB004
HER-2
108.2












DVD037
VEGF
HER-2
43.4



DVD038
HER-2
VEGF
41.2











AB001
CD-20
57



AB014
VEGF
52.4












DVD039
VEGF
CD-20
0.22



DVD040
CD-20
VEGF
0.48











AB014
VEGF
52.4



AB010
IGF1, 2
38.6












DVD041
VEGF
IGF1, 2
39.4



DVD042
IGF1, 2
VEGF
8.8











AB015
DLL-4
57.6



AB014
VEGF
52.4












DVD044
DLL4
VEGF
5.4



DVD043
VEGF
DLL4
17.4











AB014
VEGF
52.4



AB012
HGF
22.8












DVD045
VEGF
HGF
4



DVD046
HGF
VEGF
1.4











AB005
RON
67.4



AB014
VEGF
52.4












DVD048
RON
VEGF
14



DVD047
VEGF
RON
18











AB014
VEGF
52.4



AB016
NRP1
114.6












DVD049
VEGF
NRP1
13



DVD050
NRP1
VEGF
27.2











AB015
DLL-4
57.6



AB047
PlGF
23.6












DVD257
DLL4
PlGF
31.2



DVD258
PlGF
DLL4
21











AB014
VEGF
52.4



AB047
PlGF
23.6












DVD259
VEGF
PlGF
2.2



DVD260
PlGF
VEGF
7.8











AB062
ErbB3
24.6



AB033
EGFR
44.4












DVD299
ErbB3
EGFR
4.2



DVD300
EGFR
ErbB3
1.6











AB063
ErbB3
37.8



AB033
EGFR
44.4












DVD305
ErbB3
EGFR
5.6



DVD306
EGFR
ErbB3
11.2










All DVDs expressed well in 293 cells. DVDs could be easily purified over a protein A column. In most cases >5 mg/L purified DVD-Ig could be obtained easily from supernatants of 293 cells.
Example 1.4.5
Characterization and Lead Selection of A/B DVD-Igs
The binding affinities of anti-A/B DVD-Igs are analyzed on Biacore against both protein A and protein B. The tetravalent property of the DVD-Ig is examined by multiple binding studies on Biacore. Meanwhile, the neutralization potency of the DVD-Igs for protein A and protein B are assessed by bioassays, respectively, as described herein. The DVD-Ig molecules that best retain the affinity and potency of the original parent mAbs are selected for in-depth physicochemical and bio-analytical (rat PK) characterizations as described herein for each mAb. Based on the collection of analyses, the final lead DVD-Ig is advanced into CHO stable cell line development, and the CHO-derived material is employed in stability, pharmacokinetic and efficacy studies in cynomolgus monkey, and preformulation activities.
Example 2
Generation and Characterization of Dual Variable Domain Immunoglobulins (DVD-Ig)
Dual variable domain immunoglobulins (DVD-Ig) using parent antibodies with known amino acid sequences were generated by synthesizing polynucleotide fragments encoding DVD-Ig variable heavy and DVD-Ig variable light chain sequences and cloning the fragments into a pHybC-D2 vector according to Example 1.4.4.1. The DVD-Ig constructs were cloned into and expressed in 293 cells as described in Example 1.4.4.2. The DVD-Ig protein was purified according to standard methods. Functional characteristics were determined according to the methods described in Example 1.1.1 and 1.1.2 as indicated. DVD-Ig VH and VL chains for the DVD-Igs of the invention are provided below.
Example 2.1
Generation of CD-20 and CD-19 DVD-Igs












TABLE 33






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345




















99
DVD001H
AB001VH
AB006VH
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH







WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATL






TADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGD






WYFNVWGAGTTVTVSAASTKGPQVQLQQSGAELVR






PGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWI






GQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQL






SSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGT






SVTVSS


 


100
DVD001L
AB001VL
AB006VL
QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWF






QQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSY






SLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI






KRTVAAPDILLTQTPASLAVSLGQRATISCKASQS






VDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIP






PRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTED






PWTFGGGTKLEIKR


 


101
DVD002H
AB006VH
AB001VH
QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMN






WVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATL






TADESSSTAYMQLSSLASEDSAVYFCARRETTTVG






RYYYAMDYWGQGTSVTVSSASTKGPQVQLQQPGAE






LVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGL






EWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAY






MQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGT






TVTVSA


 


102
DVD002L
AB006VL
AB001VL
DILLTQTPASLAVSLGQRATISCKASQSVDYDGDS






YLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSG






SGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGG






TKLEIKRTVAAPQIVLSQSPAILSPSPGEKVTMTC






RASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVP






VRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSN






PPTFGGGTKLEIKR









Example 2.2
Generation of CD-20 and CD-3 (Seq. 1) DVD-Igs












TABLE 34






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







103
DVD003H
AB001VH
AB002VH
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH







WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATL






TADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGD






WYFNVWGAGTTVTVSAASTKGPQVQLQQSGAELAR






PGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWI






GYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQL






SSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS


 


104
DVD003L
AB001VL
AB002VL
QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWF






QQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSY






SLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI






KRTVAAPQIVLTQSPAIMSASPGEKVTMTCRASSS






VSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSG






SGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFG






SGTKLEINR


 


105
DVD004H
AB002VH
AB001VH
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH






WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL






TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC






LDYWGQGTTLTVSSASTKGPQVQLQQPGAELVKPG






ASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGA






IYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSS






LTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA


 


106
DVD004L
AB002VL
AB001VL
QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWY






QQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSY






SLTISSMEAEDAATYYCQQWSSNPLTFGSGTKLEI






NRTVAAPQIVLSQSPAILSPSPGEKVTMTCRASSS






VSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSG






SGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFG






GGTKLEIKR









Example 2.3
Generation of CD-20 and CD-80 DVD-Igs












TABLE 35






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







107
DVD005H
AB001VH
AB007VH
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH







WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATL






TADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGD






WYFNVWGAGTTVTVSAASTKGPQVQLQESGPGLVK






PSETLSLTCAVSGGSISGGYGWGWIRQPPGKGLEW






IGSFYSSSGNTYYNPSLKSQVTISTDTSKNQFSLK






LNSMTAADTAVYYCVRDRLFSVVGMVYNNWFDVWG






PGVLVTVSS


 


108
DVD005L
AB001VL
AB007VL
QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWF






QQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSY






SLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI






KRTVAAPESALTQPPSVSGAPGQKVTISCTGSTSN






IGGYDLHWYQQLPGTAPKLLIYDINKRPSGISDRF






SGSKSGTAASLAITGLQTEDEADYYCQSYDSSLNA






QVFGGGTRLTVLG


 


109
DVD006H
AB007VH
AB001VH
QVQLQESGPGLVKPSETLSLTCAVSGGSISGGYGW






GWIRQPPGKGLEWIGSFYSSSGNTYYNPSLKSQVT






ISTDTSKNQFSLKLNSMTAADTAVYYCVRDRLFSV






VGMVYNNWFDVWGPGVLVTVSSASTKGPQVQLQQP






GAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPG






RGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSS






TAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWG






AGTTVTVSA


 


110
DVD006L
AB007VL
AB001VL
ESALTQPPSVSGAPGQKVTISCTGSTSNIGGYDLH






WYQQLPGTAPKLLIYDINKRPSGISDRFSGSKSGT






AASLAITGLQTEDEADYYCQSYDSSLNAQVFGGGT






RLTVLGQPKAAPQIVLSQSPAILSPSPGEKVTMTC






RASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVP






VRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSN






PPTFGGGTKLEIKR









Example 2.4
Generation of CD-20 and CD-22 DVD-Igs












TABLE 36






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







111
DVD007H
AB001VH
AB008VH
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH







WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATL






TADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGD






WYFNVWGAGTTVTVSAASTKGPQVQLVQSGAEVKK






PGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWI






GYINPRNDYTEYNQNFKDKATITADESTNTAYMEL






SSLRSEDTAFYFCARRDITTFYWGQGTTVTVSS


 


112
DVD007L
AB001VL
AB008VL
QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWF






QQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSY






SLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI






KRTVAAPDIQLTQSPSSLSASVGDRVTMSCKSSQS






VLYSANHKNYLAWYQQKPGKAPKLLIYWASTRESG






VPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYL






SSWTFGGGTKLEIKR


 


113
DVD008H
AB008VH
AB001VH
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLH






WVRQAPGQGLEWIGYINPRNDYTEYNQNFKDKATI






TADESTNTAYMELSSLRSEDTAFYFCARRDITTFY






WGQGTTVTVSSASTKGPQVQLQQPGAELVKPGASV






KMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYP






GNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTS






EDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA


 


114
DVD008L
AB008VL
AB001VL
DIQLTQSPSSLSASVGDRVTMSCKSSQSVLYSANH






KNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSG






SGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGG






GTKLEIKRTVAAPQIVLSQSPAILSPSPGEKVTMT






CRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGV






PVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTS






NPPTFGGGTKLEIKR









Example 2.5
Generation of CD-20 and CD-40 DVD-Igs












TABLE 37






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


No.
Name
Name
Name
12345678901234567890123456789012345







115
DVD009H
AB001VH
AB009VH
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH







WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATL






TADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGD






WYFNVWGAGTTVTVSAASTKGPQVQLVESGGGVVQ






PGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV






AVISYEESNRYHADSVKGRFTISRDNSKITLYLQM






NSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTV






SS


 


116
DVD009L
AB001VL
AB009VL
QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWF






QQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSY






SLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI






KRTVAAPDIVMTQSPLSLTVTPGEPASISCRSSQS






LLYSNGYNYLDWYLQKPGQSPQVLISLGSNRASGV






PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQ






TPFTFGPGTKVDIRR


 


117
DVD010H
AB009VH
AB001VH
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH






WVRQAPGKGLEWVAVISYEESNRYHADSVKGRFTI






SRDNSKITLYLQMNSLRTEDTAVYYCARDGGIAAP






GPDYWGQGTLVTVSSASTKGPQVQLQQPGAELVKP






GASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIG






AIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLS






SLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTV






SA


 


118
DVD010L
AB009VL
AB001VL
DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGY






NYLDWYLQKPGQSPQVLISLGSNRASGVPDRFSGS






GSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFGP






GTKVDIRRTVAAPQIVLSQSPAILSPSPGEKVTMT






CRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGV






PVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTS






NPPTFGGGTKLEIKR









Example 2.6
Generation of CD-3 (Seq. 1) and HER-2 (Seq. 1) DVD-Igs












TABLE 38






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







119
DVD011H
AB002VH
AB004VH
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH







WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL






TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC






LDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR






IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


120
DVD011L
AB002VL
AB004VL
QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWY






QQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSY






SLTISSMEAEDAATYYCQQWSSNPLTFGSGTKLEI






NRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQD






VNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS






GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF






GQGTKVEIKR


 


121
DVD012H
AB004VH
AB002VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPQVQLQQSGAELARP






GASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIG






YINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLS






SLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS


 


122
DVD012L
AB004VL
AB002VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPQIVLTQSPAIMSASPGEKVTMTCRASS






SVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFS






GSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTF






GSGTKLEINR









Example 2.7
Generation of CD-3 (Seq. 1) and CD-19 DVD-Igs












TABLE 39






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







123
DVD013H
AB002VH
AB006VH
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH







WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL






TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC






LDYWGQGTTLTVSSASTKGPQVQLQQSGAELVRPG






SSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQ






IWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSS






LASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSV






TVSS


 


124
DVD013L
AB002VL
AB006VL
QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWY






QQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSY






SLTISSMEAEDAATYYCQQWSSNPLTFGSGTKLEI






NRTVAAPDILLTQTPASLAVSLGQRATISCKASQS






VDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIP






PRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTED






PWTFGGGTKLEIKR


 


125
DVD014H
AB006VH
AB002VH
QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMN






WVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATL






TADESSSTAYMQLSSLASEDSAVYFCARRETTTVG






RYYYAMDYWGQGTSVTVSSASTKGPQVQLQQSGAE






LARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGL






EWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAY






MQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTL






TVSS


 


126
DVD014L
AB006VL
AB002VL
DILLTQTPASLAVSLGQRATISCKASQSVDYDGDS






YLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSG






SGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGG






TKLEIKRTVAAPQIVLTQSPAIMSASPGEKVTMTC






RASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVP






YRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSN






PLTFGSGTKLEINR









Example 2.8
Generation of EGFR (Seq. 2) and HER-2 (Seq. 1) DVD-Igs












TABLE 40






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







127
DVD015H
AB033VH
AB004VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH







WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR






IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


128
DVD015L
AB033VL
AB004VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT






FGQGTKVEIKR


 


129
DVD016H
AB004VH
AB033VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPQVQLKQSGPGLVQP






SQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLG






VIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNS






LQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


130
DVD016L
AB004VL
AB033VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPDILLTQSPVILSVSPGERVSFSCRASQ






SIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF






SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT






FGAGTKLELKR









Example 2.9
Generation of EGFR (Seq. 2) and CD-3 (Seq. 1) DVD-Igs












TABLE 41






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







131
DVD017H
AB033VH
AB002VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH







WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPQVQLQQSGAELARPG






ASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGY






INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSS






LTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS


 


132
DVD017L
VD033VL
AB002VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPQIVLTQSPAIMSASPGEKVTMTCRASS






SVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFS






GSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTF






GSGTKLEINR


 


133
DVD018H
AB002VH
AB033VH
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH






WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL






TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC






LDYWGQGTTLTVSSASTKGPQVQLKQSGPGLVQPS






QSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV






IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSL






QSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


134
DVD018L
AB002VL
AB033VL
QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWY






QQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSY






SLTISSMEAEDAATYYCQQWSSNPLTFGSGTKLEI






NRTVAAPDILLTQSPVILSVSPGERVSFSCRASQS






IGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS






GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTF






GAGTKLELKR









Example 2.10
Generation of EGFR (Seq. 2) and IGF1,2 DVD-Igs












TABLE 42






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







135
DVD019H
AB033VH
AB010VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH







WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPQVQLVQSGAEVKKPG






ASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGW






MNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSS






LRSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSS


 


136
DVD019L
AB033VL
AB010VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPQSVLTQPPSVSAAPGQKVTISCSGSSS






NIENNHVSWYQQLPGTAPKLLIYDNNKRPSGIPDR






FSGSKSGTSATLGITGLQTGDEADYYCETWDTSLS






AGRVFGGGTKLTVLG


 


137
DVD020H
AB010VH
AB033VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDIN






WVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTM






TRNTSISTAYMELSSLRSEDTAVYYCARDPYYYYY






GMDVWGQGTTVTVSSASTKGPQVQLKQSGPGLVQP






SQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLG






VIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNS






LQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


138
DVD020L
AB010VL
AB033VL
QSVLTQPPSVSAAPGQKVTISCSGSSSNIENNHVS






WYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGT






SATLGITGLQTGDEADYYCETWDTSLSAGRVFGGG






TKLTVLGQPKAAPDILLTQSPVILSVSPGERVSFS






CRASQSIGTNIHWYQQRTNGSPRLLIKYASESISG






IPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNN






NWPTTFGAGTKLELKR









Example 2.11
Generation of EGFR (Seq. 2) and IGF1R (Seq. 1) DVD-Igs with Linker Set 1












TABLE 43






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







139
DVD021H
AB033VH
AB011VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH







WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLLESGGGLVQPG






GSLRLSCTASGFTFSSYAMNWVRQAPGKGLEWVSA






ISGSGGTTFYADSVKGRFTISRDNSRTTLYLQMNS






LRAEDTAVYYCAKDLGWSDSYYYYYGMDVWGQGTT






VTVSS


 


140
DVD021L
AB033VL
AB011VL
DILLTQSPVILSVSPGERVSESCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDIQMTQFPSSLSASVGDRVTITCRASQ






GIRNDLGWYQQKPGKAPKRLIYAASRLHRGVPSRF






SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPCS






FGQGTKLEIKR


 


141
DVD022H
AB011VH
AB033VH
EVQLLESGGGLVQPGGSLRLSCTASGFTFSSYAMN






WVRQAPGKGLEWVSAISGSGGTTFYADSVKGRFTI






SRDNSRTTLYLQMNSLRAEDTAVYYCAKDLGWSDS






YYYYYGMDVWGQGTTVTVSSASTKGPQVQLKQSGP






GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKG






LEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVF






FKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTL






VTVSA


 


142
DVD022L
AB011VL
AB033VL
DIQMTQFPSSLSASVGDRVTITCRASQGIRNDLGW






YQQKPGKAPKRLIYAASRLHRGVPSRFSGSGSGTE






FTLTISSLQPEDFATYYCLQHNSYPCSFGQGTKLE






IKRTVAAPDILLTQSPVILSVSPGERVSFSCRASQ






SIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF






SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT






FGAGTKLELKR









Example 2.12
Generation of EGFR (Seq. 2) and IGF1R (Seq. 1) DVD-Igs with Linker Set 2












TABLE 44






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







143
DVD611H
AB011VH
AB033VH
EVQLLESGGGLVQPGGSLRLSCTASGFTFSSYAMN







WVRQAPGKGLEWVSAISGSGGTTFYADSVKGRFTI






SRDNSRTTLYLQMNSLRAEDTAVYYCAKDLGWSDS






YYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPQV






QLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWV






RQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKD






NSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFA






YWGQGTLVTVSA


 


144
DVD611L
AB011VL
AB033VL
DIQMTQFPSSLSASVGDRVTITCRASQGIRNDLGW






YQQKPGKAPKRLIYAASRLHRGVPSRFSGSGSGTE






FTLTISSLQPEDFATYYCLQHNSYPCSFGQGTKLE






IKRTVAAPSVFIFPPDILLTQSPVILSVSPGERVS






FSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESI






SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQ






NNNWPTTFGAGTKLELKR


 


145
DVD612H
AB033VH
AB011VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLLESG






GGLVQPGGSLRLSCTASGFTFSSYAMNWVRQAPGK






GLEWVSAISGSGGTTFYADSVKGRFTISRDNSRTT






LYLQMNSLRAEDTAVYYCAKDLGWSDSYYYYYGMD






VWGQGTTVTVSS


 


146
DVD612L
AB033VL
AB011VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNTHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPDIQMTQFPSSLSASVGDRVT






ITCRASQGIRNDLGWYQQKPGKAPKRLIYAASRLH






RGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQ






HNSYPCSFGQGTKLEIKR









Example 2.13
Generation of EGFR (Seq. 2) and IGF1R (Seq. 1) DVD-Igs with Linker Set 3












TABLE 45






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







147
DVD613H
AB011VH
AB033VH
EVQLLESGGGLVQPGGSLRLSCTASGFTFSSYAMN







WVRQAPGKGLEWVSAISGSGGTTFYADSVKGRFTI






SRDNSRTTLYLQMNSLRAEDTAVYYCAKDLGWSDS






YYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPQV






QLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWV






RQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKD






NSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFA






YWGQGTLVTVSA


 


148
DVD613L
AB011VL
AB033VL
DIQMTQFPSSLSASVGDRVTITCRASQGIRNDLGW






YQQKPGKAPKRLIYAASRLHRGVPSRFSGSGSGTE






FTLTISSLQPEDFATYYCLQHNSYPCSFGQGTKLE






IKRTVAAPDILLTQSPVILSVSPGERVSFSCRASQ






SIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF






SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT






FGAGTKLELKR


 


149
DVD614H
AB033VH
AB011VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLLESG






GGLVQPGGSLRLSCTASGFTFSSYAMNWVRQAPGK






GLEWVSAISGSGGTTFYADSVKGRFTISRDNSRTT






LYLQMNSLRAEDTAVYYCAKDLGWSDSYYYYYGMD






VWGQGTTVTVSS


 


150
DVD614L
AB033VL
AB011VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDIQMTQFPSSLSASVGDRVTITCRASQ






GIRNDLGWYQQKPGKAPKRLIYAASRLHRGVPSRF






SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPCS






FGQGTKLEIKR









Example 2.14
Generation of EGFR (Seq. 2) and IGF1R (Seq. 1) DVD-Igs with Linker Set 4












TABLE 46






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







151
DVD615H
AB011VH
AB033VH
EVQLLESGGGLVQPGGSLRLSCTASGFTFSSYAMN







WVRQAPGKGLEWVSAISGSGGTTFYADSVKGRFTI






SRDNSRTTLYLQMNSLRAEDTAVYYCAKDLGWSDS






YYYYYGMDVWGQGTTVTVSSASTKGPQVQLKQSGP






GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKG






LEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVF






FKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTL






VTVSA


 


152
DVD615L
AB011VL
AB033VL
DIQMTQFPSSLSASVGDRVTITCRASQGIRNDLGW






YQQKPGKAPKRLIYAASRLHRGVPSRFSGSGSGTE






FTLTISSLQPEDFATYYCLQHNSYPCSFGQGTKLE






IKRTVAAPSVFIFPPDILLTQSPVILSVSPGERVS






FSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESI






SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQ






NNNWPTTFGAGTKLELKR


 


153
DVD616H
AB033VH
AB011VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLLESGGGLVQPG






GSLRLSCTASGFTFSSYAMNWVRQAPGKGLEWVSA






ISGSGGTTFYADSVKGRFTISRDNSRTTLYLQMNS






LRAEDTAVYYCAKDLGWSDSYYYYYGMDVWGQGTT






VTVSS


 


154
DVD616L
AB033VL
AB011VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPDIQMTQFPSSLSASVGDRVT






ITCRASQGIRNDLGWYQQKPGKAPKRLIYAASRLH






RGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQ






HNSYPCSFGQGTKLEIKR









Example 2.15
Generation of EGFR (Seq. 2) and IGF1R (Seq. 2) DVD-Igs with Linker Set 1












TABLE 47






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







155
DVD603H
AB075VH
AB033VH
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS







WVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI






TADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLE






WSTQDHYYYYYMDVWGKGTTVTVSSASTKGPQVQL






KQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQ






SPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS






KSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYW






GQGTLVTVSA


 


156
DVD603L
AB075VL
AB033VL
SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWY






QQKPGQAPILVIYGENKRPSGIPDRFSGSSSGNTA






SLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKL






TVLGQPKAAPDILLTQSPVILSVSPGERVSFSCRA






SQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPS






RFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWP






TTFGAGTKLELKR


 


157
DVD604H
AB033VH
AB075VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLVQSGAEVKKPG






SSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG






IIPIFGTANYAQKFQGRVTITADKSTSTAYMELSS






LRSEDTAVYYCARAPLRFLEWSTQDHYYYYYMDVW






GKGTTVTVSS


 


158
DVD604L
AB033VL
AB075VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSSELTQDPAVSVALGQTVRITCQGDSL






RSYYATWYQQKPGQAPILVIYGENKRPSGIPDRFS






GSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHL






VFGGGTKLTVLG









Example 2.16
Generation of EGFR (Seq. 2) and IGF1R (Seq. 2) DVD-Igs with Linker Set 2












TABLE 48






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







159
DVD605H
AB075VH
AB033VH
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS







WVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI






TADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLE






WSTQDHYYYYYMDVWGKGTTVTVSSASTKGPSVFP






LAPQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNY






GVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRL






SINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYY






DYEFAYWGQGTLVTVSA


 


160
DVD605L
AB075VL
AB033VL
SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWY






QQKPGQAPILVIYGENKRPSGIPDRFSGSSSGNTA






SLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKL






TVLGQPKAAPSVTLFPPDILLTQSPVILSVSPGER






VSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASE






SISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC






QQNNNWPTTFGAGTKLELKR


 


161
DVD606H
AB033VH
AB075VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLVQSG






AEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQ






GLEWMGGIIPIFGTANYAQKFQGRVTITADKSTST






AYMELSSLRSEDTAVYYCARAPLRFLEWSTQDHYY






YYYMDVWGKGTTVTVSS


 


162
DVD606L
AB033VL
AB075VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPSSELTQDPAVSVALGQTVRI






TCQGDSLRSYYATWYQQKPGQAPILVIYGENKRPS






GIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSR






DGSGQHLVFGGGTKLTVLG









Example 2.17
Generation of EGFR (Seq. 2) and IGF1R (Seq. 2) DVD-Igs with Linker Set 3












TABLE 49






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







163
DVD607H
AB075VH
AB033VH
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS







WVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI






TADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLE






WSTQDHYYYYYMDVWGKGTTVTVSSASTKGPSVFP






LAPQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNY






GVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRL






SINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYY






DYEFAYWGQGTLVTVSA


 


164
DVD607L
AB075VL
AB033VL
SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWY






QQKPGQAPILVIYGENKRPSGIPDRFSGSSSGNTA






SLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKL






TVLGQPKAAPDILLTQSPVILSVSPGERVSFSCRA






SQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPS






RFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWP






TTFGAGTKLELKR


 


165
DVD608H
AB033VH
AB075VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLVQSG






AEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQ






GLEWMGGIIPIFGTANYAQKFQGRVTITADKSTST






AYMELSSLRSEDTAVYYCARAPLRFLEWSTQDHYY






YYYMDVWGKGTTVTVSS


 


166
DVD608L
AB033VL
AB075VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSSELTQDPAVSVALGQTVRITCQGDSL






RSYYATWYQQKPGQAPILVIYGENKRPSGIPDRFS






GSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHL






VFGGGTKLTVLG









Example 2.18
Generation of EGFR (Seq. 2) and IGF1R (Seq. 2) DVD-Igs with Linker Set 4












TABLE 50






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







167
DVD609H
AB075VH
AB033VH
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS







WVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI






TADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLE






WSTQDHYYYYYMDVWGKGTTVTVSSASTKGPQVQL






KQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQ






SPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNS






KSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYW






GQGTLVTVSA


 


168
DVD609L
AB075VL
AB033VL
SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWY






QQKPGQAPILVIYGENKRPSGIPDRFSGSSSGNTA






SLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKL






TVLGQPKAAPSVTLFPPDILLTQSPVILSVSPGER






VSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASE






SISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC






QQNNNWPTTFGAGTKLELKR


 


169
DVD610H
AB033VH
AB075VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLVQSGAEVKKPG






SSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG






IIPIFGTANYAQKFQGRVTITADKSTSTAYMELSS






LRSEDTAVYYCARAPLRFLEWSTQDHYYYYYMDVW






GKGTTVTVSS


 


170
DVD610L
AB033VL
AB075VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPSSELTQDPAVSVALGQTVRI






TCQGDSLRSYYATWYQQKPGQAPILVIYGENKRPS






GIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSR






DGSGQHLVFGGGTKLTVLG









Example 2.19
Generation of EGFR (Seq. 2) and IGF1R (Seq. 3) DVD-Igs with Linker Set 1












TABLE 51






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







171
DVD625H
AB077VH
AB033VH
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSFAMH







WVRQAPGKGLEWISVIDTRGATYYADSVKGRFTIS






RDNAKNSLYLQMNSLRAEDTAVYYCARLGNFYYGM






DVWGQGTTVTVSSASTKGPQVQLKQSGPGLVQPSQ






SLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVI






WSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQ






SNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


172
DVD625L
AB077VL
AB033VL
EIVLTQSPGTLSVSPGERATLSCRASQSIGSSLHW






YQQKPGQAPRLLIKYASQSLSGIPDRFSGSGSGTD






FTLTISRLEPEDFAVYYCHQSSRLPHTFGQGTKVE






IKRTVAAPDILLTQSPVILSVSPGERVSFSCRASQ






SIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF






SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT






FGAGTKLELKR


 


173
DVD626H
AB033VH
AB077VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLVQSGGGLVKPG






GSLRLSCAASGFTFSSFAMHWVRQAPGKGLEWISV






IDTRGATYYADSVKGRFTISRDNAKNSLYLQMNSL






RAEDTAVYYCARLGNFYYGMDVWGQGTTVTVSS


 


174
DVD626L
AB033VL
AB077VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPEIVLTQSPGTLSVSPGERATLSCRASQ






SIGSSLHWYQQKPGQAPRLLIKYASQSLSGIPDRF






SGSGSGTDFTLTISRLEPEDFAVYYCHQSSRLPHT






FGQGTKVEIKR









Example 2.20
Generation of EGFR (Seq. 2) and IGF1R (Seq. 3) DVD-Igs with Linker Set 2












TABLE 52






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







175
DVD627H
AB077VH
AB033VH
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSFAMH







WVRQAPGKGLEWISVIDTRGATYYADSVKGRFTIS






RDNAKNSLYLQMNSLRAEDTAVYYCARLGNFYYGM






DVWGQGTTVTVSSASTKGPSVFPLAPQVQLKQSGP






GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKG






LEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVF






FKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTL






VTVSA


 


176
DVD627L
AB077VL
AB033VL
EIVLTQSPGTLSVSPGERATLSCRASQSIGSSLHW






YQQKPGQAPRLLIKYASQSLSGIPDRFSGSGSGTD






FTLTISRLEPEDFAVYYCHQSSRLPHTFGQGTKVE






IKRTVAAPSVFIFPPDILLTQSPVILSVSPGERVS






FSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESI






SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQ






NNNWPTTFGAGTKLELKR


 


177
DVD628H
AB033VH
AB077VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLVQSG






GGLVKPGGSLRLSCAASGFTFSSFAMHWVRQAPGK






GLEWISVIDTRGATYYADSVKGRFTISRDNAKNSL






YLQMNSLRAEDTAVYYCARLGNFYYGMDVWGQGTT






VTVSS


 


178
DVD628L
AB033VL
AB077VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPEIVLTQSPGTLSVSPGERAT






LSCRASQSIGSSLHWYQQKPGQAPRLLIKYASQSL






SGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQ






SSRLPHTFGQGTKVEIKR









Example 2.21
Generation of EGFR (Seq. 2) and IGF1R (Seq. 3) DVD-Igs with Linker Set 3












TABLE 53






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







179
DVD629H
AB077VH
AB033VH
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSFAMH







WVRQAPGKGLEWISVIDTRGATYYADSVKGRFTIS






RDNAKNSLYLQMNSLRAEDTAVYYCARLGNFYYGM






DVWGQGTTVTVSSASTKGPSVFPLAPQVQLKQSGP






GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKG






LEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVF






FKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTL






VTVSA


 


180
DVD629L
AB077VL
AB033VL
EIVLTQSPGTLSVSPGERATLSCRASQSIGSSLHW






YQQKPGQAPRLLIKYASQSLSGIPDRFSGSGSGTD






FTLTISRLEPEDFAVYYCHQSSRLPHTFGQGTKVE






IKRTVAAPDILLTQSPVILSVSPGERVSFSCRASQ






SIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF






SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT






FGAGTKLELKR


 


181
DVD630H
AB033VH
AB077VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLVQSG






GGLVKPGGSLRLSCAASGFTFSSFAMHWVRQAPGK






GLEWISVIDTRGATYYADSVKGRFTISRDNAKNSL






YLQMNSLRAEDTAVYYCARLGNFYYGMDVWGQGTT






VTVSS


 


182
DVD630L
AB033VL
AB077VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPEIVLTQSPGTLSVSPGERATLSCRASQ






SIGSSLHWYQQKPGQAPRLLIKYASQSLSGIPDRF






SGSGSGTDFTLTISRLEPEDFAVYYCHQSSRLPHT






FGQGTKVEIKR









Example 2.22
Generation of EGFR (Seq. 2) and IGF1R (Seq. 3) DVD-Igs with Linker Set 4












TABLE 54






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







183
DVD631H
AB077VH
AB033VH
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSFAMH







WVRQAPGKGLEWISVIDTRGATYYADSVKGRFTIS






RDNAKNSLYLQMNSLRAEDTAVYYCARLGNFYYGM






DVWGQGTTVTVSSASTKGPQVQLKQSGPGLVQPSQ






SLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVI






WSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQ






SNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


184
DVD631L
AB077VL
AB033VL
EIVLTQSPGTLSVSPGERATLSCRASQSIGSSLHW






YQQKPGQAPRLLIKYASQSLSGIPDRFSGSGSGTD






FTLTISRLEPEDFAVYYCHQSSRLPHTFGQGTKVE






IKRTVAAPSVFIFPPDILLTQSPVILSVSPGERVS






FSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESI






SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQ






NNNWPTTFGAGTKLELKR


 


185
DVD632H
AB033VH
AB077VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLVQSGGGLVKPG






GSLRLSCAASGFTFSSFAMHWVRQAPGKGLEWISV






IDTRGATYYADSVKGRFTISRDNAKNSLYLQMNSL






RAEDTAVYYCARLGNFYYGMDVWGQGTTVTVSS


 


186
DVD632L
AB033VL
AB077VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPEIVLTQSPGTLSVSPGERAT






LSCRASQSIGSSLHWYQQKPGQAPRLLIKYASQSL






SGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQ






SSRLPHTFGQGTKVEIKR









Example 2.23
Generation of EGFR (Seq. 2) and RON (Seq. 1) DVD-Igs with Linker Set 1












TABLE 55






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







187
DVD023H
AB033VH
AB005VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH







WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLVQSGGGLVKPG






GSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAV






ISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNS






LRAEDTAVYYCARFSGWPNNYYYYGMDVWGQGTTV






TVSS


 


188
DVD023L
AB033VL
AB005VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDVVMTQSPLSLPVTPGEPASISCRSSQ






SLLHSNGFNYVDWYLQKPGQSPHLLIYFGSYRASG






VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQAL






QTPPWTFGQGTKVEIRR


 


189
DVD024H
AB005VH
AB033VH
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYAMH






WVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCARFSGWPNN






YYYYGMDVWGQGTTVTVSSASTKGPQVQLKQSGPG






LVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGL






EWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFF






KMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLV






TVSA


 


190
DVD024L
AB005VL
AB033VL
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGF






NYVDWYLQKPGQSPHLLIYFGSYRASGVPDRFSGS






GSGTDFTLKISRVEAEDVGVYYCMQALQTPPWTFG






QGTKVEIRRTVAAPDILLTQSPVILSVSPGERVSF






SCRASQSIGTNIHWYQQRTNGSPRLLIKYASESIS






GIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQN






NNWPTTFGAGTKLELKR









Example 2.24
Generation of EGFR (Seq. 2) and RON (Seq. 1) DVD-Igs with Linker Set 2












TABLE 56






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







191
DVD535H
AB005VH
AB033VH
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYAMH







WVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCARFSGWPNN






YYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPQVQ






LKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVR






QSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDN






SKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAY






WGQGTLVTVSA


 


192
DVD535L
AB005VL
AB033VL
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGF






NYVDWYLQKPGQSPHLLIYFGSYRASGVPDRFSGS






GSGTDFTLKISRVEAEDVGVYYCMQALQTPPWTFG






QGTKVEIRRTVAAPSVFIFPPDILLTQSPVILSVS






PGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIK






YASESISGIPSRFSGSGSGTDFTLSINSVESEDIA






DYYCQQNNNWPTTFGAGTKLELKR


 


193
DVD536H
AB033VH
AB005VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLVQSG






GGLVKPGGSLRLSCAASGFTFSSYAMHWVRQAPGK






GLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNT






LYLQMNSLRAEDTAVYYCARFSGWPNNYYYYGMDV






WGQGTTVTVSS


 


194
DVD536L
AB033VL
AB005VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPDVVMTQSPLSLPVTPGEPAS






ISCRSSQSLLHSNGFNYVDWYLQKPGQSPHLLIYF






GSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV






YYCMQALQTPPWTFGQGTKVEIRR









Example 2.25
Generation of EGFR (Seq. 2) and RON (Seq. 1) DVD-Igs with Linker Set 3












TABLE 57






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







195
DVD537H
AB005VH
AB033VH
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYAMH







WVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCARFSGWPNN






YYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPQVQ






LKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVR






QSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDN






SKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAY






WGQGTLVTVSA


 


196
DVD537L
AB005VL
AB033VL
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGF






NYVDWYLQKPGQSPHLLIYFGSYRASGVPDRFSGS






GSGTDFTLKISRVEAEDVGVYYCMQALQTPPWTFG






QGTKVEIRRTVAAPDILLTQSPVILSVSPGERVSF






SCRASQSIGTNIHWYQQRTNGSPRLLIKYASESIS






GIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQN






NNWPTTFGAGTKLELKR


 


197
DVD538H
AB033VH
AB005VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLVQSG






GGLVKPGGSLRLSCAASGFTFSSYAMHWVRQAPGK






GLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNT






LYLQMNSLRAEDTAVYYCARFSGWPNNYYYYGMDV






WGQGTTVTVSS


 


198
DVD538L
AB033VL
AB005VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDVVMTQSPLSLPVTPGEPASISCRSSQ






SLLHSNGFNYVDWYLQKPGQSPHLLIYFGSYRASG






VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQAL






QTPPWTFGQGTKVEIRR









Example 2.26
Generation of EGFR (Seq. 2) and RON (Seq. 1) DVD-Igs with Linker Set 4












TABLE 58






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







199
DVD539H
AB005VH
AB033VH
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYAMH







WVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCARFSGWPNN






YYYYGMDVWGQGTTVTVSSASTKGPQVQLKQSGPG






LVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGL






EWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFF






KMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLV






TVSA


 


200
DVD539L
AB005VL
AB033VL
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGF






NYVDWYLQKPGQSPHLLIYFGSYRASGVPDRFSGS






GSGTDFTLKISRVEAEDVGVYYCMQALQTPPWTFG






QGTKVEIRRTVAAPSVFIFPPDILLTQSPVILSVS






PGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIK






YASESISGIPSRFSGSGSGTDFTLSINSVESEDIA






DYYCQQNNNWPTTFGAGTKLELKR


 


201
DVD540H
AB033VH
AB005VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLVQSGGGLVKPG






GSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAV






ISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNS






LRAEDTAVYYCARFSGWPNNYYYYGMDVWGQGTTV






TVSS


 


202
DVD540L
AB033VL
AB005VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPDVVMTQSPLSLPVTPGEPAS






ISCRSSQSLLHSNGFNYVDWYLQKPGQSPHLLIYF






GSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV






YYCMQALQTPPWTFGQGTKVEIRR









Example 2.27
Generation of EGFR (Seq. 2) and HGF (Seq. 1) DVD-Igs












TABLE 59






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







203
DVD025H
AB033VH
AB012VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH







WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPQVQLVESGGGLVKPG






GSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSY






ISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNS






LRAEDTAVYYCARDEYNSGWYVLFDYWGQGTLVTV






SS


 


204
DVD025L
AB033VL
AB012VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDIQMTQSPSSVSASVGDRVTITCRASQ






GISSWLAWYQQKPGKAPNLLIYEASSLQSGVPSRF






GGSGSGTDFTLTISSLQPEDFATYYCQQANGFPWT






FGQGTKVEIKR


 


205
DVD026H
AB012VH
AB033VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS






WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPQVQLKQSGPGLV






QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEW






LGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKM






NSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTV






SA


 


206
DVD026L
AB012VL
AB033VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPDILLTQSPVILSVSPGERVSFSCRASQ






SIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF






SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT






FGAGTKLELKR









Example 2.28
Generation of EGFR (Seq. 2) and c-MET DVD-Igs












TABLE 60






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







207
DVD027H
AB033VH
AB013VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH







WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPQVQLQQSGPELVRPG






ASVKWSCPASGYTFTSYWLHWVKKQRPGQGLEWIG






MIDPSNSDTRFNPPNFKDKATLNVDRSSNTAYNLL






SSLTSADSAVYYCATYGSYVSPLDYWGQGTSVYVSS


 


208
DVD027L
AB033VL
AB013VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDIMMSQSPSSLTVSVGEKVTVSCKSSQ






SLLVTSSQKNYLAWYQQKPQQSPKLLIYWASTRES






GVPDRFTGSGSGTDFTLTITSVKADDLAVYYCQQY






YAYPWTFGDGTKLEIKR


 


209
DVD028H
AB013VH
AB033VH
QVQLQQSGPELVRPGASVKWSCPASGYTFTSYWLH






WVKKQRPGQGLEWIGMIDPSNSDTRFNPPNFKDKA






TLNVDRSSNTAYNLLSSLTSADSAVYYCATYGSYV






SPLDYWGQGTSVYVSSASTKGPQVQLKQSGPGLVQ






PSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWL






GVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMN






SLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


210
DVD028L
AB013VL
AB033VL
DIMMSQSPSSLTVSVGEKVTVSCKSSQSLLVTSSQ






KNYLAWYQQKPQQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTITSVKADDLAVYYCQQYYAYPWTFG






DGTKLEIKRTVAAPDILLTQSPVILSVSPGERVSF






SCRASQSIGTNIHWYQQRTNGSPRLLIKYASESIS






GIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQN






NNWPTTFGAGTKLELKR









Example 2.29
Generation of HER-2 (Seq. 1) and IGF1,2 DVD-Igs












TABLE 61






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







211
DVD029H
AB004VH
AB010VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH







WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPQVQLVQSGAEVKKP






GASVKVSCKASGYTFTSYDINWVRQATGQGLEWMG






WMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELS






SLRSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSS


 


212
DVD029L
AB004VL
AB010VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKARKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPQSVLTQPPSVSAAPGQKVTISCSGSSS






NIENNHVSWYQQLPGTAPKLLIYDNNKRPSGIPDR






FSGSKSGTSATLGITGLQTGDEADYYCETWDTSLS






AGRVFGGGTKLTVLG


 


213
DVD030H
AB010VH
AB004VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDIN






WVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTM






TRNTSISTAYMELSSLRSEDTAVYYCARDPYYYYY






GMDVWGQGTTVTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA






RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN






SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


214
DVD030L
AB010VL
AB004VL
QSVLTQPPSVSAAPGQKVTISCSGSSSNIENNHVS






WYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGT






SATLGITGLQTGDEADYYCETWDTSLSAGRVFGGG






TKLTVLGQPKAAPDIQMTQSPSSLSASVGDRVTIT






CRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG






VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY






TTPPTFGQGTKVEIKR









Example 2.30
Generation of HER-2 (Seq. 1) and IGF1R DVD-Igs












TABLE 62






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







215
DVD031H
AB004VH
AB011VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH







WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPEVQLLESGGGLVQP






GGSLRLSCTASGFTFSSYAMNWVRQAPGKGLEWVS






AISGSGGTTFYADSVKGRFTISRDNSRTTLYLQMN






SLRAEDTAVYYCAKDLGWSDSYYYYYGMDVWGQGT






TVTVSS


 


216
DVD031L
AB004VL
AB011VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPDIQMTQFPSSLSASVGDRVTITCRASQ






GIRNDLGWYQQKPGKAPKRLIYAASRLHRGVPSRF






SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPCS






FGQGTKLEIKR


 


217
DVD032H
AB011VH
AB004VH
EVQLLESGGGLVQPGGSLRLSCTASGFTFSSYAMN






WVRQAPGKGLEWVSAISGSGGTTFYADSVKGRFTI






SRDNSRTTLYLQMNSLRAEDTAVYYCAKDLGWSDS






YYYYYGMDVWGQGTTVTVSSASTKGPEVQLVESGG






GLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKG






LEWVARIYPTNGYTRYADSVKGRFTISADTSKNTA






YLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT






LVTVSS


 


218
DVD032L
AB011VL
AB004VL
DIQMTQFPSSLSASVGDRVTITCRASQGIRNDLGW






YQQKPGKAPKRLIYAASRLHRGVPSRFSGSGSGTE






FTLTISSLQPEDFATYYCLQHNSYPCSFGQGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT






FGQGTKVEIKR









Example 2.31
Generation of RON (Seq. 1) and HGF (Seq. 1) DVD-Igs












TABLE 63






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







219
DVD033H
AB005VH
AB012VH
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYAMH







WVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCARFSGWPNN






YYYYGMDVWGQGTTVTVSSASTKGPQVQLVESGGG






LVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGL






EWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLY






LQMNSLRAEDTAVYYCARDEYNSGWYVLFDYWGQG






TLVTVSS


 


220
DVD033L
AB005VL
AB012VL
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGF






NYVDWYLQKPGQSPHLLIYFGSYRASGVPDRFSGS






GSGTDFTLKISRVEAEDVGVYYCMQALQTPPWTFG






QGTKVEIRRTVAAPDIQMTQSPSSVSASVGDRVTI






TCRASQGISSWLAWYQQKPGKAPNLLIYEASSLQS






GVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQQA






NGFPWTFGQGTKVEIKR


 


221
DVD034H
AB012VH
AB005VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS






WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPEVQLVQSGGGLV






KPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEW






VAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQ






MNSLRAEDTAVYYCARFSGWPNNYYYYGMDVWGQG






TTVTVSS


 


222
DVD034L
AB012VL
AB005VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPDVVMTQSPLSLPVTPGEPASISCRSSQ






SLLHSNGFNYVDWYLQKPGQSPHLLIYFGSYRASG






VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQAL






QTPPWTFGQGTKVEIRR









Example 2.32
Generation of VEGF (Seq. 1) and EGFR (Seq. 2) DVD-Igs












TABLE 64






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







223
DVD035H
AB014VH
AB033VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN







WVRQARGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLKQSGPGL






VQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE






WLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFK






MNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVT






VSA


 


224
DVD035L
AB014VL
AB033VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDILLTQSPVILSVSPGERVSFSCRASQ






SIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF






SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT






FGAGTKLELKR


 


225
DVD036H
AB033VH
AB014VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGW






INTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNS






LRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVT






VSS


 


226
DVD036L
AB033VL
AB014VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR









Example 2.33
Generation of VEGF (Seq. 1) and HER-2 (Seq. 1) DVD-Igs












TABLE 65






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







227
DVD037H
AB014VH
AB004VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN







WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPEVQLVESGGGL






VQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE






WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYL






QMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLV






TVSS


 


228
DVD037L
AB014VL
AB004VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT






FGQGTKVEIKR


 


229
DVD038H
AB004VH
AB014VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVG






WINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMN






SLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLV






TVSS


230
DVD038L
AB004VL
AB014VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR









Example 2.34
Generation of VEGF (Seq. 1) and CD-20 DVD-Igs












TABLE 66






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







231
DVD039H
AB014VH
AB001VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN







WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLQQPGAEL






VKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLE






WIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYM






QLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTT






VTVSA


 


232
DVD039L
AB014VL
AB001VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPQIVLSQSPAILSPSPGEKVTMTCRASS






SVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFS






GSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTF






GGGTKLEIKR


 


233
DVD040H
AB001VH
AB014VH
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH






WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATL






TADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGD






WYFNVWGAGTTVTVSAASTKGPEVQLVESGGGLVQ






PGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWV






GWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQM






NSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTL






VTVSS


 


234
DVD040L
AB001VL
AB014VL
QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWF






QQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSY






SLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI






KRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQD






ISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFS






GSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTF






GQGTKVEIKR









Example 2.35
Generation of VEGF (Seq. 1) and IGF1,2 DVD-Igs












TABLE 67






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







235
DVD041H
AB014VH
AB010VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN







WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLVQSGAEV






KKPGASVKVSCKASGYTFTSYDINWVRQATGQGLE






WMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYM






ELSSLRSEDTAVYYCARDPYYYYYGMDVWGQGTTV






TVSS


 


236
DVD041L
AB014VL
AB010VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPQSVLTQPPSVSAAPGQKVTISCSGSSS






NIENNHVSWYQQLPGTAPKLLIYDNNKRPSGIPDR






FSGSKSGTSATLGITGLQTGDEADYYCETWDTSLS






AGRVFGGGTKLTVLG


 


237
DVD042H
AB010VH
AB014VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDIN






WVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTM






TRNTSISTAYMELSSLRSEDTAVYYCARDPYYYYY






GMDVWGQGTTVTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVG






WINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMN






SLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLV






TVSS


 


238
DVD042L
AB010VL
AB014VL
QSVLTQPPSVSAAPGQKVTISCSGSSSNIENNHVS






WYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGT






SATLGITGLQTGDEADYYCETWDTSLSAGRVFGGG






TKLTVLGQPKAAPDIQMTQSPSSLSASVGDRVTIT






CSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSG






VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYS






TVPWTFGQGTKVEIKR









Example 2.36
Generation of VEGF (Seq. 1) and DLL4 (Seq. 1) DVD-Igs












TABLE 68






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







239
DVD043H
AB014VH
AB015VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN







WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPEVQLVESGGGL






VQPGGSLRLSCAASGFTFTDNWISWVRQAPGKGLE






WVGYISPNSGFTYYADSVKGRFTISADTSKNTAYL






QMNSLRAEDTAVYYCARDNFGGYFDYWGQGTLVTV






SS


 


240
DVD043L
AB014VL
AB015VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATTYYCQQSYTGTV






TFGQGTKVEIKR


 


241
DVD044H
AB015VH
AB014VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS






WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWI






NTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSL






RAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTV






SS


 


242
DVD044L
AB015VL
AB014VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPDIQMTQSPSSLSASVGDRVTITCSAS






QDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSR






FSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPW






TFGQGTKVEIKR









Example 2.37
Generation of VEGF (Seq. 1) and HGF (Seq. 1) DVD-Igs with Linker Set 1












TABLE 69






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







243
DVD045H
AB014VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN







WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLVESGGGL






VKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLE






WVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYL






QMNSLRAEDTAVYYCARDEYNSGWYVLFDYWGQGT






LVTVSS


 


244
DVD045L
AB014VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSVSASVGDRVTITCRASQ






GISSWLAWYQQKPGKAPNLLIYEASSLQSGVPSRF






GGSGSGTDFTLTISSLQPEDFATYYCQQANGFPWT






FGQGTKVEIKR


 


245
DVD046H
AB012VH
AB014VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS






WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPEVQLVESGGGLV






QPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEW






VGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQ






MNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGT






LVTVSS


 


246
DVD046L
AB012VL
AB014VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR









Example 2.38
Generation of VEGF (Seq. 1) and HGF (Seq. 1) DVD-Igs with Linker Set 2












TABLE 70






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







247
DVD641H
AB012VH
AB014VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS







WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPSVFPLAPEVQLV






ESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQA






PGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTS






KSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYF






DVWGQGTLVTVSS


 


248
DVD641L
AB012VL
AB014VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLH






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






YSTVPWTFGQGTKVEIKR


 


249
DVD642H
AB014VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPQVQL






VESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQ






APGKGLEWVSYISSSGSTIYYADSVKGRFTISRDN






AKNSLYLQMNSLRAEDTAVYYCARDEYNSGWYVLF






DYWGQGTLVTVSS


 


250
DVD642L
AB014VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSVSASVGDRVT






ITCRASQGISSWLAWYQQKPGKAPNLLIYEASSLQ






SGVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQQ






ANGFPWTFGQGTKVEIKR









Example 2.39
Generation of VEGF (Seq. 1) and HGF (Seq. 1) DVD-Igs with Linker Set 3












TABLE 71






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







251
DVD647H
AB012VH
AB014VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS







WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPSVFPLAPEVQLV






ESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQA






PGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTS






KSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYF






DVWGQGTLVTVSS


 


252
DVD647L
AB012VL
AB014VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR


 


253
DVD648H
AB014VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPQVQL






VESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQ






APGKGLEWVSYISSSGSTIYYADSVKGRFTISRDN






AKNSLYLQMNSLRAEDTAVYYCARDEYNSGWYVLF






DYWGQGTLVTVSS


 


254
DVD648L
AB014VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSVSASVGDRVTITCRASQ






GISSWLAWYQQKPGKAPNLLIYEASSLQSGVPSRF






GGSGSGTDFTLTISSLQPEDFATYYCQQANGFPWT






FGQGTKVEIKR









Example 2.40
Generation of VEGF (Seq. 1) and HGF (Seq. 1) DVD-Igs with Linker Set 4












TABLE 72






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







255
DVD653H
AB012VH
AB014VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS







WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPEVQLVESGGGLV






QPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEW






VGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQ






MNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGT






LVTVSS


 


256
DVD653L
AB012VL
AB014VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLH






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






YSTVPWTFGQGTKVEIKR


 


257
DVD654H
AB014VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLVESGGGL






VKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLE






WVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYL






QMNSLRAEDTAVYYCARDEYNSGWYVLFDYWGQGT






LVTVSS


 


258
DVD654L
AB014VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSVSASVGDRVT






ITCRASQGISSWLAWYQQKPGKAPNLLIYEASSLQ






SGVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQQ






ANGFPWTFGQGTKVEIKR









Example 2.41
Generation of VEGF (Seq. 1) and RON (Seq. 1) DVD-Igs












TABLE 73






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







259
DVD047H
AB014VL
AB005VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN







WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYRHYYGS






SHWYFDVWGQGTLVTVSSASTKGPEVQLVQSGGGL






VKPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLE






WVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYL






QMNSLRAEDTAVYYCARFSGWPNNYYYYGMDVWGQ






GTTVTVSS


 


260
DVD047L
AB014VH
AB005VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDVVMTQSPLSLPVTPGEPASISCRSSQ






SLLHSNGFNYVDWYLQKPGQSPHLLIYFGSYRASG






VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQAL






QTPPWTFGQGTKVEIRR


 


261
DVD048H
AB005VH
AB014VL
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYAMH






WVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCARFSGWPNN






YYYYGMDVWGQGTTVTVSSASTKGPEVQLVESGGG






LVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGL






EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAY






LQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQ






GTLVTVSS


 


262
DVD048L
AB005VL
AB014VH
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGF






NYVDWYLQKPGQSPHLLIYFGSYRASGVPDRFSGS






GSGTDFTLKISRVEAEDVGVYYCMQALQTPPWTFG






QGTKVEIRRTVAAPDIQMTQSPSSLSASVGDRVTI






TCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHS






GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQY






STVPWTFGQGTKVEIKR









Example 2.42
Generation of VEGF (Seq. 1) and NRP1 (Seq. 1) DVD-Igs












TABLE 74






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







263
DVD049H
AB014VH
AB016VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN







WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPEVQLVESGGGL






VQPGGSLRLSCAASGFSFSSEPISWVRQAPGKGLE






WVSSITGKNGYTYYADSVKGRFTISADTSKNTAYL






QMNSLRAEDTAVYYCARWGKKVYGMDVWGQGTLVT






VSS


 


264
DVD049L
AB014VL
AB016VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






SISSYLAWYQQKRGKAPKLLIYGASSRASGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYMSVPIT






FGQGTKVEIKR


 


265
DVD050H
AB016VH
AB014VH
EVQLVESGGGLVQPGGSLRLSCAASGFSFSSEPIS






WVRQAPGKGLEWVSSITGKNGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGKKVYG






MDVWGQGTLVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGW






INTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNS






LRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVT






VSS


 


266
DVD050L
AB016VL
AB014VL
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLAW






YQQKPGKAPKLLIYGASSRASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYMSVPITFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR









Example 2.43
Generation of HGF (Seq. 1) and RON (Seq. 2) DVD-Igs












TABLE 75






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







267
DVD073H
AB034VH
AB012VH
QVQLQESGPGLVKPSEILSLTCTVSGGSISSHYWS







WVRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTIS






VDTSKNQFSLNLSSVTAADTAVYYCARIPNYYDRS






GYYPGYWYFDLWGRGTLVTVSSASTKGPQVQLVES






GGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG






KGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKN






SLYLQMNSLRAEDTAVYYCARDEYNSGWYVLFDYW






GQGTLVTVSS


 


268
DVD073L
AB034VL
AB012VL
QAVLTQPSSLSAPPGASASLTCTLRSGFNVDSYRI






SWYQQKPGSPPQYLLRYKSDSDKQQGSGVPSRFSG






SKDASANAGILLISGLQSEDEADYYCMIWHSSAWV






FGGGTKLTVLRTVAAPDIQMTQSPSSVSASVGDRV






TITCRASQGISSWLAWYQQKPGKAPNLLIYEASSL






QSGVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQ






QANGFPWTFGQGTKVEIKR


 


269
DVD074H
AB012VH
AB034VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS






WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPQVQLQESGPGLV






KPSEILSLTCTVSGGSISSHYWSWVRQPPGKGLEW






IGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLNL






SSVTAADTAVYYCARIPNYYDRSGYYPGYWYFDLW






GRGTLVTVSS


 


270
DVD074L
AB012VL
AB034VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPQAVLTQPSSLSAPPGASASLTCTLRSG






FNVDSYRISWYQQKPGSPPQYLLRYKSDSDKQQGS






GVPSRFSGSKDASANAGILLISGLQSEDEADYYCM






IWHSSAWVFGGGTKLTVLR









Example 2.44
Generation of EGFR (Seq. 2) and RON (Seq. 2) DVD-Igs












TABLE 76






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







271
DVD075H
AB034VH
AB033VH
QVQLQESGPGLVKPSEILSLTCTVSGGSISSHYWS







WVRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTIS






VDTSKNQFSLNLSSVTAADTAVYYCARIPNYYDRS






GYYPGYWYFDLWGRGTLVTVSSASTKGPQVQLKQS






GPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPG






KGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQ






VFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQG






TLVTVSA


 


272
DVD075L
AB034VL
AB033VL
QAVLTQPSSLSAPPGASASLTCTLRSGFNVDSYRI






SWYQQKPGSPPQYLLRYKSDSDKQQGSGVPSRFSG






SKDASANAGILLISGLQSEDEADYYCMIWHSSAWV






FGGGTKLTVLRTVAAPDILLTQSPVILSVSPGERV






SFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASES






ISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ






QNNNWPTTFGAGTKLELKR


 


273
DVD076H
AB033VH
AB034VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPQVQLQESGPGLVKPS






EILSLTCTVSGGSISSHYWSWVRQPPGKGLEWIGY






IYYSGSTNYNPSLKSRVTISVDTSKNQFSLNLSSV






TAADTAVYYCARIPNYYDRSGYYPGYWYFDLWGRG






TLVTVSS


 


274
DVD076L
AB033VL
AB034VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPQAVLTQPSSLSAPPGASASLTCTLRSG






FNVDSYRISWYQQKPGSPPQYLLRYKSDSDKQQGS






GVPSRFSGSKDASANAGILLISGLQSEDEADYYCM






IWHSSAWVFGGGTKLTVLR









Example 2.45
Generation of VEGF (Seq. 1) and RON (Seq. 2) DVD-Igs












TABLE 77






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







275
DVD077H
AB034VH
AB014VH
QVQLQESGPGLVKPSEILSLTCTVSGGSISSHYWS







WVRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTIS






VDTSKNQFSLNLSSVTAADTAVYYCARIPNYYDRS






GYYPGYWYFDLWGRGTLVTVSSASTKGPEVQLVES






GGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPG






KGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKS






TAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDV






WGQGTLVTVSS


 


276
DVD077L
AB034VL
AB014VL
QAVLTQPSSLSAPPGASASLTCTLRSGFNVDSYRI






SWYQQKPGSPPQYLLRYKSDSDKQQGSGVPSRFSG






SKDASANAGILLISGLQSEDEADYYCMIWHSSAWV






FGGGTKLTVLRTVAAPDIQMTQSPSSLSASVGDRV






TITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSL






HSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ






QYSTVPWTFGQGTKVEIKR


 


277
DVD078H
AB014VH
AB034VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLQESGPGL






VKPSEILSLTCTVSGGSISSHYWSWVRQPPGKGLE






WIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLN






LSSVTAADTAVYYCARIPNYYDRSGYYPGYWYFDL






WGRGTLVTVSS


 


278
DVD078L
AB014VL
AB034VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPQAVLTQPSSLSAPPGASASLTCTLRSG






FNVDSYRISWYQQKPGSPPQYLLRYKSDSDKQQGS






GVPSRFSGSKDASANAGILLISGLQSEDEADYYCM






IWHSSAWVFGGGTKLTVLR









Example 2.46
Generation of EGFR (Seq. 1) and HER-2 (Seq. 1) DVD-Igs












TABLE 78






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







279
DVD079H
AB003VH
AB004VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY







WTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR






IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


280
DVD079L
AB003VL
AB004VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT






FGQGTKVEIKR


 


281
DVD080H
AB004VH
AB003VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPQVQLQESGPGLVKP






SETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEW






IGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKL






SSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS


 


282
DVD080L
AB004VL
AB003VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQ






DISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF






SGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLA






FGGGTKVEIKR









Example 2.47
Generation of EGFR (Seq. 1) and CD3 (Seq. 1) DVD-Igs












TABLE 79






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







283
DVD081H
AB003VH
AB002VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY







WTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPQVQLQQSGAELARPG






ASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGY






INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSS






LTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS


 


284
DVD081L
AB003VL
AB002VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPQIVLTQSPAIMSASPGEKVTMTCRASS






SVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFS






GSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTF






GSGTKLEINR


 


285
DVD082H
AB002VH
AB003VH
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH






WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL






TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC






LDYWGQGTTLTVSSASTKGPQVQLQESGPGLVKPS






ETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWI






GHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLS






SVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS


 


286
DVD082L
AB002VL
AB003VL
QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWY






QQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSY






SLTISSMEAEDAATYYCQQWSSNPLTFGSGTKLEI






NRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQD






ISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFS






GSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAF






GGGTKVEIKR









Example 2.48
Generation of EGFR (Seq. 1) and IGF1R DVD-Igs












TABLE 80






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







287
DVD083H
AB003VH
AB011VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY







WTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPEVQLLESGGGLVQPG






GSLRLSCTASGFTFSSYAMNWVRQAPGKGLEWVSA






ISGSGGTTFYADSVKGRFTISRDNSRTTLYLQMNS






LRAEDTAVYYCAKDLGWSDSYYYYYGMDVWGQGTT






VTVSS


 


288
DVD083L
AB003VL
AB011VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPDIQMTQFPSSLSASVGDRVTITCRASQ






GIRNDLGWYQQKPGKAPKRLIYAASRLHRGVPSRF






SGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPCS






FGQGTKLEIKR


 


289
DVD084H
AB011VH
AB003VH
EVQLLESGGGLVQPGGSLRLSCTASGFTFSSYAMN






WVRQAPGKGLEWVSAISGSGGTTFYADSVKGRFTI






SRDNSRTTLYLQMNSLRAEDTAVYYCAKDLGWSDS






YYYYYGMDVWGQGTTVTVSSASTKGPQVQLQESGP






GLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPG






KGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQ






FSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTM






VTVSS


 


290
DVD084L
AB011VL
AB003VL
DIQMTQFPSSLSASVGDRVTITCRASQGIRNDLGW






YQQKPGKAPKRLIYAASRLHRGVPSRFSGSGSGTE






FTLTISSLQPEDFATYYCLQHNSYPCSFGQGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQ






DISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF






SGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLA






FGGGTKVEIKR









Example 2.49
Generation of EGFR (Seq. 1) and RON (Seq. 1) DVD-Igs












TABLE 81






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







291
DVD085H
AB003VH
AB005VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY







WTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPEVQLVQSGGGLVKPG






GSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAV






ISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNS






LRAEDTAVYYCARFSGWPNNYYYYGMDVWGQGTTV






TVSS


 


292
DVD085L
AB003VL
AB005VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPDVVMTQSPLSLPVTPGEPASISCRSSQ






SLLHSNGFNYVDWYLQKPGQSPHLLIYFGSYRASG






VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQAL






QTPPWTFGQGTKVEIRR


 


293
DVD086H
AB005VH
AB003VH
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYAMH






WVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCARFSGWPNN






YYYYGMDVWGQGTTVTVSSASTKGPQVQLQESGPG






LVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGK






GLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQF






SLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMV






TVSS


 


294
DVD086L
AB005VL
AB003VL
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGF






NYVDWYLQKPGQSPHLLIYFGSYRASGVPDRFSGS






GSGTDFTLKISRVEAEDVGVYYCMQALQTPPWTFG






QGTKVEIRRTVAAPDIQMTQSPSSLSASVGDRVTI






TCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET






GVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHF






DHLPLAFGGGTKVEIKR









Example 2.50
Generation of EGFR (Seq. 1) and RON (Seq. 2) DVD-Igs












TABLE 82






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







295
DVD087H
AB003VH
AB034VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY







WTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPQVQLQESGPGLVKPS






EILSLTCTVSGGSISSHYWSWVRQPPGKGLEWIGY






IYYSGSTNYNPSLKSRVTISVDTSKNQFSLNLSSV






TAADTAVYYCARIPNYYDRSGYYPGYWYFDLWGRG






TLVTVSS


 


296
DVD087L
AB003VL
AB034VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPQAVLTQPSSLSAPPGASASLTCTLRSG






FNVDSYRISWYQQKPGSPPQYLLRYKSDSDKQQGS






GVPSRFSGSKDASANAGILLISGLQSEDEADYYCM






IWHSSAWVFGGGTKLTVLR


 


297
DVD088H
AB034VH
AB003VH
QVQLQESGPGLVKPSEILSLTCTVSGGSISSHYWS






WVRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTIS






VDTSKNQFSLNLSSVTAADTAVYYCARIPNYYDRS






GYYPGYWYFDLWGRGTLVTVSSASTKGPQVQLQES






GPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQS






PGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSK






TQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQG






TMVTVSS


 


298
DVD088L
AB034VL
AB003VL
QAVLTQPSSLSAPPGASASLTCTLRSGFNVDSYRI






SWYQQKPGSPPQYLLRYKSDSDKQQGSGVPSRFSG






SKDASANAGILLISGLQSEDEADYYCMIWHSSAWV






FGGGTKLTVLRTVAAPDIQMTQSPSSLSASVGDRV






TITCQASQDISNYLNWYQQKPGKAPKLLIYDASNL






ETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQ






HFDHLPLAFGGGTKVEIKR









Example 2.51
Generation of EGFR (Seq. 1) and HGF (Seq. 1) DVD-Igs












TABLE 83






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







299
DVD089H
AB003VH
AB012VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY







WTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPQVQLVESGGGLVKPG






GSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSY






ISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNS






LRAEDTAVYYCARDEYNSGWYVLFDYWGQGTLVTV






SS


 


300
DVD089L
AB003VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPDIQMTQSPSSVSASVGDRVTITCRASQ






GISSWLAWYQQKPGKAPNLLIYEASSLQSGVPSRF






GGSGSGTDFTLTISSLQPEDFATYYCQQANGFPWT






FGQGTKVEIKR


 


301
DVD090H
AB012VH
AB003VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS






WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPQVQLQESGPGLV






KPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGL






EWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSL






KLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTV






SS


 


302
DVD090L
AB012VL
AB003VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQ






DISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF






SGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLA






FGGGTKVEIKR









Example 2.52
Generation of EGFR (Seq. 1) and c-MET DVD-Igs












TABLE 84






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







303
DVD091H
AB003VH
AB013VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY







WTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPQVQLQQSGPELVRPG






ASVKWSCPASGYTFTSYWLHWVKKQRPGQGLEWIG






MIDPSNSDTRFNPPNFKDKATLNVDRSSNTAYNLL






SSLTSADSAVYYCATYGSYVSPLDYWGQGTSVYVSS


 


304
DVD091L
AB003VL
AB013VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPDIMMSQSPSSLTVSVGEKVTVSCKSSQ






SLLVTSSQKNYLAWYQQKPQQSPKLLIYWASTRES






GVPDRFTGSGSGTDFTLTITSVKADDLAVYYCQQY






YAYPWTFGDGTKLEIKR


 


305
DVD092H
AB013VH
AB003VH
QVQLQQSGPELVRPGASVKWSCPASGYTFTSYWLH






WVKKQRPGQGLEWIGMIDPSNSDTRFNPPNFKDKA






TLNVDRSSNTAYNLLSSLTSADSAVYYCATYGSYV






SPLDYWGQGTSVYVSSASTKGPQVQLQESGPGLVK






PSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLE






WIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLK






LSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS


 


306
DVD092L
AB013VL
AB003VL
DIMMSQSPSSLTVSVGEKVTVSCKSSQSLLVTSSQ






KNYLAWYQQKPQQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTITSVKADDLAVYYCQQYYAYPWTFG






DGTKLEIKRTVAAPDIQMTQSPSSLSASVGDRVTI






TCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET






GVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHF






DHLPLAFGGGTKVEIKR









Example 2.53
Generation of EGFR (Seq. 1) and VEGF (Seq. 1) DVD-Igs












TABLE 85






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







307
DVD093H
AB003VH
AB014VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY







WTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGW






INTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNS






LRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVT






VSS


 


308
DVD093L
AB003VL
AB014VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR


 


309
DVD094H
AB014VH
AB003VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLQESGPGL






VKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKG






LEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFS






LKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVT






VSS


 


310
DVD094L
AB014VL
AB003VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQ






DISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF






SGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLA






FGGGTKVEIKR









Example 2.54
Generation of NRP1 (Seq. 2) and VEGF (Seq. 1) DVD-Igs












TABLE 86






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







311
DVD107H
AB035VH
AB014VH
EVQLVESGGGLVQRGGSLRLSCAASGFTFSSYAMS







WVRQAPGKGLEWVSQISPAGGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARELRYYRM






SKVMDVQGQGTLVTVSSASTKGPEVQLVESGGGLV






QPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEW






VGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQ






MNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGT






LVTVSS


 


312
DVD107L
AB035VL
AB014VL
DIQMTQSPSSLSASVGDRVTITCRASQYFSSYLAW






YQQKPGKAPKLLIYGASSRASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYLGSPPTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR


 


313
DVD108H
AB014VH
AB035VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPEVQLVESGGGL






VQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE






WVSQISPAGGYTNYADSVKGRFTISADTSKNTAYL






QMNSLRAEDTAVYYCARELPYYRMSKVMDVQGQGT






LVTVSS


 


314
DVD108L
AB014VL
AB035VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






YFSSYLAWYQQKPGKAPKLLIYGASSRASGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYLGSPPT






FGQGTKVEIKR









Example 2.55
Generation of CD3 (Seq. 2) and CD-20 DVD-Igs












TABLE 87






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







315
DVD131H
AB039VH
AB001VH
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH







WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL






TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC






LDYWGQGTTLTVSSASTKGPQVQLQQPGAELVKPG






ASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGA






IYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSS






LTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA


 


316
DVD131L
AB039VL
AB001VL
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY






QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY






SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI






NRTVAAPQIVLSQSPAILSPSPGEKVTMTCRASSS






VSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSG






SGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFG






GGTKLEIKR


 


317
DVD132H
AB001VH
AB039VH
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH






WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATL






TADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGD






WYFNVWGAGTTVTVSAASTKGPQVQLQQSGAELAR






PGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWI






GYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQL






SSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS


 


318
DVD132L
AB001VL
AB039VL
QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWF






QQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSY






SLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI






KRTVAAPQIVLTQSPAIMSASPGEKVTMTCSASSS






VSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRG






SGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFG






SGTKLEINR









Example 2.56
Generation of CD-3 (Seq. 2) and HER-2 (Seq. 1) DVD-Igs












TABLE 88






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







319
DVD135H
AB039VH
AB004VH
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH







WVKQRRGQGLEWIGYINPSRGYTNYNQKFKDKATL






TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC






LDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR






IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


320
DVD135L
AB039VL
AB004VL
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY






QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY






SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI






NRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQD






VNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS






GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF






GQGTKVEIKR


 


321
DVD136H
AB004VH
AB039VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPQVQLQQSGAELARP






GASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIG






YINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLS






SLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS


 


322
DVD136L
AB004VL
AB039VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVRSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPQIVLTQSPAIMSASPGEKVTMTCSASS






SVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFR






GSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTF






GSGTKLEINR









Example 2.57
Generation of CD-3 (Seq. 2) and CD-19 DVD-Igs












TABLE 89






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







323
DVD137H
AB039VH
AB006VH
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH







WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL






TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC






LDYWGQGTTLTVSSASTKGPQVQLQQSGAELVRPG






SSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQ






IWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSS






LASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSV






TVSS


 


324
DVD137L
AB039VL
AB006VL
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY






QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY






SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI






NRTVAAPDILLTQTPASLAVSLGQRATISCKASQS






VDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIP






PRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTED






PWTFGGGTKLEIKR


 


325
DVD138H
AB006VH
AB039VH
QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMN






WVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATL






TADESSSTAYMQLSSLASEDSAVYFCARRETTTVG






RYYYAMDYWGQGTSVTVSSASTKGPQVQLQQSGAE






LARPGASVKMSCKASGYTFTRYTMHWVKQRRGQGL






EWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAY






MQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTL






TVSS


 


326
DVD138L
AB006VL
AB039VL
DILLTQTPASLAVSLGQRATISCKASQSVDYDGDS






YLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSG






SGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGG






TKLEIKRTVAAPQIVLTQSPAIMSASPGEKVTMTC






SASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVP






AHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSN






PFTFGSGTKLEINR









Example 2.58
Generation of CD-3 (Seq. 2) and EGFR (Seq. 2) DVD-Igs












TABLE 90






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







327
DVD139H
AB039VH
AB033VH
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH







WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL






TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC






LDYWGQGTTLTVSSASTKGPQVQLKQSGPGLVQPS






QSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV






IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSL






QSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


328
DVD139L
AB039VL
AB033VL
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY






QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY






SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI






NRTVAAPDILLTQSPVILSVSPGERVSFSCRASQS






IGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS






GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTF






GAGTKLELKR


 


329
DVD140H
AB033VH
AB039VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPQVQLQQSGAELARPG






ASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGY






INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSS






LTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS


 


330
DVD140L
AB033VL
AB039VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPQIVLTQSPAIMSASPGEKVTMTCSASS






SVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFR






GSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTF






GSGTKLEINR









Example 2.59
Generation of CD-3 (Seq. 2) and EGFR (Seq. 1) DVD-Igs












TABLE 91






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







331
DVD141H
AB039VH
AB003VH
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH







WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL






TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC






LDYWGQGTTLTVSSASTKGPQVQLQESGPGLVKPS






ETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWI






GHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLS






SVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS


 


332
DVD141L
AB039VL
AB003VL
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY






QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY






SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI






NRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQD






ISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFS






GSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAF






GGGTKVEIKR


 


333
DVD142H
AB003VH
AB039VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY






WTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPQVQLQQSGAELARPG






ASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGY






INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSS






LTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS


 


334
DVD142L
AB003VL
AB039VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPQIVLTQSPAIMSASPGEKVTMTCSASS






SVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFR






GSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTF






GSGTKLEINR









Example 2.60
Generation of EGFR (Seq. 1) and IGF1,2 DVD-Igs












TABLE 92






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







335
DVD143H
AB003VH
AB010VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY







WTWIRQSPGKGLEWIGHIYYSGNTNYNRSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPQVQLVQSGAEVKKPG






ASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGW






MNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSS






LRSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSS


 


336
DVD143L
AB003VL
AB010VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPQSVLTQPPSVSAAPGQKVTISCSGSSS






NIENNHVSWYQQLPGTAPKLLIYDNNKRPSGIPDR






FSGSKSGTSATLGITGLQTGDEADYYCETWDTSLS






AGRVFGGGTKLTVLG


 


337
DVD144H
AB010VH
AB003VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDIN






WVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTM






TRNTSISTAYMELSSLRSEDTAVYYCARDPYYYYY






GMDVWGQGTTVTVSSASTKGPQVQLQESGPGLVKP






SETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEW






IGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKL






SSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS


 


338
DVD144L
AB010VL
AB003VL
QSVLTQPPSVSAAPGQKVTISCSGSSSNIENNHVS






WYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGT






SATLGITGLQTGDEADYYCETWDTSLSAGRVFGGG






TKLTVLGQPKAAPDIQMTQSPSSLSASVGDRVTIT






CQASQDISNYLNWYQQKPGKAPKLLIYDASNLETG






VPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFD






HLPLAFGGGTKVEIKR









Example 2.61
Generation of DLL-4 (Seq. 1) and PLGF (Seq. 1) DVD-Igs












TABLE 93






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







339
VD257H
AB015VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPQVQLQQSGAELVKPGA






SVKISCKASGYTFTDYYINWVKLAPGQGLEWIGWI






YRGSGNTKYNEKFKGKATLTIDTSSSTAYMQLSSL






TSEDTAVYFCVRDSPFFDYWGQGTLLTVSS


 


340
VD257L
AB015VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPDIVLTQSPDSLAVSLGERVTMNCKSS






QSLLNSGMRKSFLAWYQQKPGQSPKLLIYWASTRE






SGVPDRFTGSGSGTDFTLTISSVQAEDVAVYYCKQ






SYHLFTFGSGTKLEIKR


 


341
VD258H
AB047VH
AB015VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN






WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFTFTDNWISWVRQAPGKGLEWVGYISP






NSGFTYYADSVKGRFTISADTSKNTAYLQMNSLRA






EDTAVYYCARDNFGGYFDYWGQGTLVTVSS


 


342
VD258L
AB047VL
AB015VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPDIQMTQSPSSLSASVGDRVTIT






CRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG






VPSRFSGSGSGTDFTLTISSLQPEDFATTYYCQQS






YTGTVTFGQGTKVEIKR









Example 2.62
Generation of VEGF (Seq. 1) and PLGF (Seq. 1) DVD-Igs with Linker Set 1












TABLE 94






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







343
VD259H
AB014VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN







WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLQQSGAEL






VKPGASVKISCKASGYTFTDYYINWVKLAPGQGLE






WIGWIYPGSGNTKYNEKFKGKATLTIDTSSSTAYM






QLSSLTSEDTAVYFCVRDSPFFDYWGQGTLLTVSS


 


344
VD259L
AB014VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIVLTQSPDSLAVSLGERVTMNCKSSQ






SLLNSGMRKSFLAWYQQKPGQSPKLLIYWASTRES






GVPDRFTGSGSGTDFTLTISSVQAEDVAVYYCKQS






YHLFTFGSGTKLEIKR


 


345
VD260H
AB047VH
AB014VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN






WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINT






YTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRA






EDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS


 


346
VD260L
AB047VL
AB014VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPDIQMTQSPSSLSASVGDRVTIT






CSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSG






VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYS






TVPWTFGQGTKVEIKR









Example 2.63
Generation of VEGF (Seq. 1) and PLGF (Seq. 1) DVD-Igs with Linker Set 2












TABLE 95






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







347
DVD579H
AB047VH
AB014VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLE






WVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYL






QMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQG






TLVTVSS


 


348
DVD579L
AB047VL
AB014VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPSVFIFPPDIQMTQSPSSLSASV






GDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYF






TSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFAT






YYCQQYSTVPWTFGQGTKVEIKR


 


349
DVD580H
AB014VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPQVQL






QQSGAELVKPGASVKISCKASGYTFTDYYINWVKL






APGQGLEWIGWIYPGSGNTKYNEKFKGKATLTIDT






SSSTAYMQLSSLTSEDTAVYFCVRDSPFFDYWGQG






TLLTVSS


 


350
DVD580L
AB014VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIVLTQSPDSLAVSLGERVT






MNCKSSQSLLNSGMRKSFLAWYQQKPGQSPKLLIY






WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDVA






VYYCKQSYHLFTFGSGTKLEIKR









Example 2.64
Generation of VEGF (Seq. 1) and PLGF (Seq. 1) DVD-Igs with Linker Set 3












TABLE 96






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







351
DVD587VH
AB047VH
AB014VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLE






WVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYL






QMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQG






TLVTVSS


 


352
DVD587VL
AB047VL
AB014VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPDIQMTQSPSSLSASVGDRVTIT






CSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSG






VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYS






TVPWTFGQGTKVEIKR


 


353
DVD588VH
AB014VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPQVQL






QQSGAELVKPGASVKISCKASGYTFTDYYINWVKL






APGQGLEWIGWIYPGSGNTKYNEKFKGKATLTIDT






SSSTAYMQLSSLTSEDTAVYFCVRDSPFFDYWGQG






TLLTVSS


 


354
DVD588VL
AB014VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIVLTQSPDSLAVSLGERVTMNCKSSQ






SLLNSGMRKSFLAWYQQKPGQSPKLLIYWASTRES






GVPDRFTGSGSGTDFTLTISSVQAEDVAVYYCKQS






YHLFTFGSGTKLEIKR









Example 2.65
Generation of VEGF (Seq. 1) and PLGF (Seq. 1) DVD-Igs with Linker Set 4












TABLE 97






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







355
DVD595VH
AB047VH
AB014VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINT






YTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRA






EDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS


 


356
DVD595VL
AB047VL
AB014VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPSVFIFPPDIQMTQSPSSLSASV






GDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYF






TSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFAT






YYCQQYSTVPWTFGQGTKVEIKR


 


357
DVD596VH
AB014VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLQQSGAEL






VKPGASVKISCKASGYTFTDYYINWVKLAPGQGLE






WIGWIYPGSGNTKYNEKFKGKATLTIDTSSSTAYM






QLSSLTSEDTAVYFCVRDSPFFDYWGQGTLLTVSS


 


358
DVD596VL
AB014VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIVLTQSPDSLAVSLGERVT






MNCKSSQSLLNSGMRKSFLAWYQQKPGQSPKLLIY






WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDVA






VYYCKQSYHLFTFGSGTKLEIKR









Example 2.66
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 1) DVD-Igs with Linker Set 1












TABLE 98






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







359
VD299H
AB062VH
AB033VH
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS







WIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTIS






VETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFD






LWGRGTLVTVSSASTKGPQVQLKQSGPGLVQPSQS






LSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIW






SGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQS






NDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


360
VD299L
AB062VL
AB033VL
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSN






RNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSG






SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFG






QGTKVEIKRTVAAPDILLTQSRVILSVSPGERVSF






SCRASQSIGTNIHWYQQRTNGSPRLLIKYASESIS






GIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQN






NNWPTTFGAGTKLELKR


 


361
VD300H
AB033VH
AB062VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPQVQLQQWGAGLLKPS






ETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGE






INHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSV






TAADTAVYYCARDKWTWYFDLWGRGTLVTVSS


 


362
VD300L
AB033VL
AB062VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDIEMIQSPDSLAVSLGERATINCRSSQ






SVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRES






GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY






YSTPRTFGQGTKVEIKR









Example 2.67
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 1) DVD-Igs with Linker Set 2












TABLE 99






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







363
DVD385H
AL062
AL033
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS







WIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTIS






VETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFD






LWGRGTLVTVSSASTKGPSVFPLAPQVQLKQSGPG






LVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGL






EWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFF






KMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLV






TVSA


 


364
DVD385L
AL062
AL033
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSN






RNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSG






SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFG






QGTKVEIKRTVAAPSVFIFPPDILLTQSPVILSVS






PGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIK






YASESISGIPSRFSGSGSGTDFTLSINSVESEDIA






DYYCQQNNNWPTTFGAGTKLELKR


 


365
DVD386H
AL033
AL062
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPQVQLQQWG






AGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGK






GLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQF






SLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLV






TVSS


 


366
DVD386L
AL033
AL062
DILLTQSPVILSVSRGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPDIEMTQSPDSLAVSLGERAT






INCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIY






WASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA






VYYCQQYYSTPRTFGQGTKVEIKR









Example 2.68
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 1) DVD-Igs with Linker Set 3












TABLE 100






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







367
DVD389H
AL062
AL033
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS







WIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTIS






VETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFD






LWGRGTLVTVSSASTKGPSVFPLAPQVQLKQSGPG






LVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGL






EWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFF






KMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLV






TVSA


 


368
DVD389L
AL062
AL033
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSN






RNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSG






SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFG






QGTKVEIKRTVAAPDILLTQSPVILSVSPGERVSF






SCRASQSIGTNIHWYQQRTNGSPRLLIKYASESIS






GIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQN






NNWPTTFGAGTKLELKR


 


369
DVD390H
AL033
AL062
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPQVQLQQWG






AGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGK






GLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQF






SLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLV






TVSS


 


370
DVD390L
AL033
AL062
DILLTQSPVILSVSRGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDIEMTQSPDSLAVSLGERATINCRSSQ






SVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRES






GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY






YSTPRTFGQGTKVEIKR









Example 2.69
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 1) DVD-Igs with Linker Set 4












TABLE 101






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







371
DVD393H
AL062
AL033
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS







WIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTIS






VETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFD






LWGRGTLVTVSSASTKGPQVQLKQSGPGLVQPSQS






LSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIW






SGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQS






NDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


372
DVD393L
AL062
AL033
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSN






RNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSG






SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFG






QGTKVEIKRTVAAPSVFIFPPDILLTQSPVILSVS






PGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIK






YASESISGIPSRFSGSGSGTDFTLSINSVESEDIA






DYYCQQNNNWPTTFGAGTKLELKR


 


373
DVD394H
AL033
AL062
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPQVQLQQWGAGLLKPS






ETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGE






INHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSV






TAADTAVYYCARDKWTWYFDLWGRGTLVTVSS


 


374
DVD394L
AL033
AL062
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPDIEMTQSPDSLAVSLGERAT






INCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIY






WASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA






VYYCQQYYSTPRTFGQGTKVEIKR









Example 2.70
Generation of EGFR (Seq. 1) and ErbB3 (Seq. 1) DVD-Igs












TABLE 102






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







375
DVD301H
AB062VH
AB003VH
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS







WIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTIS






VETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFD






LWGRGTLVTVSSASTKGPQVQLQESGPGLVKPSET






LSLTCTVSGGSVSSGDYYWTWIRQSRPKGLEWIGH






IYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSV






TAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS


 


376
DVD301L
AB062VL
AB003VL
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSN






RNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSG






SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFG






QGTKVEIKRTVAAPDIQMTQSPSSLSASVGDRVTI






TCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET






GVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHF






DHLPLAFGGGTKVEIKR


 


377
DVD302H
AB003VH
AB062VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY






WTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPQVQLQQWGAGLLKPS






ETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGE






INHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSV






TAADTAVYYCARDKWTWYFDLWGRGTLVTVSS


 


378
DVD302L
AB003VL
AB062VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPDIEMTQSPDSLAVSLGERATINCRSSQ






SVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRES






GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY






YSTPRTFGQGTKVEIKR









Example 2.71
Generation of HGF (Seq. 1) and ErbB3 (Seq. 1) DVD-Igs












TABLE 103






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







379
DVD303H
AB062VH
AB012VH
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS







WIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTIS






VETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFD






LWGRGTLVTVSSASTKGPQVQLVESGGGLVKPGGS






LRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYIS






SSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLR






AFDTAVYYCARDEYNSGWYVLFDYWGQGTLVTVSS


 


380
DVD303L
AB062VL
AB012VL
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSN






RNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSG






SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFG






QGTKVEIKRTVAAPDIQMTQSPSSVSASVGDRVTI






TCRASQGISSWLAWYQQKPGKAPNLLIYEASSLQS






GVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQQA






NGFPWTFGQGTKVEIKR


 


381
DVD304H
AB012VH
AB062VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS






WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPQVQLQQWGAGLL






KPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEW






IGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKL






SSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS


 


382
DVD304L
AB012VL
AB062VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPDIEMTQSPDSLAVSLGERATINCRSSQ






SVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRES






GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY






YSTPRTFGQGTKVEIKR









Example 2.72
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 2) DVD-Igs with Linker Set 1












TABLE 104






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







383
DVD305H
AB063VH
AB033VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMN







WVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRDEDTAVYYCARDRGDFDA






FDIWGQGTMVTVSSASTKGPQVQLKQSGPGLVQPS






QSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV






IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSL






QSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


384
DVD305L
AB063VL
AB033VL
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYNCQQCENFPITFGQGTRLE






IKRTVAAPDILLTQSPVILSVSPGERVSFSCRASQ






SIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF






SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT






FGAGTKLELKR


 


385
DVD306H
AB033VH
AB063VH
QVQLKQSGRGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFTFSIYSMNWVRQAPGKGLEWVSY






ISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNS






LRDEDTAVYYCARDRGDFDAFDIWGQGTMVTVSS


 


386
DVD306L
AB033VL
AB063VL
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQ






DITNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF






SGSGSGTDFTFTISSLQPEDIATYNCQQCENFPIT






FGQGTRLEIKR









Example 2.73
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 2) DVD-Igs with Linker Set 2












TABLE 105






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







387
DVD397H
AB063
AB033
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMN







WVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRDEDTAVYYCARDRGDFDA






EDIWGQGTMVTVSSASTKGPSVFPLAPQVQLKQSG






PGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGK






GLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQV






FFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGT






LVTVSA


 


388
DVD397L
AB063
AB033
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYNCQQCENFPITFGQGTRLE






IKRTVAAPSVFIFPPDILLTQSPVILSVSPGERVS






FSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESI






SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQ






NNNWPTTFGAGTKLELKR


 


389
DVD398H
AB033
AB063
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFTFSIYSMNWVRQAPGK






GLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNS






LYLQMNSLRDEDTAVYYCARDRGDFDAFDIWGQGT






MVTVSS


 


390
DVD398L
AB033
AB063
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FILSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCQASQDITNYLNWYQQKPGKAPKLLIYDASNLE






TGVPSRFSGSGSGTDFTFTISSLQPEDIATYNCQQ






CENFPITFGQGTRLEIKR









Example 2.74
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 2) DVD-Igs with Linker Set 3












TABLE 106






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







391
DVD401H
AB063
AB033
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMN







WVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRDEDTAVYYCARDRGDFDA






FDIWGQGTMVTVSSASTKGPSVFPLAPQVQLKQSG






RGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGK






GLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQV






FFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGT






LVTVSA


 


392
DVD401L
AB063
AB033
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYNCQQCENFPITFGQGTRLE






IKRTVAAPDILLTQSPVILSVSPGERVSFSCRASQ






SIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF






SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT






FGAGTKLELKR


 


393
DVD402H
AB033
AB063
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFTFSIYSMNWVRQAPGK






GLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNS






LYLQMNSLRDEDTAVYYCARDRGDFDAFDIWGQGT






MVTVSS


 


394
DVD402L
AB033
AB063
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQ






DITNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF






SGSGSGTDFTFTISSLQPEDIATYNCQQCENFPIT






FGQGTRLEIKR









Example 2.75
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 2) DVD-Igs with Linker Set 4












TABLE 107






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







395
DVD405H
AB063
AB033
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMN







WVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRDEDTAVYYCARDRGDFDA






FDIWGQGTMVTVSSASTKGPQVQLKQSGPGLVQPS






QSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV






IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSL






QSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


396
DVD405L
AB063
AB033
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYNCQQCENFPITFGQGTRLE






IKRTVAAPSVFIFPPDILLTQSPVILSVSRGERVS






FSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESI






SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQ






NNNWPTTFGAGTKLELKR


 


397
DVD406H
AB033
AB063
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFTFSIYSMNWVRQAPGKGLEWVSY






ISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNS






LRDEDTAVYYCARDRGDFDAFDIWGQGTMVTVSS


 


398
DVD406L
AB033
AB063
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCQASQDITNYLNWYQQKPGKAPKLLIYDASNLE






TGVPSRFSGSGSGTDFTFTISSLQPEDIATYNCQQ






CENFPITFGQGTRLEIKR









Example 2.76
Generation of EGFR (Seq. 1) and ErbB3 (Seq. 2) DVD-Igs












TABLE 108






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







399
DVD307H
AB063VH
AB003VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMN







WVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRDEDTAVYYCARDRGDFDA






FDIWGQGTMVTVSSASTKGPQVQLQESGPGLVKPS






ETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWI






GHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLS






SVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS


 


400
DVD307L
AB063VL
AB003VL
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYNCQQCENFPITFGQGTRLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQ






DISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF






SGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLA






FGGGTKVEIKR


 


401
DVD308H
AB003VH
AB063VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYY






WTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT






ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGA






FDIWGQGTMVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFTFSIYSMNWVRQAPGKGLEWVSY






ISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNS






LRDEDTAVYYCARDRGDFDAFDIWGQGTMVTVSS


 


402
DVD308L
AB003VL
AB063VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQ






DITNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF






SGSGSGTDFTFTISSLQPEDIATYNCQQCENFPIT






FGQGTRLEIKR









Example 2.77
Generation of HGF (Seq. 1) and ErbB3 (Seq. 2) DVD-Igs












TABLE 109






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







403
DVD309H
AB063VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMN







WVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRDEDTAVYYCARDRGDFDA






FDIWGQGTMVTVSSASTKGPQVQLVESGGGLVKPG






GSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSY






ISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNS






LRAEDTAVYYCARDEYNSGWYVLFDYWGQGTLVTV






SS


 


404
DVD309L
AB063VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYNCQQCENFPITFGQGTRLE






IKRTVAAPDIQMTQSPSSVSASVGDRVTITCRASQ






GISSWLAWYQQKPGKAPNLLIYEASSLQSGVPSRF






GGSGSGTDFTLTISSLQPEDFATYYCQQANGFPWT






FGQGTKVEIKR


 


405
DVD310H
AB012VH
AB063VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS






WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPEVQLVESGGGLV






QPGGSLRLSCAASGFTFSIYSMNWVRQAPGKGLEW






VSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQ






MNSLRDEDTAVYYCARDRGDFDAFDIWGQGTMVTV






SS


 


406
DVD310L
AB012VL
AB063VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQ






DITNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF






SGSGSGTDFTFTISSLQPEDIATYNCQQCENFPIT






FGQGTRLEIKR









Example 2.78
Generation of VEGF (Seq. 1) and DLL4 (Seq. 2) DVD-Igs with Linker Set 1












TABLE 110






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







407
DVD441H
AB069
AB014
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGW






INTYTGEPTYAADFKRRFTFSLDISKSTAYLQMNS






LRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVT






VSS


 


408
DVD441L
AB069
AB014
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPDIQMTQSPSSLSASVGDRVTITC






SASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGV






PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYST






VPWTFGQGTKVEIKR


 


409
DVD442H
AB014
AB069
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLVQSGAEV






KKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLE






WIGYISSYNGATNYNQKFKGRVTFTTDTSTSTAYM






ELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVT






VSS


 


410
DVD442L
AB014
AB069
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFAIYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIVMTQSPDSLAVSLGERATISCRASE






SVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGV






PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKE






VPWTFGGGTKVEIKR









Example 2.79
Generation of VEGF (Seq. 1) and DLL4 (Seq. 2) DVD-Igs with Linker Set 2












TABLE 111






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







411
DVD447H
AB069
AB014
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGK






GLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKST






AYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW






GQGTLVTVSS


 


412
DVD447L
AB069
AB014
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPSVFIFPPDIQMTQSPSSLSASVG






DRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFT






SSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATY






YCQQYSTVPWTFGQGTKVEIKR


 


413
DVD448H
AB014
AB069
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPQVQL






VQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQ






APGQGLEWIGYISSYNGATNYNQKFKGRVTFTTDT






STSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYW






GQGTLVTVSS


 


414
DVD448L
AB014
AB069
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIVMTQSPDSLAVSLGERAT






ISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAA






SNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVY






YCQQSKEVPWTFGGGTKVEIKR









Example 2.80
Generation of VEGF (Seq. 1) and DLL4 (Seq. 2) DVD-Igs with Linker Set 3












TABLE 112






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







415
DVD453H
AB069
AB014
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGK






GLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKST






AYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW






GQGTLVTVSS


 


416
DVD453L
AB069
AB014
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPDIQMTQSPSSLSASVGDRVTITC






SASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGV






PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYST






VPWTFGQGTKVEIKR


 


417
DVD454H
AB014
AB069
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPQVQL






VQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQ






APGQGLEWIGYISSYNGATNYNQKFKGRVTFTTDT






STSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYW






GQGTLVTVSS


 


418
DVD454L
AB014
AB069
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVRWTFGQGTKVE






IKRTVAAPDIVMTQSRDSLAVSLGERATISCRASE






SVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGV






PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKE






VPWTFGGGTKVEIKR









Example 2.81
Generation of VEGF (Seq. 1) and DLL4 (Seq. 2) DVD-Igs with Linker Set 4












TABLE 113






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







419
DVD459H
AB069
AB014
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGW






INTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNS






LRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVT






VSS


 


420
DVD459L
AB069
AB014
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPSVFIFPPDIQMTQSPSSLSASVG






DRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFT






SSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATY






YCQQYSTVPWTFGQGTKVEIKR


 


421
DVD460H
AB014
AB069
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLVQSGAEV






KKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLE






WIGYISSYNGATNYNQKFKGRVTFTTDTSTSTAYM






ELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVT






VSS


 


422
DVD460L
AB014
AB069
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIVMTQSPDSLAVSLGERAT






ISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAA






SNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVY






YCQQSKEVPWTFGGGTKVEIKR









Example 2.82
Generation of VEGF (Seq. 2) and DLL4 (Seq. 2) DVD-Igs with Linker Set 1












TABLE 114






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







423
DVD443H
AB069
AB070
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAG






ITPAGGYTYYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS


 


424
DVD443L
AB069
AB070
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPDIQMTQSPSSLSASVGDRVTITC






RASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGV






PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYT






TPPTFGQGTKVEIKR


 


425
DVD444H
AB070
AB069
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPQVQLVQSGAEVKKP






GASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIG






YISSYNGATNYNQKFKGRVTFTTDTSTSTAYMELR






SLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS


 


426
DVD444L
AB070
AB069
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPDIVMTQSPDSLAVSLGERATISCRASE






SVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGV






PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKE






VPWTFGGGTKVEIKR









Example 2.83
Generation of VEGF (Seq. 2) and DLL4 (Seq. 2) DVD-Igs with Linker Set 2












TABLE 115






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







427
DVD449H
AB069
AB070
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGK






GLEWVAGITPAGGYTYYADSVKGRFTISADTSKNT






AYLQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQG






TLVTVSS


 


428
DVD449L
AB069
AB070
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPSVFIFPPDIQMTQSPSSLSASVG






DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSA






SFLYSGVPSRFSGSGSGTDFTLIISSLQPEDFATY






YCQQSYTTPPTFGQGTKVEIKR


 


429
DVD450H
AB070
AB069
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLVQS






GAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPG






QGLEWIGYISSYNGATNYNQKFKGRVTFTTDTSTS






TAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQG






TLVTVSS


 


430
DVD450L
AB070
AB069
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFTFPPDIVMTQSPDSLAVSLGERAT






ISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAA






SNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVY






YCQQSKEVPWIFGGGTKVEIKR









Example 2.84
Generation of VEGF (Seq. 2) and DLL4 (Seq. 2) DVD-Igs with Linker Set 3












TABLE 116






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







431
DVD455H
AB069
AB070
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGK






GLEWVAGITPAGGYTYYADSVKGRFTISADTSKNT






AYLQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQG






TLVTVSS


 


432
DVD455L
AB069
AB070
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPDIQMTQSPSSLSASVGDRVTITC






RASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGV






PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYT






TPPTFGQGTKVEIKR


 


433
DVD456H
AB070
AB069
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLVQS






GAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPG






QGLEWIGYISSYNGATNYNQKFKGRVTFTTDTSTS






TAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQG






TLVTVSS


 


434
DVD456L
AB070
AB069
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPDIVMTQSPDSLAVSLGERATISCRASE






SVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGV






PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKE






VPWTFGGGTKVEIKR









Example 2.85
Generation of VEGF (Seq. 2) and DLL4 (Seq. 2) DVD-Igs with Linker Set 4












TABLE 117






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







435
DVD461H
AB069
AB070
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAG






ITPAGGYTYYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS


 


436
DVD461L
AB069
AB070
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPSVFIFPPDIQMTQSPSSLSASVG






DRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSA






SFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATY






YCQQSYTTPPTFGQGTKVEIKR


 


437
DVD462H
AB070
AB069
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPQVQLVQSGAEVKKP






GASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIG






YISSYNGATNYNQKFKGRVTFTTDTSTSTAYMELR






SLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS


 


438
DVD462L
AB070
AB069
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIVMTQSRDSLAVSLGERAT






ISCRASESVDNYGISFMKWFQQKRGQPPKLLIYAA






SNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVY






YCQQSKEVPWTFGGGTKVEIKR









Example 2.86
Generation of VEGF (Seq. 3) and DLL4 (Seq. 2) DVD-Igs with Linker Set 1












TABLE 118






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







439
DVD445H
AB069
AB071
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFTINASWIHWVRQAPGKGLEWVGA






IYPYSGYTNYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVS






S


 


440
DVD445L
AB069
AB071
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPDIQMTQSPSSLSASVGDRVTITC






RASQVIRRSLAWYQQKPGKAPKLLIYAASNLASGV






PSRFSGSGSGTDFTLTISSLQPEDFAIYYCQQSNT






SPLIFGQGTKVEIKR


 


441
DVD446H
AB071
AB069
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPQVQLVQSGAEVKK






PGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWI






GYISSYNGATNYNQKFKGRVTFTTDTSTSTAYMEL






RSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVS






S


 


442
DVD446L
AB071
AB069
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSRLTFGQGTKVE






IKRTVAAPDIVMTQSPDSLAVSLGERATISCRASE






SVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGV






PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKE






VPWTFGGGTKVEIKR









Example 2.87
Generation of VEGF (Seq. 3) and DLL4 (Seq. 2) DVD-Igs with Linker Set 2












TABLE 119






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







443
DVD451H
AB069
AB071
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFTINASWIHWVRQAPGK






GLEWVGAIYPYSGYTNYADSVKGRFTISADTSKNT






AYLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQ






GTLVTVSS


 


444
DVD451L
AB069
AB071
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPSVFIFPPDIQMTQSPSSLSASVG






DRVTITCRASQVIRRSLAWYQQKPGKAPKLLIYAA






SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATY






YCQQSNTSPLTFGQGTKVEIKR


 


445
DVD452H
AB071
AB069
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLVQ






SGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAP






GQGLEWIGYISSYNGATNYNQKFKGRVTFTTDTST






STAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQ






GTLVTVSS


 


446
DVD452L
AB071
AB069
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPDIVMTQSPDSLAVSLGERAT






ISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAA






SNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVY






YCQQSKEVPWTFGGGTKVEIKR









Example 2.88
Generation of VEGF (Seq. 3) and DLL4 (Seq. 2) DVD-Igs with Linker Set 3












TABLE 120






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







447
DVD457H
AB069
AB071
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFTINASWIHWVRQAPGK






GLEWVGAIYPYSGYTNYADSVKGRFTISADTSKNT






AYLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQ






GTLVTVSS


 


448
DVD457L
AB069
AB071
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPDIQMTQSPSSLSASVGDRVTITC






RASQVIRRSLAWYQQKPGKAPKLLIYAASNLASGV






PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNT






SPLTFGQGTKVEIKR


 


449
DVD458H
AB071
AB069
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLVQ






SGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAP






GQGLEWIGYISSYNGATNYNQKFKGRVTFTTDTST






STAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQ






GTLVTVSS


 


450
DVD458L
AB071
AB069
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPDIVMTQSPDSLAVSLGERATISCRASE






SVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGV






PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKE






VPWTFGGGTKVEIKR









Example 2.89
Generation of VEGF (Seq. 3) and DLL4 (Seq. 2) DVD-Igs with Linker Set 4












TABLE 121






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







451
DVD463H
AB069
AB071
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIH







WVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTF






TTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVG






MDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFTINASWIHWVRQAPGKGLEWVGA






IYPYSGYTNYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVS






S


 


452
DVD463L
AB069
AB071
DIVMTQSPDSLAVSLGERATISCRASESVDNYGIS






FMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSG






SGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGG






TKVEIKRTVAAPSVFIFPPDIQMTQSPSSLSASVG






DRVTITCRASQVIRRSLAWYQQKPGKAPKLLIYAA






SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATY






YCQQSNTSPLTFGQGTKVEIKR


 


453
DVD464H
AB071
AB069
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPQVQLVQSGAEVKK






PGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWI






GYISSYNGATNYNQKFKGRVTFTTDTSTSTAYMEL






RSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVS






S


 


454
DVD464L
AB071
AB069
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSRLTFGQGTKVE






IKRTVAAPSVFIFPPDIVMTQSPDSLAVSLGERAT






ISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAA






SNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVY






YCQQSKEVPWTFGGGTKVEIKR









Example 2.90
Generation of VEGF (Seq. 2) and DLL4 (Seq. 1) DVD-Igs with Linker Set 1












TABLE 122






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







455
DVD465H
AB015
AB070
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAGI






TPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSL






RAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS


 


456
DVD465L
AB015
AB070
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPDIQMTQSPSSLSASVGDRVTITCRAS






QDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR






FSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPP






TFGQGTKVEIKR


 


457
DVD466H
AB070
AB015
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTFTDNWISWVRQAPGKGLEWVG






YISPNSGFTYYADSVKGRFTISADTSKNTAYLQMN






SLRAEDTAVYYCARDNFGGYFDYWGQGTLVTVSS


 


458
DVD466L
AB070
AB015
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATTYYCQQSYTGTV






TFGQGTKVEIKR









Example 2.91
Generation of VEGF (Seq. 2) and DLL4 (Seq. 1) DVD-Igs with Linker Set 2












TABLE 123






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







459
DVD471H
AB015
AB070
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKG






LEWVAGITPAGGYTYYADSVKGRFTISADTSKNTA






YLQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQGT






LVTVSS


 


460
DVD471L
AB015
AB070
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRV






TITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFL






YSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ






QSYTTPPTFGQGTKVEIKR


 


461
DVD472H
AB070
AB015
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTFTDNWISWVRQAPG






KGLEWVGYISPNSGFTYYADSVKGRFTISADTSKN






TAYLQMNSLRAEDTAVYYCARDNFGGYFDYWGQGT






LVTVSS


 


462
DVD472L
AB070
AB015
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATTYYCQ






QSYTGTVTFGQGTKVEIKR









Example 2.92
Generation of VEGF (Seq. 2) and DLL4 (Seq. 1) DVD-Igs with Linker Set 3












TABLE 125






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







463
DVD477H
AB015
AB070
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKG






LEWVAGITPAGGYTYYADSVKGRFTISADTSKNTA






YLQMNSLRAEDTAVYYCARFVFFLRYAMDYWGQGT






LVTVSS


 


464
DVD477L
AB015
AB070
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPDIQMTQSPSSLSASVGDRVTITCRAS






QDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR






FSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPP






TFGQGTKVEIKR


 


465
DVD478H
AB070
AB015
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTFTDNWISWVRQAPG






KGLEWVGYISPNSGFTYYADSVKGRFTISADTSKN






TAYLQMNSLRAEDTAVYYCARDNFGGYFDYWGQGT






LVTVSS


 


466
DVD478L
AB070
AB015
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSRSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATTYYCQQSYTGTV






TFGQGTKVEIKR









Example 2.93
Generation of VEGF (Seq. 2) and DLL4 (Seq. 1) DVD-Igs with Linker Set 4












TABLE 126






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







467
DVD483H
AB015
AB070
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAGI






TPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSL






RAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS


 


468
DVD483L
AB015
AB070
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRV






TITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFL






YSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ






QSYTTPPTFGQGTKVEIKR


 


469
DVD484H
AB070
AB015
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTFTDNWISWVRQAPGKGLEWVG






YISPNSGFTYYADSVKGRFTISADTSKNTAYLQMN






SLRAEDTAVYYCARDNFGGYFDYWGQGTLVTVSS


 


470
DVD484L
AB070
AB015
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATTYYCQ






QSYTGTVTFGQGTKVEIKR









Example 2.94
Generation of VEGF (Seq. 3) and DLL4 (Seq. 1) DVD-Igs with Linker Set 1












TABLE 127






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







471
DVD467H
AB015
AB071
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGFTINASWIHWVRQAPGKGLEWVGAI






YPYSGYTNYADSVKGRFTISADTSKNTAYLQMNSL






RAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS


 


472
DVD467L
AB015
AB071
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKARKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPDIQMTQSRSSLSASVGDRVTITCRAS






QVIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSR






FSGSGSGTDFTLTISSLQPEDFATYYCQQSNTSPL






TFGQGTKVEIKR


 


473
DVD468H
AB071
AB015
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQ






PGGSLRLSCAASGFTFTDNWISWVRQAPGKGLEWV






GYISPNSGFTYYADSVKGRFTISADTSKNTAYLQM






NSLRAEDTAVYYCARDNFGGYFDYWGQGTLVTVSS


 


474
DVD468L
AB071
AB015
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSRLTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATTYYCQQSYTGTV






TFGQGTKVEIKR









Example 2.95
Generation of VEGF (Seq. 3) and DLL4 (Seq. 1) DVD-Igs with Linker Set 2












TABLE 128






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







475
DVD473H
AB015
AB071
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGFTINASWIHWVRQAPGKG






LEWVGAIYPYSGYTNYADSVKGRFTISADTSKNTA






YLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQG






TLVTVSS


 


476
DVD473L
AB015
AB071
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRV






TITCRASQVIRRSLAWYQQKPGKAPKLLIYAASNL






ASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ






QSNTSPLTFGQGTKVEIKR


 


477
DVD474H
AB071
AB015
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVE






SGGGLVQPGGSLRLSCAASGFTFTDNWISWVRQAP






GKGLEWVGYISPNSGFTYYADSVKGRFTISADTSK






NTAYLQMNSLRAEDTAVYYCARDNFGGYFDYWGQG






TLVTVSS


 


478
DVD474L
AB071
AB015
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATTYYCQ






QSYTGTVTFGQGTKVEIKR









Example 2.96
Generation of VEGF (Seq. 3) and DLL4 (Seq. 1) DVD-Igs with Linker Set 3












TABLE 129






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







479
DVD479H
AB015
AB071
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGFTINASWIHWVRQAPGKG






LEWVGAIYPYSGYTNYADSVKGRFTISADTSKNTA






YLQMNSLRAEDTAVYYCARWGHSTSRWAMDYWGQG






TLVTVSS


 


480
DVD479L
AB015
AB071
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPDIQMTQSPSSLSASVGDRVTITCRAS






QVIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSR






FSGSGSGTDFTLTISSLQPEDFATYYCQQSNTSPL






TFGQGTKVEIKR


 


481
DVD480H
AB071
AB015
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVE






SGGGLVQPGGSLRLSCAASGFTFTDNWISWVRQAP






GKGLEWVGYISPNSGFTYYADSVKGRFTISADTSK






NTAYLQMNSLRAEDTAVYYCARDNFGGYFDYWGQG






TLVTVSS


 


482
DVD480L
AB071
AB015
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATTYYCQQSYTGTV






TFGQGTKVEIKR









Example 2.97
Generation of VEGF (Seq. 3) and DLL4 (Seq. 1) DVD-Igs with Linker Set 4












TABLE 130






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







483
DVD485H
AB015
AB071
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGFTINASWIHWVRQAPGKGLEWVGAI






YPYSGYTNYADSVKGRFTISADTSKNTAYLQMNSL






RAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS


 


484
DVD485L
AB015
AB071
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRV






TITCRASQVIRRSLAWYQQKPGKAPKLLIYAASNL






ASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ






QSNTSPLTFGQGTKVEIKR


 


485
DVD486H
AB071
AB015
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQ






PGGSLRLSCAASGFTFTDNWISWVRQAPGKGLEWV






GYISPNSGFTYYADSVKGRFTISADTSKNTAYLQM






NSLRAEDTAVYYCARDNFGGYFDYWGQGTLVTVSS


 


486
DVD486L
AB071
AB015
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATTYYCQ






QSYTGTVTFGQGTKVEIKR









Example 2.98
Generation of VEGF (Seq. 1) and DLL4 (Seq. 1) DVD-Igs with Linker Set 1












TABLE 131






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







487
DVD469H
AB015
AB014
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKG






LEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTA






YLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWG






QGTLVTVSS


 


488
DVD469L
AB015
AB014
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPSVFIFPPDIQMTQSRSSLSASVGDRV






TITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSL






HSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ






QYSTVPWTFGQGTKVEIKR


 


489
DVD470H
AB014
AB015
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPEVQL






VESGGGLVQPGGSLRLSCAASGFTFTDNWISWVRQ






ARGKGLEWVGYISPNSGFTYYADSVKGRFTISADT






SKNTAYLQMNSLRAEDTAVYYCARDNFGGYFDYWG






QGTLVTVSS


 


490
DVD470L
AB014
AB015
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATTYYCQ






QSYTGTVTFGQGTKVEIKR









Example 2.99
Generation of VEGF (Seq. 1) and DLL4 (Seq. 1) DVD-Igs with Linker Set 2












TABLE 132






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







491
DVD475H
AB015
AB014
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKG






LEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTA






YLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWG






QGTLVTVSS


 


492
DVD475L
AB015
AB014
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPDIQMTQSPSSLSASVGDRVTITCSAS






QDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSR






FSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPW






TFGQGTKVEIKR


 


493
DVD476H
AB014
AB015
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPEVQL






VESGGGLVQPGGSLRLSCAASGFTFTDNWISWVRQ






APGKGLEWVGYISPNSGFTYYADSVKGRFTISADT






SKNTAYLQMNSLRAEDTAVYYCARDNFGGYFDYWG






QGTLVTVSS


 


494
DVD476L
AB014
AB015
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATTYYCQQSYTGTV






TFGQGTKVEIKR









Example 2.100
Generation of VEGF (Seq. 1) and DLL4 (Seq. 1) DVD-Igs with Linker Set 3












TABLE 133






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







495
DVD481H
AB015
AB014
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWIS







WVRQAPGKGLEWVGYISPNSGFTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDNFGGYF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWI






NTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSL






RAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTV






SS


 


496
DVD481L
AB015
AB014
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATTYYCQQSYTGTVTFGQGTKV






EIKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRV






TITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSL






HSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ






QYSTVPWTFGQGTKVEIKR


 


497
DVD482H
AB014
AB015
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPEVQLVESGGGL






VQPGGSLRLSCAASGFTFTDNWISWVRQAPGKGLE






WVGYISPNSGFTYYADSVKGRFTISADTSKNTAYL






QMNSLRAEDTAVYYCARDNFGGYFDYWGQGTLVTV






SS


 


498
DVD482L
AB014
AB015
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATTYYCQ






QSYTGTVTFGQGTKVEIKR









Example 2.101
Generation of VEGF (Seq. 1) and DLL4 (Seq. 3) DVD-Igs with Linker Set 1












TABLE 134






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







499
DVD487H
AB072
AB014
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIN







WVKQKPGQGLEWIGLINPYNDGTKYNEKEKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVG






WINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMN






SLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLV






TVSS


 


500
DVD487L
AB072
AB014
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLE






LKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR


 


501
DVD488H
AB014
AB072
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPEVQLQQSGPEL






VKPGASVKMSCKASGYTFTSYVINWVKQKPGQGLE






WIGLINPYNDGTKYNEKFKVKATLTSDKSSSTAYM






ELSSLTSEDSAVYYCASYYYGSRYYFDYWGQGTTL






TVSS


 


502
DBD488L
AB014
AB072
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSSSYLSVSLGGRVTITCKASD






HINNWLAWYQQKPGNAPRLLISGATSLETGVPSRF






SGSGSGKDYTLSITSLQTEDVATYYCQQYWSIPLT






FGAGTKLELKR









Example 2.102
Generation of VEGF (Seq. 1) and DLL4 (Seq. 3) DVD-Igs with Linker Set 2












TABLE 135






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







503
DVD493H
AB072
AB014
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIN







WVKQKPGQGLEWIGLINPYNDGTKYNEKFKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPG






KGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKS






TAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDV






WGQGTLVTVSS


 


504
DVD493L
AB072
AB014
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLE






LKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLH






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






YSTVPWTFGQGTKVEIKR


 


505
DVD494H
AB014
AB072
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPEVQL






QQSGPELVKPGASVKMSCKASGYTFTSYVINWVKQ






KPGQGLEWIGLINPYNDGTKYNEKFKVKATLTSDK






SSSTAYMELSSLTSEDSAVYYCASYYYGSRYYFDY






WGQGTTLTVSS


 


506
DVD494L
AB014
AB072
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSSSYLSVSLGGRVT






ITCKASDHINNWLAWYQQKPGNAPRLLISGATSLE






TGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQ






YWSIPLTFGAGTKLELKR









Example 2.103
Generation of VEGF (Seq. 1) and DLL4 (Seq. 3) DVD-Igs with Linker Set 3












TABLE 136






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







507
DVD499H
AB072
AB014
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIN







WVKQKPGQGLEWIGLINPYNDGTKYNEKFKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPG






KGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKS






TAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDV






WGQGTLVTVSS


 


508
DVD499L
AB072
AB014
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLE






LKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR


 


509
DVD500H
AB014
AB072
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPEVQL






QQSGPELVKPGASVKMSCKASGYTFTSYVINWVKQ






KPGQGLEWIGLINPYNDGTKYNEKFKVKATLTSDK






SSSTAYMELSSLTSEDSAVYYCASYYYGSRYYFDY






WGQGTTLTVSS


 


510
DVD500L
AB014
AB072
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSSSYLSVSLGGRVTITCKASD






HINNWLAWYQQKPGNAPRLLISGATSLETGVPSRF






SGSGSGKDYTLSITSLQTEDVATYYCQQYWSIPLT






FGAGTKLELKR









Example 2.104
Generation of VEGF (Seq. 1) and DLL4 (Seq. 3) DVD-Igs with Linker Set 4












TABLE 137






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







511
DVD505H
AB072
AB014
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIN







WVKQKPGQGLEWIGLINPYNDGTKYNEKFKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVG






WINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMN






SLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLV






TVSS


 


512
DVD505L
AB072
AB014
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVAIYYCQQYWSIPLTFGAGTKLE






LKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLH






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






YSTVPWTFGQGTKVEIKR


 


513
DVD506H
AB014
AB072
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYRHYYGS






SHWYFDVWGQGTLVTVSSASTKGPEVQLQQSGPEL






VKPGASVKMSCKASGYTFTSYVINWVKQKPGQGLE






WIGLINPYNDGTKYNEKFKVKATLTSDKSSSTAYM






ELSSLTSEDSAVYYCASYYYGSRYYFDYWGQGTTL






TVSS


 


514
DVD506L
AB014
AB072
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVRSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSSSYLSVSLGGRVT






ITCKASDHINNWLAWYQQKPGNAPRLLISGATSLE






TGVRSRFSGSGSGKDYTLSITSLQTEDVATYYCQQ






YWSIPLTFGAGTKLELKR









Example 2.105
Generation of VEGF (Seq. 2) and DLL4 (Seq. 3) DVD-Igs with Linker Set 1












TABLE 138






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







515
DVD489H
AB072
AB070
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIN







WVKQKPGQGLEWIGLINPYNDGTKYNEKFKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVA






GITPAGGYTYYADSVKGRFTISADTSKNTAYLQMN






SLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVS






S


 


516
DVD489L
AB072
AB070
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLE






LKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPT






FGQGTKVEIKR


 


517
DVD490H
AB070
AB072
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPEVQLQQSGPELVKP






GASVKMSCKASGYTFISYVINWVKQKPGQGLEWIG






LINPYNDGTKYNEKFKVKATLTSDKSSSTAYMELS






SLTSEDSAVYYCASYYYGSRYYFDYWGQGTTLTVS






S


 


518
DVD490L
AB070
AB072
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFAIYYCQQSYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSSSYLSVSLGGRVTITCKASD






HINNWLAWYQQKPGNAPRLLISGAISLETGVPSRF






SGSGSGKDYTLSITSLQTEDVATYYCQQYWSIPLT






FGAGTKLELKR









Example 2.106
Generation of VEGF (Seq. 2) and DLL4 (Seq. 3) DVD-Igs with Linker Set 2












TABLE 139






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







519
DVD495H
AB072
AB070
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIN







WVKQKPGQGLEWIGLINPYNDGTKYNEKFKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPG






KGLEWVAGITPAGGYTYYADSVKGRFTISADTSKN






TAYLQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQ






GTLVTVSS


 


520
DVD495L
AB072
AB070
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLE






LKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SYTTPPTFGQGTKVEIKR


 


521
DVD496H
AB070
AB072
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLQQS






GPELVKPGASVKMSCKASGYTFTSYVINWVKQKPG






QGLEWIGLINPYNDGTKYNEKFKVKATLTSDKSSS






TAYMELSSLTSEDSAVYYCASYYYGSRYYFDYWGQ






GTTLTVSS


 


522
DVD496L
AB070
AB072
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSSSYLSVSLGGRVT






ITCKASDHINNWLAWYQQKPGNAPRLLISGATSLE






TGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQ






YWSIPLTFGAGTKLELKR









Example 2.107
Generation of VEGF (Seq. 2) and DLL4 (Seq. 3) DVD-Igs with Linker Set 3












TABLE 140






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







523
DVD501H
AB072
AB070
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIN







WVKQKPGQGLEWIGLINPYNDGTKYNEKFKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPG






KGLEWVAGITPAGGYTYYADSVKGRFTISADTSKN






TAYLQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQ






GTLVTVSS


 


524
DVD501L
AB072
AB070
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLE






LKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPT






FGQGTKVEIKR


 


525
DVD502H
AB070
AB072
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLQQS






GPELVKPGASVKMSCKASGYTFTSYVINWVKQKPG






QGLEWIGLINPYNDGTKYNEKFKVKATLTSDKSSS






TAYMELSSLTSEDSAVYYCASYYYGSRYYFDYWGQ






GTTLTVSS


 


526
DVD502L
AB070
AB072
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSSSYLSVSLGGRVTITCKASD






HINNWLAWYQQKPGNAPRLLISGATSLETGVPSRF






SGSGSGKDYTLSITSLQTEDVATYYCQQYWSIPLT






FGAGTKLELKR









Example 2.108
Generation of VEGF (Seq. 2) and DLL4 (Seq. 3) DVD-Igs with Linker Set 4












TABLE 141






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







527
DVD507H
AB072
AB070
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIN







WVKQKPGQGLEWIGLINPYNDGTKYNEKFKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVA






GITPAGGYTYYADSVKGRFTISADTSKNTAYLQMN






SLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVS






S


 


528
DVD507L
AB072
AB070
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLE






LKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SYTTPPTFGQGTKVEIKR


 


529
DVD508H
AB070
AB072
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPEVQLQQSGPELVKP






GASVKMSCKASGYTFISYVINWVKQKPGQGLEWIG






LINPYNDGTKYNEKFKVKATLTSDKSSSTAYMELS






SLTSEDSAVYYCASYYYGSRYYFDYWGQGTTLTVS






S


 


530
DVD508L
AB070
AB072
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSSSYLSVSLGGRVT






ITCKASDHINNWLAWYQQKPGNAPRLLISGATSLE






TGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQ






YWSIPLTFGAGTKLELKR









Example 2.109
Generation of VEGF (Seq. 3) and DLL4 (Seq. 3) DVD-Igs with Linker Set 1












TABLE 142






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







531
DVD491H
AB072
AB071
EVQLQQSGRELVKPGASVKMSCKASGYTFTSYVIN







WVKQKRGQGLEWIGLINPYNDGTKYNEKFKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTINASWIHWVRQAPGKGLEWVG






AIYPYSGYTNYADSVKGRFTISADTSKNTAYLQMN






SLRAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTV






SS


 


532
DVD491L
AB072
AB071
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLE






LKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






VIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSNTSPLT






FGQGTKVEIKR


 


533
DVD492H
AB071
AB072
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPEVQLQQSGPELVK






PGASVKMSCKASGYTFTSYVINWVKQKPGQGLEWI






GLINPYNDGTKYNEKFKVKATLTSDKSSSTAYMEL






SSLTSEDSAVYYCASYYYGSRYYFDYWGQGTTLTV






SS


 


534
DVD492L
AB071
AB072
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPDIQMTQSSSYLSVSLGGRVTITCKASD






HINNWLAWYQQKPGNAPRLLISGATSLETGVPSRF






SGSGSGKDYTLSITSLQTEDVATYYCQQYWSIPLT






FGAGTKLELKR









Example 2.110
Generation of VEGF (Seq. 3) and DLL4 (Seq. 3) DVD-Igs with Linker Set 2












TABLE 143






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







535
DVD497H
AB072
AB071
EVQLQQSGPELVKRGASVKMSCKASGYTFTSYVIN







WVKQKPGQGLEWIGLINPYNDGTKYNEKFKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTINASWIHWVRQAPG






KGLEWVGAIYPYSGYTNYADSVKGRFTISADTSKN






TAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWG






QGTLVTVSS


 


536
DVD497L
AB072
AB071
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLE






LKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQVIRRSLAWYQQKPGKAPKLLIYAASNLA






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SNTSPLTFGQGTKVEIKR


 


537
DVD498H
AB071
AB072
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLQQ






SGPELVKPGASVKMSCKASGYTFTSYVINWVKQKP






GQGLEWIGLINPYNDGTKYNEKFKVKATLTSDKSS






STAYMELSSLTSEDSAVYYCASYYYGSRYYFDYWG






QTTILTVSS


 


538
DVD498L
AB071
AB072
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPDIQMIQSSSYLSVSLGGRVT






ITCKASDHINNWLAWYQQKPGNAPRLLISGATSLE






TGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQ






YWSIPLTFGAGTKLELKR









Example 2.111
Generation of VEGF (Seq. 3) and DLL4 (Seq. 3) DVD-Igs with Linker Set 3












TABLE 144






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







539
DVD503H
AB072
AB071
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIN







WVKQKPGQGLEWIGLINPYNDGTKYNEKFKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQRGGSLRLSCAASGFTINASWIHWVRQAPG






KGLEWVGAIYPYSGYTNYADSVKGRFTISADTSKN






TAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWG






QGTLVTVSS


 


540
DVD503L
AB072
AB071
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLE






LKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






VIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSNTSPLT






FGQGTKVEIKR


 


541
DVD504H
AB071
AB072
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLQQ






SGPELVKPGASVKMSCKASGYTFTSYVINWVKQKP






GQGLEWIGLINPYNDGTKYNEKFKVKATLTSDKSS






STAYMELSSLTSEDSAVYYCASYYYGSRYYFDYWG






QGTTLTVSS


 


542
DVD504L
AB071
AB072
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPDIQMTQSSSYLSVSLGGRVTITCKASD






HINNWLAWYQQKPGNAPRLLISGATSLETGVPSRF






SGSGSGKDYTLSITSLQTEDVATYYCQQYWSIPLT






FGAGTKLELKR









Example 2.112
Generation of VEGF (Seq. 3) and DLL4 (Seq. 3) DVD-Igs with Linker Set 4












TABLE 145






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







543
DVD509H
AB072
AB071
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIN







WVKQKPGQGLEWIGLINPYNDGTKYNEKFKVKATL






TSDKSSSTAYMELSSLTSEDSAVYYCASYYYGSRY






YFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTINASWIHWVRQAPGKGLEWVG






AIYPYSGYTNYADSVKGRFTISADTSKNTAYLQMN






SLRAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTV






SS


 


544
DVD509L
AB072
AB071
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAW






YQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKD






YTLSITSLQTEDVATYYCQQYWSIPLTFGAGTKLE






LKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQVIRRSLAWYQQKPGKAPKLLIYAASNLA






SGVPSRFSGSGSGIDFTLTISSLQPEDFATYYCQQ






SNTSPLTFGQGTKVEIKR


 


545
DVD510H
AB071
AB072
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPEVQLQQSGPELVK






PGASVKMSCKASGYTFTSYVINWVKQKPGQGLEWI






GLINPYNDGTKYNEKFKVKATLTSDKSSSTAYMEL






SSLTSEDSAVYYCASYYYGSRYYFDYWGQGTTLTV






SS


 


546
DVD510L
AB071
AB072
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSSSYLSVSLGGRVT






ITCKASDHINNWLAWYQQKPGNAPRLLISGATSLE






TGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQ






YWSIPLTFGAGTKLELKR









Example 2.113
Generation of VEGF (Seq. 1) and DLL4 (Seq. 4) DVD-Igs with Linker Set 1












TABLE 146






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







547
DVD511H
AB073
AB014
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDRYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLTSEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGYTFTNYGMNWVRQARGKGLEWVG






WINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMN






SLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLV






TVSS


 


548
DVD511L
AB073
AB014
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPDIQMTQSPSSLSASVGDRVTITCS






ASQDISNYLNWYQQKRGKAPKVLIYFTSSLHSGVP






SRFSGSGSGTDFTLTISSLQREDFATYYCQQYSTV






PWTFGQGTKVEIKR


 


549
DVD512H
AB014
AB073
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPEVQLQQSGPEL






EKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLE






WIGNIDPYFGGTNYNQKFKGKATLTVDKSSSTAYM






QLKSLTSEDSAVYYCARNYDYDGGCFDYWGQGTTL






TVSS


 


550
DVD512L
AB014
AB073
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVRSRFSGSGSGTD






FTLTISSLQREDFATYYCQQYSTVRWTFGQGTKVE






IKRTVAAPQAVVTQESALTTSPGETVTLTCRSSTG






AVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPA






RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNH






WVFGGGTKLTVLG









Example 2.114
Generation of VEGF (Seq. 1) and DLL4 (Seq. 4) DVD-Igs with Linker Set 2












TABLE 147






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







551
DVD517H
AB073
AB014
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDPYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLTSEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQRGGSLRLSCAASGYTFTNYGMNWVRQAPG






KGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKS






TAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDV






WGQGTLVTVSS


 


552
DVD517L
AB073
AB014
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVRAPFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPSVTLFPPDIQMTQSRSSLSASVGD






RVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTS






SLHSGVRSRFSGSGSGTDFTLTISSLQPEDFATYY






CQQYSTVRWTFGQGTKVEIKR


 


553
DVD518H
AB014
AB073
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPEVQL






QQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQ






SNGKSLEWIGNIDPYFGGTNYNQKFKGKATLTVDK






SSSTAYMQLKSLTSEDSAVYYCARNYDYDGGCFDY






WGQGTTLTVSS


 


554
DVD518L
AB014
AB073
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPQAVVTQESALTTSPGETVTL






TCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNN






RAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC






ALWYSNHWVFGGGTKLTVLG









Example 2.115
Generation of VEGF (Seq. 1) and DLL4 (Seq. 4) DVD-Igs with Linker Set 3












TABLE 148






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







555
DVD523H
AB073
AB014
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDPYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLTSEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPG






KGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKS






TAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDV






WGQGTLVTVSS


 


556
DVD523L
AB073
AB014
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPDIQMTQSPSSLSASVGDRVTITCS






ASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVP






SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTV






PWTFGQGTKVEIKR


 


557
DVD524H
AB014
AB073
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPEVQL






QQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQ






SNGKSLEWIGNIDPYFGGTNYNQKFKGKATLTVDK






SSSTAYMQLKSLTSEDSAVYYCARNYDYDGGCFDY






WGQGTTLTVSS


 


558
DVD524L
AB014
AB073
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVRWTFGQGTKVE






IKRTVAAPQAVVTQESALTTSPGETVTLTCRSSTG






AVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPA






RFSGSLIGDKAALTITGAQIEDEAIYFCALWYSNH






WVFGGGTKLTVLG









Example 2.116
Generation of VEGF (Seq. 1) and DLL4 (Seq. 4) DVD-Igs with Linker Set 4












TABLE 149






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







559
DVD529H
AB073
AB014
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDPYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLTSEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVG






WINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMN






SLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLV






TVSS


 


560
DVD529L
AB073
AB014
QAVVTQESALTTSRGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPSVTLFPPDIQMTQSPSSLSASVGD






RVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTS






SLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY






CQQYSTVPWTFGQGTKVEIKR


 


561
DVD530H
AB014
AB073
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLTIVSSASTKGPEVQLQQSGPEL






EKRGASVKISCKASGYSFTGYNMNWVKQSNGKSLE






WIGNIDPYFGGTNYNQKFKGKATLTVDKSSSTAYM






QLKSLTSEDSAVYYCARNYDYDGGCFDYWGQGTTL






TVSS


 


562
DVD530L
AB014
AB073
DIQMTQSRSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVRSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVRWTFGQGTKVE






IKRTVAAPSVFIFPPQAVVTQESALTTSPGETVTL






TCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNN






RAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC






ALWYSNHWVFGGGTKLTVLG









Example 2.117
Generation of VEGF (Seq. 2) and DLL4 (Seq. 4) DVD-Igs with Linker Set 1












TABLE 150






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







563
DVD513H
AB073
AB070
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDPYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLTSEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVA






GITPAGGYTYYADSVKGRFTISADTSKNTAYLQMN






SLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVS






S


 


564
DVD513L
AB073
AB070
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPDIQMTQSPSSLSASVGDRVTITCR






ASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVP






SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTT






PPTFGQGTKVEIKR


 


565
DVD514H
AB070
AB073
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPEVQLQQSGPELEKP






GASVKISCKASGYSFTGYNMNWVKQSNGKSLEWIG






NIDPYFGGTNYNQKFKGKATLTVDKSSSTAYMQLK






SLTSEDSAVYYCARNYDYDGGCFDYWGQGTTLTVS






S


 


566
DVD514L
AB070
AB073
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPQAVVTQESALTTSPGETVTLTCRSSTG






AVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPA






RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNH






WVFGGGTKLTVLG









Example 2.118
Generation of VEGF (Seq. 2) and DLL4 (Seq. 4) DVD-Igs with Linker Set 2












TABLE 151






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







567
DVD519H
AB073
AB070
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDPYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLTSEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPG






KGLEWVAGITPAGGYTYYADSVKGRFTISADTSKN






TAYLQMNSLRAEDTAVYYCARFVEFLPYAMDYWGQ






GTLVTVSS


 


568
DVD519L
AB073
AB070
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPSVTLFPPDIQMTQSPSSLSASVGD






RVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSAS






FLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY






CQQSYTTPPTFGQGTKVEIKR


 


569
DVD520H
AB070
AB073
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLQQS






GPELEKPGASVKISCKASGYSFTGYNMNWVKQSNG






KSLEWIGNIDPYFGGTNYNQKFKGKATLTVDKSSS






TAYMQLKSLTSEDSAVYYCARNYDYDGGCFDYWGQ






GTTLTVSS


 


570
DVD520L
AB070
AB073
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPQAVVTQESALTTSPGETVTL






TCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNN






RAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC






ALWYSNHWVFGGGTKLTVLG









Example 2.119
Generation of VEGF (Seq. 2) and DLL4 (Seq. 4) DVD-Igs with Linker Set 3












TABLE 152






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







571
DVD525H
AB073
AB070
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDPYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLISEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPG






KGLEWVAGITPAGGYTYYADSVKGRFTISADTSKN






TAYLQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQ






GTLVTVSS


 


572
DVD525L
AB073
AB070
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPDIQMTQSPSSLSASVGDRVTITCR






ASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVP






SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTT






PPTFGQGTKVEIKR


 


573
DVD526H
AB070
AB073
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLQQS






GPELEKPGASVKISCKASGYSFTGYNMNWVKQSNG






KSLEWIGNIDPYFGGTNYNQKFKGKATLTVDKSSS






TAYMQLKSLTSEDSAVYYCARNYDYDGGCFDYWGQ






GTTLTVSS


 


574
DVD526L
AB070
AB073
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFAIYYCQQSYTTPPTFGQGTKVE






IKRTVAAPQAVVTQESALTTSPGETVTLTCRSSTG






AVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPA






RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNH






WVFGGGTKLTVLG









Example 2.120
Generation of VEGF (Seq. 2) and DLL4 (Seq. 4) DVD-Igs with Linker Set 4












TABLE 153






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







575
DVD531H
AB073
AB070
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDPYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLTSEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVA






GITPAGGYTYYADSVKGRFTISADTSKNTAYLQMN






SLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVS






S


 


576
DVD531L
AB073
AB070
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPSVTLFPPDIQMTQSPSSLSASVGD






RVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSAS






FLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY






CQQSYTTPPTFGQGTKVEIKR


 


577
DVD532H
AB070
AB073
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPEVQLQQSGPELEKP






GASVKISCKASGYSFTGYNMNWVKQSNGKSLEWIG






NIDPYFGGTNYNQKFKGKATLTVDKSSSTAYMQLK






SLTSEDSAVYYCARNYDYDGGCFDYWGQGTTLTVS






S


 


578
DVD532L
AB070
AB073
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPQAVVTQESALTTSPGETVTL






TCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNN






RAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC






ALWYSNHWVFGGGTKLTVLG









Example 2.121
Generation of VEGF (Seq. 3) and DLL4 (Seq. 4) DVD-Igs with Linker Set 1












TABLE 154






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







579
DVD515H
AB073
AB071
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDPYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLTSEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTINASWIHWVRQAPGKGLEWVG






AIYPYSGYTNYADSVKGRFTISADTSKNTAYLQMN






SLRAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTV






SS


 


580
DVD515L
AB073
AB071
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPDIQMTQSPSSLSASVGDRVTITCR






ASQVIRRSLAWYQQKPGKAPKLLIYAASNLASGVP






SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNTS






PLTFGQGTKVEIKR


 


581
DVD516H
AB071
AB073
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPEVQLQQSGPELEK






PGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWI






GNIDPYFGGTNYNQKFKGKATLTVDKSSSTAYMQL






KSLTSEDSAVYYCARNYDYDGGCFDYWGQGTTLTV






SS


 


582
DVD516L
AB071
AB073
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPQAVVTQESALTTSPGETVTLTCRSSTG






AVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPA






RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNH






WVFGGGTKLTVLG









Example 2.122
Generation of VEGF (Seq. 3) and DLL4 (Seq. 4) DVD-Igs with Linker Set 2












TABLE 155






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







583
DVD521H
AB073
AB071
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDPYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLTSEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTINASWIHWVRQAPG






KGLEWVGAIYPYSGYTNYADSVKGRFTISADTSKN






TAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWG






QGTLVTVSS


 


584
DVD521L
AB073
AB071
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPSVTLFPPDIQMTQSPSSLSASVGD






RVTITCRASQVIRRSLAWYQQKPGKAPKLLIYAAS






NLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYY






CQQSNTSPLTFGQGTKVEIKR


 


585
DVD522H
AB071
AB073
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLQQ






SGPELEKPGASVKISCKASGYSFTGYNMNWVKQSN






GKSLEWIGNIDPYFGGTNYNQKFKGKATLTVDKSS






STAYMQLKSLTSEDSAVYYCARNYDYDGGCFDYWG






QGTTLTVSS


 


586
DVD522L
AB071
AB073
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPQAVVTQESALTTSPGETVTL






TCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNN






RAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC






ALWYSNHWVFGGGTKLTVLG









Example 2.123
Generation of VEGF (Seq. 3) and DLL4 (Seq. 4) DVD-Igs with Linker Set 3












TABLE 156






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







587
DVD527H
AB073
AB071
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDPYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLTSEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTINASWIHWVRQAPG






KGLEWVGAIYPYSGYTNYADSVKGRFTISADTSKN






TAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWG






QGTLVTVSS


 


588
DVD527L
AB073
AB071
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPDIQMTQSPSSLSASVGDRVTITCR






ASQVIRRSLAWYQQKPGKAPKLLIYAASNLASGVP






SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNTS






PLTFGQGTKVEIKR


 


589
DVD528H
AB071
AB073
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLQQ






SGPELEKPGASVKISCKASGYSFTGYNMNWVKQSN






GKSLEWIGNIDPYFGGTNYNQKFKGKATLTVDKSS






STAYMQLKSLTSEDSAVYYCARNYDYDGGCFDYWG






QGTTLTVSS


 


590
DVD528L
AB071
AB073
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPQAVVTQESALTTSPGETVTLTCRSSTG






AVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPA






RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNH






WVFGGGTKLTVLG









Example 2.124
Generation of VEGF (Seq. 3) and DLL4 (Seq. 4) DVD-Igs with Linker Set 4












TABLE 157






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







591
DVD533H
AB073
AB071
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN







WVKQSNGKSLEWIGNIDPYFGGTNYNQKFKGKATL






TVDKSSSTAYMQLKSLTSEDSAVYYCARNYDYDGG






CFDYWGQGTTLTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTINASWIHWVRQAPGKGLEWVG






AIYPYSGYTNYADSVKGRFTISADTSKNTAYLQMN






SLRAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTV






SS


 


592
DVD533L
AB073
AB071
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYA






NWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG






DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK






LTVLGQPKAAPSVTLFPPDIQMTQSPSSLSASVGD






RVTITCRASQVIRRSLAWYQQKPGKAPKLLIYAAS






NLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYY






CQQSNTSPLTFGQGTKVEIKR


 


593
DVD534H
AB071
AB073
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPEVQLQQSGPELEK






PGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWI






GNIDPYFGGTNYNQKFKGKATLTVDKSSSTAYMQL






KSLTSEDSAVYYCARNYDYDGGCFDYWGQGTTLTV






SS


 


594
DVD534L
AB071
AB073
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPQAVVTQESALTTSPGETVTL






TCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNN






RAPGVPARFSGSLIGDKAALTITGAQIEDEAIYFC






ALWYSNHWVFGGGTKLTVLG









Example 2.125
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 1) DVD-Igs with Linker Set 1












TABLE 158






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







595
DVD387H
AB062
AB004
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS







WIRQRRGKGLEWIGEINHSGSTNYNRSLKSRVTIS






VETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFD






LWGRGTLVTVSSASTKGPSVFPLAPEVQLVESGGG






LVQRGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL






EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY






LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTL






VTVSS


 


596
DVD387L
AB062
AB004
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSN






RNYLAWYQQNPGQPPKLLIYWASTRESGVRDRFSG






SGSGTDETLTISSLQAEDVAVYYCQQYYSTRRTFG






QGTKVEIKRTVAAPSVFIFPPDIQMTQSPSSLSAS






VGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY






SASFLYSGVPSRFSGSRSGIDFTLTISSLQPEDFA






TYYCQQHYTTPPTFGQGTKVEIKR


 


597
DVD388H
AB004
AB062
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLQQW






GAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPG






KGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQ






FSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTL






VTVSS


 


598
DVD388L
AB004
AB062
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIEMTQSPDSLAVSLGERAT






INCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIY






WASTRESGVRDRFSGSGSGTDFTLTISSLQAEDVA






VYYCQQYYSTPRTFGQGTKVEIKR









Example 2.126
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 1) DVD-Igs with Linker Set 2












TABLE 159






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







599
DVD391H
AB062
AB004
QVQLQQWGAGLLKPSETLSLTCAVYGGSESGYYWS







WIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTIS






VETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFD






LWGRGTLVTVSSASTKGPSVFPLAPEVQLVESGGG






LVQPGGSLRLSCAASGFNIKDIYIHWVRQAPGKGL






EWVARIYPINGYTRYADSVKGRFTISADTSKNTAY






LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTL






VTVSS


 


600
DVD391L
AB062
AB004
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSN






RNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSG






SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFG






QGTKVEIKRTVAAPDIQMTQSPSSLSASVGDRVTI






TCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS






GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH






YTTPPTFGQGTKVEIKR


 


601
DVD392H
AB004
AB062
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLQQW






GAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPG






KGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQ






FSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTL






VTVSS


 


602
DVD392L
AB004
AB062
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPDIEMTQSPDSLAVSLGERATINCRSSQ






SVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRES






GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY






YSTPRTFGQGTKVEIKR









Example 2.127
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 1) DVD-Igs with Linker Set 3












TABLE 160






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







603
DVD395H
AB062
AB004
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS







WIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTIS






VETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFD






LWGRGTLVTVSSASTKGPEVQLVESGGGLVQPGGS






LRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIY






PTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLR






AEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


604
DVD395L
AB062
AB004
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSN






RNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSG






SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFG






QGTKVEIKRTVAAPSVFIFPPDIQMTQSPSSLSAS






VGDRVTITCRASQDVNTAVAWYQQKRGKAPKLLIY






SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA






TYYCQQHYTTPPTFGQGTKVEIKR


 


605
DVD396H
AB004
AB062
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDIYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPQVQLQQWGAGLLKP






SETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIG






EINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSS






VTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS


 


606
DVD396L
AB004
AB062
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIEMTQSRDSLAVSLGERAT






INCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIY






WASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA






VYYCQQYYSTPRTFGQGTKVEIKR









Example 2.128
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 1) DVD-Igs with Linker Set 4












TABLE 161






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







607
DVD683H
AB062
AB004
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS







WIRQPPGKGLEWIGEINHSGSTNYNRSLKSRVTIS






VETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFD






LWGRGTLVTVSSASTKGPEVQLVESGGGLVQPGGS






LRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIY






PTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLR






AEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


608
DVD683L
AB062
AB004
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSN






RNYLAWYQQNPGQPPKLLIYWASTRESGVRDRFSG






SGSGTDFTLTISSLQAEDVAVYYCQQYYSTRRTFG






QGTKVEIKRTVAAPDIQMTQSRSSLSASVGDRVTI






TCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS






GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQH






YTTPPTFGQGTKVEIKR


 


609
DVD684H
AB004
AB062
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPQVQLQQWGAGLLKP






SETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIG






EINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSS






VTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS


 


610
DVD684L
AB004
AB062
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPDIEMTQSPDSLAVSLGERATINCRSSQ






SVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRES






GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY






YSTPRTFGQGTKVEIKR









Example 2.129
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 2) DVD-Igs with Linker Set 1












TABLE 162






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







611
DVD399H
AB063
AB004
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMN







WVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRDEDTAVYYCARDRGDFDA






FDIWGQGTMVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGK






GLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT






AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG






TLVTVSS


 


612
DVD399L
AB063
AB004
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYNCQQCENFPITFGQGTRLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ






HYTTPPTFGQGTKVEIKR


 


613
DVD400H
AB004
AB063
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTFSIYSMNWVRQAPG






KGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKN






SLYLQMNSLRDEDTAVYYCARDRGDFDAFDIWGQG






TMVTVSS


 


614
DVD400L
AB004
AB063
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCQASQDITNYLNWYQQKPGKAPKLLIYDASNLE






TGVPSRFSGSGSGTDFTFTISSLQPEDIATYNCQQ






CENFPITFGQGTRLEIKR









Example 2.130
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 2) DVD-Igs with Linker Set 2












TABLE 163






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







615
DVD403H
AB063
AB004
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMN







WVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRDEDTAVYYCARDRGDFDA






FDIWGQGTMVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGK






GLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT






AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG






TLVTVSS


 


616
DVD403L
AB063
AB004
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYNCQQCENFPITFGQGTRLE






IKRTVAAPDIQMTQSRSSLSASVGDRVTITCRASQ






DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT






FGQGTKVEIKR


 


617
DVD404H
AB004
AB063
EVQLVESGGGLVQRGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTFSIYSMNWVRQAPG






KGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKN






SLYLQMNSLRDEDTAVYYCARDRGDFDAFDIWGQG






TMVTVSS


 


618
DVD404L
AB004
AB063
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQ






DITNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF






SGSGSGTDFTFTISSLQPEDIATYNCQQCENFPIT






FGQGTRLEIKR









Example 2.131
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 2) DVD-Igs with Linker Set 3












TABLE 164






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







619
DVD407H
AB063
AB004
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMN







WVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRDEDTAVYYCARDRGDFDA






FDIWGQGTMVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR






IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


620
DVD407L
AB063
AB004
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FIFTISSLQPEDIATYNCQQCENFPITFGQGTRLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ






HYTTPPTFGQGTKVEIKR


 


621
DVD408H
AB004
AB063
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTFSIYSMNWVRQAPGKGLEWVS






YISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMN






SLRDEDTAVYYCARDRGDFDAFDIWGQGTMVTVSS


 


622
DVD408L
AB004
AB063
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCQASQDITNYLNWYQQKPGKAPKLLIYDASNLE






TGVPSRFSGSGSGTDFTFTISSLQPEDIATYNCQQ






CENFPITFGQGTRLEIKR









Example 2.132
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 2) DVD-Igs with Linker Set 4












TABLE 165






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







623
DVD685H
AB063
AB004
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMN







WVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRDEDTAVYYCARDRGDFDA






FDIWGQGTMVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR






IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


624
DVD685L
AB063
AB004
DIQMTQSRSSLSASVGDRVTITCQASQDITNYLNW






YQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTD






FTFTISSLQPEDIATYNCQQCENFPITFGQGTRLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT






FGQGTKVEIKR


 


625
DVD686H
AB004
AB063
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTFSIYSMNWVRQAPGKGLEWVS






YISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMN






SLRDEDTAVYYCARDRGDFDAFDIWGQGTMVTVSS


 


626
DVD686L
AB004
AB063
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCQASQ






DITNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF






SGSGSGTDFTFTISSLQPEDIATYNCQQCENFPIT






FGQGTRLEIKR









Example 2.133
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 3) DVD-Igs with Linker Set 1












TABLE 166






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







627
DVD409H
AB067
AB033
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMA







WVRQAPGKGLEWVSSISSSGGWTLYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCTRGLKMATI






FDYWGQGTLVTVSSASTKGPQVQLKQSGPGLVQPS






QSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV






IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSL






QSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA


 


628
DVD409L
AB067
AB033
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVV






SWYQQHPGKAPKLIIYEVSQRPSGVSNRFSGSKSG






NTASLTISGLQTEDEADYYCCSYAGSSIFVIFGGG






TKVTVLGQPKAAPDILLTQSPVILSVSPGERVSFS






CRASQSIGTNIHWYQQRTNGSPRLLIKYASESISG






IPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNN






NWPTTFGAGTKLELKR


 


629
DVD410H
AB033
AB067
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLLESGGGLVQPG






GSLRLSCAASGFTFSHYVMAWVRQAPGKGLEWVSS






ISSSGGWTLYADSVKGRFTISRDNSKNTLYLQMNS






LRAEDTAVYYCTRGLKMATIFDYWGQGTLVTVSS


 


630
DVD410L
AB033
AB067
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPQSALTQPASVSGSPGQSITISCTGTSS






DVGSYNVVSWYQQHPGKAPKLIIYEVSQRPSGVSN






RFSGSKSGNTASLTISGLQTEDEADYYCCSYAGSS






IFVIFGGGTKVTVLG









Example 2.134
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 3) DVD-Igs with Linker Set 2












TABLE 167






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







631
DVD413H
AB067
AB033
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMA







WVRQAPGKGLEWVSSISSSGGWTLYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCTRGLKMATI






FDYWGQGTLVTVSSASTKGPSVFPLAPQVQLKQSG






PGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGK






GLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQV






FFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGT






LVTVSA


 


632
DVD413L
AB067
AB033
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVV






SWYQQHPGKAPKLIIYEVSQRPSGVSNRFSGSKSG






NTASLTISGLQTEDEADYYCCSYAGSSIFVIFGGG






TKVTVLGQPKAAPSVTLFPPDILLTQSPVILSVSP






GERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY






ASESISGIPSRFSGSGSGTDFTLSINSVESEDIAD






YYCQQNNNWPTTFGAGTKLELKR


 


633
DVD414H
AB033
AB067
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLLESG






GGLVQPGGSLRLSCAASGFTFSHYVMAWVRQAPGK






GLEWVSSISSSGGWTLYADSVKGRFTISRDNSKNT






LYLQMNSLRAEDTAVYYCTRGLKMATIFDYWGQGT






LVTVSS


 


634
DVD414L
AB033
AB067
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FILSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPQSALTQPASVSGSPGQSITI






SCTGTSSDVGSYNVVSWYQQHPGKAPKLIIYEVSQ






RPSGVSNRFSGSKSGNIASLTISGLQTEDEADYYC






CSYAGSSIFVIFGGGTKVTVLG









Example 2.135
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 3) DVD-Igs with Linker Set 3












TABLE 168






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







635
DVD417H
AB067
AB033
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMA







WVRQAPGKGLEWVSSISSSGGWTLYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCTRGLKMATI






FDYWGQGTLVTVSSASTKGPSVFPLAPQVQLKQSG






PGLVQPSQSLSITCTVSGFSLTNYGVHWVRQPRGK






GLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQV






FFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGT






LVTVSA


 


636
DVD417L
AB067
AB033
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVV






SWYQQHPGKAPKLIIYEVSQRPSGVSNRFSGSKSG






NTASLTISGLQTEDEADYYCCSYAGSSIFVIFGGG






TKVTVLGQPKAAPDILLTQSPVILSVSPGERVSFS






CRASQSIGTNIHWYQQRTNGSPRLLIKYASESISG






IPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNN






NWPTTFGAGTKLELKR


 


637
DVD418H
AB033
AB067
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPSVFPLAPEVQLLESG






GGLVQPGGSLRLSCAASGFTFSHYVMAWVRQAPGK






GLEWVSSISSSGGWTLYADSVKGRFTISRDNSKNT






LYLQMNSLRAEDTAVYYCTRGLKMATIFDYWGQGT






LVTVSS


 


638
DVD418L
AB033
AB067
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPQSALTQPASVSGSPGQSITISCIGISS






DVGSYNVVSWYQQHPGKAPKLIIYEVSQRPSGVSN






RFSGSKSGNTASLTISGLQTEDEADYYCCSYAGSS






IFVIFGGGTKVTVLG









Example 2.136
Generation of EGFR (Seq. 2) and ErbB3 (Seq. 3) DVD-Igs with Linker Set 4












TABLE 169






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







639
DVD421H
AB067
AB033
EVQLLESGGGLVQPGGSLRLSCAASGETESHYVMA







WVRQAPGKGLEWVSSISSSGGWTLYADSVKGRETI






SRDNSKNTLYLQMNSLRAEDTAVYYCTRGLKMATI






FDYWGQGTLVTVSSASTKGPQVQLKQSGPGLVQPS






QSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV






IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSL






QSNDTAIYYCARALTYYDYEEAYWGQGTLVTVSA


 


640
DVD421L
AB067
AB033
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVV






SWYQQHPGKAPKLIIYEVSQRPSGVSNRFSGSKSG






NTASLTISGLQTEDEADYYCCSYAGSSIFVIFGGG






TKVTVLGQPKAAPSVTLFPPDILLTQSPVILSVSP






GERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY






ASESISGIPSRFSGSGSGTDFTLSINSVESEDIAD






YYCQQNNNWPTTFGAGTKLELKR


 


641
DVD422H
AB033
AB067
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH






WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN






KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE






FAYWGQGTLVTVSAASTKGPEVQLLESGGGLVQPG






GSLRLSCAASGFTFSHYVMAWVRQAPGKGLEWVSS






ISSSGGWTLYADSVKGRFTISRDNSKNTLYLQMNS






LRAEDTAVYYCTRGLKMATIFDYWGQGTLVTVSS


 


642
DVD422L
AB033
AB067
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW






YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD






FTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE






LKRTVAAPSVFIFPPQSALTQPASVSGSPGQSITI






SCTGTSSDVGSYNVVSWYQQHPGKAPKLIIYEVSQ






RPSGVSNRFSGSKSGNTASLTISGLQTEDEADYYC






CSYAGSSIFVIFGGGTKVTVLG









Example 2.137
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 3) DVD-Igs with Linker Set 1












TABLE 170






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







643
DVD411H
AB067
AB004
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMA







WVRQAPGKGLEWVSSISSSGGWTLYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCTRGLKMATI






FDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR






IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


644
DVD411L
AB067
AB004
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVV






SWYQQHPGKAPKLIIYEVSQRPSGVSNRESGSKSG






NTASLTISGLQTEDEADYYCCSYAGSSIFVIFGGG






TKVTVLGQPKAAPDIQMTQSPSSLSASVGDRVTIT






CRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG






VRSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY






TTPPTFGQGTKVEIKR


 


645
DVD412H
AB004
AB067
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPEVQLLESGGGLVQP






GGSLRLSCAASGFTFSHYVMAWVRQAPGKGLEWVS






SISSSGGWTLYADSVKGRFTISRDNSKNTLYLQMN






SLRAEDTAVYYCTRGLKMATIFDYWGQGTLVTVSS


 


646
DVD412L
AB004
AB067
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPQSALTQPASVSGSPGQSITISCTGTSS






DVGSYNVVSWYQQHPGKAPKLIIYEVSQRPSGVSN






RFSGSKSGNTASLTISGLQTEDEADYYCCSYAGSS






IFVIFGGGTKVTVLG









Example 2.138
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 3) DVD-Igs with Linker Set 2












TABLE 171






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







647
DVD415H
AB067
AB004
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMA







WVRQAPGKGLEWVSSISSSGGWTLYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCTRGLKMATI






FDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGK






GLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT






AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG






TLVTVSS


 


648
DVD415L
AB067
AB004
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVV






SWYQQHPGKARKLIIYEVSQRRSGVSNRFSGSKSG






NTASLTISGLQTEDEADYYCCSYAGSSIFVIFGGG






TKVTVLGQPKAAPSVTLFPPDIQMTQSPSSLSASV






GDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS






ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFAT






YYCQQHYTTPPTFGQGTKVEIKR


 


649
DVD416H
AB004
AB067
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLLES






GGGLVQPGGSLRLSCAASGFTFSHYVMAWVRQAPG






KGLEWVSSISSSGGWTLYADSVKGRFTISRDNSKN






TLYLQMNSLRAEDTAVYYCTRGLKMATIFDYWGQG






TLVTVSS


 


650
DVD416L
AB004
AB067
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPQSALTQPASVSGSPGQSITI






SCTGTSSDVGSYNVVSWYQQHPGKAPKLIIYEVSQ






RPSGVSNRFSGSKSGNTASLTISGLQTEDEADYYC






CSYAGSSIFVIFGGGTKVTVLG









Example 2.139
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 3) DVD-Igs with Linker Set 3












TABLE 172






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







651
DVD419H
AB067
AB004
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMA







WVRQAPGKGLEWVSSISSSGGWTLYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCTRGLKMATI






FDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGK






GLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT






AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG






TLVTVSS


 


652
DVD419L
AB067
AB004
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVV






SWYQQHPGKAPKLIIYEVSQRPSGVSNRFSGSKSG






NTASLTISGLQTEDEADYYCCSYAGSSIFVIFGGG






TKVTVLGQPKAAPDIQMTQSPSSLSASVGDRVTIT






CRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG






VRSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY






TTPPTFGQGTKVEIKR


 


653
DVD420H
AB004
AB067
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLLES






GGGLVQPGGSLRLSCAASGFTFSHYVMAWVRQAPG






KGLEWVSSISSSGGWTLYADSVKGRFTISRDNSKN






TLYLQMNSLRAEDTAVYYCTRGLKMATIFDYWGQG






TLVTVSS


 


654
DVD420L
AB004
AB067
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPQSALTQPASVSGSPGQSITISCTGTSS






DVGSYNVVSWYQQHPGKAPKLIIYEVSQRPSGVSN






RFSGSKSGNTASLTISGLQTEDEADYYCCSYAGSS






IFVIFGGGTKVTVLG









Example 2.140
Generation of HER2 (Seq. 1) and ErbB3 (Seq. 3) DVD-Igs with Linker Set 4












TABLE 173






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







655
DVD423H
AB067
AB004
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMA







WVRQAPGKGLEWVSSISSSGGWTLYADSVKGRFTI






SRDNSKNTLYLQMNSLRAEDTAVYYCTRGLKMATI






FDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGENIKDTYIHWVRQAPGKGLEWVAR






IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


656
DVD423L
AB067
AB004
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVV






SWYQQHPGKAPKLIIYEVSQRPSGVSNRFSGSKSG






NTASLTISGLQTEDEADYYCCSYAGSSIFVIFGGG






TKVTVLGQPKAAPSVTLFPPDIQMTQSRSSLSASV






GDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS






ASFLYSGVRSRFSGSRSGTDFTLTISSLQPEDFAT






YYCQQHYTTPPTFGQGTKVEIKR


 


657
DVD424H
AB004
AB067
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPEVQLLESGGGLVQP






GGSLRLSCAASGFTFSHYVMAWVRQAPGKGLEWVS






SISSSGGWTLYADSVKGRFTISRDNSKNTLYLQMN






SLRAEDTAVYYCTRGLKMATIFDYWGQGTLVTVSS


 


658
DVD424L
AB004
AB067
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPQSALTQPASVSGSPGQSITI






SCTGTSSDVGSYNVVSWYQQHPGKAPKLIIYEVSQ






RPSGVSNRFSGSKSGNTASLTISGLQTEDEADYYC






CSYAGSSIFVIFGGGTKVTVLG









Example 2.141
Generation of VEGF (Seq. 1) and PLGF (Seq. 2) DVD-Igs with Linker Set 1












TABLE 174






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







659
DVD541H
AB074VH
AB014VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINT






YTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRA






EDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS


 


660
DVD541L
AB074VL
AB014VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR


 


661
DVD542H
AB014VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPQVQLKQSGPGL






VQPSQSLSITCTVSGFSLTTYGIHWVRQSPGKGLE






WLGVMWSGGDTDYDAAFISRLSISKDNSKSQVFFK






MNSLQANDTGIYYCARYRFYGMDYWGQGTSVTVSS


 


662
DVD542L
AB014VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPAIQMTQSSSSFSVSLGDRVTITCKASE






DIYNRFAWYQQKPGNAPRLLISGAASLEAGVPSRF






SGSGSGQDYTLSITSLQTEDVATYYCQQYWSTPWT






FGGGTKLEIKR









Example 2.142
Generation of VEGF (Seq. 1) and PLGF (Seq. 2) DVD-Igs with Linker Set 2












TABLE 175






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







663
DVD549H
AB074VH
AB014VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLE






WVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYL






QMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQG






TLVTVSS


 


664
DVD549L
AB074VL
AB014VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLH






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






YSTVPWTFGQGTKVEIKR


 


665
DVD550H
AB014VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPQVQL






KQSGPGLVQPSQSLSITCTVSGFSLTTYGIHWVRQ






SPGKGLEWLGVMWSGGDTDYDAAFISRLSISKDNS






KSQVFFKMNSLQANDTGIYYCARYRFYGMDYWGQG






TSVTVSS


 


666
DVD550L
AB014VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPAIQMTQSSSSFSVSLGDRVT






ITCKASEDIYNRFAWYQQKPGNAPRLLISGAASLE






AGVPSRFSGSGSGQDYTLSITSLQTEDVATYYCQQ






YWSTPWTFGGGTKLEIKR









Example 2.143
Generation of VEGF (Seq. 1) and PLGF (Seq. 2) DVD-Igs with Linker Set 3












TABLE 176






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







667
DVD557H
AB074VH
AB014VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGYTFTNYGMNWVHQAPGKGLE






WVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYL






QMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQG






TLVTVSS


 


668
DVD557L
AB074VL
AB014VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR


 


669
DVD558H
AB014VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPQVQL






KQSGPGLVQPSQSLSITCTVSGFSLTTYGIHWVRQ






SPGKGLEWLGVMWSGGDTDYDAAFISRLSISKDNS






KSQVFFKMNSLQANDTGIYYCARYRFYGMDYWGQG






TSVTVSS


 


670
DVD558L
AB014VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPAIQMTQSSSSFSVSLGDRVTITCKASE






DIYNRFAWYQQKPGNAPRLLISGAASLEAGVPSRF






SGSGSGQDYTLSITSLQTEDVATYYCQQYWSTPWT






FGGGTKLEIKR









Example 2.144
Generation of VEGF (Seq. 1) and PLGF (Seq. 2) DVD-Igs with Linker Set 4












TABLE 177






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







671
DVD565H
AB074VH
AB014VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINT






YTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRA






EDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS


 


672
DVD565L
AB074VL
AB014VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLH






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






YSTVPWTFGQGTKVEIKR


 


673
DVD566H
AB014VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYEDVWGQGTLVTVSSASTKGPQVQLKQSGPGL






VQPSQSLSITCTVSGFSLTTYGIHWVRQSPGKGLE






WLGVMWSGGDTDYDAAFISRLSISKDNSKSQVFFK






MNSLQANDTGIYYCARYRFYGMDYWGQGTSVTVSS


 


674
DVD566L
AB014VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPAIQMTQSSSSFSVSLGDRVT






ITCKASEDIYNRFAWYQQKPGNAPRLLISGAASLE






AGVPSRFSGSGSGQDYTLSITSLQTEDVATYYCQQ






YWSTPWTFGGGTKLEIKR









Example 2.145
Generation of VEGF (Seq. 2) and PLGF (Seq. 2) DVD-Igs with Linker Set 1












TABLE 178






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







675
DVD543H
AB074VH
AB070VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFTISDYWIHWVRQAPGKGLEWVAGITP






AGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRA






EDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS


 


676
DVD543L
AB074VL
AB070VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPT






FGQGTKVEIKR


 


677
DVD544H
AB070VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPQVQLKQSGPGLVQP






SQSLSITCTVSGFSLTTYGIHWVRQSPGKGLEWLG






VMWSGGDTDYDAAFISRLSISKDNSKSQVFFKMNS






LQANDTGIYYCARYRFYGMDYWGQGTSVTVSS


 


678
DVD544L
AB070VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPAIQMTQSSSSFSVSLGDRVTITCKASE






DIYNRFAWYQQKPGNAPRLLISGAASLEAGVPSRF






SGSGSGQDYTLSITSLQTEDVATYYCQQYWSTPWT






FGGGTKLEIKR









Example 2.146
Generation of VEGF (Seq. 2) and PLGF (Seq. 2) DVD-Igs with Linker Set 2












TABLE 179






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







679
DVD551H
AB074VH
AB070VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFTISDYWIHWVHQAPGKGLE






WVAGITPAGGYTYYADSVKGRFTISADTSKNTAYL






QMNSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLV






TVSS


 


680
DVD551L
AB074VL
AB070VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SYTTPPTFGQGTKVEIKR


 


681
DVD552H
AB070VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLKQS






GPGLVQPSQSLSITCTVSGFSLTTYGIHWVRQSPG






KGLEWLGVMWSGGDTDYDAAFISRLSISKDNSKSQ






VFFKMNSLQANDTGIYYCARYRFYGMDYWGQGTSV






TVSS


 


682
DVD552L
AB070VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPAIQMTQSSSSFSVSLGDRVT






ITCKASEDIYNRFAWYQQKPGNAPRLLISGAASLE






AGVPSRFSGSGSGQDYTLSITSLQTEDVATYYCQQ






YWSTPWTFGGGTKLEIKR









Example 2.147
Generation of VEGF (Seq. 2) and PLGF (Seq. 2) DVD-Igs with Linker Set 3












TABLE 180






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







683
DVD559H
AB074VH
AB070VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLE






WVAGITPAGGYTYYADSVKGRFTISADTSKNTAYL






QMNSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLV






TVSS


 


684
DVD559L
AB074VL
AB070VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPT






FGQGTKVEIKR


 


685
DVD560H
AB070VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLKQS






GPGLVQPSQSLSITCTVSGFSLTTYGIHWVRQSPG






KGLEWLGVMWSGGDTDYDAAFISRLSISKDNSKSQ






VFFKMNSLQANDTGIYYCARYRFYGMDYWGQGTSV






TVSS


 


686
DVD560L
AB070VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPAIQMTQSSSSFSVSLGDRVTITCKASE






DIYNRFAWYQQKPGNAPRLLISGAASLEAGVPSRF






SGSGSGQDYTLSITSLQTEDVATYYCQQYWSTPWT






FGGGTKLEIKR









Example 2.148
Generation of VEGF (Seq. 2) and PLGF (Seq. 2) DVD-Igs with Linker Set 4












TABLE 181






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







687
DVD567H
AB074VH
AB070VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFTISDYWIHWVRQAPGKGLEWVAGITP






AGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRA






EDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS


 


688
DVD567L
AB074VL
AB070VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SYTTPPTFGQGTKVEIKR


 


689
DVD568VH
AB070VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPQVQLKQSGPGLVQP






SQSLSITCTVSGFSLTTYGIHWVRQSPGKGLEWLG






VMWSGGDTDYDAAFISRLSISKDNSKSQVFFKMNS






LQANDTGIYYCARYRFYGMDYWGQGTSVTVSS


 


690
DVD568VL
AB070VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPAIQMTQSSSSFSVSLGDRVT






ITCKASEDIYNRFAWYQQKPGNAPRLLISGAASLE






AGVPSRFSGSGSGQDYTLSITSLQTEDVATYYCQQ






YWSTPWTFGGGTKLEIKR









Example 2.149
Generation of VEGF (Seq. 3) and PLGF (Seq. 2) DVD-Igs with Linker Set 1












TABLE 182






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







691
DVD545H
AB074VH
AB071VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFTINASWIHWVRQAPGKGLEWVGAIYP






YSGYTNYADSVKGRFTISADTSKNTAYLQMNSLRA






EDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS


 


692
DVD545L
AB074VL
AB071VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPDIQMTQSRSSLSASVGDRVTITCRASQ






VIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSNTSPLT






FGQGTKVEIKR


 


693
DVD546H
AB071VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPQVQLKQSGPGLVQ






PSQSLSITCTVSGFSLTTYGIHWVRQSPGKGLEWL






GVMWSGGDTDYDAAFISRLSISKDNSKSQVFFKMN






SLQANDTGIYYCARYRFYGMDYWGQGTSVTVSS


 


694
DVD546L
AB071VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPAIQMTQSSSSFSVSLGDRVTITCKASE






DIYNRFAWYQQKRGNAPRLLISGAASLEAGVPSRF






SGSGSGQDYTLSITSLQTEDVATYYCQQYWSTPWT






FGGGTKLEIKR









Example 2.150
Generation of VEGF (Seq. 3) and PLGF (Seq. 2) DVD-Igs with Linker Set 2












TABLE 183






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







695
DVD553H
AB074VH
AB071VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFTINASWIHWVRQAPGKGLE






WVGAIYPYSGYTNYADSVKGRFTISADISKNTAYL






QMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQGTL






VTVSS


 


696
DVD553L
AB074VL
AB071VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQVIRRSLAWYQQKPGKAPKLLIYAASNLA






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SNTSPLTFGQGTKVEIKR


 


697
DVD554H
AB071VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLKQ






SGPGLVQPSQSLSITCTVSGFSLTTYGIHWVRQSP






GKGLEWLGVMWSGGDTDYDAAFISRLSISKDNSKS






QVFFKMNSLQANDTGIYYCARYRFYGMDYWGQGTS






VTVSS


 


698
DVD554L
AB071VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPAIQMTQSSSSFSVSLGDRVT






ITCKASEDIYNRFAWYQQKPGNAPRLLISGAASLE






AGVPSRFSGSGSGQDYTLSITSLQTEDVATYYCQQ






YWSTPWTFGGGTKLEIKR









Example 2.151
Generation of VEGF (Seq. 3) and PLGF (Seq. 2) DVD-Igs with Linker Set 3












TABLE 184






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







699
DVD561H
AB074VH
AB071VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFTINASWIHWVRQAPGKGLE






WVGAIYPYSGYTNYADSVKGRFTISADISKNTAYL






QMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQGTL






VTVSS


 


700
DVD561L
AB074VL
AB071VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






VIRRSLAWYQQKRGKAPKLLIYAASNLASGVPSRF






SGSGSGTDElLIISSLQPEDFATYYCQQSNTSPLT






FGQGTKVEIKR


 


701
DVD562H
AB071VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLKQ






SGPGLVQPSQSLSITCTVSGFSLTTYGIHWVRQSP






GKGLEWLGVMWSGGDTDYDAAFISRLSISKDNSKS






QVFFKMNSLQANDTGIYYCARYRFYGMDYWGQGTS






VTVSS


 


702
DVD562L
AB071VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPAIQMTQSSSSFSVSLGDRVTITCKASE






DIYNRFAWYQQKPGNAPRLLISGAASLEAGVPSRF






SGSGSGQDYTLSITSLQTEDVATYYCQQYWSTPWT






FGGGTKLEIKR









Example 2.152
Generation of VEGF (Seq. 3) and PLGF (Seq. 2) DVD-Igs with Linker Set 4












TABLE 185






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







703
DVD569H
AB074VH
AB071VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFTINASWIHWVRQAPGKGLEWVGAIYP






YSGYTNYADSVKGRFTISADTSKNTAYLQMNSLRA






EDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS


 


704
DVD569L
AB074VL
AB071VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQVIRRSLAWYQQKPGKAPKLLIYAASNLA






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SNTSPLTFGQGTKVEIKR


 


705
DVD570H
AB071VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADISKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPQVQLKQSGPGLVQ






PSQSLSITCTVSGFSLTTYGIHWVRQSPGKGLEWL






GVMWSGGDTDYDAAFISRLSISKDNSKSQVFFKMN






SLQANDTGIYYCARYRFYGMDYWGQGTSVTVSS


 


706
DVD570L
AB071VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPAIQMTQSSSSFSVSLGDRVT






ITCKASEDIYNRFAWYQQKPGNAPRLLISGAASLE






AGVPSRFSGSGSGQDYTLSITSLQTEDVATYYCQQ






YWSTPWTFGGGTKLEIKR









Example 2.153
Generation of HER2 (Seq. 1) and PLGF (Seq. 2) DVD-Igs with Linker Set 1












TABLE 186






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







707
DVD547H
AB074VH
AB004VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYP






TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA






FDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


708
DVD547L
AB074VL
AB004VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT






FGQGTKVEIKR


 


709
DVD548H
AB004VH
AB074VH
EVQLVESGGGLVQRGGSLRLSCAASGFNIKDTYIH






WVRQARGKGLEWVARIYRTNGYTRYADSVKGRFTI






SADISKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPQVQLKQSGRGLVQP






SQSLSITCTVSGFSLTTYGIHWVRQSPGKGLEWLG






VMWSGGDTDYDAAFISRLSISKDNSKSQVFFKMNS






LQANDTGIYYCARYRFYGMDYWGQGTSVTVSS


 


710
DVD548L
AB004VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FILlISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPAIQMTQSSSSFSVSLGDRVTIICKASE






DIYNRFAWYQQKPGNAPRLLISGAASLEAGVPSRF






SGSGSGQDYTLSITSLQTEDVATYYCQQYWSTPWT






FGGGTKLEIKR









Example 2.154
Generation of HER2 (Seq. 1) and PLGF (Seq. 2) DVD-Igs with Linker Set 2












TABLE 187






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







711
DVD555H
AB074VH
AB004VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE






WVARIYPTNGYTRYADSVKGRFTISADISKNTAYL






QMNSLRAEDIAVYYCSRWGGDGFYAMDYWGQGTLV






TVSS


 


712
DVD555L
AB074VL
AB004VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ






HYTTPPTFGQGTKVEIKR


 


713
DVD556H
AB004VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLKQS






GPGLVQPSQSLSITCTVSGFSLTTYGIHWVRQSPG






KGLEWLGVMWSGGDTDYDAAFISRLSISKDNSKSQ






VFFKMNSLQANDTGIYYCARYRFYGMDYWGQGTSV






TVSS


 


714
DVD556L
AB004VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPAIQMTQSSSSFSVSLGDRVT






ITCKASEDIYNRFAWYQQKPGNARRLLISGAASLE






AGVPSRFSGSGSGQDYTLSITSLQTEDVATYYCQQ






YWSTPWTFGGGTKLEIKR









Example 2.155
Generation of HER2 (Seq. 1) and PLGF (Seq. 2) DVD-Igs with Linker Set 3












TABLE 188






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







715
DVD563H
AB074VH
AB004VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE






WVARIYPTNGYTRYADSVKGRFTISADISKNTAYL






QMNSLRAEDIAVYYCSRWGGDGFYAMDYWGQGTLV






TVSS


 


716
DVD563L
AB074VL
AB004VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVNTAVAWYQQKPGKARKLLIYSASFLYSGVPSRF






SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT






FGQGTKVEIKR


 


717
DVD564H
AB004VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLKQS






GPGLVQPSQSLSITCTVSGFSLTTYGIHWVRQSPG






KGLEWLGVMWSGGDTDYDAAFISRLSISKDNSKSQ






VFFKMNSLQANDTGIYYCARYRFYGMDYWGQGTSV






TVSS


 


718
DVD564L
AB004VL
AB074VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPAIQMTQSSSSFSVSLGDRVTIICKASE






DIYNRFAWYQQKPRGNAPRLLISGAASLEAGVPSRF






SGSGSGQDYTLSITSLQTEDVATYYCQQYWSTPWT






FGGGTKLEIKR









Example 2.156
Generation of HER2 (Seq. 1) and PLGF (Seq. 2) DVD-Igs with Linker Set 4












TABLE 189






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







719
DVD571H
AB074VH
AB004VH
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIH







WVRQSPGKGLEWLGVMWSGGDTDYDAAFISRLSIS






KDNSKSQVFFKMNSLQANDTGIYYCARYRFYGMDY






WGQGTSVTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYP






TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA






FDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


720
DVD571L
AB074VL
AB004VL
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAW






YQQKPGNAPRLLISGAASLEAGVPSRFSGSGSGQD






YTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ






HYTTPPTFGQGTKVEIKR


 


721
DVD572H
AB004VH
AB074VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPQVQLKQSGPGLVQP






SQSLSITCTVSGFSLTTYGIHWVRQSPGKGLEWLG






VMWSGGDTDYDAAFISRLSISKDNSKSQVFFKMNS






LQANDTGIYYCARYRFYGMDYWGQGTSVTVSS


 


722
DVD572L
AB004VL
AB004VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPAIQMTQSSSSFSVSLGDRVT






ITCKASEDIYNRFAWYQQKPGNAPRLLISGAASLE






AGVPSRFSGSGSGQDYTLSITSLQTEDVATYYCQQ






YWSTPWTFGGGTKLEIKR









Example 2.157
Generation of PLGF (Seq. 1) and VEGF (Seq. 2) DVD-Igs with Linker Set 1












TABLE 191






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345




















723
DVD573H
AB047VH
AB07VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYINWV







KLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTIDT






SSSTAYMQLSSLISEDTAVYECVRDSPFFDYWGQGTL






LTVSSASTKGPEVQLVESGGGLVQPGGSLRLSCAASG






FTISDYWIHWVRQAPGKGLEWVAGITPAGGYTYYADS






VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARFV






FFLPYAMDYWGQGTLVTVSS


 


724
DVD573L
AB047VL
AB070VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMRKS






FLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSG






TDFTLTISSVQAEDVAVYYCKQSYHLFTFGSGTKLEI






KRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQDVS






TAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS






GTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKV






EIKR


 


725
DVD574H
AB070VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWV






RQAPGKGLEWVAGITPAGGYTYYADSVKGRFTISADT






SKNTAYLQMNSLRAEDTAVYYCARFVFFLPYAMDYWG






QGTLVTVSSASTKGPQVQLQQSGAELVKPGASVKISC






KASGYTFTDYYINWVKLAPGQGLEWIGWIYPGSGNTK






YNEKFKGKATLTIDTSSSTAYMQLSSLTSEDTAVYFC






VRDSPFFDYWGQGTLLTVSS


 


726
DVD574L
AB070VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQ






QKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT






ISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKRTVA






APDIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSG






SGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGSGTKL






EIKR









Example 2.158
Generation of PLGF (Seq. 1) and VEGF (Seq. 2) DVD-Igs with Linker Set 2












TABLE 192






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







727
DVD581H
AB047VH
AB070VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYRGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLE






WVAGITPAGGYTYYADSVKGRFTISADISKNTAYL






QMNSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLV






TVSS


 


728
DVD581L
AB047VL
AB070VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPSVFIFPPDIQMTQSPSSLSASV






GDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS






ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFAT






YYCQQSYTTPPTFGQGTKVEIKR


 


729
DVD582H
AB070VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLQQS






GAELVKPGASVKISCKASGYTFTDYYINWVKLAPG






QGLEWIGWIYPGSGNTKYNEKFKGKATLTIDTSSS






TAYMQLSSLISEDTAVYFCVRDSPFFDYWGQGTLL






TVSS


 


730
DVD582L
AB070VL
AB047VL
DIQMTQSRSSLSASVGDRVTITCRASQDVSTAVAW






YQQKRGKARKLLIYSASFLYSGVPSRFSGSGSGTD






FILlISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIVLTQSPDSLAVSLGERVT






MNCKSSQSLLNSGMRKSFLAWYQQKPGQSPKLLIY






WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDVA






VYYCKQSYHLFTFGSGTKLEIKR









Example 2.159
Generation of PLGF (Seq. 1) and VEGF (Seq. 2) DVD-Igs with Linker Set 3












TABLE 193






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







731
DVD589VH
AB047VH
AB070VH
QVQLQQSGAELVKRGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLE






WVAGITPAGGYTYYADSVKGRFTISADISKNTAYL






QMNSLRAEDIAVYYCARFVFFLPYAMDYWGQGTLV






TVSS


 


732
DVD589VL
AB047VL
AB070VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPDIQMTQSPSSLSASVGDRVTIT






CRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSG






VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSY






TTPPTFGQGTKVEIKR


 


733
DVD590VH
AB070VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLQQS






GAELVKPGASVKISCKASGYTFTDYYINWVKLAPG






QGLEWIGWIYPGSGNTKYNEKFKGKATLTIDTSSS






TAYMQLSSLTSEDTAVYFCVRDSPFFDYWGQGTLL






TVSS


 


734
DVD590VL
AB070VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPDIVLTQSPDSLAVSLGERVTMNCKSSQ






SLLNSGMRKSFLAWYQQKPGQSPKLLIYWASTRES






GVPDRFTGSGSGTDFTLTISSVQAEDVAVYYCKQS






YHLFTFGSGTKLEIKR









Example 2.160
Generation of PLGF (Seq. 1) and VEGF (Seq. 2) DVD-Igs with Linker Set 4












TABLE 194






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







735
DVD597VH
AB047VH
AB070VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFTISDYWIHWVRQAPGKGLEWVAGITP






AGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRA






EDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS


 


736
DVD597VL
AB047VL
AB070VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPSVFIFPPDIQMTQSPSSLSASV






GDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS






ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFAT






YYCQQSYTTPPTFGQGTKVEIKR


 


737
DVD598VH
AB070VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADISKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPQVQLQQSGAELVKP






GASVKISCKASGYTFTDYYINWVKLAPGQGLEWIG






WIYPGSGNTKYNEKFKGKATLTIDTSSSTAYMQLS






SLTSEDTAVYFCVRDSPFFDYWGQGTLLTVSS


 


738
DVD598VL
AB070VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FILlISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIVLTQSPDSLAVSLGERVT






MNCKSSQSLLNSGMRKSFLAWYQQKPGQSPKLLIY






WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDVA






VYYCKQSYHLFTFGSGTKLEIKR









Example 2.161
Generation of PLGF (Seq. 1) and VEGF (Seq. 3) DVD-Igs with Linker Set 1












TABLE 195






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







739
DVD575H
AB047VH
AB071VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFTINASWIHWVRQARGKGLEWVGAIYP






YSGYTNYADSVKGRFTISADTSKNTAYLQMNSLRA






EDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS


 


740
DVD575L
AB047VL
AB071VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVRDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPDIQMTQSPSSLSASVGDRVTIT






CRASQVIRRSLAWYQQKPGKAPKLLIYAASNLASG






VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSN






TSPLTFGQGTKVEIKR


 


741
DVD576H
AB071VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPQVQLQQSGAELVK






PGASVKISCKASGYTFTDYYINWVKLAPGQGLEWI






GWIYPGSGNTKYNEKFKGKATLTIDTSSSTAYMQL






SSLTSEDTAVYFCVRDSPFFDYWGQGTLLTVSS


 


742
DVD576L
AB071VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPDIVLTQSPDSLAVSLGERVTMNCKSSQ






SLLNSGMRKSFLAWYQQKPGQSPKLLIYWASTRES






GVPDRFTGSGSGTDFTLTISSVQAEDVAVYYCKQS






YHLFTFGSGTKLEIKR









Example 2.162
Generation of PLGF (Seq. 1) and VEGF (Seq. 3) DVD-Igs with Linker Set 2












TABLE 196






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







743
DVD583H
AB047VH
AB071VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFTINASWIHWVRQAPGKGLE






WVGAIYPYSGYTNYADSVKGRFTISADISKNTAYL






QMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQGTL






VTVSS


 


744
DVD583L
AB047VL
AB071VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPSVFIFPPDIQMTQSPSSLSASV






GDRVTITCRASQVIRRSLAWYQQKPGKAPKLLIYA






ASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFAT






YYCQQSNTSPLTFGQGTKVEIKR


 


745
DVD584H
AB071VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADISKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLQQ






SGAELVKPGASVKISCKASGYTFTDYYINWVKLAP






GQGLEWIGWIYPGSGNTKYNEKFKGKATLTIDTSS






STAYMQLSSLISEDTAVYFCVRDSPFFDYWGQGTL






LTVSS


 


746
DVD584L
AB071VL
AB047VL
DIQMIQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPDIVLTQSRDSLAVSLGERVT






MNCKSSQSLLNSGMRKSFLAWYQQKPGQSPKLLIY






WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDVA






VYYCKQSYHLFTFGSGTKLEIKR









Example 2.163
Generation of PLGF (Seq. 1) and VEGF (Seq. 3) DVD-Igs with Linker Set 3












TABLE 197






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







747
DVD591VH
AB047VH
AB071VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFTINASWIHWVRQAPGKGLE






WVGAIYPYSGYTNYADSVKGRFTISADTSKNTAYL






QMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQGTL






VTVSS


 


748
DVD591VL
AB047VL
AB071VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPDIQMTQSPSSLSASVGDRVTIT






CRASQVIRRSLAWYQQKPGKAPKLLIYAASNLASG






VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSN






TSPLTFGQGTKVEIKR


 


749
DVD592VH
AB071VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLQQ






SGAELVKPGASVKISCKASGYTFTDYYINWVKLAP






GQGLEWIGWIYPGSGNTKYNEKFKGKATLTIDTSS






STAYMQLSSLTSEDTAVYFCVRDSPFFDYWGQGTL






LTVSS


 


750
DVD592VL
AB071VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPDIVLTQSPDSLAVSLGERVTMNCKSSQ






SLLNSGMRKSFLAWYQQKPGQSPKLLIYWASTRES






GVPDRFTGSGSGTDFTLTISSVQAEDVAVYYCKQS






YHLFTFGSGTKLEIKR









Example 2.164
Generation of PLGF (Seq. 1) and VEGF (Seq. 3) DVD-Igs with Linker Set 4












TABLE 198






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







751
DVD599VH
AB047VH
AB071VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFTINASWIHWVRQAPGKGLEWVGAIYP






YSGYTNYADSVKGRFTISADTSKNTAYLQMNSLRA






EDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS


 


752
DVD599VL
AB047VL
AB071VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPSVFIFPPDIQMTQSPSSLSASV






GDRVTITCRASQVIRRSLAWYQQKPGKAPKLLIYA






ASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFAT






YYCQQSNTSPLTFGQGTKVEIKR


 


753
DVD600VH
AB071VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPQVQLQQSGAELVK






PGASVKISCKASGYTFTDYYINWVKLAPGQGLEWI






GWIYPGSGNTKYNEKFKGKATLTIDTSSSTAYMQL






SSLTSEDTAVYFCVRDSPFFDYWGQGTLLTVSS


 


754
DVD600VL
AB071VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPDIVLTQSRDSLAVSLGERVT






MNCKSSQSLLNSGMRKSFLAWYQQKPGQSPKLLIY






WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDVA






VYYCKQSYHLFTFGSGTKLEIKR









Example 2.165
Generation of HER2 (Seq. 1) and PLGF (Seq. 1) DVD-Igs with Linker Set 1












TABLE 199






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







755
DVD577H
AB047VH
AB004VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYP






TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA






EDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


756
DVD577L
AB047VL
AB004VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPDIQMTQSPSSLSASVGDRVTIT






CRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG






VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY






TTPPTFGQGTKVEIKR


 


757
DVD578H
AB004VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPQVQLQQSGAELVKP






GASVKISCKASGYTFTDYYINWVKLAPGQGLEWIG






WIYPGSGNTKYNEKFKGKATLTIDTSSSTAYMQLS






SLTSEDTAVYFCVRDSPFFDYWGQGTLLTVSS


 


758
DVD578L
AB004VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPDIVLTQSPDSLAVSLGERVTMNCKSSQ






SLLNSGMRKSFLAWYQQKRGQSPKLLIYWASTRES






GVPDRFTGSGSGTDFTLTISSVQAEDVAVYYCKQS






YHLFTFGSGTKLEIKR









Example 2.166
Generation of HER2 (Seq. 1) and PLGF (Seq. 1) DVD-Igs with Linker Set 2












TABLE 200






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







759
DVD585H
AB047VH
AB004VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE






WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYL






QMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLV






TVSS


 


760
DVD585L
AB047VL
AB004VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVRDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPSVFIFPPDIQMTQSPSSLSASV






GDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS






ASFLYSGVRSPFSGSRSGTDFTLTISSLQPEDFAT






YYCQQHYTTPPTFGQGTKVEIKR


 


761
DVD586H
AB004VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLQQS






GAELVKPGASVKISCKASGYTFTDYYINWVKLAPG






QGLEWIGWIYPGSGNTKYNEKFKGKATLTIDTSSS






TAYMQLSSLTSEDTAVYFCVRDSPFFDYWGQGTLL






TVSS


 


762
DVD586L
AB004VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIVLTQSPDSLAVSLGERVT






MNCKSSQSLLNSGMRKSFLAWYQQKPGQSPKLLIY






WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDVA






VYYCKQSYHLFTFGSGTKLEIKR









Example 2.167
Generation of HER2 (Seq. 1) and PLGF (Seq. 1) DVD-Igs with Linker Set 3












TABLE 201






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







763
DVD593VH
AB047VH
AB004VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPSVFPLAPEVQLVESGGGL






VQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE






WVARIYPTNGYTRYADSVKGRFTISADISKNTAYL






QMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLV






TVSS


 


764
DVD593VL
AB047VL
AB004VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPDIQMTQSPSSLSASVGDRVTIT






CRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG






VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY






TTPPTFGQGTKVEIKR


 


765
DVD594VH
AB004VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLQQS






GAELVKPGASVKISCKASGYTFTDYYINWVKLAPG






QGLEWIGWIYPGSGNTKYNEKFKGKATLTIDTSSS






TAYMQLSSLTSEDTAVYFCVRDSPFFDYWGQGTLL






TVSS


 


766
DVD594VL
AB004VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPDIVLTQSPDSLAVSLGERVTMNCKSSQ






SLLNSGMRKSFLAWYQQKPGQSPKLLIYWASTRES






GVPDRFTGSGSGTDFTLTISSVQAEDVAVYYCKQS






YHLFTFGSGTKLEIKR









Example 2.168
Generation of HER2 (Seq. 1) and PLGF (Seq. 1) DVD-Igs with Linker Set 4












TABLE 202






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







767
DVD601VH
AB047VH
AB004VH
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYIN







WVKLAPGQGLEWIGWIYPGSGNTKYNEKFKGKATL






TIDTSSSTAYMQLSSLTSEDTAVYFCVRDSPFFDY






WGQGTLLTVSSASTKGPEVQLVESGGGLVQPGGSL






RLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYP






TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA






FDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


768
DVD601VL
AB047VL
AB004VL
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMR






KSFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG






SGSGTDFTLTISSVQAEDVAVYYCKQSYHLFTFGS






GTKLEIKRTVAAPSVFIFPPDIQMTQSPSSLSASV






GDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS






ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFAT






YYCQQHYTTPPTFGQGTKVEIKR


 


769
DVD602VH
AB004VH
AB047VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYRTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPQVQLQQSGAELVKP






GASVKISCKASGYTFTDYYINWVKLAPGQGLEWIG






WIYPGSGNTKYNEKFKGKATLTIDTSSSTAYMQLS






SLTSEDTAVYFCVRDSPFFDYWGQGTLLTVSS


 


770
DVD602VL
AB004VL
AB047VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIVLTQSPDSLAVSLGERVT






MNCKSSQSLLNSGMRKSFLAWYQQKPGQSPKLLIY






WASTRESGVPDRFTGSGSGTDFTLTISSVQAEDVA






VYYCKQSYHLFTFGSGTKLEIKR









Example 2.169
Generation of HGF (Seq. 1) and VEGF (Seq. 2) DVD-Igs with Linker Set 1












TABLE 203






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







771
DVD643H
AB012VH
AB070VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS







WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPSVFPLAPEVQLV






ESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQA






PGKGLEWVAGITPAGGYTYYADSVKGRFTISADIS






KNTAYLQMNSLRAEDTAVYYCARFVFFLPYAMDYW






GQGTLVTVSS


 


772
DVD643L
AB012VL
AB070VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SYTTPPTFGQGTKVEIKR


 


773
DVD644H
AB070VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLVES






GGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG






KGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKN






SLYLQMNSLRAEDTAVYYCARDEYNSGWYVLFDYW






GQGTLVTVSS


 


774
DVD644L
AB070VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSVSASVGDRVT






ITCRASQGISSWLAWYQQKPGKAPNLLIYEASSLQ






SGVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQQ






ANGFPWTFGQGTKVEIKR









Example 2.170
Generation of HGF (Seq. 1) and VEGF (Seq. 2) DVD-Igs with Linker Set 2












TABLE 204






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







775
DVD649H
AB012VH
AB070VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS







WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPSVFPLAPEVQLV






ESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQA






PGKGLEWVAGITPAGGYTYYADSVKGRFTISADTS






KNTAYLQMNSLRAEDTAVYYCARFVFFLPYAMDYW






GQGTLVTVSS


 


776
DVD649L
AB012VL
AB070VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPT






FGQGTKVEIKR


 


777
DVD650H
AB070VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLVES






GGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPG






KGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKN






SLYLQMNSLRAEDTAVYYCARDEYNSGWYVLFDYW






GQGTLVTVSS


 


778
DVD650L
AB070VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSPSSVSASVGDRVTITCRASQ






GISSWLAWYQQKPGKAPNLLIYEASSLQSGVPSRF






GGSGSGTDFTLTISSLQPEDFATYYCQQANGFPWT






EGQGTKVEIKR









Example 2.171
Generation of HGF (Seq. 1) and VEGF (Seq. 2) DVD-Igs with Linker Set 3












TABLE 205






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







779
DVD655H
AB012VH
AB070VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS







WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPEVQLVESGGGLV






QPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEW






VAGITPAGGYTYYADSVKGRFTISADTSKNTAYLQ






MNSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVT






VSS


 


780
DVD655L
AB012VL
AB070VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SYTTPPTFGQGTKVEIKR


 


781
DVD656H
AB070VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPQVQLVESGGGLVKP






GGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS






YISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMN






SLRAEDTAVYYCARDEYNSGWYVLFDYWGQGTLVT






VSS


 


782
DVD656L
AB070VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSVSASVGDRVT






ITCRASQGISSWLAWYQQKPGKAPNLLIYEASSLQ






SGVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQQ






ANGFPWTFGQGTKVEIKR









Example 2.172
Generation of HGF (Seq. 1) and VEGF (Seq. 2) DVD-Igs with Linker Set 4












TABLE 206






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







783
DVD709VH
AB012VH
AB070VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS







WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPEVQLVESGGGLV






QPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEW






VAGITPAGGYTYYADSVKGRFTISADTSKNTAYLQ






MNSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVT






VSS


 


784
DVD709VL
AB012VL
AB070VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPDIQMTQSRSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPT






EGQGTKVEIKR


 


785
DVD710VH
AB070VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPQVQLVESGGGLVKP






GGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS






YISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMN






SLRAEDTAVYYCARDEYNSGWYVLFDYWGQGTLVT






VSS


 


786
DVD710VL
AB070VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSPSSVSASVGDRVTITCRASQ






GISSWLAWYQQKPGKAPNLLIYEASSLQSGVPSRF






GGSGSGTDFTLTISSLQPEDFATYYCQQANGFPWT






FGQGTKVEIKR









Example 2.173
Generation of HGF (Seq. 1) and VEGF (Seq. 3) DVD-Igs with Linker Set 1












TABLE 207






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







787
DVD645H
AB012VH
AB071VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS







WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPSVFPLAPEVQLV






ESGGGLVQPGGSLRLSCAASGFTINASWIHWVRQA






PGKGLEWVGAIYPYSGYTNYADSVKGRFTISADTS






KNTAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDY






WGQGTLVTVSS


 


788
DVD645L
AB012VL
AB071VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQVIRRSLAWYQQKPGKAPKLLIYAASNLA






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SNTSPLTFGQGTKVEIKR


 


789
DVD646H
AB071VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLVE






SGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP






GKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAK






NSLYLQMNSLRAEDTAVYYCARDEYNSGWYVLFDY






WGQGTLVTVSS


 


790
DVD646L
AB071VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSVSASVGDRVT






ITCRASQGISSWLAWYQQKPGKAPNLLIYEASSLQ






SGVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQQ






ANGFPWTFGQGTKVEIKR









Example 2.174
Generation of HGF (Seq. 1) and VEGF (Seq. 3) DVD-Igs with Linker Set 2












TABLE 208






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







791
DVD651H
AB012VH
AB071VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS







WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPSVFPLAPEVQLV






ESGGGLVQPGGSLRLSCAASGFTINASWIHWVRQA






PGKGLEWVGAIYPYSGYTNYADSVKGRFTISADTS






KNTAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDY






WGQGTLVTVSS


 


792
DVD651L
AB012VL
AB071VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






VIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSNTSPLT






FGQGTKVEIKR


 


793
DVD652H
AB071VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPQVQLVE






SGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAP






GKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAK






NSLYLQMNSLRAEDTAVYYCARDEYNSGWYVLFDY






WGQGTLVTVSS


 


794
DVD652L
AB071VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPDIQMTQSPSSVSASVGDRVTITCRASQ






GISSWLAWYQQKPGKAPNLLIYEASSLQSGVPSRF






GGSGSGTDFTLTISSLQPEDFATYYCQQANGFPWT






FGQGTKVEIKR









Example 2.175
Generation of HGF (Seq. 1) and VEGF (Seq. 3) DVD-Igs with Linker Set 3












TABLE 209






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







795
DVD657H
AB012VH
AB071VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS







WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPEVQLVESGGGLV






QPGGSLRLSCAASGFTINASWIHWVRQAPGKGLEW






VGAIYPYSGYTNYADSVKGRFTISADTSKNTAYLQ






MNSLRAEDTAVYYCARWGHSTSRWAMDYWGQGTLV






TVSS


 


796
DVD657L
AB012VL
AB071VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSRSSLSASVGDRVT






ITCRASQVIRRSLAWYQQKPGKAPKLLIYAASNLA






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SNTSPLTFGQGTKVEIKR


 


797
DVD658H
AB071VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPQVQLVESGGGLVK






PGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWV






SYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCARDEYNSGWYVLFDYWGQGTLV






TVSS


 


798
DVD658L
AB071VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSVSASVGDRVT






ITCRASQGISSWLAWYQQKPGKAPNLLIYEASSLQ






SGVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQQ






ANGFPWTFGQGTKVEIKR









Example 2.176
Generation of HGF (Seq. 1) and VEGF (Seq. 3) DVD-Igs with Linker Set 4












TABLE 210






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







799
DVD711VH
AB012VH
AB071VH
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMS







WIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI






SRDNAKNSLYLQMNSLRAEDTAVYYCARDEYNSGW






YVLFDYWGQGTLVTVSSASTKGPEVQLVESGGGLV






QPGGSLRLSCAASGFTINASWIHWVRQAPGKGLEW






VGAIYPYSGYTNYADSVKGRFTISADTSKNTAYLQ






MNSLRAEDTAVYYCARWGHSTSPWAMDYWGQGTLV






TVSS


 


800
DVD711VL
AB012VL
AB071VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAW






YQQKPGKAPNLLIYEASSLQSGVPSRFGGSGSGTD






FTLTISSLQPEDFATYYCQQANGFPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






VIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSNTSPLT






FGQGTKVEIKR


 


801
DVD712VH
AB071VH
AB012VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQARGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPQVQLVESGGGLVK






PGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWV






SYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQM






NSLRAEDTAVYYCARDEYNSGWYVLFDYWGQGTLV






TVSS


 


802
DVD712VL
AB071VL
AB012VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPDIQMIQSPSSVSASVGDRVTITCRASQ






GISSWLAWYQQKPGKAPNLLIYEASSLQSGVPSRF






GGSGSGTDFTLTISSLQPEDFATYYCQQANGFPWT






FGQGTKVEIKR









Example 2.177
Generation of HGF (Seq. 2) and VEGF (Seq. 1) DVD-Igs with Linker Set 1












TABLE 211






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







803
DVD659H
AB079VH
AB014VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH







WVRQMRGKGLEWMGEINPTNGHTNYNPSFQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWI






NTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSL






RAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTV






SS


 


804
DVD659L
AB079VL
AB014VL
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSW






YQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCSASQ






DISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWT






FGQGTKVEIKR


 


805
DVD660H
AB014VH
AB07DVH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPEVQLVQSGAEV






KKPGESLKISCKGSGYSFTTYWMHWVRQMPGKGLE






WMGEINPTNGHTNYNPSFQGQVTISADKSISTAYL






QWSSLKASDTAMYYCARNYVGSIFDYWGQGTLVTV






SS


 


806
DVD660L
AB014VL
AB07DVL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCKASE






NVVSYVSWYQQKPGKAPKLLIYGASNRNTGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCGQSYNYPYT






FGQGTKLEIKR









Example 2.178
Generation of HGF (Seq. 2) and VEGF (Seq. 1) DVD-Igs with Linker Set 2












TABLE 211






DVD
Outer
Inner




SEQ
Variable
Variable
Variable


ID
Domain
Domain
Domain
Sequence


NO
Name
Name
Name
12345678901234567890123456789012345







807
DVD665H
AB079VH
AB014VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH







WVRQMRGKGLEWMGEINPTNGHTNYNPSFQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKG






LEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTA






YLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWG






QGTLVTVSS


 


808
DVD665L
AB079VL
AB014VL
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSW






YQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLH






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






YSTVPWTFGQGTKVEIKR


 


809
DVD666H
AB014VH
AB079VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPEVQL






VQSGAEVKKPGESLKISCKGSGYSFTTYWMHWVRQ






MPGKGLEWMGEINPTNGHTNYNPSFQGQVTISADK






SISTAYLQWSSLKASDTAMYYCARNYVGSIFDYWG






QGTLVTVSS


 


810
DVD666L
AB014VL
AB079VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCKASENVVSYVSWYQQKPGKAPKLLIYGASNRN






TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQ






SYNYPYTFGQGTKLEIKR









Example 2.179
Generation of HGF (Seq. 2) and VEGF (Seq. 1) DVD-Igs with Linker Set 3











TABLE 213





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







811
DVD671VH
AB079VH
AB014VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH






WVRQMPGKGLEWMGEINPTNGHTNYNPSFQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKG






LEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTA






YLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWG






QGTLVTVSS


 


812
DVD671VL
AB079VL
AB014VL
DIQMTQSPSSLSAVSVGDRVTITCKASENVVSYVS






WYQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGT






DFTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKL






EIKRTVAAPDIQMTQSPSSLASASVGDRVTITCSA






SQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPS






RFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVP






WTFGQGTKVEIKR


 


813
DVD672VH
AB014VH
AB079VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPEVQL






VQSGAEVKKPGESLKISCKGSGYSFTTYWMHWVRQ






MPGKGLEWMGEINPTNGHTNYNPSFQGQVTISADK






SISTAYLQWSSLKASDTAMYYCARNYVGSIFDYWG






QGTLVTVSS


 


814
DVD672VL
AB014VL
AB079VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCKASE






NVVSYVSWYQQKPGKAPKLLIYGASNRNTGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCGQSYNYPYT






FGQGTKLEIKR









Example 2.180
Generation of HGF (Seq. 2) and VEGF (Seq. 1) DVD-Igs with Linker Set 4











TABLE 214





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







815
DVD677VH
AB079VH
AB014VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH






WVRQMPGKGLEWMGEINPTNGHTNYNPSGQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWI






NTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSL






RAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTV






SS


 


816
DVD677VL
AB079VL
AB014VL
DIQMTSQPSSLSASVGDRVTITCKASENVVSYVSW






YQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKLE






IKRTVAAPSVFIFPPDIQMTSQPSSLSASVGDRVT






ITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLH






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






YSTVPWTFGQGTKVEIKR


 


817
DVD678VH
AB014VH
AB079VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMN






WVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTF






SLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGS






SHWYFDVWGQGTLVTVSSASTKGPEVQLVQSGAEV






KKPGESLKISCKGSGYSFTTYWMHWVRQMPGKGLE






WMGEINPTNGHTNYNPSFQGQVTISADKSISTAYL






QWSSLKASDTAMYYCARNYVGSIFDYWGQGTLVTV






SS


 


818
DVD678VL
AB014VL
AB079VL
DIQMTQSPSSLSASVGDRVTITCSASQDSINYLNW






YQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCKASENVVSYVSWYQQKPGKAPKLLIYGASNRN






TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQ






SYNYPYTFGQGTKLEIKR









Example 2.181
Generation of HGF (Seq. 2) and VEGF (Seq. 2) DVD-Igs with Linker Set 1











TABLE 215





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







819
DVD661H
AB079VH
AB070VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH






WVRQMPGKGLEWMGEINPTNGHTNYNPSFQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAGI






TPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSL






RAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS


 


820
DVD661L
AB079VL
AB070VL
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSW






YQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVWAYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPT






FGQGTKVEIKR


 


821
DVD662H
AB070VH
AB079VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPEVQLVQSGAEVKKP






GESLKISCKGSGYSFTTYWMHWVRQMPGKGLEWMG






EINPTNGHTNYNPSFQGQVTISADKSISTAYLQWS






SLKASDTAMYYCARNYVGSIFDYWGQGTLVTVSS


 


822
DVD662L
AB070VL
AB079VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCKASE






NVVSYNSWYQQKPGKAPKLLIYGASNRNTGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCGQSYNYPYT






FGQGTKLEIKR









Example 2.182
Generation of HGF (Seq. 2) and VEGF (Seq. 2) DVD-Igs with Linker Set 2











TABLE 216





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







823
DVD667H
AB079VH
AB070VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH






WVRQMPGKGLEWMGEINPTNGHTNYNPSFQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKG






LEWVAGITPAGGYTYYADSVKGRFTISADTSKNTA






YLQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQGT






LVTVSS


 


824
DVD667L
AB079VL
AB070VL
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSW






YQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SYTTPPTFGQGTKVEIKR


 


825
DVD668H
AB070VH
AB079VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVQS






GAEVKKPGESLKISCKGSGYSFTTYWMHWVRQMPG






KGLEWMGEINPTNGHTNYNPSFQGQVTISADKSIS






TAYLQWSSLKASDTAMYYCARNYVGSIFDYWGQGT






LVTVSS


 


826
DVD668L
AB070L
AB079VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCKASENVVSYVSWYQQKPGKAPKLLIYGASNRN






TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQ






SYNYPYTFGQGTKLEIKR









Example 2.183
Generation of HGF (Seq. 2) and VEGF (Seq. 2) DVD-Igs with Linker Set 3











TABLE 217





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







827
DVD673VH
AB079VH
AB070VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH






WVRQMPGKGLEWMGEINPTNGHTNYNPSFQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKG






LEWVAGITPAGGYTYYADSVKGRFTISADTSKNTA






YLQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQGT






LVTVSS


 


828
DVD673VL
AB079VL
AB070VL
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSW






YQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPT






FGQGTKVEIKR


 


829
DVD674VH
AB070VH
AB079VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVQS






GAEVKKPGESLKISCKGSGYSFTTYWMHWVRQMPG






KGLEWMGEINPTNGHTNYNPSFQGQVTISADKSIS






TAYLQWSSLKASDTAMYYCARNYVGSIFDYWGQGT






LVTVSS


 


830
DVD674VL
AB070VL
AB079VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCKASE






NVVSYVSWYQQKPGKAPKLLIYGASNRNTGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCGQSYNYPYT






FGQGTKLEIKR









Example 2.184
Generation of HGF (Seq. 2) and VEGF (Seq. 2) DVD-Igs with Linker Set 4











TABLE 218





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







831
DVD679VH
AB079VH
AB070VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH






WVRQMPGKGLEWMGEINPTNGHTNYNPSFQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAGI






TPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSL






RAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS


 


832
DVD679VL
AB079VL
AB070VL
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSW






YQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SYTTPPTFGQGTKVEIKR


 


833
DVD680VH
AB070VH
AB079VH
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIH






WVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARDVFFLPY






AMDYWGQGTLVTVSSASTKGPEVQLVQSGAEVKKP






GESLKISCKGSGYSFTTYWMHWVRQMPGKGLEWMG






EINPTNGHTNYNPSFQGQVTISADKSISTAYLQWS






SLKASDTAMYYCARNYVGSIFDYWGQGTLVTVSS


 


834
DVD680VL
AB070VL
AB079VL
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCKASENVVSYVSWYQQKPGKAPKLLIYGASNRN






TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQ






SYNYPYTFGQGTKLEIKR









Example 2.185
Generation of HGF (Seq. 2) and VEGF (Seq. 3) DVD-Igs with Linker Set 1











TABLE 219





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







835
DVD663H
AB079VH
AB071VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH






WVRQMPGKGLEWMGEINPTNGHTNYNPSFQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGFTINASWIHWVRQAPGKGLEWVGAI






YPYSGYTNYADSVKGRFTISADTSKNTAYLQMNSL






RAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS


 


836
DVD663L
AB079VL
AB071VL
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSW






YQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






VIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSNTSPLT






FGQGTKVEIKR


 


837
DVD664H
AB071VH
AB079VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPEVQLVQSGAEVKK






PGESLKISCKGSGYSFTTYWMHWVRQMPGKGLEWM






GEINPTNGHTNYNPSFQGQVTISADKSISTAYLQW






SSLKASDTAMYYCARNYVGSIFDYWGQGTLVTVSS


 


838
DVD664L
AB071VL
AB079VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCKASE






NVVSYVSWYQQKPGKAPKLLIYGASNRNTGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCGQSYNYPYT






FGQGTKLEIKR









Example 2.186
Generation of HGF (Seq. 2) and VEGF (Seq. 3) DVD-Igs with Linker Set 2











TABLE 220





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







839
DVD669H
AB079VH
AB071VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH






WVRQMPGKGLEWMGEINPTNGHTNYNPSFQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGFTINASWIHWVRQAPGKG






LEWVGAIYPYSGYTNYADSVKGRFTISADTSKNTA






YLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQG






TLVTVSS


 


840
DVD669L
AB079VL
AB071VL
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSW






YQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQVIRRSLAWYQQKPGKAPKLLIYAASNLA






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SNTSPLTFGQGTKVEIKR


 


841
DVD670H
AB071VH
AB079VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVQ






SGAEVKKPGESLKISCKGSGYSFTTYWMHWVRQMP






GKGLEWMGEINPTNGHTNYNPSFQGQVTISADKSI






STAYLQWSSLKASDTAMYYCARNYVGSIFDYWGQG






TLVTVSS


 


842
DVD670L
AB071VL
AB079VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCKASENVVSYVSWYQQKPGKAPKLLIYGASNRN






TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQ






SYNYPYTFGQGTKLEIKR









Example 2.187
Generation of HGF (Seq. 2) and VEGF (Seq. 3) DVD-Igs with Linker Set 3











TABLE 221





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







843
DVD675VH
AB079VH
AB071VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH






WVRQMPGKGLEWMGEINPTNGHTNYNPSFQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESGG






GLVQPGGSLRLSCAASGFTINASWIHWVRQAPGKG






LEWVGAIYPYSGYTNYADSVKGRFTISADTSKNTA






YLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQG






TLVTVSS


 


844
DVD675VL
AB079VL
AB071VL
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSW






YQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKLE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






VIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQSNTSPLT






FGQGTKVEIKR


 


845
DVD676VH
AB071VH
AB079VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVQ






SGAEVKKPGESLKISCKGSGYSFTTYWMHWVRQMP






GKGLEWMGEINPTNGHTNYNPSFQGQVTISADKSI






STAYLQWSSLKASDTAMYYCARNYVGSIFDYWGQG






TLVTVSS


 


846
DVD676VL
AB071VL
AB079VL
DIQMTQSPSSLSASVGDRVTITCRASWVIRRSLAW






YQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCKASE






NVVSYVSWYQQKPGKAPKLLIYGASNRNTGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCGQSYNYPYT






FGQGTKLEIKR









Example 2.188
Generation of HGF (Seq. 2) and VEGF (Seq. 3) DVD-Igs with Linker Set 4











TABLE 222





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







847
DVD681VH
AB079VH
AB071VH
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMH






WVRQMPGKGLEWMGEINPTNGHTNYNPSFQGQVTI






SADKSISTAYLQWSSLKASDTAMYYCARNYVGSIF






DYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPGG






SLRLSCAASGFTINASWIHWVRQAPGKGLEWVGAI






YPYSGYTNYADSVKGRFTISADTSKNTAYLQMNSL






RAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS


 


848
DVD681VL
AB079VL
AB071VL
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSW






YQQKPGKAPKLLIYGASNRNTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCGQSYNYPYTFGQGTKLE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITVRASQVIRRSLAWYQQKPGKAPKLLIYAASNLA






SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






SNTSPLTFGQGTKVEIKR


 


849
DVD682VH
AB071VH
AB079VH
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIH






WVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSP






WAMDYWGQGTLVTVSSASTKGPEVQLVQSGAEVKK






PGESLKISCKGSGYSFTTYWMHWVRQMPGKGLEWM






GEINPTNGHTNYNPSFQGQVTISADKSISTAYLQW






SSLKASDTAMYYCARNYVGSIFDYWGQGTLVTVSS


 


850
DVD682VL
AB071VL
AB079VL
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAW






YQQKPGKAPKLLIYAASNALSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCKASENVVSYVSWYQQKPGKAPKLLIYGASNRN






TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQ






SYNYPYTFGQGTKLEIKR









Example 2.189
Generation of HER2 (Seq. 1) and HER2 (Seq. 2) DVD-Igs with Linker Set 1











TABLE 223





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







851
DVD687H
AB004VH
AB080VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIDKTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVA






DVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMN






SLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS


 


852
DVD687L
AB004VL
AB080VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPFTGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCKASQ






DVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYT






FGQGTKVEIKR


 


853
DVD688H
AB080VH
AB004VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMD






WVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTL






SVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFY






FDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR






IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYSCRWGGDGFYAMDYWGQGTLVTVSS


 


854
DVD688L
AB080VL
AB004VL
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAW






YQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT






FGQGTKVEIKR









Example 2.190
Generation of HER2 (Seq. 1) and HER2 (Seq. 2) DVD-Igs with Linker Set 2











TABLE 224





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







855
DVD689H
AB004VH
AB080VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYSCRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPG






KGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKN






TLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG






TLVTVSS


 


856
DVD689L
AB004VL
AB080VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRY






TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






YYIYPYTFGQGTKVEIKR


 


857
DVD690H
AB080VH
AB004VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMD






WVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTL






SVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFY






FDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGK






GLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT






AYLQMNSLRAEDTAVYYCSRWGGDGFYADMYWGQG






TLVTVSS


 


858
DVD690L
AB080VL
AB004VL
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAW






YQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY






SGVPSRFSGSGSGTDGTLTISSLQPEDFATYYCQQ






HYTTPPTFGQGTKVEIKR









Example 2.191
Generation of HER2 (Seq. 1) and HER2 (Seq. 2) DVD-Igs with Linker Set 3











TABLE 225





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







859
DVD691H
AB004VH
AB080VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY






AMDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVES






GGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPG






KGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKN






TLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG






TLVTVSS


 


860
DVD691L
AB004VL
AB080VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQHYTTPPFTGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCKASQ






DVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRF






SGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYT






FGQGTKVEIKR


 


861
DVD692H
AB080VH
AB004VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMD






WVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTL






SVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFY






FDYWGQGTLVTVSSASTKGPSVFPLAPEVQLVESG






GGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGK






GLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT






AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG






TLVTVSS


 


862
DVD692L
AB080VL
AB004VL
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAW






YQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVE






IKRTVAAPDIQMTQSPSSLSASVGDRVTITCRASQ






DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF






SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT






FGQGTKVEIKR









Example 2.192
Generation of HER2 (Seq. 1) and HER2 (Seq. 2) DVD-Igs with Linker Set 4











TABLE 226





SEQ
DVD Variable
Outer Variable
Inner Variable
Sequence


ID NO
Domain Name
Domain Name
Domain Name
12345678901234567890123456789012345







863
DVD693H
AB004VH
AB080VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH






WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI






SADTSKNTAYLQMNSLRAEDTAVYYSCRWGGDGFY






AMDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQP






GGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVA






DVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMN






SLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS


 


864
DVD693L
AB004VL
AB080VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW






YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD






FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRY






TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ






YYIYPYTFGQGTKVEIKR


 


865
DVD694H
AB080VH
AB004VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMD






WVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTL






SVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFY






FDYWGQGTLVTVSSASTKGPEVQLVESGGGLVQPG






GSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR






IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS






LRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS


 


866
DVD694L
AB080VL
AB004VL
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAW






YQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTD






FTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVE






IKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT






ITCRASQDVNTAVAWYQQKPGKAPKLLOYSASFLY






SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ






HYTTPPTFGQGTKVEIKR









Example 2.193
Cloning Vector Sequences Used to Clone Parent Antibody and DVD-Ig Sequences









TABLE 226





Vector

Nucleotide sequences


name
SEQ ID NO
123456789012345678901234567890123456789012345678901







V1
867
GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC




ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC




GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC




ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG




GTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG




AATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCT




TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG




GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC




TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC




CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC




AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC




GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC




AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA




GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGC




GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC




TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC




AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC




TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC




TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC




CTCTCCCTGTCTCCGGGTAAATGAGCGGCCGCTCGAGGCCGGCAAGGCCGG




ATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAA




TAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGG




CAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCC




CCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCG




GGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGC




CCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGA




CTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTG




GCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTG




CCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACAT




GTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATC




AGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCA




ATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTC




CCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGT




TACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTA




AGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATG




GGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGG




CTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCT




TCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAA




GGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATA




AAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAA




CCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCT




GAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACT




GGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGT




GCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACA




GGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGAC




GCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAA




CGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTT




TTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTG




CGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCC




GCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCC




GGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGC




TGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAG




GGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATG




TTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCC




TAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCAT




ATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCT




GGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAA




TCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATG




CTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGG




TAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATAT




CTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCT




AATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATA




TGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTG




GGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAAT




CTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGA




ATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAA




TGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAA




TGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA




TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAG




GAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGC




GGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAA




AGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCT




CAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAAT




GATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGA




CGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTT




GGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGT




AAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAA




CTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA




CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAA




TGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGC




AACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCG




GCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCT




GCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGG




TGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCC




CTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA




ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTA




ACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCA




TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGAC




CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGA




AAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG




CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA




AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGAT




ACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAA




CTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGC




TGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATA




GTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA




GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA




GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC




GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG




AAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA




GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGC




CAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA




CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGC




CTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGA




GTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCC




CGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTG




GAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTA




GGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAAT




TGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGC




CAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAA




GCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCA




TCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGAC




TAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTAT




TCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGC




TTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATG




GACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTG




GGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGG




CAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGA




TGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATAT




AAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAG




AACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGG




TTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGAT




TCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTG




CGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCG




CTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGC




TTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCT




TTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGG




TATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCG




CACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACG




GGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCC




GTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGC




GTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATG




GAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAA




AAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCG




GGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTC




TTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTG




GGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGA




ATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGT




GGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAG




ATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGGAGTTTGGG




CTGAGCTGGCTTTTTCTTGTCGCGATTTTAAAAGGTGTCCAGTGC


 


V2
868
ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG




AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA




GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC




CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC




AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC




TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC




AGGGGAGAGTGTTGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGA




CCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTT




GGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATT




TGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGG




ACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGC




ATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCA




CATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTG




ACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATC




CTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGT




GTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCC




AGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCC




TGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTT




ATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGT




AGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACG




GGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGC




GATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGA




TTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATC




AAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCC




TTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGT




GAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGA




CGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAA




ACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTT




TAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCA




TCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGA




TACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCA




TGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGC




AGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCC




ACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAA




TTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGG




ACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCAC




TGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATAC




CTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTG




GAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGG




TCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGG




GTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATA




TCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCC




TAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCAT




AGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCT




GGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAA




TAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATA




CTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGC




ATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATAT




CTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCT




AATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATA




TGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCG




GGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTG




AAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGAT




AATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGG




AACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT




GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAT




GAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG




CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA




AGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGG




TAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCAC




TTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCA




AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTA




CTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATT




ATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT




GACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGG




GGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT




ACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTT




GCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATT




AATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGC




CCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGG




GTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTAT




CGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAG




ACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA




CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT




TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC




TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA




AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC




AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC




AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC




TGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGC




ACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG




TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA




TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTT




GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGA




AAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG




CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG




GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATT




TTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGC




GGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTT




TCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTG




AGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAG




CGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTG




GCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGG




CAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCA




GGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGG




ATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTA




GCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCTAC




TCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCC




TAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTT




TTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGT




AGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAG




ATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTA




GGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCG




CACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAAC




CGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA




CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGT




AGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGT




AAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCC




TTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCC




CGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGG




AGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCG




CCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAA




GTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTG




GCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGT




TTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTC




GGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTC




TCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGC




CCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGA




AAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCG




GCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTT




TCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTC




CAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTG




GGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGAC




TGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCT




TTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAG




TTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGA




CCTCGAGATCCATTGTGCCCGGGCGCACCATGGACATGCGCGTGCCCGCCC




AGCTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGCTCGCGATGC


 


V3
869
CAACCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAG




CTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCG




GGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGA




GTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGC




AGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGC




TGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACA




GAATGTTCATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCT




CGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGA




ATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGG




TCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACG




AACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATG




TAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACAT




GTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACA




TCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTG




GAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTA




ACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGG




GGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGT




GTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATA




AGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGT




ATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGA




AGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGAT




ATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTC




CACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAG




GAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTC




GTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAG




GTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGG




GGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACC




CCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAA




CAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCT




CACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATAC




TGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGT




TGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGC




GGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACG




CCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTG




TGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACT




GTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGC




GGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTG




CATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAG




GACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCC




TCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTA




GCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCT




GGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAA




TCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGG




CTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGG




TAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAG




AGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTA




CCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATA




TGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTG




GGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAAT




TTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGC




TATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGT




AGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAG




ACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT




AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAAC




CCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG




ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAG




TATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCT




TCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA




TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAA




GATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTT




TAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGA




GCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTC




ACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATG




CAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGAC




AACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGA




TCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACC




AAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCG




CAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAAT




AGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT




TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC




TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT




AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA




GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA




AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAA




AAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTA




ACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGG




ATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA




AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC




TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGT




TCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC




GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGG




CGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA




GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA




GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAG




CGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG




GGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTA




TCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTT




GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGC




CTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCC




TGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGC




TGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGA




GGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC




GATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAG




TGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGC




TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATA




ACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCT




AGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCA




ATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAA




CTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTA




TTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGT




GAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATG




GATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGT




CTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCAC




ATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGG




TGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTG




GCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGT




CGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAG




TGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTG




CGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGA




GCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGC




CCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCG




CGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTC




TCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCA




AGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTT




TGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGC




GAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCA




AGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCC




GCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAG




ATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCG




CTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCC




GTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAG




GCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGG




GGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGA




AGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTT




TGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTT




TTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCT




CGAGATCCATTGTGCCCGGGCGCCACCATGACTTGGACCCCACTCCTCTTC




CTCACCCTCCTCCTCCACTGCACAGGAAGCTTATCG


 


V4
870
ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG




AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA




GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC




CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC




AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC




TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC




AGGGGAGAGTGTTGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGA




CCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTT




GGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATT




TGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGG




ACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGC




ATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCA




CATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTG




ACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATC




CTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGT




GTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCC




AGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCC




TGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTT




ATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGT




AGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACG




GGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGC




GATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGA




TTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATC




AAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCC




TTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGT




GAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGA




CGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAA




ACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTT




TAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCA




TCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGA




TACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCA




TGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGC




AGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCC




ACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAA




TTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGG




ACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCAC




TGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATAC




CTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTG




GAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGG




TCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGG




GTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATA




TCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCC




TAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCAT




AGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCT




GGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAA




TAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATA




CTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGC




ATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATAT




CTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCT




AATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATA




TGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCG




GGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTG




AAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGAT




AATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGG




AACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT




GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAT




GAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG




CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA




AGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGG




TAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCAC




TTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCA




AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTA




CTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATT




ATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT




GACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGG




GGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT




ACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTT




GCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATT




AATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGC




CCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGG




GTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTAT




CGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAG




ACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA




CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT




TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC




TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA




AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC




AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC




AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC




TGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGC




ACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG




TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA




TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTT




GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGA




AAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG




CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG




GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATT




TTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGC




GGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTT




TCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTG




AGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAG




CGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTG




GCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGG




CAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCA




GGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGG




ATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTA




GCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATC




TCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCC




TAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTT




TTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGT




AGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAG




ATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTA




GGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCG




CACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAAC




CGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA




CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGT




AGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGT




AAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCC




TTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCC




CGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGG




AGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCG




CCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAA




GTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTG




GCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGT




TTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTC




GGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTC




TCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGC




CCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGA




AAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCG




GCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTT




TCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTC




CAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTG




GGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGAC




TGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCT




TTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAG




TTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGA




CCTCGAGATCCATTGTGCCCGGGCGCACCATGACTTGGACCCCACTCCTCT




TCCTCACCCTCCTCCTCCACTGCACAGGAAGCTTATCG


 


V5
871
CAACCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAG




CTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCG




GGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGA




GTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGC




AGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGC




TGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACA




GAATGTTCATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCT




CGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGA




ATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGG




TCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACG




AACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATG




TAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACAT




GTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACA




TCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTG




GAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTA




ACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGG




GGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGT




GTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATA




AGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGT




ATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGA




AGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGAT




ATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTC




CACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAG




GAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTC




GTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAG




GTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGG




GGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACC




CCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAA




CAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCT




CACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATAC




TGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGT




TGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGC




GGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACG




CCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTG




TGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACT




GTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGC




GGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTG




CATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAG




GACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCC




TCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTA




GCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCT




GGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAA




TCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGG




CTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGG




TAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAG




AGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTA




CCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATA




TGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTG




GGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAAT




TTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGC




TATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGT




AGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAG




ACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT




AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAAC




CCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG




ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAG




TATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCT




TCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA




TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAA




GATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTT




TAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGA




GCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTC




ACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATG




CAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGAC




AACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGA




TCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACC




AAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCG




CAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAAT




AGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT




TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC




TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT




AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA




GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA




AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAA




AAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTA




ACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGG




ATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA




AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC




TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGT




TCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC




GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGG




CGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA




GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA




GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAG




CGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG




GGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTA




TCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTT




GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGC




CTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCC




TGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGC




TGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGA




GGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC




GATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAG




TGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGC




TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATA




ACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCT




AGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCA




ATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAA




CTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTA




TTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGT




GAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATG




GATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGT




CTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCAC




ATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGG




TGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTG




GCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGT




CGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAG




TGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTG




CGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGA




GCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGC




CCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCG




CGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTC




TCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCA




AGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTT




TGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGC




GAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCA




AGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCC




GCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAG




ATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCG




CTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCC




GTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAG




GCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGG




GGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGA




AGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTT




TGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTT




TTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCT




CGAGATCCATTGTGCCCGGGCGCCACCATGGACATGCGCGTGCCCGCCCAG




CTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGCTCGCGATGC


 


V7
872
GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC




ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC




GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC




ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG




GTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG




AATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCT




TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGG




GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC




TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC




CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC




AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC




GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC




AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA




GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGC




GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC




TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC




AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC




TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC




TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC




CTCTCCCTGTCTCCGGGTAAATGAGCGGCCGCTCGAGGCCGGCAAGGCCGG




ATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAA




TAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGG




CAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCC




CCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCG




GGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGC




CCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGA




CTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTG




GCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTG




CCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACAT




GTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATC




AGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCA




ATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTC




CCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGT




TACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTA




AGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATG




GGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGG




CTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCT




TCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAA




GGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATA




AAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAA




CCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCT




GAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACT




GGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGT




GCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACA




GGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGAC




GCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAA




CGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTT




TTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTG




CGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCC




GCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCC




GGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGC




TGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAG




GGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATG




TTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCC




TAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCAT




ATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCT




GGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAA




TCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATG




CTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGG




TAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATAT




CTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCT




AATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATA




TGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTG




GGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAAT




CTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGA




ATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAA




TGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAA




TGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA




TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAG




GAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGC




GGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAA




AGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCT




CAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAAT




GATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGA




CGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTT




GGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGT




AAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAA




CTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA




CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAA




TGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGC




AACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCG




GCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCT




GCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGG




TGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCC




CTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA




ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTA




ACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCA




TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGAC




CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGA




AAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG




CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA




AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGAT




ACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAA




CTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGC




TGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATA




GTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA




GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA




GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC




GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG




AAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA




GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGC




CAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA




CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGC




CTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGA




GTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCC




CGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTG




GAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTA




GGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAAT




TGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGC




CAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAA




GCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCA




TCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGAC




TAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTAT




TCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGC




TTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATG




GACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTG




GGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGG




CAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGA




TGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATAT




AAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAG




AACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGG




TTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGAT




TCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTG




CGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCG




CTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGC




TTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCT




TTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGG




TATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCG




CACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACG




GGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCC




GTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGC




GTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATG




GAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAA




AAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCG




GGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTC




TTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTG




GGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGA




ATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGT




GGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAG




ATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGGAGTTTGGG




CTGAGCTGGCTTTTTCTTGTCGCGATTTTAAAAGGTGTCCAGTGC









The present invention incorporates by reference in their entirety techniques well known in the field of molecular biology and drug delivery. These techniques include, but are not limited to, techniques described in the following publications:

    Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &Sons, NY (1993);
    Ausubel, F. M. et al. eds., Short Protocols In Molecular Biology (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X).
    Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
    Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999);
    Goodson, in Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984);
    Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981;
    Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
    Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991);
    Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242;
    Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
    Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990);
    Lu and Weiner eds., Cloning and Expression Vectors for Gene Function Analysis (2001) BioTechniques Press. Westborough, Mass. 298 pp. (ISBN 1-881299-21-X).
    Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);
    Old, R. W. & S. B. Primrose, Principles of Gene Manipulation: An Introduction To Genetic Engineering (3d Ed. 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology; V.2:409 pp. (ISBN 0-632-01318-4).
    Sambrook, J. et al. eds., Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6).
    Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978
    Winnacker, E. L. From Genes To Clones: Introduction To Gene Technology (1987) VCH Publishers, NY (translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).


INCORPORATION BY REFERENCE
The contents of all cited references (including literature references, patents, patent applications, and websites) that may be cited throughout this application are hereby expressly incorporated by reference in their entirety, as are the references cited therein. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology and cell biology, which are well known in the art.
EQUIVALENTS
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.

## Claims

1. A binding protein comprising first and second polypeptide chains, each independently comprising VD1-(X1)n-VD2-C-(X2)n, wherein
VD1 is a first variable domain;
VD2 is a second variable domain;
C is a constant domain;
X1 is a linker;
X2 is an Fc region;
n is 0 or 1;

wherein the VD1 domains on the first and second polypeptide chains form a first functional target binding site and the VD2 domains on the first and second polypeptide chains form a second functional target binding site, and wherein the binding protein is capable of binding VEGF and DLL4, wherein the first and second polypeptide chains of the binding protein comprise:
SEQ ID NOs: 239 and 240,
SEQ ID NOs: 411 and 412,
SEQ ID NOs: 413 and 414,
SEQ ID NOs: 419 and 420,
SEQ ID NOs: 435 and 436,
SEQ ID NOs: 445 and 446,
SEQ ID NOs: 449 and 450,
SEQ ID NOs: 453 and 454,
SEQ ID NOs: 461 and 462,
SEQ ID NOs: 465 and 466,
SEQ ID NOs: 469 and 470,
SEQ ID NOs: 473 and 474,
SEQ ID NOs: 475 and 476,
SEQ ID NOs: 477 and 478,
SEQ ID NOs: 479 and 480,
SEQ ID NOs: 481 and 482,
SEQ ID NOs: 483 and 484,
SEQ ID NOs: 485 and 486,
SEQ ID NOs: 489 and 490,
SEQ ID NOs: 493 and 494,
SEQ ID NOs: 497 and 498,
SEQ ID NOs: 501 and 502,
SEQ ID NOs: 503 and 504,
SEQ ID NOs: 507 and 508,
SEQ ID NOs: 509 and 510,
SEQ ID NOs: 511 and 512,
SEQ ID NOs: 513 and 514,
SEQ ID NOs: 517 and 518,
SEQ ID NOs: 525 and 526,
SEQ ID NOs: 529 and 530,
SEQ ID NOs: 533 and 534,
SEQ ID NOs: 537 and 538,
SEQ ID NOs: 541 and 542,
SEQ ID NOs: 543 and 544,
SEQ ID NOs: 545 and 546,
SEQ ID NOs: 557 and 558,
SEQ ID NOs: 559 and 560,
SEQ ID NOs: 561 and 562,
SEQ ID NOs: 585 and 586,
SEQ ID NOs: 589 and 590, or
SEQ ID NOs: 593 and 594.

2. The binding protein according to claim 1, wherein the first polypeptide chain is a heavy chain and n is 0 or 1 for (X2)n, and wherein the second polypeptide chain is a light chain and n is 0 for (X2)n.

3. The binding protein according to claim 1, wherein the first and second polypeptide chains of the binding protein comprise:
SEQ ID NOs: 239 and 240,
SEQ ID NOs: 445 and 446,
SEQ ID NOs: 449 and 450,
SEQ ID NOs: 453 and 454,
SEQ ID NOs: 465 and 466,
SEQ ID NOs: 473 and 474,
SEQ ID NOs: 477 and 478,
SEQ ID NOs: 481 and 482,
SEQ ID NOs: 485 and 486,
SEQ ID NOs: 489 and 490,
SEQ ID NOs: 493 and 494,
SEQ ID NOs: 497 and 498,
SEQ ID NOs: 503 and 504,
SEQ ID NOs: 507 and 508,
SEQ ID NOs: 509 and 510,
SEQ ID NOs: 511 and 512,
SEQ ID NOs: 513 and 514,
SEQ ID NOs: 517 and 518,
SEQ ID NOs: 525 and 526,
SEQ ID NOs: 529 and 530,
SEQ ID NOs: 533 and 534,
SEQ ID NOs: 537 and 538,
SEQ ID NOs: 541 and 542,
SEQ ID NOs: 543 and 544,
SEQ ID NOs: 545 and 546,
SEQ ID NOs: 557 and 558,
SEQ ID NOs: 559 and 560,
SEQ ID NOs: 561 and 562,
SEQ ID NOs: 585 and 586,
SEQ ID NOs: 589 and 590, or
SEQ ID NOs: 593 and 594.

4. The binding protein according to claim 1, comprising two first polypeptide chains and two second polypeptide chains and four functional target binding sites.

5. The binding protein according to claim 1, wherein the Fc region is a variant sequence Fc region.

6. The binding protein according to claim 1, wherein the Fc region is an Fc region from an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

7. The binding protein according to claim 1, wherein the binding protein is a crystallized binding protein.

8. A binding protein conjugate comprising a binding protein according to claim 1, the binding protein conjugate further comprising an immunoadhesion molecule, an imaging agent, a therapeutic agent, or a cytotoxic agent.

9. The binding protein conjugate according to claim 8, wherein the imaging agent is a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin.

10. The binding protein conjugate according to claim 9, wherein the radiolabel is 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm.

11. The binding protein conjugate according to claim 8, wherein the therapeutic or cytotoxic agent is an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, a toxin, or an apoptotic agent.

12. An isolated nucleic acid encoding the binding protein amino acid sequence according to claim 1.

13. A vector comprising the isolated nucleic acid according to claim 12.

14. A host cell comprising the vector according to claim 13.

15. The host cell according to claim 14, wherein the host cell is a prokaryotic cell, Escherichia coli, a eukaryotic cell, an animal cell, a plant cell, a fungal cell, a yeast cell, an Sf9 cell, a mammalian cell, an avian cell, an insect cell, a CHO cell or a COS cell.

16. A method of producing a binding protein, comprising culturing the host cell of claim 14 in culture medium under conditions sufficient to produce the binding protein.

17. A pharmaceutical composition comprising the binding protein according to claim 1 and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17, further comprising at least one additional therapeutic agent.

19. A method of determining the presence, amount, or concentration of VEGF and/or DLL4 in a test sample by an immunoassay comprising contacting the test sample with at least one binding protein of claim 1 and at least one detectable label.

20. A kit for assaying a test sample for the presence, amount, or concentration of VEGF and/or DLL4, said kit comprising (a) instructions for assaying the test sample for VEGF and/or DLL4 and (b) at least one binding protein comprising the binding protein of claim 1.

21. The binding protein of claim 1, wherein the first and second polypeptide chains of the binding protein comprise:
SEQ ID NOs: 489 and 490,
SEQ ID NOs: 517 and 518,
SEQ ID NOs: 503 and 504,
SEQ ID NOs: 507 and 508,
SEQ ID NOs: 525 and 526,
SEQ ID NOs: 511 and 512,
SEQ ID NOs: 529 and 530, or
SEQ ID NOs: 543 and 544.

22. The binding protein of claim 1, wherein the first and second polypeptide chains of the binding protein comprise:
SEQ ID NOs: 411 and 412,
SEQ ID NOs: 413 and 414,
SEQ ID NOs: 475 and 476,
SEQ ID NOs: 477 and 478,
SEQ ID NOs: 479 and 480,
SEQ ID NOs: 481 and 482,
SEQ ID NOs: 483 and 484,
SEQ ID NOs: 485 and 486,
SEQ ID NOs: 511 and 512,
SEQ ID NOs: 513 and 514,
SEQ ID NOs: 543 and 544,
SEQ ID NOs: 545 and 546,
SEQ ID NOs: 559 and 560, or
SEQ ID NOs: 561 and 562.