Patent Publication Number: US-2022213224-A1

Title: T cell activating bispecific antigen binding molecules

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional of U.S. patent application Ser. No. 16/541,258 filed Aug. 15, 2019, which is a divisional of U.S. patent application Ser. No. 15/600,015, filed May 19, 2017, now abandoned, which is a continuation of International Application No. PCT/EP2015/076745, Publication No. WO2016/079081, filed Nov. 17, 2015, which claims priority to European Patent Application No. 14194097.3, filed Nov. 20, 2014, the disclosures of which are incorporated herein by reference in their entirety. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 2, 2021, is named 51177-010003_Sequence_Listing_9_2_21_ST25.txt and is 537,784 bytes in size. 
     FIELD OF THE INVENTION 
     The present invention generally relates to bispecific antigen binding molecules for activating T cells. In addition, the present invention relates to polynucleotides encoding such bispecific antigen binding molecules, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the bispecific antigen binding molecules of the invention, and to methods of using these bispecific antigen binding molecules in the treatment of disease. 
     BACKGROUND 
     The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues intact and undamaged. 
     An attractive way of achieving this is by inducing an immune response against the tumor, to make immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumor cells. CTLs constitute the most potent effector cells of the immune system, however they cannot be activated by the effector mechanism mediated by the Fc domain of conventional therapeutic antibodies. 
     In this regard, bispecific antibodies designed to bind with one “arm” to a surface antigen on target cells, and with the second “arm” to an activating, invariant component of the T cell receptor (TCR) complex, have become of interest in recent years. The simultaneous binding of such an antibody to both of its targets forces a temporary interaction between target cell and T cell, causing activation of any cytotoxic T cell and subsequent lysis of the target cell. Hence, the immune response is re-directed to the target cells and is independent of peptide antigen presentation by the target cell or the specificity of the T cell as would be relevant for normal MHC-restricted activation of CTLs. In this context it is crucial that CTLs are only activated when the bispecific antibody binds to a target cell and the CTL, i.e. the immunological synapse is mimicked. Particularly desirable are bispecific antibodies that do not require lymphocyte preconditioning or co-stimulation in order to elicit efficient lysis of target cells. 
     Several bispecific antibody formats have been developed and their suitability for T cell mediated immunotherapy investigated. Out of these, the so-called BiTE (bispecific T cell engager) molecules have been very well characterized and already shown some promise in the clinic (reviewed in Nagorsen and Bsuerle, Exp Cell Res 317, 1255-1260 (2011)). BiTEs are tandem scFv molecules wherein two scFv molecules are fused by a flexible linker. Further bispecific formats being evaluated for T cell engagement include diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies (Kipriyanov et al., J Mol Biol 293, 41-66 (1999)). A more recent development are the so-called DART (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117, 4542-51 (2011)). The so-called triomabs, which are whole hybrid mouse/rat IgG molecules and also currently being evaluated in clinical trials, represent a larger sized format (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). 
     The variety of formats that are being developed shows the great potential attributed to T cell re-direction and activation in immunotherapy. The task of generating bispecific antibodies suitable therefor is, however, by no means trivial, but involves a number of challenges that have to be met related to efficacy, toxicity, applicability and produceability of the antibodies. 
     Small constructs such as, for example, BiTE molecules—while being able to efficiently crosslink effector and target cells—have a very short serum half life requiring them to be administered to patients by continuous infusion. IgG-like formats on the other hand—while having the great benefit of a long half life—suffer from toxicity associated with the native effector functions inherent to IgG molecules. Their immunogenic potential constitutes another unfavorable feature of IgG-like bispecific antibodies, especially non-human formats, for successful therapeutic development. Finally, a major challenge in the general development of bispecific antibodies has been the production of bispecific antibody constructs at a clinically sufficient quantity and purity, due to the mispairing of antibody heavy and light chains of different specificities upon co-expression, which decreases the yield of the correctly assembled construct and results in a number of non-functional side products from which the desired bispecific antibody may be difficult to separate. 
     Given the difficulties and disadvantages associated with currently available bispecific antibodies for T cell mediated immunotherapy, there remains a need for novel, improved formats of such molecules. 
     SUMMARY OF THE INVENTION 
     In a first aspect the present invention provides a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, wherein the first antigen binding moiety comprises a first light chain and wherein the first antigen binding moiety is capable of specific binding to an activating T cell antigen and the second antigen binding moiety comprises a second light chain and wherein the second antigen binding moiety is capable of specific binding to a target cell antigen, wherein the amino acid sequence of the first and the second light chain is identical. In one embodiment, the first antigen binding moiety is a Fab. In one embodiment, the second antigen binding moiety is a Fab. In one embodiment, the first and the second antigen binding moiety is a Fab. 
     In one aspect the invention provides a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, one of which is a Fab molecule capable of specific binding to an activating T cell antigen and the other one of which is a Fab molecule capable of specific binding to a target cell antigen, wherein the first and the second Fab molecule have identical VLCL light chains. 
     In one embodiment said T cell activating bispecific antigen binding molecule further comprises an Fc domain composed of a first and a second subunit capable of stable association. 
     In one embodiment said T cell activating bispecific antigen binding molecule comprises a light chain comprising the light chain CDRs of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. 
     In one embodiment said T cell activating bispecific antigen binding molecule comprises a light chain comprising SEQ ID NO: 31. 
     In one embodiment said Fab molecule capable of specific binding to an activating T cell antigen comprises a heavy chain comprising the heavy chain CDR of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39. 
     In a particular embodiment, not more than one antigen binding moiety capable of specific binding to an activating T cell antigen is present in the T cell activating bispecific antigen binding molecule (i.e. the T cell activating bispecific antigen binding molecule provides monovalent binding to the activating T cell antigen). 
     In some embodiments, the first and the second antigen binding moiety of the T cell activating bispecific antigen binding molecule are fused to each other, optionally via a peptide linker. In one such embodiment, the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In another such embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. 
     In yet another such embodiment, the second antigen binding moiety is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen binding moiety. In yet another such embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the second antigen binding moiety. 
     In one embodiment, the first antigen binding moiety of the T cell activating bispecific antigen binding molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. 
     In one embodiment, the second antigen binding moiety of the T cell activating bispecific antigen binding molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. 
     In another embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. 
     In one embodiment, the first and the second antigen binding moiety of the T cell activating bispecific antigen binding molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. 
     In certain embodiments, the T cell activating bispecific antigen binding molecule comprises a third antigen binding moiety which is a Fab molecule capable of specific binding to a target cell antigen. In one embodiment said third antigen binding moiety is a Fab molecule comprising an identical VLCL light chain as the first and the second antigen binding moiety. 
     In one such embodiment the first, second and third antigen binding moiety are each a Fab molecule comprising the light chain CDRs of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. 
     In one such embodiment the first, second and third antigen binding moiety are each a Fab molecule comprising a light chain comprising SEQ ID NO: 31. 
     In one embodiment, the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In a particular embodiment, the second and the third antigen binding moiety of the T cell activating antigen binding molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. In another particular embodiment, the first and the third antigen binding moiety of the T cell activating antigen binding molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. The components of the T cell activating bispecific antigen binding molecule may be fused directly or through suitable peptide linkers. In one embodiment the second and the third antigen binding moiety and the Fc domain are part of an immunoglobulin molecule. In one embodiment the first and the third antigen binding moiety and the Fc domain are part of an immunoglobulin molecule. In a particular embodiment the immunoglobulin molecule is an IgG class immunoglobulin. In an even more particular embodiment the immunoglobulin is an IgG 1  subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG 4  subclass immunoglobulin. 
     In a particular embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG 1  Fc domain. In another specific embodiment, the Fc domain is an IgG 4  Fc domain. In an even more specific embodiment, the Fc domain is an IgG 4  Fc domain comprising the amino acid substitution S228P. In an even more specific embodiment, the Fc domain is an IgG 4  Fc domain comprising the amino acid substitutions L235E and S228P (SPLE). In particular embodiments the Fc domain is a human Fc domain. 
     In particular embodiments the Fc domain comprises a modification promoting the association of the first and the second Fc domain subunit. In a specific such embodiment, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. 
     In a particular embodiment the Fc domain exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG 1  Fc domain. In certain embodiments the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In one embodiment, the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function. In one embodiment, the one or more amino acid substitution in the Fc domain that reduces binding to an Fc receptor and/or effector function is at one or more position selected from the group of L234, L235, and P329 (Kabat numbering). In particular embodiments, each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an Fc receptor and/or effector function wherein said amino acid substitutions are L234A, L235A and P329G. In one such embodiment, the Fc domain is an IgG 1  Fc domain, particularly a human IgG 1  Fc domain. In other embodiments, each subunit of the Fc domain comprises two amino acid substitutions that reduce binding to an Fc receptor and/or effector function wherein said amino acid substitutions are L235E and P329G. In one such embodiment, the Fc domain is an IgG 4  Fc domain, particularly a human IgG 4  Fc domain. 
     In one embodiment the Fc receptor is an Fcγ receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is human FcγRIIa, FcγRI, and/or FcγRIIIa. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). 
     In a particular embodiment, the activating T cell antigen that the bispecific antigen binding molecule is capable of binding is CD3. In other embodiments, the target cell antigen that the bispecific antigen binding molecule is capable of binding is a tumor cell antigen. In one embodiment, the target cell antigen is selected from the group consisting of: Folate Receptor 1 (FoIR1), Mucin-1 (MUC1), and B Cell Maturation Antigen (BCMA). In one specific embodiment, the target cell antigen is not BCMA. 
     In another aspect, the invention provides for a light chain comprising the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 for use in a T cell activating bispecific antigen binding molecule. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 31. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 35. 
     In another aspect, the invention provides for a light chain comprising the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34 for use in a library for production of T cell activating bispecific antigen binding molecule. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 31. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 35. 
     In another aspect, the invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 31. 
     In another aspect, the invention provides for an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 35. 
     According to another aspect of the invention there is provided an isolated polynucleotide encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof. The invention also encompasses polypeptides encoded by the polynucleotides of the invention. The invention further provides an expression vector comprising the isolated polynucleotide of the invention, and a host cell comprising the isolated polynucleotide or the expression vector of the invention. In some embodiments the host cell is a eukaryotic cell, particularly a mammalian cell. 
     In another aspect is provided a method of producing the T cell activating bispecific antigen binding molecule of the invention, comprising the steps of a) culturing the host cell of the invention under conditions suitable for the expression of the T cell activating bispecific antigen binding molecule and b) recovering the T cell activating bispecific antigen binding molecule. The invention also encompasses a T cell activating bispecific antigen binding molecule produced by the method of the invention. 
     The invention further provides a pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of the invention and a pharmaceutically acceptable carrier. 
     Also encompassed by the invention are methods of using the T cell activating bispecific antigen binding molecule and pharmaceutical composition of the invention. In one aspect the invention provides a T cell activating bispecific antigen binding molecule or a pharmaceutical composition of the invention for use as a medicament. In one aspect is provided a T cell activating bispecific antigen binding molecule or a pharmaceutical composition according to the invention for use in the treatment of a disease in an individual in need thereof. In a specific embodiment the disease is cancer. 
     Also provided is the use of a T cell activating bispecific antigen binding molecule of the invention for the manufacture of a medicament for the treatment of a disease in an individual in need thereof; as well as a method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the T cell activating bispecific antigen binding molecule according to the invention in a pharmaceutically acceptable form. In a specific embodiment the disease is cancer. In any of the above embodiments the individual preferably is a mammal, particularly a human. 
     The invention also provides a method for inducing lysis of a target cell, particularly a tumor cell, comprising contacting a target cell with a T cell activating bispecific antigen binding molecule of the invention in the presence of a T cell, particularly a cytotoxic T cell. 
     In another aspect, the invention provides for a method for identifying a variable heavy chain for use in a bispecific antigen binding molecule specific for a T cell activation antigen and a target cell antigen, comprising the step of screening a combinatorial library comprising variable heavy chains with a light chain comprising the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 31. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 35. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A-I  illustrate exemplary configurations of the T cell activating bispecific antigen binding molecules (TCBs) disclosed herein. All constructs except the kappa-lambda format in ( FIG. 11 ) have P329G LALA mutations and comprise knob-into-hole Fc fragments with knob-into-hole modifications. ( FIG. 1A ) Illustration of the “FoIR1 TCB 2+1 inverted (common light chain)”. The FoIR1 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification. These constructs are not crossed and have three times the same VLCL light chain. ( FIG. 1B ) Illustration of the “FoIR1 TCB 1+1 head-to-tail (common light chain)”. These constructs are not crossed and have two times the same VLCL light chain. ( FIG. 1C ) Illustration of the “FoIR1 TCB 1+1 classical (common light chain)”. These constructs are not crossed and have two times the same VLCL light chain. ( FIG. 1D ) Illustration of the “FoIR1 TCB 2+1 classical (common light chain)”. The CD3 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification. These constructs are not crossed and have three times the same VLCL light chain. ( FIG. 1E ) Illustration of the “FoIR1 TCB 2+1 crossfab classical”. These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain. The CD3 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification. ( FIG. 1F ) Illustration of the “FoIR1 TCB 2+1 crossfab inverted”. These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain. The FoIR1 binder is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain comprising the knob modification. ( FIG. 1G ) Illustration of the “FoIR1 TCB 1+1 crossfab head-to-tail”. These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain. ( FIG. 1H ) Illustration of the “FoIR1 TCB 1+1 crossfab classical”. These constructs comprise a Ck-VH chain for the CD3 binder instead of the conventional CH1-VH chain.  FIG. 11  illustrates the CD3/FoIR1 kappa-lambda antibody format. These constructs comprise a crossed common light chain VLCH1 and one crossed VHCL chain specific for CD3 and one crossed VHCL chain specific for FoIR1. 
         FIGS. 2A-C  depict graphs summarizing Binding of FoLR1 IgG binders to HeLa cells. Binding of newly generated FoIR1 binders to FoIR1 expressed on HeLa cells were determined by flow cytometry. Bound antibodies were detected with a fluorescently labeled anti-human secondary antibody. 
         FIGS. 3A-B  depict graphs summarizing specificity of FoIR1 binders for FoIR1. Binding of FoIR1 IgGs to HEK cells transiently transfected with either FoIR1 or FoIR2 was analyzed by flow cytometry to identify clones which bind specifically to FoIR1 and not to FoIR2. The antibodies were detected with a fluorescently labeled anti-human secondary antibody. 
         FIGS. 4A-B  depict graphs summarizing cross-reactivity of FoIR1 binders to cyFoLR1. Cross-reactivity of the FoIR1 antibodies to cyno FoIR1 was addressed on HEK cells transiently transfected with cyFoIR1 by flow cytometry. The antibodies were detected with a fluorescently labeled anti-human secondary antibody. 
         FIG. 5  depicts a graph illustrating internalization of FoIR1 TCBs after binding. Internalization of the four FoIR1 TCBs after binding to FoIR1 was tested on HeLa cells. Remaining FoIR1 TCBs on the surface were detected with a fluorescently labeled anti-human secondary antibody after indicated time points of incubation at 37° C. Percentage of internalization was calculated. 
         FIGS. 6A-E  depict graphs summarizing binding of FoIR1 IgGs to cells with different FoIR1 expression levels. Binding of 9D11, 16D5 and Mov19 IgG to tumor cells with different FoIR1 expression levels was analyzed by flow cytometry. DP47 IgG was included as isotype control and MKN-45 were included as FoIR1 negative cell line. The antibodies were detected with a fluorescently labeled anti-human secondary antibody. 
         FIGS. 7A-L  depict graphs summarizing T cell mediated killing of HT-29 and SKOV3 cells. FoIR1 TCBs were used to test T cell mediated killing of HT-29 and SKOV3 tumor cells and upregulation of activation marker on T cells upon killing. ( FIGS. 7A-D ) T cell mediated killing of HT-29 and SKOV3 cells in the presence of 9D11 FoIR1 TCB and 16D5 FoIR1 TCB was measured by LDH release after 24 h and 48 h. DP47 TCB was included as negative control. After 48 h incubation upregulation of the activation marker CD25 and CD69 on CD8 T cells and CD4 T cells upon killing of SKOV3 ( FIGS. 7E-H ) or HT-29 ( FIGS. 7I-L ) tumor cells was assessed by flow cytometry. 
         FIG. 8  depicts a graph showing absence of anti-FoIR1 binding to erythrocytes. Erythrocytes were gated as CD235a positive population and binding of 9D11 IgG, 16D5 IgG, Mov19 IgG and DP47 IgG to this population was determined by flow cytometry. The antibodies were detected with a fluorescently labeled anti-human secondary antibody. 
         FIGS. 9A-D  depict graphs summarizing activation marker upregulation in whole blood. CD25 and CD69 activation marker upregulation of CD4 T cells and CD8 T cells 24 h after addition of 9D11 FoIR1 TCB, 16D5 FoIR1 TCB, Mov19 FoIR1 TCB and DP47 TCB was analyzed by flow cytometry. 
         FIG. 10  Binding of 9D11 TCB a-glyco variants to HeLa cells. Binding of 9D11 FoIR1 TCB a-glyco variants to Hela cells was compared to binding of the original 9D11 TCB on HeLa cells. The antibodies were detected with a fluorescently labeled anti-human secondary antibody and binding was determined by flow cytometry. 
         FIGS. 11A-F  depict graphs summarizing T cell mediated killing with 9D11 FoIR1 TCB a-glyco variants of tumor cells. 9D11 FoIR1 TCB a-glyco variants were used to test T cell mediated killing of ( FIGS. 11A-D ) SKOV3, MKN-45 (as FoIR1 negative control) and ( FIGS. 11E-F ) HT-29 tumor cells in comparison to killing with the original 9D11 FoIR1 TCB. As read-out LDH release after 24 h and 48 h was used. 
         FIGS. 12A-X  depict graphs summarizing T cell mediated killing of primary epithelial cells. Primary epithelial cells with very low levels of FoIR1 were used to test T cell mediated killing with 16D5 FoIR1 TCB and 9D11 FoIR1 TCB, DP47 TCB was included as a negative control and HT29 cells were included as positive control. ( FIGS. 12A-H ) LDH release of human retinal pigment (HRP), human renal cortical (HRC), human bronchial (HB) and of HT29 cells was determined after 24 h and 48 h. CD25 and CD69 activation marker upregulation on CD4 T cells and CD8 T cells upon killing of (FIGS. 121-L) HRP, ( FIGS. 12M-P ) HRC, ( FIGS. 12Q-T ) HB and ( FIGS. 12  U-X) HT29 was determined after 48 h by flow cytometry. 
         FIGS. 13A-C  show a comparison of different TCB formats with 16D5. Four different TCB formats containing the FoIR1 binder 16D5 were compared in  FIG. 13A  binding to HeLa cells, in  FIG. 14  B T cell mediated killing of SKOV3 cells after 24 h and 48 h and in  FIG. 14C  CD25 and CD69 activation marker upregulation on CD4 T cells and CD8 T cells 48 h after killing. 
         FIGS. 14A-C  depict a comparison of different TCB formats with 9D11. Three different TCB formats containing the FoIR1 binder 9D11 were compared in A) binding to HeLa cells, in B) T cell mediated killing of SKOV3 cells after 24 h and 48 h and in C) CD25 and CD69 activation marker upregulation on CD4 T cells and CD8 T cells 48 h after killing. 
         FIG. 15  depicts a PK-profile of FOLR1 TCB in NOG mice for three different doses. 
         FIG. 16  illustrates an experimental protocol for efficacy study with FOLR1 TCB. 
         FIGS. 17A-B  depict tumor growth curves. ( FIG. 17A ) Mean values and SEM of tumor volumes in the different treatment groups. ( FIG. 17B ) Tumor growth of single mice in all treatment groups. TGI (tumor growth inhibition) give the percentage of the Mean tumor volume compared to vehicle group. 
         FIG. 18  shows tumor weights at study termination. 
         FIGS. 19A-B  show FACS analysis of tumor infiltrating T-cells at study day 32. ( FIG. 19A ) Tumor single cells suspensions were stained with anti-human CD3/CD4/CD8 and analyzed by flow cytometry. ( FIG. 19B ) Mean values and SEM of T-cell counts per mg tumor tissue in different treatment groups. 
         FIGS. 20A-B  show FACS analysis for T-cell activation/degranulation and cytokine secretion at study day 32. CD4+ ( FIG. 20A ) and CD8+ ( FIG. 20B ) tumor infiltrating T-cells were stained for cytokines, activation and degranulation markers. Displayed are the mean values and SEM of T-cell counts per mg tumor tissue in different treatment groups. 
         FIGS. 21A-B  show percent tumor lysis. SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 24 h ( FIG. 21A ) and 48 h ( FIG. 21B ) killing of tumor cells was determined by measuring LDH release 
         FIGS. 22A-D  show CD25 and CD69 upregulation on CD4 T cells. SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 48 h CD25 and CD69 upregulation on CD4 T cells ( FIGS. 22A-B ) and CD8 T cells ( FIGS. 22C-D ) was measured by flow cytometry. 
         FIGS. 23A-B  show percent tumor lysis. T-cell killing of SKov-3 cells (medium FoIR1) induced by 36F2 TCB, Mov19 TCB and 21A5 TCB after 24 h ( FIG. 23A ) and 48 h ( FIG. 23B ) of incubation (E:T=10:1, effectors human PBMCs). 
         FIGS. 24A-C  show T-cell killing induced by 36F2 TCB, 16D5 TCB, 16D5 TCB classical, 16D5 TCB 1+1 and 16D5 TCB HT of Hela (high FoIR1) ( FIG. 24A ), Skov-3 (medium FoIR1) ( FIG. 24B ) and HT-29 (low FoIR1) ( FIG. 24C ) human tumor cells (E:T=10:1, effectors human PBMCs, incubation time 24 h). DP47 TCB was included as non-binding control. 
         FIGS. 25A-C  show upregulation of CD25 and CD69 on human CD8+ ( FIGS. 25A , B) and CD4+ ( FIG. 25C ), T cells after T cell-mediated killing of Hela cells (high FoIR1) ( FIG. 25A ), SKov-3 cells (medium FoIR1) ( FIG. 25B ) and HT-29 cells (low FoIR1) ( FIG. 25C ) (E:T=10:1, 48 h incubation) induced by 36F2 TCB, 16D5 TCB and DP47 TCB (non-binding control). 
         FIGS. 26A-F  show T-cell killing induced by 36F2 TCB, 16D5 TCB and DP47 TCB of human Renal Cortical Epithelial Cells ( FIGS. 26A , B), human Retinal Pigment Epithelial Cells ( FIGS. 26C , D) and HT-29 cells ( FIGS. 26E , F) cells after 24 h ( FIGS. 26A , C, E) and 48 h ( FIGS. 26B , D, F) of incubation (E:T=10:1, effectors human PBMCs). 
         FIG. 27  depicts a table summarizing quantification of FoIR1 binding sites on various normal and cancer cells lines. 
         FIGS. 28A-B  show binding of 16D5 TCB and its corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and its demidation variants 9D11 TCB N100A and 9D11 TCB S100aA to human CD3 expressed on Jurkat cells. 
         FIGS. 29A-B  show T-cell killing of SKov-3 (medium FoIR1) human tumor cells induced by 16D5 TCB and its corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA ( FIG. 29A ) and 9D11 TCB and its demidation variants 9D11 TCB N100A and 9D11 TCB S100aA ( FIG. 29B ) (E:T=10:1, effectors human PBMCs, incubation time 24 h). DP47 TCB was included as non-binding control. 
         FIGS. 30A-B  show T-cell killing of HT-29 (low FoIR1) human tumor cells induced by 16D5 TCB and its corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA ( FIG. 30A ) and 9D11 TCB and its demidation variants 9D11 TCB N100A and 9D11 TCB S100aA ( FIG. 30B ) (E:T=10:1, effectors human PBMCs, incubation time 24 h). DP47 TCB was included as non-binding control. 
         FIG. 31  shows a sequence alignment of the VH domains of the 3 identified MUC1-specific binders. All three clones are derivatives of the IGHV3-23 germline (SEQ ID NO: 136). Clone 58D6 (SEQ ID NO: 60) and 110A5 (SEQ ID NO: 64) originate from a library that was only randomized in CDR3, while clone 106D2 (SEQ ID NO: 62) was identified from a library randomized in all 3 CDRs. Positions in CDR1 and 2 that deviate from the germline sequence are printed italic. 
         FIGS. 32A-B  shows results of characterization of CLC binders. ( FIG. 32A ) SPR analysis. SPR-based kinetic analyses of 3 clones specifically binding to MUC1. Smooth lines represent a global fit of the data to a 1:1 interaction model. ( FIG. 32B ) Summary of kinetic and thermodynamic parameters. 
         FIG. 33  depicts a schematic diagram of the generated TCB construct. The CLC TCB construct consists of 3 different immunoglobulin chains: 1) an IgG heavy chain harbouring the “hole mutations” in the Fc part and containing the target-specific VH domain; 2) an Ig chain consisting of the target-specific VH and a CH1 domain, followed by the CD3-specific VH domain and a CH1 domain, followed by the Fc part containing the “knob” mutations; and 3) the common light chain that anneals to both the MUC1-specific and the CD3-specific sequences. 
         FIGS. 34A-B  depicts purification and analytical characterization of the produced MUC1-specific TCBs ( FIG. 34A  and  FIG. 34B ). The purification method involved an affinity step (protein A) followed by size exclusion chromatography (Superdex 200, GE Healthcare). The final product was analyzed and characterized by analytical size exclusion chromatography (Superdex 200 column) and by capillary electrophoresis. 
         FIG. 35  depicts SPR analysis of the MUC1-specific binders in the TCB format. Shown is the binding of 2 MUC1-specific TCBs at different concentrations (see text) to either MUC1 or an unrelated antigen. Smooth lines represent a global fit of the data to a 1:1 interaction model 
         FIGS. 36A-G  depict bispecific bivalent and trivalent antibodies comprising only the Fab fragments (specific to CD3 and BCMA) with or without an Fc part as specified: (A) Fab BCMA-Fc-Fab CD3; (B) Fab BCMA-Fc-Fab CD3-Fab BCMA; (C) Fab BCMA-Fc-Fab BCMA-Fab CD3; (D) Fc-Fab CD3-Fab BCMA; (E) Fc-Fab BCMA-Fab CD3; (F) Fab CD3-Fab BCMA-Fab BCMA; (G) Fab CD3-Fab BCMA. Preferably, the LC of Fab CD3 and Fab BCMA are identical (common LC) to avoid LC mispairing and reduce side-products. Fab CD3 and Fab BCMA are linked to each other with flexible linkers. 
         FIG. 37  depicts lack of binding of BCMA IgG antibody to TACI receptor as detected by surface plasmon resonance (SPR). Curve 1 corresponds to the signal on reference channel, curve 2 to the channel where the binding occurs (binding channel) and the curve 2-1 is the subtracted signal (binding channel—reference channel), meaning that this is the signal due to the binding event. SPR binding assay clearly demonstrated that pSCHLI372 IgG did not bind to human TACI receptor. 
         FIGS. 38A-C  show production and purification of BCMA-TCB CLC. CE-SDS graphs (non-reduced (top) and reduced (bottom)) of the final protein preparations after Protein A (PA) affinity chromatography and size exclusion chromatographic (SEC) purification steps applied to (A) pSCHLI333-TCB CLC, (B) pSCHLI372-TCB CLC, (C) pSCHLI373-TCB CLC. All three molecules are of molecular format as described in  FIG. 36B . 
         FIGS. 39A-B  show binding of BCMA-TCB CLC antibodies on BCMAhi-positive H929 cells by flow cytometry. The median fluorescence intensity of BCMA-TCB CLC antibodies were plotted in function of antibody concentrations (0.12 to 500 nM); (A) pSCHLI372-TCB CLC and pSCHLI373-TCB CLC on H929 cells (A) and MKN45 cells (B). DP47-TCB is a negative control TCB which did not bind to BCMA at concentrations below 100 nM (see Example 7). 
         FIGS. 40A-B  show binding of BCMA-TCB CLC antibodies on CD3-positive Jurkat T cells as measured by flow cytometry. Median fluorescence intensity for BCMA-TCB CLC antibodies (pSCHLI372-TCB CLC and pSCHLI373-TCB CLC) binding to Jurkat T cells and plotted in function of antibody concentration. Non-binding to BCMA-negative and CD3-negative MKN45 cells at concentrations below 100 nM. 
         FIG. 41  shows T-cell activation mediated by BCMA-TCB CLC antibodies in presence of H929 cells as detected by flow cytometry. Expression level of the early activation marker CD69 and the late activation marker CD25 on CD4+ and CD8+ T cells after 48 hours of incubation. pSCHL1372-TCB CLC and pSCHL1373-TCB CLC antibodies induced an up-regulation of CD69 and CD25 activation markers in a concentration-dependent and specific manner in the presence of BCMA-positive target cells. E:T ratio used as 10 PBMCs:1 H929 cell; cells were incubated for 48 h before measurement of CD69 and CD25 upregulation. DP47-TCB which is a negative control TCB did not induce T-cell activation. Representative results are from two independent experiments. 
         FIGS. 42A-B  show BCMA-TCB CLC antibodies induce T-cell redirected killing of BCMAhi-positive H929 myeloma cells as detected by colorimetric LDH release assay. BCMA-TCB CLC antibodies pSCHLI372-TCB CLC (A, B) and pSCHLI373-TCB CLC (A) induced a concentration-dependent killing of BCMAhi-positive H929 myeloma cells as measured by LDH release. DP47-TCB which is a negative control TCB that does not bind to BCMA but only to CD3 did not induce H929 cell killing. E:T ratio used as 10 PBMCs:1 H929 cell; cells were incubated for 24 h before measurement of LDH release. Representative results are from three independent experiments. 
         FIG. 43  shows BCMA-TCB CLC antibodies induce T-cell redirected killing of BCMAmed/lo-positive U266 myeloma cells as detected by colorimetric LDH release assay. BCMA-TCB CLC antibodies pSCHLI372-TCB CLC and pSCHLI373-TCB CLC induced a concentration-dependent killing of BCMAmed/lo-positive U266 myeloma cells as measured by LDH release. DP47-TCB which is a negative control TCB that does not bind to BCMA but only to CD3 did not induce H929 cell killing. E:T ratio used as 10 PBMCs:1 U266 cell; cells were incubated for 24 h before measurement of LDH release. Representative results are from two independent experiments. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Definitions 
     Terms are used herein as generally used in the art, unless otherwise defined in the following. 
     As used herein, the term “antigen binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and derivatives, e.g. fragments, thereof. 
     The term “bispecific” means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells. 
     The term “valent” as used herein denotes the presence of a specified number of antigen binding sites in an antigen binding molecule. As such, the term “monovalent binding to an antigen” denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the antigen binding molecule. 
     An “antigen binding site” refers to the site, i.e. one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site. 
     As used herein, the term “antigen binding moiety” refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen binding moiety is able to direct the entity to which it is attached (e.g. a second antigen binding moiety) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant. In another embodiment an antigen binding moiety is able to activate signaling through its target antigen, for example a T cell receptor complex antigen. Antigen binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen binding moieties include an antigen binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding moieties may comprise antibody constant regions as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of the two isotypes: κ and λ. 
     As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope,” and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM). The proteins referred to as antigens herein (e.g. MCSP, FAP, CEA, EGFR, CD33, CD3) can be any native form the proteins from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated. In a particular embodiment the antigen is a human protein. Where reference is made to a specific protein herein, the term encompasses the “full-length”, unprocessed protein as well as any form of the protein that results from processing in the cell. The term also encompasses naturally occurring variants of the protein, e.g. splice variants or allelic variants. Exemplary human proteins useful as antigens include, but are not limited to Folate Receptor 1 (FoIR1, Folate receptor alpha (FRA); Folate binding protein (FBP); human FoIR1 UniProt no.: P15328; murine FoIR1 UniProt no.: P35846; cynomolgus FoIR1 UniProt no.: G7PR14), Mucin-1 (MUC1), and B Cell Maturation Antigen (BCMA), and CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1 for the human sequence; or UniProt no. Q95L15 (version 49), NCBI GenBank no. BAB71849.1 for the cynomolgus [ Macaca fascicularis ] sequence). 
     In certain embodiments the T cell activating bispecific antigen binding molecule of the invention binds to an epitope of an activating T cell antigen or a target cell antigen that is conserved among the activating T cell antigen or target antigen from different species. 
     By “specific binding” is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antigen binding moiety to an unrelated protein is less than about 10% of the binding of the antigen binding moiety to the antigen as measured, e.g., by SPR. In certain embodiments, an antigen binding moiety that binds to the antigen, or an antigen binding molecule comprising that antigen binding moiety, has a dissociation constant (K D ) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10 −8  M or less, e.g. from 10 −8  M to 10 −13  M, e.g., from 10 −9  M to 10 −13  M). 
     “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ), which is the ratio of dissociation and association rate constants (k off  and k on , respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by well established methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR). 
     “Reduced binding”, for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction. 
     An “activating T cell antigen” as used herein refers to an antigenic determinant expressed on the surface of a T lymphocyte, particularly a cytotoxic T lymphocyte, which is capable of inducing T cell activation upon interaction with an antigen binding molecule. Specifically, interaction of an antigen binding molecule with an activating T cell antigen may induce T cell activation by triggering the signaling cascade of the T cell receptor complex. In a particular embodiment the activating T cell antigen is CD3. 
     “T cell activation” as used herein refers to one or more cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. The T cell activating bispecific antigen binding molecules of the invention are capable of inducing T cell activation. Suitable assays to measure T cell activation are known in the art described herein. 
     A “target cell antigen” as used herein refers to an antigenic determinant presented on the surface of a target cell, for example a cell in a tumor such as a cancer cell or a cell of the tumor stroma. 
     As used herein, the terms “first” and “second” with respect to antigen binding moieties etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the T cell activating bispecific antigen binding molecule unless explicitly so stated. 
     The term “BCMA” as used herein relates to human B cell maturation target, also known as BCMA, TR17_HUMAN, TNFRSF17 (UniProt Q02223), which is a member of the tumor necrosis receptor superfamily that is preferentially expressed in differentiated plasma cells. The extracellular domain of BCMA consists according to UniProt of amino acids 1-54 (or 5-51). The term “antibody against BCMA, anti BCMA antibody” as used herein relates to an antibody specifically binding to BCMA. 
     The term “CD3ε or CD3” as used herein relates to human CD3ε described under UniProt P07766 (CD3E_HUMAN). The term “antibody against CD3, anti CD3 antibody” relates to an antibody binding to CD3ε. 
     A “Fab molecule” refers to a protein consisting of the VH and CH1 domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin. The term “Fab molecules having identical VLCL light chains” as used therein refers to binders that share one light chain but still have separate specificities. T-cell activating bispecific molecules of the invention comprise at least two Fab molecules having identical VLCL light chains. The corresponding heavy chains are remodeled and confer specific binding to a T cell activating bispecific antigen and a target cell antigen, respectively. 
     By “fused” is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers. 
     The term “common light chain” as used herein refers to a light chain that within a bispecific or multispecific molecule pairs with more than one heavy chain or fragment thereof to form at least a first and a second antigen binding site, e.g., a Fab, each specific for a different antigen. For example, the common light chain pairs with a first heavy chain or fragment thereof within an antigen binding molecule to form a first binding site specific for a tumor antigen, and another copy of the common light chain pairs with a second heavy chain or fragment thereof within an antigen binding molecule to form a second binding site specific for a T cell activating antigen, e.g., CD3. 
     The term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes, e.g. γ 1  (IgG 1 ), γ 2  (IgG 2 ), γ 3  (IgG 3 ), γ 4  (IgG 4 ), α 1  (IgA 1 ) and α 2  (IgA 2 ). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region. 
     The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity. 
     An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′) 2 , diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g. Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab′) 2  fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g.  E. coli  or phage), as described herein. 
     The term “antigen binding domain” refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Particularly, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). 
     The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6 th  ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. 
     The term “hypervariable region” or “HVR”, as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the complementarity determining regions (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as “complementarity determining regions” (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen binding regions. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table A as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody. 
     
       
         
           
               
             
               
                 TABLE A 
               
             
            
               
                   
               
               
                 CDR Definitions 1   
               
            
           
           
               
               
               
               
               
            
               
                   
                 CDR 
                 Kabat 
                 Chothia 
                 AbM 2   
               
               
                   
                   
               
               
                   
                 V H  CDR1 
                 31-35 
                 26-32 
                 26-35 
               
               
                   
                 V H  CDR2 
                 50-65 
                 52-58 
                 50-58 
               
               
                   
                 V H  CDR3 
                 95-102 
                 95-102 
                 95-102 
               
               
                   
                 V L  CDR1 
                 24-34 
                 26-32 
                 24-34 
               
               
                   
                 V L  CDR2 
                 50-56 
                 50-52 
                 50-56 
               
               
                   
                 V L  CDR3 
                 89-97 
                 91-96 
                 89-97 
               
               
                   
                   
               
               
                   
                   1 Numbering of all CDR definitions in Table A is according to the numbering conventions set forth by Kabat et al. (see below). 
               
               
                   
                   2 “AbM” with a lowercase “b” as used in Table A refers to the CDRs as defined by Oxford Molecular&#39;s “AbM” antibody modeling software. 
               
            
           
         
       
     
     Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable region sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region are according to the Kabat numbering system. 
     The polypeptide sequences of the sequence listing are not numbered according to the Kabat numbering system. However, it is well within the ordinary skill of one in the art to convert the numbering of the sequences of the Sequence Listing to Kabat numbering. 
     “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. 
     The “class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 . The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. 
     The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. A “subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain. 
     A “modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which might be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding moieties) are not the same. In some embodiments the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a particular embodiment, the modification promoting association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain. 
     The term “effector functions” refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation. 
     As used herein, the terms “engineer, engineered, engineering”, are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches. 
     The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and insertions of amino acids. Particular amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an Fc region, non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and/or chemical properties, are particularly preferred. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc domain to glycine can be indicated as 329G, G329, G329, P329G, or Pro329Gly. 
     As used herein, term “polypeptide” refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded. 
     By an “isolated” polypeptide or a variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. 
     “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 
       100 times the fraction X/Y 
     where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program&#39;s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. 
     The term “polynucleotide” refers to an isolated nucleic acid molecule or construct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g. an amide bond, such as found in peptide nucleic acids (PNA). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, e.g. DNA or RNA fragments, present in a polynucleotide. 
     By “isolated” nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in cells that ordinarily contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator. 
     By a nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs, such as the ones discussed above for polypeptides (e.g. ALIGN-2). 
     The term “expression cassette” refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the invention comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof. 
     The term “vector” or “expression vector” is synonymous with “expression construct” and refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated in a target cell. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette that comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof. 
     The terms “host cell”, “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A host cell is any type of cellular system that can be used to generate the bispecific antigen binding molecules of the present invention. Host cells include cultured cells, e.g. mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. 
     An “activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). 
     Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. As used herein, the term “reduced ADCC” is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006/082515 or PCT patent application no. PCT/EP2012/055393). 
     An “effective amount” of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered. 
     A “therapeutically effective amount” of an agent, e.g. a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease. 
     An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non-human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human. 
     The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. 
     A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. 
     As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, T cell activating bispecific antigen binding molecules of the invention are used to delay development of a disease or to slow the progression of a disease. 
     The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products. 
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     The present invention provides bispecific antigen binding molecules designed for T cell activation and re-direction that combine good efficacy and produceability and methods of making and using the same. In particular this invention relates to bispecific molecules wherein at least two binding moieties have identical light chains and, in some embodiments, corresponding remodeled heavy chains that confer the specific binding to a T cell activating bispecific antigen and a target cell antigen, respectively. The use of this so-called ‘common light chain’ principle, i.e. combining two binders that share one light chain but still have separate specificities, prevents light chain mispairing. Thus, there are less side products during production, facilitating the homogenous preparation of T cell activating bispecific antigen binding molecules. 
     The present invention particularly pertains to a predefined rare light chain which contributes significantly to antigen binding and heteromultimeric pairing with different binding partners, e.g., heavy chains and fragments thereof. This common light chain is, thus, suitable as for use in a library from which new bi- or multispecific antigen binding molecules can be prepared. Advantageously, the common light chain used in connection with antigen binding molecules and methods disclosed herein can be used to form an antigen binding molecule useful for T cell activation. One of skill in the art can recognize the advantageous efficiency of having such light chain that can function in both, T cell activating antigen binding moieties and target antigen binding moieties. This allows for efficient production of T cell activating bispecific antigen binding molecules that comprise the T cell activation component and a target antigen binding component. In a particular embodiment, the common light chain a lambda constant light chain domain. In a particular embodiment, the common light chain is a human or humanized lambda light chain. In a particular embodiment, the common light chain is of the rare human lambda 7 family of light chain. Using a light chain of the lambda, particularly, the rare lambda 7 family was an uncommon approach and would have been expected to decrease the likelihood of identifying suitable heavy chain binding partners to create antigen binding molecules specific for a variety of targets. Thus, prior to the inventor&#39;s work disclosed herein, it was not known that a lambda 7 light chain with suitable properties could be developed for a variety of different unrelated target antigens such as, e.g., FoIR1, MUC1, and BCMA. It was further not known that a lambda light chain could be developed to generate stable, functional, high affinity binders to improve production of T cell activating bispecific antigen binding molecules with CD3 specificity and a target antigen specificity, where the target antigens are unrelated, e.g., FoIR1, MUC1, and BCMA. In one embodiment, such common light chain can be used to construct a common light chain (CLC) library which is based on a specific CD3 binder, not a germline antibody, to generate specifically CD3 binders contributing to the binder, as described below. The advantage of this approach is that it allows for maintaining the previously identified and validated CD3 binder such that merely a new target antigen binder for the target antigen binding moiety of a T cell activating bispecific antigen binding molecules has to be identified based on the heavy chain. This allows for a module approach to generating different T cell activating bispecific antigen binding molecules with identical or highly homologous chains. While the light chain is identical within a given T cell activating antigen binding molecule, the light chains of different T cell activating bispecific antigen binding molecules might be identical or highly homologous. By “highly homologous” is meant that the light chains of different T cell activating bispecific antigen binding molecules produced by this module approach comprise an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical. Preferably, highly homologous light chains of the invention have identical variable light chain regions and differ only on their constant region. For example, in some embodiments, the amino acid variance is confined to the linker region. In some embodiment, the common light chain comprises a kappa constant light chain domain. 
     In addition to the foregoing advantages, yield of correctly paired heteromultimeric molecules using this approach is enhanced, as explained above, because the light chain used within the antigen binding moieties of a T cell activating bispecific antigen binding molecule is identical. Stated another way, using a common light chain the production of these molecules is facilitated as any mispairing of a light chain to the incorrect heavy chain is abolished. Thus, the isolation of a highly pure T cell activating bispecific antigen binding molecule species is facilitated. In a particular embodiment, the T cell activating bispecific antigen binding molecule uses Fabs as building blocks. Compared to other formats the use of Fab fragments as building blocks as opposed to, e.g., the use of scFv fragments results in higher thermal stability and the lack of scFv aggregation and intermolecular scFv formation. 
     Prior to the inventors&#39; work described herein, it was not known that a common light chain could be generated that can not only serve as common light chain in a bi- or multispecific molecule but also support the functional property of T cell activation of a T cell activating bispecific antigen binding molecule. Furthermore, prior to the inventors&#39; work described herein, it was not known that a common light chain could be generated that significantly contributes to antigen binding properties of the antigen binding moiety within a T cell activating bispecific antigen binding molecule. A well-established strategy is to identify heavy chains or fragments thereof that contribute most of the binding properties, such as strength and specificity. According to the present invention, the common light chain significantly contributes to the binding properties. 
     Accordingly, in a first aspect the invention provides a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, wherein the first antigen binding moiety comprises a first light chain and wherein the first antigen binding moiety is capable of specific binding to an activating T cell antigen, wherein the second antigen binding moiety comprises a second light chain and wherein the second antigen binding moiety is capable of specific binding to a target cell antigen, wherein the amino acid sequence of the first and the second light chain is identical. In one embodiment, the first antigen binding moiety is a Fab. In one embodiment, the second antigen binding moiety is a Fab. In one aspect, the invention provides a T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, one of which is a Fab molecule capable of specific binding to an activating T cell antigen and the other one of which is a Fab molecule capable of specific binding to a target cell antigen, wherein the first and the second Fab molecule have identical light chains (variable light chain and constant light chain region, VLCL). In one embodiment, the light chain (VLCL) comprises the light chain CDRs of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. In one embodiment said identical light chain (VLCL) comprises SEQ ID NO. 35. 
     T Cell Activating Bispecific Antigen Binding Molecule Formats 
     The components of the T cell activating bispecific antigen binding molecule can be fused to each other in a variety of configurations. Exemplary configurations include but are not limited to those depicted in  FIGS. 1A-D . 
     In some embodiments, said T cell activating bispecific antigen binding molecule further comprises an Fc domain composed of a first and a second subunit capable of stable association. Below exemplary embodiments of T cell activating bispecific antigen binding molecule comprising an Fc domain are described. All these T cell activating bispecific antigen binding molecules comprise at least two Fab fragments having identical light chains (VLCL) and having different heavy chains (VHCL) which confer the specifities to two different antigens, i.e. one Fab fragment is capable of specific binding to a T cell activating antigen and the other Fab fragment is capable of specific binding to a target cell antigen. 
     In some embodiments, the first and the second antigen binding moiety of the T cell activating bispecific antigen binding molecule are fused to each other, optionally via a peptide linker. In one such embodiment, the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In another such embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. In yet another such embodiment, the second antigen binding moiety is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen binding moiety. In yet another such embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the second antigen binding moiety. 
     In one embodiment the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. 
     In a particular such embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first and a second antigen binding moiety which comprise identical (VLCL) light chains, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. 
     In an alternative such embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first and a second antigen binding moiety which comprise identical (VLCL) light chains, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first and the second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. 
     In yet another such embodiment, the second antigen binding moiety is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen binding moiety. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first and a second antigen binding moiety which comprise identical (VLCL) light chains, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first antigen binding moiety is fused at the N-terminus of the Fab light chain to the C-terminus of the Fab light chain of the second antigen binding moiety, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. 
     In other embodiments, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. 
     In a particular such embodiment, the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first and a second antigen binding moiety which comprise identical (VLCL) light chains, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. 
     In particular of these embodiments, the first antigen binding moiety is capable of specific binding to an activating T cell antigen. In other embodiments, the first antigen binding moiety is capable of specific binding to a target cell antigen. 
     The antigen binding moieties may be fused to the Fc domain or to each other directly or through a peptide linker, comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable, non-immunogenic peptide linkers include, for example, (G 4 S) n  (SEQ ID NO: 41), (SG 4 ) n  (SEQ ID NO: 42), (G 4 S) n  (SEQ ID NO: 41) or G 4 (SG 4 ) n  (SEQ ID NO: 43) peptide linkers. “n” is generally a number between 1 and 10, typically between 2 and 4. A particularly suitable peptide linker for fusing the Fab light chains of the first and the second antigen binding moiety to each other is (G 4 S) 2  (SEQ ID NO: 44). Additionally, linkers may comprise (a portion of) an immunoglobulin hinge region. Particularly where an antigen binding moiety is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker. 
     A T cell activating bispecific antigen binding molecule with a single antigen binding moiety capable of specific binding to a target cell antigen is useful, particularly in cases where internalization of the target cell antigen is to be expected following binding of a high affinity antigen binding moiety. In such cases, the presence of more than one antigen binding moiety specific for the target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availability. 
     In many other cases, however, it will be advantageous to have a T cell activating bispecific antigen binding molecule comprising two or more antigen binding moieties specific for a target cell antigen, for example to optimize targeting to the target site or to allow crosslinking of target cell antigens. 
     Accordingly, in certain embodiments, the T cell activating bispecific antigen binding molecule of the invention further comprises a third antigen binding moiety which is a Fab molecule capable of specific binding to a target cell antigen. In one embodiment, the third antigen binding moiety is capable of specific binding to the same target cell antigen as the first or second antigen binding moiety. In a particular embodiment, the first antigen binding moiety is capable of specific binding to an activating T cell antigen, and the second and third antigen binding moieties are capable of specific binding to a target cell antigen. In a preferred embodiment, the first, second and third antigen binding moeity comprise identical (VLCL) light chains. 
     In one embodiment, the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. 
     In one embodiment, the first and the third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In a specific such embodiment, the T cell activating bispecific antigen binding molecule essentially consists of a first, a second and a third antigen binding moiety (Fab fragment), an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Preferably in said embodiment the first antigen binding moiety is capable of specific binding to an activating T cell antigen, and the second and third antigen binding moieties are capable of specific binding to a target cell antigen, wherein the first, second and third antigen binding moeities are Fab fragments which comprise identical (VLCL) light chains. 
     In one embodiment, the second and the third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. 
     In one embodiment, the T cell activating bispecific antigen binding molecule essentially consists of an immunoglobulin molecule capable of specific binding to a target cell antigen, and a Fab molecule capable of specific binding to an activating T cell antigen, fused to the N-terminus of one of the immunoglobulin heavy chains, optionally via a peptide linker. Preferably in said embodiment the immunoglobulin molecule capable of specific binding to a target cell antigen and the Fab molecule capable of specific binding to an activating T cell antigen comprise identical (VLCL) light chains. 
     The first and the third antigen binding moiety (or the second and the third antigen binding moiety, respectively) may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment first and the third antigen binding moiety (or the second and the third antigen binding moiety, respectively) are each fused to the Fc domain through an immunoglobulin hinge region. 
     In a specific embodiment, the immunoglobulin hinge region is a human IgG 1  hinge region. In one embodiment first and the third antigen binding moiety (or the second and the third antigen binding moiety, respectively) and the Fc domain are part of an immunoglobulin molecule. In a particular embodiment the immunoglobulin molecule is an IgG class immunoglobulin. In an even more particular embodiment the immunoglobulin is an IgG 1  subclass immunoglobulin. In another embodiment the immunoglobulin is an IgG 4  subclass immunoglobulin. In a further particular embodiment the immunoglobulin is a human immunoglobulin. In other embodiments the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. 
     Fc Domain 
     The Fc domain of the T cell activating bispecific antigen binding molecule consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment the T cell activating bispecific antigen binding molecule of the invention comprises not more than one Fc domain. 
     In one embodiment according the invention the Fc domain of the T cell activating bispecific antigen binding molecule is an IgG Fc domain. In a particular embodiment the Fc domain is an IgG 1  Fc domain. In another embodiment the Fc domain is an IgG 4  Fc domain. In a more specific embodiment, the Fc domain is an IgG 4  Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG 4  antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment the Fc domain is human. 
     Fc Domain Modifications Promoting Heterodimerization 
     T cell activating bispecific antigen binding molecules according to the invention comprise different antigen binding moieties, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of T cell activating bispecific antigen binding molecules in recombinant production, it will thus be advantageous to introduce in the Fc domain of the T cell activating bispecific antigen binding molecule a modification promoting the association of the desired polypeptides. 
     Accordingly, in particular embodiments the Fc domain of the T cell activating bispecific antigen binding molecule according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment said modification is in the CH3 domain of the Fc domain. 
     In a specific embodiment said modification is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain. 
     The knob-into-hole technology is described e.g. in U.S. Pat. Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). 
     Accordingly, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the T cell activating bispecific antigen binding molecule an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. 
     The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis. 
     In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A). 
     In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). 
     In a particular embodiment the antigen binding moiety capable of binding to an activating T cell antigen is fused (optionally via the antigen binding moiety capable of binding to a target cell antigen) to the first subunit of the Fc domain (comprising the “knob” modification). Without wishing to be bound by theory, fusion of the antigen binding moiety capable of binding to an activating T cell antigen to the knob-containing subunit of the Fc domain will (further) minimize the generation of antigen binding molecules comprising two antigen binding moieties capable of binding to an activating T cell antigen (steric clash of two knob-containing polypeptides). 
     In an alternative embodiment a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. 
     Fc Domain Modifications Reducing Fc Receptor Binding and/or Effector Function 
     The Fc domain confers to the T cell activating bispecific antigen binding molecule favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the T cell activating bispecific antigen binding molecule to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with the T cell activating properties and the long half-life of the antigen binding molecule, results in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce efficacy of the T cell activating bispecific antigen binding molecule due to the potential destruction of T cells e.g. by NK cells. 
     Accordingly, in particular embodiments the Fc domain of the T cell activating bispecific antigen binding molecules according to the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG 1  Fc domain. In one such embodiment the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgG 1  Fc domain (or a T cell activating bispecific antigen binding molecule comprising a native IgG 1  Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgG 1  Fc domain domain (or a T cell activating bispecific antigen binding molecule comprising a native IgG 1  Fc domain). In one embodiment, the Fc domain domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function. In a particular embodiment the Fc receptor is an Fcγ receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one embodiment the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment the effector function is ADCC. In one embodiment the Fc domain domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgG 1  Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgG 1  Fc domain (or the T cell activating bispecific antigen binding molecule comprising a native IgG 1  Fc domain) to FcRn. 
     In certain embodiments the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In particular embodiments, the Fc domain of the T cell activating bispecific antigen binding molecule comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment the T cell activating bispecific antigen binding molecule comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to a T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain. In a particular embodiment the Fc receptor is an Fcγ receptor. In some embodiments the Fc receptor is a human Fc receptor. In some embodiments the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments binding affinity to a complement component, specifically binding affinity to C1q, is also reduced. In one embodiment binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or the T cell activating bispecific antigen binding molecule comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or T cell activating bispecific antigen binding molecules of the invention comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments the Fc domain of the T cell activating bispecific antigen binding molecule is engineered to have reduced effector function, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a T cell activating bispecific antigen binding molecule comprising a non-engineered Fc domain). 
     In one embodiment the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In one embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329. In a more specific embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329. In some embodiments the Fc domain comprises the amino acid substitutions L234A and L235A. In one such embodiment, the Fc domain is an IgG 1  Fc domain, particularly a human IgG 1  Fc domain. In one embodiment the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331. In a more specific embodiment the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments the Fc domain comprises amino acid substitutions at positions P329, L234 and L235. In more particular embodiments the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”). In one such embodiment, the Fc domain is an IgG 1  Fc domain, particularly a human IgG 1  Fc domain. The “P329G LALA” combination of amino acid substitutions almost completely abolishes Fcγ receptor binding of a human IgG 1  Fc domain, as described in PCT patent application no. PCT/EP2012/055393, incorporated herein by reference in its entirety. PCT/EP2012/055393 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions. 
     IgG 4  antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgG 1  antibodies. Hence, in some embodiments the Fc domain of the T cell activating bispecific antigen binding molecules of the invention is an IgG 4  Fc domain, particularly a human IgG 4  Fc domain. In one embodiment the IgG 4  Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P. To further reduce its binding affinity to an Fc receptor and/or its effector function, in one embodiment the IgG 4  Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E. In another embodiment, the IgG 4  Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G. In a particular embodiment, the IgG 4  Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G. Such IgG 4  Fc domain mutants and their Fcγ receptor binding properties are described in PCT patent application no. PCT/EP2012/055393, incorporated herein by reference in its entirety. 
     In a particular embodiment the Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG 1  Fc domain, is a human IgG 1  Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG 4  Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G. 
     In certain embodiments N-glycosylation of the Fc domain has been eliminated. In one such embodiment the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D). 
     In addition to the Fc domains described hereinabove and in PCT patent application no. PCT/EP2012/055393, Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581). 
     Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing. 
     Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. A suitable such binding assay is described herein. Alternatively, binding affinity of Fc domains or cell activating bispecific antigen binding molecules comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing FcγIIIa receptor. 
     Effector function of an Fc domain, or a T cell activating bispecific antigen binding molecule comprising an Fc domain, can be measured by methods known in the art. A suitable assay for measuring ADCC is described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in a animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998). 
     In some embodiments, binding of the Fc domain to a complement component, specifically to C1q, is reduced. Accordingly, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. C1q binding assays may be carried out to determine whether the T cell activating bispecific antigen binding molecule is able to bind C1q and hence has CDC activity. See e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)). 
     Antigen Binding Moieties 
     The antigen binding molecule of the invention is bispecific, i.e. it comprises at least two antigen binding moieties capable of specific binding to two distinct antigenic determinants. According to one embodiment of the invention, the antigen binding moieties are Fab molecules (i.e. antigen binding domains composed of a heavy and a light chain, each comprising a variable and a constant region), wherein the light chain (VLCL) of the at least two Fab molecules comprises identical sequences. In one embodiment said VLCL light chain of the Fab molecules capable of specific binding to a target cell antigen and a T cell activating antigen, respectively, comprises the light chain CDRs of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. 
     In one embodiment said VLCL light chain of the Fab molecules capable of specific binding to a target cell antigen and a T cell activating antigen, respectively, comprises SEQ ID NO: 31. 
     In one embodiment said Fab molecules are human. In another embodiment said Fab molecules are humanized. In yet another embodiment said Fab molecules comprise human heavy and light chain constant regions. 
     In a particular embodiment according to the invention, the T cell activating bispecific antigen binding molecule is capable of simultaneous binding to a target cell antigen, particularly a tumor cell antigen, and an activating T cell antigen. In one embodiment, the T cell activating bispecific antigen binding molecule is capable of crosslinking a T cell and a target cell by simultaneous binding to a target cell antigen and an activating T cell antigen. In an even more particular embodiment, such simultaneous binding results in lysis of the target cell, particularly a tumor cell. In one embodiment, such simultaneous binding results in activation of the T cell. In other embodiments, such simultaneous binding results in a cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from the group of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. In one embodiment, binding of the T cell activating bispecific antigen binding molecule to the activating T cell antigen without simultaneous binding to the target cell antigen does not result in T cell activation. 
     In one embodiment, the T cell activating bispecific antigen binding molecule is capable of re-directing cytotoxic activity of a T cell to a target cell. In a particular embodiment, said re-direction is independent of MHC-mediated peptide antigen presentation by the target cell and and/or specificity of the T cell. 
     Particularly, a T cell according to any of the embodiments of the invention is a cytotoxic T cell. In some embodiments the T cell is a CD4 +  or a CD8 +  T cell, particularly a CD8 +  T cell. 
     Activating T Cell Antigen Binding Moiety 
     The T cell activating bispecific antigen binding molecule of the invention comprises at least one antigen binding moiety capable of binding to an activating T cell antigen (also referred to herein as an “activating T cell antigen binding moiety”). In a particular embodiment, the T cell activating bispecific antigen binding molecule comprises not more than one antigen binding moiety capable of specific binding to an activating T cell antigen. In one embodiment the T cell activating bispecific antigen binding molecule provides monovalent binding to the activating T cell antigen. The activating T cell antigen binding moiety is a Fab molecule and comprises an identical VLCL light chain as the antigen binding moiety capable of specific binding to a target cell antigen. 
     In a particular embodiment the activating T cell antigen is CD3, particularly human CD3 or cynomolgus CD3, most particularly human CD3. In a particular embodiment the activating T cell antigen binding moiety is cross-reactive for (i.e. specifically binds to) human and cynomolgus CD3. In some embodiments, the activating T cell antigen is the epsilon subunit of CD3 (SEQ ID NO: 56). 
     In one embodiment, the activating T cell antigen binding moiety can compete with monoclonal antibody H2C (described in PCT publication no. WO2008/119567) for binding an epitope of CD3. In another embodiment, the activating T cell antigen binding moiety can compete with monoclonal antibody V9 (described in Rodrigues et al., Int J Cancer Suppl 7, 45-50 (1992) and U.S. Pat. No. 6,054,297) for binding an epitope of CD3. In yet another embodiment, the activating T cell antigen binding moiety can compete with monoclonal antibody FN18 (described in Nooij et al., Eur J Immunol 19, 981-984 (1986)) for binding an epitope of CD3. In a particular embodiment, the activating T cell antigen binding moiety can compete with monoclonal antibody SP34 (described in Pessano et al., EMBO J 4, 337-340 (1985)) for binding an epitope of CD3. In one embodiment, the activating T cell antigen binding moiety binds to the same epitope of CD3 as monoclonal antibody SP34. 
     In one embodiment, the activating T cell antigen binding moiety comprises the heavy chain CDR1 of SEQ ID NO: 14, the heavy chain CDR2 of SEQ ID NO: 15, the heavy chain CDR3 of SEQ ID NO: 16, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO: 34. In a further embodiment, the activating T cell antigen binding moiety comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, or variants thereof that retain functionality. 
     In a further embodiment, the activating T cell antigen binding moiety comprises a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31, or variants thereof that retain functionality. 
     In one embodiment the activating T cell antigen binding moiety comprises a heavy chain comprising the sequence of SEQ ID NO: 36 and a light chain comprising the sequence of SEQ ID NO: 31. 
     In one embodiment the activating T cell antigen binding moiety comprises a heavy chain of SEQ ID NO: 40 and a light chain comprising of SEQ ID NO: 35. 
     Target Cell Antigen Binding Moiety 
     The T cell activating bispecific antigen binding molecule of the invention comprises at least one antigen binding moiety capable of binding to a target cell antigen (also referred to herein as an “target cell antigen binding moiety”). In certain embodiments, the T cell activating bispecific antigen binding molecule comprises two antigen binding moieties capable of binding to a target cell antigen. In a particular such embodiment, each of these antigen binding moieties specifically binds to the same antigenic determinant. In one embodiment, the T cell activating bispecific antigen binding molecule comprises an immunoglobulin molecule capable of specific binding to a target cell antigen. In one embodiment the T cell activating bispecific antigen binding molecule comprises not more than two antigen binding moieties capable of binding to a target cell antigen. 
     The target cell antigen binding moiety is generally a Fab molecule that binds to a specific antigenic determinant and is able to direct the T cell activating bispecific antigen binding molecule to a target site, for example to a specific type of tumor cell that bears the antigenic determinant. Said Fab molecule has an identical VLCL light chain as the Fab molecule capable of specific binding to a T cell activating antigen. In a preferred embodiment said VLCL light chain of the Fab molecule capable of specific binding to a target cell antigen and the Fab molecule capable of specific binding to a T cell activating antigen comprise the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO: 34. In a preferred embodiment said VLCL light chain of the Fab molecule capable of specific binding to a target cell antigen and the Fab molecule capable of specific binding to a T cell activating antigen comprise a VLCL light chain of SEQ ID NO. 31. 
     In a further embodiment, the target cell antigen binding moiety comprises a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31, or variants thereof that retain functionality. 
     In a further embodiment, the target cell antigen binding moiety comprises a light chain sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35, or variants thereof that retain functionality. 
     In certain embodiments the target cell antigen binding moiety is directed to an antigen associated with a pathological condition, such as an antigen presented on a tumor cell or on a virus-infected cell. Suitable antigens are cell surface antigens, for example, but not limited to, cell surface receptors. In particular embodiments the antigen is a human antigen. In a specific embodiment the target cell antigen is selected from the group of Folate Receptor 1 (FoIR1), Mucin-1 (MUC1), and B Cell Maturation Antigen (BCMA). 
     In a specific embodiment the T cell activating bispecific antigen binding molecule comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 183, SEQ ID NO: 193, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 191, SEQ ID NO: 198, SEQ ID NO: 267, and SEQ ID NO: 272, including functional fragments or variants thereof. 
     Polynucleotides 
     The invention further provides isolated polynucleotides encoding a T cell activating bispecific antigen binding molecule as described herein or a fragment thereof. 
     Polynucleotides of the invention include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences set forth in SEQ ID NO: 183, SEQ ID NO: 193, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 191, SEQ ID NO: 198, SEQ ID NO: 267, and SEQ ID NO: 272, including functional fragments or variants thereof. 
     The polynucleotides encoding T cell activating bispecific antigen binding molecules of the invention may be expressed as a single polynucleotide that encodes the entire T cell activating bispecific antigen binding molecule or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional T cell activating bispecific antigen binding molecule. For example, the light chain portion of an antigen binding moiety may be encoded by a separate polynucleotide from the portion of the T cell activating bispecific antigen binding molecule comprising the heavy chain portion of the antigen binding moiety, an Fc domain subunit and optionally (part of) another antigen binding moiety. When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the antigen binding moiety. In another example, the portion of the T cell activating bispecific antigen binding molecule comprising one of the two Fc domain subunits and optionally (part of) one or more antigen binding moieties could be encoded by a separate polynucleotide from the portion of the T cell activating bispecific antigen binding molecule comprising the other of the two Fc domain subunits and optionally (part of) an antigen binding moiety. When co-expressed, the Fc domain subunits will associate to form the Fc domain. 
     In certain embodiments the polynucleotide or nucleic acid is DNA. In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). RNA of the present invention may be single stranded or double stranded. 
     Recombinant Methods 
     T cell activating bispecific antigen binding molecules of the invention may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production one or more polynucleotide encoding the T cell activating bispecific antigen binding molecule (fragment), e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one embodiment a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of a T cell activating bispecific antigen binding molecule (fragment) along with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the T cell activating bispecific antigen binding molecule (fragment) (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements. As used herein, a “coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5′ and 3′ untranslated regions, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g. a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the T cell activating bispecific antigen binding molecule (fragment) of the invention, or variant or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g. a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are “operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit A-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracyclins). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence). The expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs). 
     Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. For example, if secretion of the T cell activating bispecific antigen binding molecule is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding a T cell activating bispecific antigen binding molecule of the invention or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or “mature” form of the polypeptide. In certain embodiments, the native signal peptide, e.g. an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase. 
     DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the T cell activating bispecific antigen binding molecule may be included within or at the ends of the T cell activating bispecific antigen binding molecule (fragment) encoding polynucleotide. 
     In a further embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments a host cell comprising one or more vectors of the invention is provided. The polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one such embodiment a host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide that encodes (part of) a T cell activating bispecific antigen binding molecule of the invention. As used herein, the term “host cell” refers to any kind of cellular system which can be engineered to generate the T cell activating bispecific antigen binding molecules of the invention or fragments thereof. Host cells suitable for replicating and for supporting expression of T cell activating bispecific antigen binding molecules are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the T cell activating bispecific antigen binding molecule for clinical applications. Suitable host cells include prokaryotic microorganisms, such as  E. coli , or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like. For example, polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern. See Gerngross,  Nat Biotech  22, 1409-1414 (2004), and Li et al.,  Nat Biotech  24, 210-215 (2006). Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of  Spodoptera frugiperda  cells. Plant cell cultures can also be utilized as hosts. See e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham et al.,  J Gen Virol  36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather,  Biol Reprod  23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et al.,  Annals N.Y. Acad Sci  383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr −  CHO cells (Urlaub et al.,  Proc Natl Acad Sci USA  77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu,  Methods in Molecular Biology , Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., YO, NS0, Sp20 cell). 
     Standard technologies are known in the art to express foreign genes in these systems. Cells expressing a polypeptide comprising either the heavy or the light chain of an antigen binding domain such as an antibody, may be engineered so as to also express the other of the antibody chains such that the expressed product is an antibody that has both a heavy and a light chain. 
     In one embodiment, a method of producing a T cell activating bispecific antigen binding molecule according to the invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding the T cell activating bispecific antigen binding molecule, as provided herein, under conditions suitable for expression of the T cell activating bispecific antigen binding molecule, and recovering the T cell activating bispecific antigen binding molecule from the host cell (or host cell culture medium). 
     The components of the T cell activating bispecific antigen binding molecule are genetically fused to each other. T cell activating bispecific antigen binding molecule can be designed such that its components are fused directly to each other or indirectly through a linker sequence. The composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. Examples of linker sequences between different components of T cell activating bispecific antigen binding molecules are found in the sequences provided herein. Additional sequences may also be included to incorporate a cleavage site to separate the individual components of the fusion if desired, for example an endopeptidase recognition sequence. 
     In certain embodiments the one or more antigen binding moieties of the T cell activating bispecific antigen binding molecules comprise at least an antibody variable region capable of binding an antigenic determinant. Variable regions can form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods to produce polyclonal antibodies and monoclonal antibodies are well known in the art (see e.g. Harlow and Lane, “Antibodies, a laboratory manual”, Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see e.g. U.S. Pat. No. 5,969,108 to McCafferty). 
     Any animal species of antibody, antibody fragment, antigen binding domain or variable region can be used in the T cell activating bispecific antigen binding molecules of the invention. Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions useful in the present invention can be of murine, primate, or human origin. If the T cell activating bispecific antigen binding molecule is intended for human use, a chimeric form of antibody may be used wherein the constant regions of the antibody are from a human. A humanized or fully human form of the antibody can also be prepared in accordance with methods well known in the art (see e. g. U.S. Pat. No. 5,565,332 to Winter). Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human specificity-determining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) transplanting the entire non-human variable domains, but “cloaking” them with a human-like section by replacement of surface residues. Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson,  Front Biosci  13, 1619-1633 (2008), and are further described, e.g., in Riechmann et al.,  Nature  332, 323-329 (1988); Queen et al.,  Proc Natl Acad Sci USA  86, 10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al.,  Nature  321, 522-525 (1986); Morrison et al.,  Proc Natl Acad Sci  81, 6851-6855 (1984); Morrison and Oi,  Adv Immunol  44, 65-92 (1988); Verhoeyen et al.,  Science  239, 1534-1536 (1988); Padlan,  Molec Immun  31(3), 169-217 (1994); Kashmiri et al.,  Methods  36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,  Mol Immunol  28, 489-498 (1991) (describing “resurfacing”); Dall&#39;Acqua et al.,  Methods  36, 43-60 (2005) (describing “FR shuffling”); and Osbourn et al.,  Methods  36, 61-68 (2005) and Klimka et al.,  Br J Cancer  83, 252-260 (2000) (describing the “guided selection” approach to FR shuffling). Human antibodies and human variable regions can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel,  Curr Opin Pharmacol  5, 368-74 (2001) and Lonberg,  Curr Opin Immunol  20, 450-459 (2008). Human variable regions can form part of and be derived from human monoclonal antibodies made by the hybridoma method (see e.g.  Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions may also be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge (see e.g. Lonberg,  Nat Biotech  23, 1117-1125 (2005). Human antibodies and human variable regions may also be generated by isolating Fv clone variable region sequences selected from human-derived phage display libraries (see e.g., Hoogenboom et al. in  Methods in Molecular Biology  178, 1-37 (O&#39;Brien et al., ed.,  Human Press , Totowa, N.J., 2001); and McCafferty et al.,  Nature  348, 552-554; Clackson et al.,  Nature  352, 624-628 (1991)). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. 
     In certain embodiments, the antigen binding moieties useful in the present invention are engineered to have enhanced binding affinity according to, for example, the methods disclosed in U.S. Pat. Appl. Publ. No. 2004/0132066, the entire contents of which are hereby incorporated by reference. The ability of the T cell activating bispecific antigen binding molecule of the invention to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance technique (analyzed on a BIACORE T100 system) (Liljeblad, et al.,  Glyco J  17, 323-329 (2000)), and traditional binding assays (Heeley,  Endocr Res  28, 217-229 (2002)). Competition assays may be used to identify an antibody, antibody fragment, antigen binding domain or variable domain that competes with a reference antibody for binding to a particular antigen, e.g. an antibody that competes with the V9 antibody for binding to CD3. In certain embodiments, such a competing antibody binds to the same epitope (e.g. a linear or a conformational epitope) that is bound by the reference antibody. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). In an exemplary competition assay, immobilized antigen (e.g. CD3) is incubated in a solution comprising a first labeled antibody that binds to the antigen (e.g. V9 antibody) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen. The second antibody may be present in a hybridoma supernatant. As a control, immobilized antigen is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed, and the amount of label associated with immobilized antigen is measured. If the amount of label associated with immobilized antigen is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988)  Antibodies: A Laboratory Manual  ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). 
     T cell activating bispecific antigen binding molecules prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the T cell activating bispecific antigen binding molecule binds. For example, for affinity chromatography purification of T cell activating bispecific antigen binding molecules of the invention, a matrix with protein A or protein G may be used. Sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate a T cell activating bispecific antigen binding molecule essentially as described in the Examples. The purity of the T cell activating bispecific antigen binding molecule can be determined by any of a variety of well known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like. For example, the heavy chain fusion proteins expressed as described in the Examples were shown to be intact and properly assembled as demonstrated by reducing SDS-PAGE. Three bands were resolved at approximately Mr 25,000, Mr 50,000 and Mr 75,000, corresponding to the predicted molecular weights of the T cell activating bispecific antigen binding molecule light chain, heavy chain and heavy chain/light chain fusion protein. 
     Assays 
     T cell activating bispecific antigen binding molecules provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art. 
     Affinity Assays 
     The affinity of the T cell activating bispecific antigen binding molecule for an Fc receptor or a target antigen can be determined in accordance with the methods set forth in the Examples by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare), and receptors or target proteins such as may be obtained by recombinant expression. Alternatively, binding of T cell activating bispecific antigen binding molecules for different receptors or target antigens may be evaluated using cell lines expressing the particular receptor or target antigen, for example by flow cytometry (FACS). A specific illustrative and exemplary embodiment for measuring binding affinity is described in the following and in the Examples below. 
     According to one embodiment, K D  is measured by surface plasmon resonance using a BIACORE® T100 machine (GE Healthcare) at 25° C. 
     To analyze the interaction between the Fc-portion and Fc receptors, His-tagged recombinant Fc-receptor is captured by an anti-Penta His antibody (Qiagen) (“Penta His” disclosed as SEQ ID NO: 45) immobilized on CM5 chips and the bispecific constructs are used as analytes. Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier&#39;s instructions. Anti Penta-His antibody (“Penta His” disclosed as SEQ ID NO: 45) is diluted with 10 mM sodium acetate, pH 5.0, to 40 μg/ml before injection at a flow rate of 5 μl/min to achieve approximately 6500 response units (RU) of coupled protein. Following the injection of the ligand, 1 M ethanolamine is injected to block unreacted groups. Subsequently the Fc-receptor is captured for 60 s at 4 or 10 nM. For kinetic measurements, four-fold serial dilutions of the bispecific construct (range between 500 nM and 4000 nM) are injected in HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20, pH 7.4) at 25° C. at a flow rate of 30 μl/min for 120 s. 
     To determine the affinity to the target antigen, bispecific constructs are captured by an anti human Fab specific antibody (GE Healthcare) that is immobilized on an activated CM5-sensor chip surface as described for the anti Penta-His antibody (“Penta His” disclosed as SEQ ID NO: 45). The final amount of coupled protein is approximately 12000 RU. The bispecific constructs are captured for 90 s at 300 nM. The target antigens are passed through the flow cells for 180 s at a concentration range from 250 to 1000 nM with a flowrate of 30 μl/min. The dissociation is monitored for 180 s. 
     Bulk refractive index differences are corrected for by subtracting the response obtained on reference flow cell. The steady state response was used to derive the dissociation constant K D  by non-linear curve fitting of the Langmuir binding isotherm. Association rates (k on ) and dissociation rates (k off ) are calculated using a simple one-to-one Langmuir binding model (BIACORE® T100 Evaluation Software version 1.1.1) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K D ) is calculated as the ratio k off /k on . See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999). 
     Activity Assays 
     Biological activity of the T cell activating bispecific antigen binding molecules of the invention can be measured by various assays as described in the Examples. Biological activities may for example include the induction of proliferation of T cells, the induction of signaling in T cells, the induction of expression of activation markers in T cells, the induction of cytokine secretion by T cells, the induction of lysis of target cells such as tumor cells, and the induction of tumor regression and/or the improvement of survival. 
     Compositions, Formulations, and Routes of Administration 
     In a further aspect, the invention provides pharmaceutical compositions comprising any of the T cell activating bispecific antigen binding molecules provided herein, e.g., for use in any of the below therapeutic methods. In one embodiment, a pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises any of the T cell activating bispecific antigen binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below. 
     Further provided is a method of producing a T cell activating bispecific antigen binding molecule of the invention in a form suitable for administration in vivo, the method comprising (a) obtaining a T cell activating bispecific antigen binding molecule according to the invention, and (b) formulating the T cell activating bispecific antigen binding molecule with at least one pharmaceutically acceptable carrier, whereby a preparation of T cell activating bispecific antigen binding molecule is formulated for administration in vivo. 
     Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more T cell activating bispecific antigen binding molecule dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one T cell activating bispecific antigen binding molecule and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by  Remington&#39;s Pharmaceutical Sciences,  18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards or corresponding authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example,  Remington&#39;s Pharmaceutical Sciences,  18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. 
     The composition may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. T cell activating bispecific antigen binding molecules of the present invention (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, by inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g. liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example,  Remington&#39;s Pharmaceutical Sciences,  18th Ed. Mack Printing Company, 1990, incorporated herein by reference). Parenteral administration, in particular intravenous injection, is most commonly used for administering polypeptide molecules such as the T cell activating bispecific antigen binding molecules of the invention. 
     Parenteral compositions include those designed for administration by injection, e.g. subcutaneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal or intraperitoneal injection. For injection, the T cell activating bispecific antigen binding molecules of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks&#39; solution, Ringer&#39;s solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the T cell activating bispecific antigen binding molecules may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the T cell activating bispecific antigen binding molecules of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes. 
     Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in  Remington&#39;s Pharmaceutical Sciences  (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof. 
     In addition to the compositions described previously, the T cell activating bispecific antigen binding molecules may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the T cell activating bispecific antigen binding molecules may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. 
     Pharmaceutical compositions comprising the T cell activating bispecific antigen binding molecules of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. 
     The T cell activating bispecific antigen binding molecules may be formulated into a composition in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms. 
     Therapeutic Methods and Compositions 
     Any of the T cell activating bispecific antigen binding molecules provided herein may be used in therapeutic methods. T cell activating bispecific antigen binding molecules of the invention can be used as immunotherapeutic agents, for example in the treatment of cancers. 
     For use in therapeutic methods, T cell activating bispecific antigen binding molecules of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. 
     In one aspect, T cell activating bispecific antigen binding molecules of the invention for use as a medicament are provided. In further aspects, T cell activating bispecific antigen binding molecules of the invention for use in treating a disease are provided. In certain embodiments, T cell activating bispecific antigen binding molecules of the invention for use in a method of treatment are provided. In one embodiment, the invention provides a T cell activating bispecific antigen binding molecule as described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, the invention provides a T cell activating bispecific antigen binding molecule for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the T cell activating bispecific antigen binding molecule. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In further embodiments, the invention provides a T cell activating bispecific antigen binding molecule as described herein for use in inducing lysis of a target cell, particularly a tumor cell. In certain embodiments, the invention provides a T cell activating bispecific antigen binding molecule for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual comprising administering to the individual an effective amount of the T cell activating bispecific antigen binding molecule to induce lysis of a target cell. An “individual” according to any of the above embodiments is a mammal, preferably a human. 
     In a further aspect, the invention provides for the use of a T cell activating bispecific antigen binding molecule of the invention in the manufacture or preparation of a medicament. In one embodiment the medicament is for the treatment of a disease in an individual in need thereof. In a further embodiment, the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further embodiment, the medicament is for inducing lysis of a target cell, particularly a tumor cell. In still a further embodiment, the medicament is for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual comprising administering to the individual an effective amount of the medicament to induce lysis of a target cell. An “individual” according to any of the above embodiments may be a mammal, preferably a human. 
     In a further aspect, the invention provides a method for treating a disease. In one embodiment, the method comprises administering to an individual having such disease a therapeutically effective amount of a T cell activating bispecific antigen binding molecule of the invention. In one embodiment a composition is administered to said individual, comprising the T cell activating bispecific antigen binding molecule of the invention in a pharmaceutically acceptable form. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. An “individual” according to any of the above embodiments may be a mammal, preferably a human. 
     In a further aspect, the invention provides a method for inducing lysis of a target cell, particularly a tumor cell. In one embodiment the method comprises contacting a target cell with a T cell activating bispecific antigen binding molecule of the invention in the presence of a T cell, particularly a cytotoxic T cell. In a further aspect, a method for inducing lysis of a target cell, particularly a tumor cell, in an individual is provided. In one such embodiment, the method comprises administering to the individual an effective amount of a T cell activating bispecific antigen binding molecule to induce lysis of a target cell. In one embodiment, an “individual” is a human. 
     In certain embodiments the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferation disorders that can be treated using a T cell activating bispecific antigen binding molecule of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. In certain embodiments the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. A skilled artisan readily recognizes that in many cases the T cell activating bispecific antigen binding molecule may not provide a cure but may only provide partial benefit. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of T cell activating bispecific antigen binding molecule that provides a physiological change is considered an “effective amount” or a “therapeutically effective amount”. The subject, patient, or individual in need of treatment is typically a mammal, more specifically a human. 
     In some embodiments, an effective amount of a T cell activating bispecific antigen binding molecule of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of a T cell activating bispecific antigen binding molecule of the invention is administered to an individual for the treatment of disease. 
     For the prevention or treatment of disease, the appropriate dosage of a T cell activating bispecific antigen binding molecule of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the type of T cell activating bispecific antigen binding molecule, the severity and course of the disease, whether the T cell activating bispecific antigen binding molecule is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient&#39;s clinical history and response to the T cell activating bispecific antigen binding molecule, and the discretion of the attending physician. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. 
     The T cell activating bispecific antigen binding molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of T cell activating bispecific antigen binding molecule can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the T cell activating bispecific antigen binding molecule would be in the range from about 0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg body weight, about 5 microgram/kg body weight, about 10 microgram/kg body weight, about 50 microgram/kg body weight, about 100 microgram/kg body weight, about 200 microgram/kg body weight, about 350 microgram/kg body weight, about 500 microgram/kg body weight, about 1 milligram/kg body weight, about 5 milligram/kg body weight, about 10 milligram/kg body weight, about 50 milligram/kg body weight, about 100 milligram/kg body weight, about 200 milligram/kg body weight, about 350 milligram/kg body weight, about 500 milligram/kg body weight, to about 1000 mg/kg body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 microgram/kg body weight to about 500 milligram/kg body weight, etc., can be administered, based on the numbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the T cell activating bispecific antigen binding molecule). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. 
     The T cell activating bispecific antigen binding molecules of the invention will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, the T cell activating bispecific antigen binding molecules of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. 
     For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC 50  as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. 
     Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data. 
     Dosage amount and interval may be adjusted individually to provide plasma levels of the T cell activating bispecific antigen binding molecules which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC. 
     In cases of local administration or selective uptake, the effective local concentration of the T cell activating bispecific antigen binding molecules may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation. 
     A therapeutically effective dose of the T cell activating bispecific antigen binding molecules described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of a T cell activating bispecific antigen binding molecule can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50  (the dose lethal to 50% of a population) and the ED 50  (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 /ED 50 . T cell activating bispecific antigen binding molecules that exhibit large therapeutic indices are preferred. In one embodiment, the T cell activating bispecific antigen binding molecule according to the present invention exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED 50  with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient&#39;s condition (see, e.g., Fingl et al., 1975, in: The  Pharmacological Basis of Therapeutics , Ch. 1, p. 1, incorporated herein by reference in its entirety). 
     The attending physician for patients treated with T cell activating bispecific antigen binding molecules of the invention would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient. 
     Other Agents and Treatments 
     The T cell activating bispecific antigen binding molecules of the invention may be administered in combination with one or more other agents in therapy. For instance, a T cell activating bispecific antigen binding molecule of the invention may be co-administered with at least one additional therapeutic agent. The term “therapeutic agent” encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers. In a particular embodiment, the additional therapeutic agent is an anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent. 
     Such other agents are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of T cell activating bispecific antigen binding molecule used, the type of disorder or treatment, and other factors discussed above. The T cell activating bispecific antigen binding molecules are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate. 
     Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the T cell activating bispecific antigen binding molecule of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. T cell activating bispecific antigen binding molecules of the invention can also be used in combination with radiation therapy. 
     Articles of Manufacture 
     In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a T cell activating bispecific antigen binding molecule of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a T cell activating bispecific antigen binding molecule of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer&#39;s solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. 
     Screening Methods 
     Described herein is the advantageous efficiency of using a single light chain or highly homologous variants thereof for methods of identifying appropriate heavy chain variable regions to construct a T cell activating bispecific antigen binding molecules. To construct these binders, the light chain not only has to be able to pair with various heavy chains to produce binding moieties of different specificity but also retain the ability to form a binding moiety that can activate the T cells to which it binds. 
     The light chain described herein can be used as a common light chain (CLC) for identifying appropriate heavy chain variable regions, e.g., by screeining a library of heavy chain variable regions. This allows for maintaining the previously identified and validated CD3 binder such that merely a new target antigen binder for the target antigen binding moiety of a T cell activating bispecific antigen binding molecules has to be identified. 
     Accordingly, in another aspect, the invention provides for a method for identifying a variable heavy chain for use in a bispecific antigen binding molecule specific for a T cell activation antigen and a target cell antigen, comprising the step of screening a combinatorial library comprising variable heavy chains with a light chain comprising the amino acid sequence of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 31. In one embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 35. This method can be used to develop stable, functional, high affinity binders to improve production of T cell activating bispecific antigen binding molecules with, e.g., CD3 specificity and a target antigen specificity, where the target antigens are unrelated, e.g., FoIR1, MUC1, and BCMA. 
     Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference. 
     EXAMPLES 
     General Methods 
     Recombinant DNA Techniques 
     Standard methods were used to manipulate DNA as described in Sambrook et al.,  Molecular cloning: A laboratory manual ; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The molecular biological reagents were used according to the manufacturers&#39; instructions. General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A. et al., (1991)  Sequences of Proteins of Immunological Interest,  5 th  ed., NIH Publication No. 91-3242. 
     DNA Sequencing 
     DNA sequences were determined by standard double strand sequencing at Synergene (Schlieren). 
     Gene Synthesis 
     Desired gene segments where required were either generated by PCR using appropriate templates or were synthesized by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. In cases where no exact gene sequence was available, oligonucleotide primers were designed based on sequences from closest homologues and the genes were isolated by RT-PCR from RNA originating from the appropriate tissue. The gene segments flanked by singular restriction endonuclease cleavage sites were cloned into standard cloning/sequencing vectors. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments were designed with suitable restriction sites to allow sub-cloning into the respective expression vectors. All constructs were designed with a 5′-end DNA sequence coding for a leader peptide which targets proteins for secretion in eukaryotic cells. 
     Isolation of Primary Human Pan T Cells from PBMCs 
     Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation from enriched lymphocyte preparations (buffy coats) obtained from local blood banks or from fresh blood from healthy human donors. Briefly, blood was diluted with sterile PBS and carefully layered over a Histopaque gradient (Sigma, H8889). After centrifugation for 30 minutes at 450×g at room temperature (brake switched off), part of the plasma above the PBMC containing interphase was discarded. The PBMCs were transferred into new 50 ml Falcon tubes and tubes were filled up with PBS to a total volume of 50 ml. The mixture was centrifuged at room temperature for 10 minutes at 400×g (brake switched on). The supernatant was discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps at 4° C. for 10 minutes at 350×g). The resulting PBMC population was counted automatically (ViCell) and stored in RPM11640 medium, containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37° C., 5% CO 2  in the incubator until assay start. 
     T cell enrichment from PBMCs was performed using the Pan T Cell Isolation Kit II (Miltenyi Biotec #130-091-156), according to the manufacturer&#39;s instructions. Briefly, the cell pellets were diluted in 40 μl cold buffer per 10 million cells (PBS with 0.5% BSA, 2 mM EDTA, sterile filtered) and incubated with 10 μl Biotin-Antibody Cocktail per 10 million cells for 10 min at 4° C. 30 μl cold buffer and 20 μl Anti-Biotin magnetic beads per 10 million cells were added, and the mixture incubated for another 15 min at 4° C. Cells were washed by adding 10-20× the current volume and a subsequent centrifugation step at 300×g for 10 min. Up to 100 million cells were resuspended in 500 μl buffer. Magnetic separation of unlabeled human pan T cells was performed using LS columns (Miltenyi Biotec #130-042-401) according to the manufacturer&#39;s instructions. The resulting T cell population was counted automatically (ViCell) and stored in AIM-V medium at 37° C., 5% CO 2  in the incubator until assay start (not longer than 24 h). 
     Isolation of Primary Human Naive T Cells from PBMCs 
     Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation from enriched lymphocyte preparations (buffy coats) obtained from local blood banks or from fresh blood from healthy human donors. T-cell enrichment from PBMCs was performed using the Naive CD8 +  T cell isolation Kit from Miltenyi Biotec (#130-093-244), according to the manufacturer&#39;s instructions, but skipping the last isolation step of CD8 +  T cells (also see description for the isolation of primary human pan T cells). 
     Isolation of Murine Pan T Cells from Splenocytes 
     Spleens were isolated from C57BL/6 mice, transferred into a GentleMACS C-tube (Miltenyi Biotech #130-093-237) containing MACS buffer (PBS+0.5% BSA+2 mM EDTA) and dissociated with the GentleMACS Dissociator to obtain single-cell suspensions according to the manufacturer&#39;s instructions. The cell suspension was passed through a pre-separation filter to remove remaining undissociated tissue particles. After centrifugation at 400×g for 4 min at 4° C., ACK Lysis Buffer was added to lyse red blood cells (incubation for 5 min at room temperature). The remaining cells were washed with MACS buffer twice, counted and used for the isolation of murine pan T cells. The negative (magnetic) selection was performed using the Pan T Cell Isolation Kit from Miltenyi Biotec (#130-090-861), following the manufacturer&#39;s instructions. The resulting T cell population was automatically counted (ViCell) and immediately used for further assays. 
     Isolation of Primary Cynomolgus PBMCs from Heparinized Blood 
     Peripheral blood mononuclar cells (PBMCs) were prepared by density centrifugation from fresh blood from healthy cynomolgus donors, as follows: Heparinized blood was diluted 1:3 with sterile PBS, and Lymphoprep medium (Axon Lab #1114545) was diluted to 90% with sterile PBS. Two volumes of the diluted blood were layered over one volume of the diluted density gradient and the PBMC fraction was separated by centrifugation for 30 min at 520×g, without brake, at room temperature. The PBMC band was transferred into a fresh 50 ml Falcon tube and washed with sterile PBS by centrifugation for 10 min at 400×g at 4° C. One low-speed centrifugation was performed to remove the platelets (15 min at 150×g, 4° C.), and the resulting PBMC population was automatically counted (ViCell) and immediately used for further assays. 
     Exemplary Antigen Generation 
     The antigen is expressed in two different versions. For a non-Fc containing construct the extracellular domain is fused to an avi-His tag attached to its C-terminus. For an Fc containing antigen an Fc fusion using the knob-into-hole technology to the N-terminus of a heterodimeric Fc part is applied (Merchant et al.) to have only one molecule of the protein of interest per Fc dimer. This is done to avoid the formation of any artificial dimeric structures of the protein of interest. The avi-tag is here attached to the C-terminus of the Fc. Those proteins can easily be transiently expressed in mammalian cells like HEK or CHO, purified via ProteinA chromatography, biotinylted and can be attached to streptavidin beads for phage selections via the biotinylated avi-tag according to standard methods. 
     Affinity Maturation 
     Affinity maturation of the Fab fragments coming out of a CLC-library has to be limited to the heavy chain only, in order to retain the light chain in an unchanged manner to have the CD3e binding affinity retained. Since our initial library is randomized in the CDR3 of the heavy chain only, we focus for the maturation step on the CDRs 1 and 2. For this we design PCR primers for each framework that will introduce randomization in to CDR1 and CDR2 separately. Each primer is designed to bind to one of the six heavy chain frameworks and can be used in a generic way for each antibody clone coming from that particular phage library. The process of maturation is carried out as described by Knappik, or by Steidl (Knappik et al.,  J. Mol. Biol . (2000) 296, 57-86). S. Steidl et al.;  Molecular Immunology  46 (2008) 135-144). 
     Phage panning is carried out as described above, with the difference that the concentrations of the soluble antigen is used at 2×10{circumflex over ( )}−8M and is decreased to a final concentration to 2×10{circumflex over ( )}−10M. 
     Cloning, Production, Purification and Biochemical Characterization of CLC TCB 
     The resulting variable region of heavy and light chain DNA sequences are subcloned in frame with either the constant heavy chain or the constant light chain pre-inserted into the respective recipient mammalian expression vector. The antibody expression is driven by an MPSV promoter and carries a synthetic polyA signal sequence at the 3′ end of the CDS. In addition each vector contains an EBV OriP sequence for transient expression in HEK293-EBNA cells. As antibody isotypes IgG1 P329G LALA or IgG4 SPLE PG are used. 
     The CLC TCB is produced by co-transfecting HEK293-EBNA cells with the mammalian expression vectors using polyethylenimine. The cells are transfected with the corresponding expression vectors in a 1:1:3 ratio (“vector heavy chain Fc(hole)”:“vector heavy chain Fc(knob)-FabCrossfab”):“vector common light chain”. 
     For transfection HEK293 EBNA cells are cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells are seeded 24 hours before transfection. For transfection cells are centrifuged for 5 min by 210×g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 200 g DNA. After addition of 540 μl PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium is added and cell are cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 is added. After 7 days cultivation supernatant is collected for purification by centrifugation for 15 min at 210×g, the solution is sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% w/v is added, and kept at 4° C. 
     The secreted protein is purified from cell culture supernatants by affinity chromatography using ProteinA. Supernatant is loaded on a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibrated with 40 ml 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein is removed by washing with at least 10 column volume 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Target protein is eluted during a gradient over 20 column volume from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 to 20 mM sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solution is neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Target protein is concentrated and filtrated prior loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine, 140 mM sodium chloride solution of pH 6.0. 
     The protein concentration of purified protein samples is determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. 
     Purity and molecular weight of molecules are analyzed by CE-SDS analyses in the presence and absence of a reducing agent. The Caliper LabChip GXII system (Caliper lifescience) is used according to the manufacturer&#39;s instruction. 2 ug sample is used for analyses. 
     The aggregate content of antibody samples is analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25° C. 
     CLC TCB Characterization in Cell-Based Assays 
     Binding of CLC TCB to ECD Tumor Antigen- and CD3-Expressing Cells 
     The binding of CLC TCB is tested using tumor cells expressing the antigen X of interest and a CD3e-expressing immortalized T lymphocyte line (Jurkat). Briefly, cells are harvested, counted, checked for viability and resuspended at 2×106 cells/ml in FACS buffer (100 μl PBS 0.1% BSA). 100 μl of cell suspension (containing 0.2×106 cells) is incubated in round-bottom 96-well plate for 30 min at 4° C. with increasing concentrations of the CLC TCB (3 pM-200 nM), washed twice with cold PBS 0.1% BSA, re-incubated for further 30 min at 4° C. with the PE-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgG Fcg Fragment Specific secondary antibody (Jackson Immuno Research Lab PE #109-116-170), washed twice with cold PBS 0.1% BSA and immediately analyzed by FACS using a FACS Cantoll (Software FACS Diva) by gating live, DAPI-negative, cells. Binding curves are obtained using GraphPadPrism5. 
     Example 1 
     Purification of Biotinylated Folate Receptor-Fc Fusions 
     To generate new antibodies against human FoIR1 the following antigens and screening tools were generated as monovalent Fc fusion proteins (the extracellular domain of the antigen linked to the hinge region of Fc-knob which is co-expressed with an Fc-hole molecule). The antigen genes were synthesized (Geneart, Regensburg, Germany) based on sequences obtained from GenBank or SwissProt and inserted into expression vectors to generate fusion proteins with Fc-knob with a C-terminal Avi-tag for in vivo or in vitro biotinylation. In vivo biotinylation was achieved by co-expression of the bacterial birA gene encoding a bacterial biotin ligase during production. Expression of all genes was under control of a chimeric MPSV promoter on a plasmid containing an oriP element for stable maintenance of the plasmids in EBNA containing cell lines. 
     For preparation of the biotinylated monomeric antigen/Fc fusion molecules, exponentially growing suspension HEK293 EBNA cells were co-transfected with three vectors encoding the two components of fusion protein (knob and hole chains) as well as BirA, an enzyme necessary for the biotinylation reaction. 30 The corresponding vectors were used at a 9.5:9.5:1 ratio (“antigen ECD-Fc knob-avi tag”:“Fc hole”:“BirA”). 
     For protein production in 500 ml shake flasks, 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 minutes at 210 g, and supernatant was replaced by pre-warmed CD CHO medium. Expression vectors were resuspended in 20 mL of CD CHO medium containing 200 μg of vector DNA. After addition of 540 μL of polyethylenimine (PEI), the solution was mixed for 15 seconds and incubated for 10 minutes at room temperature. Afterwards, cells were mixed with the DNA/PEI solution, transferred to a 500 mL shake flask and incubated for 3 hours at 37° C. in an incubator with a 5% CO 2  atmosphere. After the incubation, 160 mL of F17 medium was added and cells were cultured for 24 hours. One day after transfection, 1 mM valproic acid and 7% Feed 1 (Lonza) were added to the culture. The production medium was also supplemented with 100 μM biotin. After 7 days of culturing, the cell supernatant was collected by spinning down cells for 15 min at 210 g. The solution was sterile filtered (0.22 μm filter), supplemented with sodium azide to a final concentration of 0.01% (w/v), and kept at 4° C. 
     Secreted proteins were purified from cell culture supernatants by affinity chromatography using Protein A, followed by size exclusion chromatography. For affinity chromatography, the supernatant was loaded on a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibrated with 40 mL 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Unbound protein was removed by washing with at least 10 column volumes of 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. The bound protein was eluted using a linear pH-gradient created over 20 column volumes of 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0. The column was then washed with 10 column volumes of 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0. 
     pH of collected fractions was adjusted by adding 1/10 (v/v) of 0.5 M sodium phosphate, pH 8.0. The protein was concentrated and filtered prior to loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.0. 
     The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the FoIR1-Fc-fusion was analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). The aggregate content of samples was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25° C. 
     Purified antigen-Fc-fusion proteins were analyzed by surface plasmon resonance assays using commercially available antibodies to confirm correct and natural conformation of the antigens (data not shown). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Antigens produced for isolation, selection and 
               
               
                 counter selection of human FoIR1 antibodies 
               
            
           
           
               
               
               
               
               
            
               
                   
                 ECD 
                 Accession 
                   
                 Seq ID 
               
               
                 Antigen 
                 (aa) 
                 number 
                 Sequence 
                 No 
               
               
                   
               
               
                 human 
                 25-234 
                 P15328 
                 RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQ 
                 227 
               
               
                 FoIR1 
                   
                   
                 CRPWRKNACCSTNTSQEAHKDVSYLYRFNWNH 
                   
               
               
                   
                   
                   
                 CGEMAPACKRHFIQDTCLYECSPNLGPWIQQV 
                   
               
               
                   
                   
                   
                 DQSWRKERVLNVPLCKEDCEQVWVEDCRTSYT 
                   
               
               
                   
                   
                   
                 CKSNWHKGWNVVTSGFNKCAVGAACQPFHFYF 
                   
               
               
                   
                   
                   
                 PTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWF 
                   
               
               
                   
                   
                   
                 DPAQGNPNEEVARFYAAAM 
                   
               
               
                   
               
               
                 human 
                 17-230 
                 P14207 
                 TMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLH 
                 228 
               
               
                 FoIR2 
                   
                   
                 DQCSPWKKNACCTASTSQELHKDTSRLYNFNW 
                   
               
               
                   
                   
                   
                 DHCGKMEPACKRHFIQDTCLYECSPNLGPWIQ 
                   
               
               
                   
                   
                   
                 QVNQSWRKERFLDVPLCKEDCQRVWVEDCHTS 
                   
               
               
                   
                   
                   
                 HTCKSNWHRGWDVVTSGVNKCPAGALCRTFES 
                   
               
               
                   
                   
                   
                 YFPTPAALCEGLWSHSYKVSNYSRGSGRCIQM 
                   
               
               
                   
                   
                   
                 WFDSAQGNPNEEVARFYAAAMHVN 
                   
               
               
                   
               
               
                 human 
                 24-243 
                 P41439 
                 SARARTDLLNVCMNAKHHKTQPSPEDELYGQC 
                 229 
               
               
                 FoIR3 
                   
                   
                 SPWKKNACCTASTSQELHKDTSRLYNFNWDHC 
                   
               
               
                   
                   
                   
                 GKMEPTCKRHFIQDSCLYECSPNLGPWIRQVN 
                   
               
               
                   
                   
                   
                 QSWRKERILNVPLCKEDCERVWVEDCRTSYTC 
                   
               
               
                   
                   
                   
                 KSNWHKGWNVVTSGINECPAGALCSTFESYFP 
                   
               
               
                   
                   
                   
                 TPAALCEGLWSHSFKVSNYSRGSGRCIQMWFD 
                   
               
               
                   
                   
                   
                 SAQGNPNEEVAKFYAAAMNAGAPSRGIIDS 
                   
               
               
                   
               
               
                 murine 
                 25-232 
                 P35846 
                 TRARTELLNVCMDAKHHKEKPGPEDNLHDQCS 
                 230 
               
               
                 FoIR1 
                   
                   
                 PWKTNSCCSTNTSQEAHKDISYLYRFNWNHCG 
                   
               
               
                   
                   
                   
                 TMTSECKRHFIQDTCLYECSPNLGPWIQQVDQ 
                   
               
               
                   
                   
                   
                 SWRKERILDVPLCKEDCQQVWVEDCQSSFTCK 
                   
               
               
                   
                   
                   
                 SNWHKGWNWSSGHNECPVGASCHPFTFYFPTS 
                   
               
               
                   
                   
                   
                 AALCEEIWSHSYKLSNYSRGSGRCIQMWFDPA 
                   
               
               
                   
                   
                   
                 QGNPNEEVARFYAEAMS 
                   
               
               
                   
               
               
                 cynomolgus 
                 25-234 
                 G7PR14 
                 EAQTRTARARTELLNVCMNAKHHKEKPGPEDK 
                 231 
               
               
                 FoIR1 
                   
                   
                 LHEQCRPWKKNACCSTNTSQEAHKDVSYLYRF 
                   
               
               
                   
                   
                   
                 NWNHCGEMAPACKRHFIQDTCLYECSPNLGPW 
                   
               
               
                   
                   
                   
                 IQQVDQSWRKERVLNVPLCKEDCERVWVEDCR 
                   
               
               
                   
                   
                   
                 TSYCKSNWHKGWNWTSGFNKCPVGAACQPFHF 
                   
               
               
                   
                   
                   
                 YFPTPTVLCNEIWTYSYKVSNYSRGSGRCIQM 
                   
               
               
                   
                   
                   
                 WFDPAQGNPNEEVARFYAAAMS 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Summary of the yield and final monomer content of  
               
               
                 the FoIR- Fc- fusions. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Monomer 
                   
               
               
                   
                   
                 [%] 
                   
               
               
                   
                 Antigen 
                 (SEC) 
                 Yield 
               
               
                   
                   
               
               
                   
                 huFoIR1 
                 100 
                 30 mg/L 
               
               
                   
                 cyFoIR1 
                 100 
                 32 mg/L 
               
               
                   
                 muFoIR1 
                 100 
                 31 mg/L 
               
               
                   
                 huFoIR2 
                 100 
                 16 mg/L 
               
               
                   
                 huFoIR3 
                  95 
                 38 mg/L 
               
               
                   
                   
               
            
           
         
       
     
     Example 2 
     Generation of Common Light Chain with CD3ε Specificity 
     The T cell activating bispecific molecules described herein comprise at least one CD3 binding moiety. This moiety can be generated by immunizing laboratory animals, screening phage library or using known anti-CD3 antibodies. The common light chain with CD3ε specificity was generated by humanizing the light chain of a murine parental anti-CD3ε antibody (CH2527). For humanization of an antibody of non-human origin, the CDR residues from the non-human antibody (donor) have to be transplanted onto the framework of a human (acceptor) antibody. Generally, acceptor framework sequences are selected by aligning the sequence of the donor to a collection of potential acceptor sequences and choosing one that has either reasonable homology to the donor, or shows similar amino acids at some positions critical for structure and activity. In the present case, the search for the antibody acceptor framework was performed by aligning the mouse VL-domain sequence of the parental antibody to a collection of human germline sequences and choosing the human sequence that showed high sequence identity. Surprisingly, a good match in terms of framework sequence homology was found in a rather infrequent human light chain belonging to the V-domain family 7 of the lambda type, more precisely, hVL_7_46 (IMGT nomenclature, GenBank Acc No. Z73674). This infrequent human light chain was subsequently chosen as acceptor framework for humanization of the light chain of CH2527. The three complementarity determining regions (CDRs) of the mouse light chain variable domain were grafted onto this acceptor framework. Since the framework 4 region is not part of the variable region of the germline V-gene, the alignment for this region (J-element) was done individually. Hence the IGLJ3-02 sequence was chosen for humanization of this light chain. 
     Thirteen humanized variants were generated (CH2527-VL7_46-1 to VL7_46-10, VL7_46-12 to VL7_46-14). These differ in framework residues (and combinations thereof) that were back-mutated to the murine V-domain sequence or in CDR-residues (Kabat definition) that could be kept identical to the human germline sequence. The following framework residues outside the CDRs were back-mutated to the murine residues in the final humanized VL-domain variant VL7_46-13 (murine residues listed): V36, E38, F44, G46, G49, and G57, respectively. The human J-element IGLJ3-02 was 100% identical to the J-element of the murine parental antibody. 
     Example 3 
     SPR Assessment of Humanized Variants with CD3ε Specificity 
     Humanized VL variants were assessed as chimera in a 2+1 classical format ( FIG. 1D ), i.e. humanized light chain V-domains were paired with murine heavy chain V-domains. SPR assessment was carried out on a ProteOn XPR36 instrument (Bio-Rad). More precisely, the variants were captured directly from the culture supernatant on an anti-Fab derivatized GLM sensorchip (Goat Anti-Human IgG, F(ab′)2 Fragment Specific, Jackson ImmunoResearch) in vertical orientation. The following analytes were subsequently injected horizontally as single concentrations to assess binding to human and cynomolgus CD3ε: 3 μM hu CD3ε(-1-26)-Fc(knob)-avi (ID807) and 2.5 μM cy CD3ε-(-1-26)-Fc(knob)-Avi-Fc(hole) (ID873), respectively. Binding responses were qualitatively compared to binding of the murine control construct and graded + (comparable binding observed), +/− (reduced binding observed) and − (no binding observed). The capture antibody was regenerated after each cycle of ligand capture and analyte binding and the murine construct was re-injected at the end of the study to confirm the activity of the capture surface. The results are summarized in Table 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Qualitative binding assessment based on SPR for the humanized light 
               
               
                 chain variants combined with the murine heavy chain of CH2527. 
               
               
                 Only the humanized light chain variant that was finally chosen, 
               
               
                 CH2527-VL7_46-13, highlighted in bold letters,  
               
               
                 exhibited comparable binding to human and cynomolgus CD3ε. 
               
            
           
           
               
               
            
               
                 Humanized VL variant 
                 Binding to CD3 ε   
               
               
                   
               
               
                 Murine CH2527-VL 
                 
                   + 
                 
               
               
                 CH2527-VL7 46-1 
                 
                   − 
                 
               
               
                 CH2527-VL7 46-2 
                 
                   − 
                 
               
               
                 CH2527-VL7 46-3 
                 
                   − 
                 
               
               
                 CH2527-VL7 46-4 
                 
                   − 
                 
               
               
                 CH2527-VL7 46-5 
                 
                   − 
                 
               
               
                 CH2527-VL7 46-6 
                 
                   − 
                 
               
               
                 CH2527-VL7 46-7 
                 
                   − 
                 
               
               
                 CH2527-VL7 46-8 
                 
                   − 
                 
               
               
                 CH2527-VL7 46-9 
                 
                   − 
                 
               
               
                 CH2527-VL7 46-10 
                 
                   − 
                 
               
               
                 CH2527-VL7 46-12 
                 
                   +/− 
                 
               
               
                 
                   CH2527-VL7 46-13 
                 
                 
                   
                     + 
                   
                 
               
               
                 CH2527-VL7 46-14 
                 
                   − 
                 
               
               
                   
               
            
           
         
       
     
     Example 4 
     Properties of Humanized Common Light Chain with CD3ε Specificity 
     The light chain V-domain variant that was chosen for the humanized lead molecule is VL7_46-13. The degree of humanness, i.e. the sequence homology of the humanized V-domain to the human germline V-domain sequence was determined. For VL7_46-13, the overall sequence identity with the closest human germline homolog is 65% before humanization and 80% afterwards. Omitting the CDR regions, the sequence identity is 92% to the closest human germline homolog. As can be seen from Table 3, VL7_46-13 is the only humanized VL variant out of a panel of 13 variants that showed comparable binding to the parental murine antibody and also retained its cross-reactivity to cynomolgus CD3ε. This result indicates that it was not trivial to humanize the murine VL-domain without losing binding affinity to CD3ε which required several back-mutations to murine framework residues (in particular G46) while retaining G24 in CDR1. In addition, this result shows that the VL-domain plays a crucial role in target recognition. Importantly, the humanized VL-domain VL7_46-13 based on an infrequent human germline belonging to the V-domain family 7 of the lambda type and retaining affinity and specificity for CD3ε, is also suitable to be used as a common light chain in phage-displayed antibody libraries of the Fab-format and enables successful selection for novel specificities which greatly facilitates the generation and production of bispecific molecules binding to CD3ε and e.g. a tumor target and sharing the same ‘common’ light chain. 
     Example 5 
     Generation of a Phage Displayed Antibody Library Using a Human Germ-Line Common Light Chain Derived from HVK1-39 
     Several approaches to generate bispecific antibodies that resemble full length human IgG utilize modifications in the Fc region that induce heterodimerization of two distinct heavy chains. Such examples include knobs-into-holes (Merchant et al.,  Nat Biotechnol.  1998 July; 16(7):677-81) SEED (Davis et al.,  Protein Eng Des Sel.  2010 April; 23(4):195-202) and electrostatic steering technologies (Gunasekaran et al.,  J Biol Chem.  2010 Jun. 18; 285(25):19637-46). Although these approaches enable effective heterodimerization of two distinct heavy chains, appropriate pairing of cognate light and heavy chains remains a problem. Usage of a common light chain (LC) can solve this issue (Merchant, et al. Nat Biotech 16, 677-681 (1998)). 
     Here, we describe the generation of an antibody library for the display on a M13 phage. Essentially, we designed a multi framework library for the heavy chain with one constant (or “common”) light chain. This library is designed for generating multispecific antibodies without the need to use sophisticated technologies to avoid light chain mispairing. 
     By using a common light chain the production of these molecules can be facilitated as no mispairing occurs any longer and the isolation of a highly pure bispecific antibody is facilitated. As compared to other formats the use of Fab fragments as building blocks as opposed to e.g. the use of scFv fragments results in higher thermal stability and the lack of scFv aggregation and intermolecular scFv formation. 
     Library Generation 
     In the following the generation of an antibody library for the display on M13 phage is described. Essentially, we designed a multi framework library for the heavy chain with one constant (or “common”) light chain. 
     We used these heavy chains in the library (GenBank Accession Numbers in brackets): 
     IGHV1-46*01 (X92343) (SEQ ID NO:104), 
     IGHV1-69*06 (L22583), (SEQ ID NO:105) 
     IGHV3-15*01 (X92216), (SEQ ID NO:106) 
     IGHV3-23*01 (M99660), (SEQ ID NO:107) 
     IGHV4-59*01 (AB019438), (SEQ ID NO:108) 
     IGHV5-51*01 (M99686), (SEQ ID NO:109) 
     All heavy chains use the IGHJ2 as J-element, except the IGHV1-69*06 which uses IGHJ6 sequence. The design of the randomization included the CDR-H1, CDR-H2, and CDR-H3. For CDR-H1 and CDR-H2 a “soft” randomization strategy was chosen, and the randomization oligonucleotides were such that the codon for the amino acid of the germ-line sequence was present at 50%. All other amino acids, except cysteine, were summing up for the remaining 50%. In CDR-H3, where no germ-line amino acid is present due to the presence of the genetic D-element, oligonucleotides were designed that allow for the usage of randomized inserts between the V-element and the J-element of 4 to 9 amino acids in length. Those oligonucleotides contained in their randomized part e.g. The three amino acids G/Y/S are present to 15% each, those amino acids A/D/T/R/P/L/V/N/W/F/I/E are present to 4,6% each. 
     Exemplary methods for generation of antibody libraries are described in Hoogenboom et al.,  Nucleic Acids Res.  1991, 19, 4133-413; Lee et., al  J. Mol. Biol . (2004) 340, 1073-1093. 
     The light chain is derived from the human sequence hVK1-39, and is used in an unmodified and non-randomized fashion. This will ensure that the same light chain can be used for other projects without additional modifications. 
     Exemplary Library Selection: 
     Selections with all affinity maturation libraries are carried out in solution according to the following procedure using a monomeric and biotinylated extracellular domain of a target antigen X. 
     1. 10{circumflex over ( )}12 phagemid particles of each library are bound to 100 nM biotinylated soluble antigen for 0.5 h in a total volume of 1 ml. 2. Biotinylated antigen is captured and specifically bound phage particles are isolated by addition of ˜5×10{circumflex over ( )}7 streptavidin-coated magnetic beads for 10 min. 3. Beads are washed using 5-10×1 ml PBS/Tween20 and 5-10×1 ml PBS. 4. Elution of phage particles is done by addition of 1 ml 100 mM TEA (triethylamine) for 10 min and neutralization by addition of 500 ul 1 M Tris/HCl pH 7.4 and 5. Re-infection of exponentially growing  E. coli  TG1 bacteria, infection with helper phage VCSM13 and subsequent PEG/NaCl precipitation of phagemid particles is applied in subsequent selection rounds. Selections are carried out over 3-5 rounds using either constant or decreasing (from 10{circumflex over ( )}−7M to 2×10{circumflex over ( )}−9M) antigen concentrations. In round 2, capture of antigen/phage complexes is performed using neutravidin plates instead of streptavidin beads. All binding reactions are supplemented either with 100 nM bovine serum albumin, or with non-fat milk powder in order to compete for unwanted clones arising from mere sticky binding of the antibodies to the plastic support. 
     Selections are being carried out over three or four rounds using decreasing antigen concentrations of the antigen starting from 100 nM and going down to 5 nM in the final selection round. Specific binders are defined as signals ca. 5× higher than background and are identified by ELISA. Specific binders are identified by ELISA as follows: 100 μl of 10 nM biotinylated antigen per well are coated on neutravidin plates. Fab-containing bacterial supernatants are added and binding Fabs are detected via their Flag-tags by using an anti-Flag/HRP secondary antibody. ELISA-positive clones are bacterially expressed as soluble Fab fragments in 96-well format and supernatants are subjected to a kinetic screening experiment by SPR-analysis using ProteOn XPR36 (BioRad). Clones expressing Fabs with the highest affinity constants are identified and the corresponding phagemids are sequenced. For further characterization, the Fab sequences are amplified via PCR from the phagemid and cloned via appropriate restriction sites into human IgG1 expression vectors for mammalian production. 
     Generation of a Phage Displayed Antibody Library Using a Humanized CD3ε Specific Common Light Chain 
     Here, the generation of an antibody library for the display on M13 phage is described. Essentially, we designed a multi framework library for the heavy chain with one constant (or “common”) light chain. This library was designed for the generation of Fc-containing, but FcgR binding inactive T cell bispecific antibodies of IgG1 P329G LALA or IgG4 SPLE PG isotype in which one or two Fab recognize a tumor surface antigen expressed on a tumor cell whereas the remaining Fab arm of the antibody recognizes CD3e on a T cell. 
     Library Generation 
     In the following the generation of an antibody library for the display on M13 phage is described. Essentially, we designed a multi framework library for the heavy chain with one constant (or “common”) light chain. This library is designed solely for the generation of Fc-containing, but FcgR binding inactive T cell bispecific antibodies of IgG1 P329G LALA or IgG4 SPLE PG isotype. 
     Diversity was introduced via randomization oligonucleotides only in the CDR3 of the different heavy chains. Methods for generation of antibody libraries are well known in the art and are described in (Hoogenboom et al., Nucleic Acids Res. 1991, 19, 4133-413; or in: Lee et., al J. Mol. Biol. (2004) 340, 1073-1093). 
     We used these heavy chains in the library: 
     IGHV1-46*01 (X92343), (SEQ ID NO:104) 
     IGHV1-69*06 (L22583), (SEQ ID NO:105) 
     IGHV3-15*01 (X92216), (SEQ ID NO:106) 
     IGHV3-23*01 (M99660), (SEQ ID NO:107) 
     IGHV4-59*01 (AB019438), (SEQ ID NO:108) 
     IGHV5-51*01 (M99686), (SEQ ID NO:109) 
     We used the light chain derived from the humanized human and Cynomolgus CD3 ε specific antibody CH2527 in the library: (VL7_46-13; SEQ ID NO:112). This light chain was not randomized and used without any further modifications in order to ensure compatibility with different bispecific binders. 
     All heavy chains use the IGHJ2 as J-element, except the IGHV1-69*06 which uses IGHJ6 sequence. The design of the randomization focused on the CDR-H3 only, and PCR oligonucleotides were designed that allow for the usage of randomized inserts between the V-element and the J-element of 4 to 9 amino acids in length. 
     Example 6 
     Selection of Antibody Fragments from Common Light Chain Libraries (Comprising Light Chain with CD3ε Specificity) to FoIR1 
     The antibodies 16A3, 15A1, 18D3, 19E5, 19A4, 15H7, 15B6, 16D5, 15E12, 21D1, 16F12, 21A5, 21G8, 19H3, 20G6, and 20H7 comprising the common light chain VL7_46-13 with CD3ε specificity were obtained by phage display selections against different species (human, cynomolgus and murine) of FoIR1. Clones 16A3, 15A1, 18D3, 19E5, 19A4, 15H7, 15B6, 21D1, 16F12, 19H3, 20G6, and 20H7 were selected from a sub-library in which the common light chain was paired with a heavy chain repertoire based on the human germline VH1_46. In this sub-library, CDR3 of VH1_46 has been randomized based on 6 different CDR3 lengths. Clones 16D5, 15E12, 21A5, and 21G8 were selected from a sub-library in which the common light chain was paired with a heavy chain repertoire based on the human germline VH3_15. In this sub-library, CDR3 of VH3_15 has been randomized based on 6 different CDR3 lengths. In order to obtain species cross-reactive (or murine FoIR1-reactive) antibodies, the different species of FoIR1 were alternated (or kept constant) in different ways over 3 rounds of biopanning: 16A3 and 15A1 (human—cynomolgus—human FoIR1); 18D3 (cynomolgus—human—murine FoIR1); 19E5 and 19A4 (3 rounds against murine FoIR1); 15H7, 15B6, 16D5, 15E12, 21D1, 16F12, 21A5, 21G8 (human—cynomolgus—human FoIR1); 19H3, 20G6, and 20H7 (3 rounds against murine FoIR1). 
     Human, murine and cynomolgus FoIR1 as antigens for the phage display selections as well as ELISA- and SPR-based screenings were transiently expressed as N-terminal monomeric Fc-fusion in HEK EBNA cells and in vivo site-specifically biotinylated via co-expression of BirA biotin ligase at the avi-tag recognition sequence located at the C-terminus of the Fc portion carrying the receptor chain (Fc knob chain). In order to assess the specificity to FoIR1, two related receptors, human FoIR2 and FoIR3 were generated in the same way. 
     Selection rounds (biopanning) were performed in solution according to the following pattern: 
     1. Pre-clearing of ˜1012 phagemid particles on maxisorp plates coated with 10 ug/ml of an unrelated human IgG to deplete the libraries of antibodies recognizing the Fc-portion of the antigen. 
     2. Incubating the non-Fc-binding phagemid particles with 100 nM biotinylated human, cynomolgus, or murine FoIR1 for 0.5h in the presence of 100 nM unrelated non-biotinylated Fc knob-into-hole construct for further depletion of Fc-binders in a total volume of 1 ml. 
     3. Capturing the biotinylated FoIR1 and attached specifically binding phage by transfer to 4 wells of a neutravidin pre-coated microtiter plate for 10 min (in rounds 1 &amp; 3). 
     4. Washing the respective wells using 5×PBS/Tween20 and 5×PBS. 
     5. Eluting the phage particles by addition of 250 ul 100 mM TEA (triethylamine) per well for 10 min and neutralization by addition of 500 ul 1 M Tris/HCl pH 7.4 to the pooled eluates from 4 wells. 
     6. Post-clearing of neutralized eluates by incubation on neutravidin pre-coated microtiter plate with 100 nM biotin-captured FoIR2 or FoIR3 for final removal of Fc- and unspecific binders. 
     7. Re-infection of log-phase  E. coli  TG1 cells with the supernatant of eluted phage particles, infection with helperphage VCSM13, incubation on a shaker at 30° C. over night and subsequent PEG/NaCl precipitation of phagemid particles to be used in the next selection round. 
     Selections were carried out over 3 rounds using constant antigen concentrations of 100 nM. In round 2, in order to avoid enrichment of binders to neutravidin, capture of antigen:phage complexes was performed by addition of 5.4×10 7  streptavidin-coated magnetic beads. Specific binders were identified by ELISA as follows: 100 ul of 25 nM biotinylated human, cynomolgus, or murine FoIR1 and 10 ug/ml of human IgG were coated on neutravidin plates and maxisorp plates, respectively. Fab-containing bacterial supernatants were added and binding Fabs were detected via their Flag-tags using an anti-Flag/HRP secondary antibody. Clones exhibiting signals on human FoIR1 and being negative on human IgG were short-listed for further analyses and were also tested in a similar fashion against the remaining two species of FoIR1. They were bacterially expressed in a 0.5 liter culture volume, affinity purified and further characterized by SPR-analysis using BioRad&#39;s ProteOn XPR36 biosensor. 
     Affinities (K D ) of selected clones were measured by surface plasmon resonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25° C. with biotinylated human, cynomolgus, and murine FoIR1 as well as human FoIR2 and FoIR3 (negative controls) immobilized on NLC chips by neutravidin capture. Immobilization of antigens (ligand): Recombinant antigens were diluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween 20) to 10 μg/ml, then injected at 30 μl/minute in vertical orientation. Injection of analytes: For ‘one-shot kinetics’ measurements, injection direction was changed to horizontal orientation, two-fold dilution series of purified Fab (varying concentration ranges) were injected simultaneously along separate channels 1-5, with association times of 200 s, and dissociation times of 600 s. Buffer (PBST) was injected along the sixth channel to provide an “in-line” blank for referencing. Association rate constants (k on ) and dissociation rate constants (k off ) were calculated using a simple one-to-one Langmuir binding model in ProteOn Manager v3.1 software by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K D ) was calculated as the ratio k off /k on . Table 4 lists the equilibrium dissociation constants (K D ) of the selected clones specific for FoIR1. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Equilibrium dissociation constants (KD) for anti-FoIR1 antibodies (Fab-format) selected by phage 
               
               
                 display from common light chain sub-libraries comprising VL7_46-13, a humanized light chain specific for 
               
               
                 CD3ε KD in nM. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Clone 
                 huFoIR1 [nM] 
                 oyFoIR1 [nM] 
                 muFoIR1 [nm] 
                 huFoIR2 [nM] 
                 huFoIR3 [nM] 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 16A3 
                 21.7 
                 18 
                 very weak 
                 no binding 
                 no binding 
               
               
                 15A1 
                 30.9 
                 17.3 
                 very weak 
                 no binding 
                 no binding 
               
               
                 18D3 
                 93.6 
                 40.2 
                 very weak 
                 no binding 
                 no binding 
               
               
                 19E5 
                 522 
                 276 
                 19.4 
                 no binding 
                 no binding 
               
               
                 19A4 
                 2050 
                 4250 
                 43.1 
                 no binding 
                 no binding 
               
               
                 15H7 
                 13.4 
                 72.5 
                 no binding 
                 no binding 
                 no binding 
               
               
                 15B6 
                 19.1 
                 13.9 
                 no binding 
                 no binding 
                 no binding 
               
               
                 16D5 
                 39.5 
                 114 
                 no binding 
                 no binding 
                 no binding 
               
               
                 15E12 
                 55.7 
                 137 
                 no binding 
                 no binding 
                 no binding 
               
               
                 21D1 
                 62.6 
                 32.1 
                 no binding 
                 no binding 
                 no binding 
               
               
                 16F12 
                 68 
                 90.9 
                 no binding 
                 no binding 
                 no binding 
               
               
                 21A5 
                 68.8 
                 131 
                 no binding 
                 no binding 
                 no binding 
               
               
                 21G8 
                 130 
                 261 
                 no binding 
                 no binding 
                 no binding 
               
               
                 19H3 
                 no binding 
                 no binding 
                 89.7 
                 no binding 
                 no binding 
               
               
                 20G6 
                 no binding 
                 no binding 
                 78.5 
                 no binding 
                 no binding 
               
               
                   
               
            
           
         
       
     
     Example 7 
     Selection of Antibody Fragments from Generic Multi-Framework Libraries to FoIR1 
     The antibodies 11F8, 36F2, 9D11, 5D9, 6B6, and 14E4 were obtained by phage display selections based on generic multi-framework sub-libraries against different species (human, cynomolgus and murine) of FoIR1. In these multi-framework sub-libraries, different VL-domains with randomized CDR3 (3 different lengths) are paired with different VH-domains with randomized CDR3 (6 different lengths). The selected clones are of the following VL/VH pairings: 11F8 (Vk_1_5/VH_1_69), 36F2 (Vk_3_20/VH_1_46), 9D11 (Vk2D_28/VH1_46), 5D9 (Vk3_20/VH1_46), 6B6 (Vk3_20/VH1_46), and 14E4 (Vk3_20/VH3_23). In order to obtain species cross-reactive (or murine FoIR1-reactive) antibodies, the different species of FoIR1 were alternated (or kept constant) in different ways over 3 or 4 rounds of biopanning: 11F8 (cynomolgus—murine—human FoIR1); 36F2 (human—murine—cynomolgus—murine FoIR1); 9D11 (cynomolgus—human—cynomolgus FoIR1); 5D9 (human—cynomolgus—human FoIR1); 6B6 (human—cynomolgus—human FoIR1) and 14E4 (3 rounds against murine FoIR1). 
     Human, murine and cynomolgus FoIR1 as antigens for the phage display selections as well as ELISA- and SPR-based screenings were transiently expressed as N-terminal monomeric Fc-fusion in HEK EBNA cells and in vivo site-specifically biotinylated via co-expression of BirA biotin ligase at the avi-tag recognition sequence located at the C-terminus of the Fc portion carrying the receptor chain (Fc knob chain). In order to assess the specificity to FoIR1, two related receptors, human FoIR2 and FoIR3 were generated in the same way. 
     Selection rounds (biopanning) were performed in solution according to the following pattern: 
     1. Pre-clearing of ˜10 12  phagemid particles on maxisorp plates coated with 10 ug/ml of an unrelated human IgG to deplete the libraries of antibodies recognizing the Fc-portion of the antigen. 
     2. Incubating the non-Fc-binding phagemid particles with 100 nM biotinylated human, cynomolgus, or murine FoIR1 for 0.5h in the presence of 100 nM unrelated non-biotinylated Fc knob-into-hole construct for further depletion of Fc-binders in a total volume of 1 ml. 
     3. Capturing the biotinylated FoIR1 and attached specifically binding phage by transfer to 4 wells of a neutravidin pre-coated microtiter plate for 10 min (in rounds 1 &amp; 3). 
     4. Washing the respective wells using 5×PBS/Tween20 and 5×PBS. 
     5. Eluting the phage particles by addition of 250 ul 100 mM TEA (triethylamine) per well for 10 min and neutralization by addition of 500 ul 1 M Tris/HCl pH 7.4 to the pooled eluates from 4 wells. 
     6. Post-clearing of neutralized eluates by incubation on neutravidin pre-coated microtiter plate with 100 nM biotin-captured FoIR2 or FoIR3 for final removal of Fc- and unspecific binders. 
     7. Re-infection of log-phase  E. coli  TG1 cells with the supernatant of eluted phage particles, infection with helperphage VCSM13, incubation on a shaker at 30° C. over night and subsequent PEG/NaCl precipitation of phagemid particles to be used in the next selection round. 
     Selections were carried out over 3 rounds using constant antigen concentrations of 100 nM. In round 2 and 4, in order to avoid enrichment of binders to neutravidin, capture of antigen:phage complexes was performed by addition of 5.4×10 7  streptavidin-coated magnetic beads. Specific binders were identified by ELISA as follows: 100 ul of 25 nM biotinylated human, cynomolgus, or murine FoIR1 and 10 ug/ml of human IgG were coated on neutravidin plates and maxisorp plates, respectively. Fab-containing bacterial supernatants were added and binding Fabs were detected via their Flag-tags using an anti-Flag/HRP secondary antibody. Clones exhibiting signals on human FoIR1 and being negative on human IgG were short-listed for further analyses and were also tested in a similar fashion against the remaining two species of FoIR1. They were bacterially expressed in a 0.5 liter culture volume, affinity purified and further characterized by SPR-analysis using BioRad&#39;s ProteOn XPR36 biosensor. Affinities (K D ) of selected clones were measured by surface plasmon resonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25° C. with biotinylated human, cynomolgus, and murine FoIR1 as well as human FoIR2 and FoIR3 (negative controls) immobilized on NLC chips by neutravidin capture. Immobilization of antigens (ligand): Recombinant antigens were diluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween 20) to 10 μg/ml, then injected at 30 μl/minute in vertical orientation. Injection of analytes: For ‘one-shot kinetics’ measurements, injection direction was changed to horizontal orientation, two-fold dilution series of purified Fab (varying concentration ranges) were injected simultaneously along separate channels 1-5, with association times of 150 or 200 s, and dissociation times of 200 or 600 s, respectively. Buffer (PBST) was injected along the sixth channel to provide an “in-line” blank for referencing. Association rate constants (k on ) and dissociation rate constants (k off ) were calculated using a simple one-to-one Langmuir binding model in ProteOn Manager v3.1 software by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K D ) was calculated as the ratio k off /k on . Table 5 lists the equilibrium dissociation constants (K D ) of the selected clones specific for FoIR1. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Equilibrium dissociation constants (K D ) for anti-FoIR1 antibodies (Fab-format) selected by phage 
               
               
                 display from generic multi-framework sub-libraries. K D  in nM. 
               
            
           
           
               
               
            
               
                   
                 KD (nM) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Clone 
                 huFoIR1 
                 cyFoIR1 
                 muFoIR1 
                 huFOIR2 
                 huFOIR3 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 11F8 
                 632 
                 794 
                 1200 
                 no binding 
                 no binding 
               
               
                 36F2 
                 1810 
                 1640 
                 737 
                 no binding 
                 no binding 
               
               
                 9D11 
                 8.64 
                 5.29 
                 no binding 
                 no binding 
                 no binding 
               
               
                 5D9 
                 8.6 
                 5.9 
                 no binding 
                 no binding 
                 no binding 
               
               
                 6B6 
                 14.5 
                 9.4 
                 no binding 
                 no binding 
                 no binding 
               
               
                 14E4 
                 no binding 
                 no binding 
                 6.09 
                 no binding 
                 no binding 
               
               
                   
               
            
           
         
       
     
     Example 8 
     Production and Purification of Novel FoIR1 Binders in IgG and T-Cell Bispecific Formats 
     To identify FoIR1 binders which are able to induce T-cell dependent killing of selected target cells the antibodies isolated from a common light chain- or Fab-library were converted into the corresponding human IgG 1  format. In brief, the variable heavy and variable light chains of unique FoIR1 binders from phage display were amplified by standard PCR reactions using the Fab clones as the template. The PCR products were purified and inserted (either by restriction endonuclease and ligase based cloning, or by ‘recombineering’ using the InFusion kit from Invitrogen) into suitable expression vectors in which they are fused to the appropriate human constant heavy or human constant light chain. The expression cassettes in these vectors consist of a chimeric MPSV promoter and a synthetic polyadenylation site. In addition, the plasmids contain the oriP region from the Epstein Barr virus for the stable maintenance of the plasmids in HEK293 cells harboring the EBV nuclear antigen (EBNA). After PEI mediated transfection the antibodies were transiently produced in HEK293 EBNA cells and purified by standard ProteinA affinity chromatography followed by size exclusion chromatography as described: 
     Transient Transfection and Production 
     All (bispecific) antibodies (if not obtained from a commercial source) used herein were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below. HEK293 EBNA cells are cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells are seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly). For transfection cells are centrifuged for 5 min by 210×g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixed in 20 ml CD CHO medium to a final amount of 200 μg DNA. After addition of 540 μl PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells are mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium is added and cell are cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 is added. After 7 days cultivation supernatant is collected for purification by centrifugation for 15 min at 210×g, the solution is sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% w/v is added, and kept at 4° C. After production the supernatants were harvested and the antibody containing supernatants were filtered through 0.22 μm sterile filters and stored at 4° C. until purification. 
     Antibody Purification 
     All molecules were purified in two steps using standard procedures, such as protein A affinity purification (Äkta Explorer) and size exclusion chromatography. The supernatant obtained from transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to HiTrap PA FF (GE Healthcare, column volume (cv)=5 ml) equilibrated with 8 column volumes (cv) buffer A (20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5). After washing with 10 cv of buffer A, the protein was eluted using a pH gradient to buffer B (20 mM sodium citrate pH 3, 100 mM NaCl, 100 mM glycine) over 12 cv. Fractions containing the protein of interest were pooled and the pH of the solution was gently adjusted to pH 6.0 (using 0.5 M Na 2 HPO 4  pH 8.0). Samples were concentrated to 2 ml using ultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius) and subsequently applied to a HiLoad™ 16/60 Superdex™ 200 preparative grade (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween-20. The aggregate content of eluted fractions was analyzed by analytical size exclusion chromatography. Therefore, 30 μl of each fraction was applied to a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K 2 HPO 4 , 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN 3 , pH 6.7 running buffer at 25° C. Fractions containing less than 2% oligomers were pooled and concentrated to final concentration of 1-1.5 mg/ml using ultra concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius). The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N 2  and stored at −80° C. 
     Based on in vitro characterization results selected binders were converted into a T-cell bispecific format. In these molecules the FoIR1:CD3 binding moieties are arranged in a 2:1 order with the FoIR1 Fabs being located at the N-terminus. For clones isolated from the standard Fab library the CD3 binding part was generated as a CrossFab (CH1CK crossing) while for the clones from the common light chain library no crossing was necessary. These bispecific molecules were produced and purified analogously to the IgGs. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Yield and monomer content of novel FolR1 binders in  
               
               
                 IgG and TCB format, respectively. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 IgG 
                 TCB 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Yield  
                 Monomer 
                 Yield 
                 Monomer 
               
               
                 # 
                 Clone 
                 Library 
                 [mg/L] 
                 [%] 
                 [mg/L} 
                 [%] 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 11F8 
                 Fab 
                 8.03 
                 96.26 
                 — 
                 — 
               
               
                 2 
                 14E4 
                 Fab 
                 8.90 
                 98.12 
                 — 
                 — 
               
               
                 3 
                 15B6 
                 CLC 
                 7.72 
                 100.00 
                 — 
                 — 
               
               
                 4 
                 15E12 
                 CLC 
                 6.19 
                 100.00 
                 — 
                 — 
               
               
                 5 
                 15H7 
                 CLC 
                 8.94 
                 100.00 
                 — 
                 — 
               
               
                 6 
                 16A3 
                 CLC 
                 0.60 
                 n.d. 
                 — 
                 — 
               
               
                 7 
                 16D5 
                 CLC 
                 36.50 
                 96.96 
                 4.36 
                 97.19 
               
               
                 8 
                 16F12 
                 CLC 
                 5.73 
                 97.17 
                 — 
                 — 
               
               
                 9 
                 18D3 
                 CLC 
                 0.90 
                 n.d. 
                 — 
                 — 
               
               
                 10 
                 19A4 
                 CLC 
                 38.32 
                 100.00 
                 37.50 
                 100.00 
               
               
                 11 
                 19E5 
                 CLC 
                 46.09 
                 100.00 
                 — 
                 — 
               
               
                 12 
                 19H3 
                 CLC 
                 7.64 
                 100.00 
                 — 
                 — 
               
               
                 13 
                 20G6 
                 CLC 
                 24.00 
                 100.00 
                 — 
                 — 
               
               
                 14 
                 20H7 
                 CLC 
                 45.39 
                 100.00 
                 — 
                 — 
               
               
                 15 
                 21A5 
                 CLC 
                 1.38 
                 98.56 
                 47.31 
                 95.08 
               
               
                 16 
                 21D1 
                 CLC 
                 5.47 
                 100.00 
                 — 
                 — 
               
               
                 17 
                 21G8 
                 CLC 
                 6.14 
                 97.28 
                 9.27 
                 100.00 
               
               
                 18 
                 36F2 
                 Fab 
                 11.22 
                 100.00 
                 18.00 
                 100.00 
               
               
                 19 
                 5D9 
                 Fab 
                 20.50 
                 100.00 
                 0.93 
                 97.32 
               
               
                 20 
                 6B6 
                 Fab 
                 3.83 
                 100.00 
                 4.17 
                 91.53 
               
               
                 21 
                 9D11 
                 Fab 
                 14.61 
                 100.00 
                 2.63 
                 100.00 
               
               
                   
               
               
                 CLC: Common light chain 
               
            
           
         
       
     
     Example 9 
     2+1 and 1+1 T-Cell Bispecific Formats 
     Four different T-cell bispecific formats were prepared for one common light chain binder (1605) and three formats for one binder from the Fab library (9011) to compare their killing properties in vitro. 
     The standard format is the 2+1 inverted format as already described (FoIR1:CD3 binding moieties arranged in a 2:1 order with the FoIR1 Fabs located at the N-terminus). In the 2+1 classical format the FoIR1:CD3 binding moieties are arranged in a 2:1 order with the CD3 Fab being located at the N-terminus. Two monovalent formats were also prepared. The 1+1 head-to-tail has the FoIR1:CD3 binding moieties arranged in a 1:1 order on the same arm of the molecule with the FoIR1 Fab located at the N-terminus. In the 1+1 classical format the FoIR1:CD3 binding moieties are present once, each on one arm of the molecule. For the 9D11 clone isolated from the standard Fab library the CD3 binding part was generated as a CrossFab (CH1Cκ crossing) while for the 16D5 from the common light chain library no crossing was necessary. These bispecific molecules were produced and purified analogously to the standard inverted T-cell bispecific format. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Summary of the yield and final monomer content of the  
               
               
                 different T-cell bispecific formats. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Monomer 
                   
               
               
                   
                   
                 [%] 
                   
               
               
                   
                 Construct 
                 (SEC) 
                 Yield 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 16D5 FoIR1 TCB 2+1 (inverted) 
                 96% 
                 5.4 mg/L 
               
               
                   
                 16D5 FoIR1 TCB 2+1 (classical) 
                 90% 
                 4.6 mg/L 
               
               
                   
                 16D5 FoIR1 TCB 1+1 (head-to-tail) 
                 100% 
                 5.4 mg/L 
               
               
                   
                 16D5 FoIR1 TCB 1+1 (classical) 
                 100% 
                 0.7 mg/L 
               
               
                   
                 9D11 FoIR1 TCB 2+1 (inverted) 
                 100% 
                 2.6 mg/L 
               
               
                   
                 9D11 FoIR1 TCB 1+1 (head-to-tail) 
                 100% 
                 6.1 mg/L 
               
               
                   
                 9D11 FoIR1 TCB 1+1 (classical) 
                 96% 
                 1.3 mg/L 
               
               
                   
                 Mov19 FoIR1 TCB 2+1 (inverted) 
                 98% 
                 3 mg/L 
               
               
                   
                 Mov19 FoIR1 TCB 1+1 (head-to-tail) 
                 100% 
                 5.2 mg/L 
               
               
                   
                   
               
            
           
         
       
     
     Example 10 
     Biochemical Characterization of FoIR1 Binders by Surface Plasmon Resonance 
     Binding of FoIR1 binders as IgG or in the T-cell bispecific format to different recombinant folate receptors (human FoIR1, 2 and 3, murine FoIR1 and cynomolgus FoIR1; all as Fc fusions) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany). 
     Single Injections 
     First the anti-FoIR1 IgGs were analyzed by single injections (Table 1) to characterize their crossreactivity (to human, murine and cyno FoIR1) and specificity (to human FoIR1, human FoIR2, human FoIR3). Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FoIR1-Fc) or human Folate Receptor 2 and 3 (FoIR2-Fc, FoIR3-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany). The immobilization level was about 300-400 RU. The IgGs were injected for 60 seconds at a concentration of 500 nM. IgGs binding to huFoIR2 and huFoIR3 were rejected for lack of specificity. Most of the binders are only crossreactive between human and cyno FoIR1, additional crossreactivity to murine FoIR1 went most of the time hand in hand with loss of specificity. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Crossreactivity and specificity of 25 new folate receptor 1 binders (as IgGs) as well as of two 
               
               
                 control IgGs (Mov19 and Farletuzumab). 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Binding to 
                 Binding to 
                 Binding to 
                 Binding to 
                 Binding to 
               
               
                 Clone name 
                 huFoIR1 
                 cyFoIR1 
                 muFoIR1 
                 huFoIR2 
                 huFoIR3 
               
               
                   
               
               
                 Mov19 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 Farletuzumab 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 16A3 
                 + 
                 + 
                 +/− 
                 − 
                 − 
               
               
                 18D3 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 19E5 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                 19A4 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                 15H7 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 15B6 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 16D5 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 15E12 
                 + 
                 + 
                 +/− 
                 + 
                 + 
               
               
                 21D1 
                 + 
                 + 
                 +/− 
                 − 
                 − 
               
               
                 16F12 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 21A5 
                 + 
                 + 
                 − 
                 − 
                 +/− 
               
               
                 21G8 
                 + 
                 + 
                 − 
                 + 
                 + 
               
               
                 19H3 
                 − 
                 − 
                 + 
                 − 
                 − 
               
               
                 20G6 
                 − 
                 − 
                 + 
                 − 
                 − 
               
               
                 20H7 
                 − 
                 − 
                 + 
                 − 
                 − 
               
               
                 9D11 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                 5D9 
                 + 
                 + 
                 − 
                 + 
                 + 
               
               
                 6B6 
                 + 
                 + 
                 − 
                 + 
                 + 
               
               
                 11F8 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                 36F2 
                 + 
                 + 
                 + 
                 − 
                 − 
               
               
                 14E4 
                 − 
                 − 
                 + 
                 − 
                 − 
               
               
                   
               
               
                 + means binding, − means no binding, +/− means weak binding. 
               
            
           
         
       
     
     Avidity to Folate Receptor 1 
     The avidity of the interaction between the anti-FoIR1 IgGs or T cell bispecifics and the recombinant folate receptors was determined as described below (Table 9). 
     Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FoIR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany). The immobilization level was about 300-400 RU. The anti-FoIR1 IgGs or T cell bispecifics were passed at a concentration range from 2.1 to 500 nM with a flow of 30 μL/minutes through the flow cells over 180 seconds. The dissociation was monitored for 600 seconds. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated IL2 receptor Fc fusion. For the analysis of the interaction of 19H3 IgG and murine folate receptor 1, folate (Sigma F7876) was added in the HBS-EP running buffer at a concentration of 2.3 μM. The binding curves resulting from the bivalent binding of the IgGs or T cell bispecifics were approximated to a 1:1 Langmuir binding and fitted with that model (which is not correct, but gives an idea of the avidity). The apparent avidity constants for the interactions were derived from the rate constants of the fitting using the Bia Evaluation software (GE Healthcare). 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Bivalent binding (avidity with apparent KD) of selected FoIR1 binders as  
               
               
                 IgGs or as T-cell bispecifics (TCB) on human and cyno FoIR1. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Apparent KD 
               
               
                 Analyte 
                 Ligand 
                 ka (1/Ms) 
                 kd (1/s) 
                 (M) 
               
               
                   
               
               
                 16D5 TCB 
                 huFoIR1 
                 8.31E+04 
                 3.53E−04 
                 4.24E−09 
               
               
                   
                 cyFoIR1 
                 1.07E+05 
                 3.70E−04 
                 3.45E−09 
               
               
                 9D11 TCB 
                 huFoIR1 
                 1.83E+05 
                 9.83E−05 
                 5.36E−10 
               
               
                   
                 cyFoIR1 
                 2.90E+05 
                 6.80E−05 
                 2.35E−10 
               
               
                 21A5 TCB 
                 huFoIR1 
                 2.43E+05 
                 2.64E−04 
                 1.09E−09 
               
               
                   
                 cyFoIR1 
                 2.96E+05 
                 2.76E−04 
                 9.32E−10 
               
               
                 36F2 IgG 
                 huFoIR1 
                 2.62E+06 
                 1.51E−02 
                 5.74E−9 
               
               
                   
                 cyFoIR1 
                 3.02E+06 
                 1.60E−02 
                 5.31E−9 
               
               
                   
                 muFoIR1 
                 3.7E+05 
                 6.03E−04 
                 1.63E−9 
               
               
                 Mov19 IgG 
                 huFoIR1 
                 8.61E+05 
                 1.21E−04 
                 1.4E−10 
               
               
                   
                 cyFoIR1 
                 1.29E+06 
                 1.39E−04 
                 1.08E−10 
               
               
                 Farletuzumab 
                 huFoIR1 
                 1.23E+06 
                 9E−04 
                 7.3E−10 
               
               
                   
                 cyFoIR1 
                 1.33E+06 
                 8.68E−04 
                 6.5E−10 
               
               
                 19H3 IgG 
                 muFoIR1 
                 7.1E+05 
                 1.1E−03 
                 1.55E−09 
               
               
                   
               
            
           
         
       
     
     1. Affinity to Folate Receptor 1 
     The affinity of the interaction between the anti-FoIR1 IgGs or the T cell bispecifics and the recombinant folate receptors was determined as described below (Table 10). 
     For affinity measurement, direct coupling of around 6000-7000 resonance units (RU) of the anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). Anti-FoIR1 IgGs or T cell bispecifics were captured at 20 nM with a flow rate of 10 μl/min for 20 or 40 sec, the reference flow cell was left without capture. Dilution series (6.17 to 500 nM or 12.35 to 3000 nM) of human or cyno Folate Receptor 1 Fc fusion were passed on all flow cells at 30 μl/min for 120 or 240 sec to record the association phase. The dissociation phase was monitored for 240 s and triggered by switching from the sample solution to HBS-EP. The chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 2.1 or pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1. The affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare). 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Monovalent binding (affinity) of selected FoIR1 binders as IgGs or  
               
               
                 as T-cell bispecifics (TCB) on human and cyno FoIR1. 
               
            
           
           
               
               
               
               
               
            
               
                 Ligand 
                 Analyte 
                 ka (1/Ms) 
                 kd (1/s) 
                 KD (M) 
               
               
                   
               
               
                 16D5 TCB 
                 huFoIR1 
                 1.53E+04 
                 6.88E−04 
                 4.49E−08 
               
               
                   
                 cyFoIR1 
                 1.32E+04 
                 1.59E−03 
                 1.21E−07 
               
               
                 9D11 TCB 
                 huFoIR1 
                 3.69E+04 
                 3.00E−04 
                 8.13E−09 
               
               
                   
                 cyFoIR1 
                 3.54E+04 
                 2.06E−04 
                 5.82E−09 
               
               
                 21A5 TCB 
                 huFoIR1 
                 1.79E+04 
                 1.1E−03 
                 6.16E−08 
               
               
                   
                 cyFoIR1 
                 1.48E+04 
                 2.06E−03 
                 1.4E−07 
               
               
                 Mov19 IgG 
                 huFoIR1 
                 2.89E+05 
                 1.59E−04 
                 5.5E−10 
               
               
                   
                 cyFoIR1 
                 2.97E+05 
                 1.93E−04 
                 6.5E−10 
               
               
                 Farletuzumab 
                 huFoIR1 
                 4.17E+05 
                 2.30E−02 
                 5.53E−08 
               
               
                   
                 cyFoIR1 
                 5.53E+05 
                 3.73E−02 
                 6.73E−08 
               
               
                   
               
            
           
         
       
     
     2. Affinity to CD3 
     The affinity of the interaction between the anti-FoIR1 T cell bispecifics and the recombinant human CD386-Fc was determined as described below (Table 11). 
     For affinity measurement, direct coupling of around 9000 resonance units (RU) of the anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). Anti-FoIR1 T cell bispecifics were captured at 20 nM with a flow rate of 10 μl/min for 40 sec, the reference flow cell was left without capture. Dilution series (6.17 to 500 nM) of human CD386-Fc fusion were passed on all flow cells at 30 μl/min for 240 sec to record the association phase. The dissociation phase was monitored for 240 s and triggered by switching from the sample solution to HBS-EP. The chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 2.1. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1. The affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare). 
                     TABLE 11                  Monovalent binding (affinity) of selected FoIR1 T-cell bispecifics (TCB) on        human CD3-Fc.                                 Ligand   Analyte   ka (1/Ms)   kd (1/s)   KD (M)               16D5 TCB   huCD3   4.25E+04   3.46E−03   8.14E−08       21A5 TCB   huCD3   3.72E+04   3.29E−03   8.8E−08                    
The CD3 binding part is identical for all constructs and the affinity is similar for the measured T cell bispecifics (K D  range between 60 and 90 nM).
 
     Example 11 
     Simultaneous Binding T Cell Bispecifics on Folate Receptor 1 and CD3 
     Simultaneous binding of the anti-FoIR1 T cell bispecifics on recombinant Folate Receptor 1 and recombinant human CD386-Fc was determined by surface plasmon resonance as described below. Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FoIR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, Freiburg/Germany). The immobilization level was about 300-400 RU. The anti-FoIR1 T cell bispecifics were injected for 60 s at 500 nM with a flow of 30 μL/minutes through the flow cells, followed by an injection of hu CD88-Fc for 60 s at 500 nM. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated IL2 receptor Fc fusion. The four T cell bispecifics tested (16D5 TCB, 21A5 TCB, 51C7 TCB and 45D2 TCB) were able to bind simultaneously to Folate Receptor 1 and human CD3 as expected. 
     Example 12 
     Epitope Binning 
     For epitope binning, the anti-FoIR1 IgGs or T cell bispecifics were directly immobilized on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare), with a final response around 700 RU. 500 nM huFoIR1-Fc was then captured for 60 s, followed by 500 nM of the different binders for 30 s. The surface was regenerated with two injections of 10 mM glycine pH 2 for 30 s each. It is assessed if the different binders can bind to huFoIR1 captured on immobilized binders (Table 12). 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Epitope characterization of selected FolR1 binders as IgGs  
               
               
                 or as T-cell bispecifics (TCB) on human FolR1.  
               
            
           
           
               
               
               
            
               
                   
                   
                 Analytes in solution 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 On 
                 16D5 
                 21A5 
                 9D11 
                 36F2 
                 Mov19 
                   
               
               
                   
                 huFolR1 
                 TCB 
                 TCB 
                 TCB 
                 IgG 
                 IgG 
                 Farletuzumab 
               
               
                   
               
               
                 Immo- 
                 16D5 TCB 
                 − 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                 bilized 
                 21A5 TCB 
                 − 
                 − 
                 − 
                 + 
                 + 
                 + 
               
            
           
           
               
               
               
            
               
                   
                 9D11 TCB 
                 No additional binding on FolR1 possible once  
               
               
                   
                   
                 captured on 9D11 
               
               
                   
                 36F2 IgG 
                 Measure not possible, huFolR1  
               
               
                   
                   
                 dissociates too rapidly 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Mov19 
                 + 
                 + 
                 +/− 
                 − 
                 − 
                 − 
               
               
                   
                 IgG 
               
               
                   
               
               
                 + means binding, − means no binding, +/− means weak binding 
               
            
           
         
       
     
     Based on these results and additional data with simultaneous binding on immobilized huFoIR1, the binders were separated in three groups. It is not clear if 9D11 has a separate epitope because it displaces all the other binders. 16D5 and 21A5 seem to be in the same group and Mov19, Farletuzumab (Coney et al., Cancer Res. 1991 Nov. 15; 51(22):6125-32; Kalli et al., Curr Opin Investig Drugs. 2007 December; 8(12):1067-73) and 36F2 in another (Table 13). However, 36F2 binds to a different epitope than Mov 19 and Farletuzumab as it binds to human, cynomous and murine FoIR1. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Epitope grouping of selected FolR1 binders as 
               
               
                 IgGs or as T-cell bispecifics (TCB) on human 
               
               
                 FolR1 
               
            
           
           
               
               
               
            
               
                 Epitope 1 
                 Epitope 2 
                 Epitope 3 
               
               
                   
               
               
                 16D5 
                 9D11 
                 Mov19 
               
               
                 21A5 
                   
                 Farletuzumab 
               
               
                   
                   
                 36F2 
               
               
                   
               
            
           
         
       
     
     Example 13 
     Selection of Binders 
     FoIR1 binders in the IgG formats were screened by surface plasmon resonance (SPR) and by in vitro assay on cells to select the best candidates. 
     The anti-FoIR1 IgGs were analyzed by SPR to characterize their crossreactivity (to human, murine and cynomolgus FoIR1) and specificity (to human FoIR1, human FoIR2, human FoIR3). Unspecific binding to human FoIR2 and 3 was considered an exclusion factor. Binding and specificity to human FoIR1 was confirmed on cells. Some binders did not bind on cells expressing FoIR1 even though they recognized the recombinant human FoIR1 in SPR. Aggregation temperature was determined but was not an exclusion factor because the selected binders were all stable. Selected binders were tested in a polyreactivity ELISA to check for unspecific binding, which led to the exclusion of four binders. This process resulted in an initial selection of three binders: 36F2 (Fab library), 9D11 (Fab library) and 16D5 (common light chain). 36F2 dissociated rapidly from huFoIR1 and was, therefore, initially not favored. 
     Example 14 
     Specific Binding of Newly Generated FoIR1 Binders to Human FoIR1 Positive Tumor Cells 
     New FoIR1 binders were generated via Phage Display using either a Fab library or a common light chain library using the CD3 light chain. The identified binders were converted into a human IgG1 format and binding to FoIR1 high expressing HeLa cells was addressed. As reference molecule the human FoIR1 binder Mov19 was included. Most of the binders tested in this assay showed intermediate to good binding to FoIR1 with some clones binding equally well as Mov19 (see  FIG. 2 ). The clones 16A3, 18D3, 15H7, 15B6, 21D1, 14E4 and 16F12 were excluded because binding to FoIR1 on cells could not be confirmed by flow cytometry. In a next step the selected clones were tested for specificity to human FoIR1 by excluding binding to the closely related human FoIR2. HEK cells were transiently transfected with either human FoIR1 or human FoIR2 to address specificity. The clones 36F2 and 9D11 derived from the Fab library and the clones 16D5 and 21A5 derived from the CLC library bind specifically to human FoIR1 and not to human FoIR2 (see  FIGS. 3A-B ). All the other tested clones showed at least some binding to human FoIR2 (see  FIGS. 3A-B ). Therefore these clones were excluded from further characterization. In parallel cross-reactivity of the FoIR1 clones to cyno FoIR1 was addressed by performing binding studies to HEK cells transiently transfected with cyno FoIR1. All tested clones were able to bind cyno FoIR1 and the four selected human FoLR1 specific clones 36F2, 9D11, 16D5 and 21A5 bind comparably well human and cyno FoLR1 ( FIG. 4 ). Subsequently three human FoIR1 specific cyno cross-reactive binders were converted into TCB format and tested for induction of T cell killing and T cell activation. These clones were 9D11 from the Fab library and 16D5 and 21A5 from the CLC library. As reference molecule Mov19 FoIR1 TCB was included in all studies. These FoIR1 TCBs were then used to compare induction of internalization after binding to FoIR1 on HeLa cells. All three tested clones are internalized upon binding to FoIR1 comparable to internalization upon binding of Mov19 FoLR1 TCB ( FIG. 5 ). 21A5 FoIR1 TCB was discontinued due to signs of polyreactivity. 
     Example 15 
     T Cell-Mediated Killing of FoIR1-Expressing Tumor Target Cells Induced by FoIR1 TCB Antibodies 
     The FoIR1 TCBs were used to determine T cell mediated killing of tumor cells expressing FoLR1. A panel of potential target cell lines was used to determine FoLR1 binding sites by Qifikit analysis. 
     The used panel of tumor cells contains FoIR1 high, intermediate and low expressing tumor cells and a FoIR1 negative cell line. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 FolR1 binding sites on tumor cells 
               
            
           
           
               
               
               
               
            
               
                   
                 Cell line 
                 Origin 
                 FolR1 binding sites 
               
               
                   
                   
               
               
                   
                 Hela 
                 Cervix adenocarcinoma 
                 2′240′716 
               
               
                   
                 Skov3 
                 Ovarian adenocarcinoma 
                    91′510 
               
               
                   
                 OVCAR5 
                 Ovarian adenocarcinoma 
                    22′077 
               
               
                   
                 HT29 
                 Colorectal adenocarcinoma 
                    10′135 
               
               
                   
                 MKN45 
                 Gastric adenocarcinoma 
                      54 
               
               
                   
                   
               
            
           
         
       
     
     Binding of the three different FoLR1 TCBs (containing 9D11, 16D5 and Mov19 binders) to this panel of tumor cell lines was determined showing that the FoIR1 TCBs bind specifically to FoIR1 expressing tumor cells and not to a FoLR1 negative tumor cell line. The amount of bound construct is proportional to the FoIR1 expression level and there is still good binding of the constructs to the FoIR1 low cell line HT-29 detectable. In addition there is no binding of the negative control DP47 TCB to any of the used cell lines ( FIGS. 6A-E ). 
     The intermediate expressing cell line SKOV3 and the low expressing cell line HT-29 were further on used to test T cell mediated killing and T cell activation using 16D5 TCB and 9D11 TCB; DP47 TCB was included as negative control. Both cell lines were killed in the presence of already very low levels of 16D5 TCB and 9D11 TCB and there was no difference in activity between both TCBs even though 9D11 TCB binds stronger to FoIR1 than 16D5 TCB. Overall killing of SKOV3 cells was higher compared to HT-29 which reflects the higher expression levels of FoIR1 on SKOV3 cells ( FIGS. 7A-D ). In line with this, a strong upregulation of the activation marker CD25 and CD69 on CD4 +  T cells and CD8 +  T cells was detected. Activation of T cells was very similar in the presence of SKOV3 cells and HT-29 cells. The negative control DP47 TCB does not induce any killing at the used concentrations and there was no significant upregulation of CD25 and CD69 on T cells. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 EC50 values of tumor cell killing and T cell activation with SKOV3 cells 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Killing 
                 Killing 
                 CD4+ 
                 CD4+ 
                 CD8+ 
                 CD8+ 
               
               
                 Construct 
                 24 h (pM) 
                 48 h (pM) 
                 CD69+ (%) 
                 CD25+ (%) 
                 CD69+ (%) 
                 CD25+ (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 9D11 
                 1.1 
                 0.03 
                 0.51 
                 0.46 
                 0.019 
                 0.03 
               
               
                 FolR1 TCB 
                   
                   
                   
                   
                   
                   
               
               
                 16D5 
                 0.7 
                 0.04 
                 0.34 
                 0.33 
                 0.025 
                 0.031 
               
               
                 FolR1 TCB 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 EC50 values of tumor cell killing and T cell activation with HT-29 cells 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Killing 
                 Killing 
                 CD4+ 
                 CD4+ 
                 CD8+ 
                 CD8+ 
               
               
                 Construct 
                 24 h (pM) 
                 48 h (pM) 
                 CD69+ (%) 
                 CD25+ (%) 
                 CD69+ (%) 
                 CD25+ (%) 
               
               
                   
               
               
                 9D11 
                 2.3 
                 0.1 
                 1.22 
                 1.11 
                 0.071 
                 0.084 
               
               
                 FolR1 TCB 
                   
                   
                   
                   
                   
                   
               
               
                 16D5 
                 2.8 
                 0.1 
                 0.69 
                 0.62 
                 0.021 
                 0.028 
               
               
                 FolR1 TCB 
               
               
                   
               
            
           
         
       
     
     Example 16 
     Binding to Erythrocytes and T Cell Activation in Whole Blood 
     To prove that there is no spontaneous activation in the absence of FoLR1 expressing tumor cells we tested if there is binding of the FoIR1 clones to erythrocytes which might potentially express FoIR1. We could not observe any specific binding of 9D11 IgG, 16D5 IgG and Mov19 IgG to erythrocytes, as negative control DP47 IgG was included ( FIG. 8 ). 
     To exclude any further unspecific binding to blood cells or unspecific activation via FoLR1 TCB, 9D11 TCB, 16D5 TCB and Mov19 TCB were added into whole blood and upregulation of CD25 and CD69 on CD4 +  T cells and CD8 +  T cells was analyzed by flow cytometry. DP47 TCB was included as negative control. No activation of T cells with any of the tested constructs could be observed by analyzing upregulation of CD25 and CD69 on CD4 +  T cells and CD8 +  T cells ( FIG. 9 ). 
     Example 17 
     Removal of the N-Glycosylation Site in 9D11 Light Chain 
     During analysis of the different FoIR1 binders to identify potential sequence hot spots, at the end of CDR L3 of the clone 9D11 a putative N-glycosylation site was identified. Usually the consensus motif for N-glycosylation is defined as N-X-S/T-X (where X is not P). The sequence of CDR L3 (MQASIMNRT (SEQ ID NO: 46)) perfectly matches this consensus motif having the sequence N-R-T. Since glycosylation might not be completely reproducible among different production batches this could have an impact on FoIR1 binding, if the glycosylation in CDR L3 contributes to antigen binding. To evaluate if this N-glycosylation site is important for FoIR1 binding, or could be replaced without impairing binding, different variants of the 9D11 light chain were generated in which the N-glycosylation site was exchanged by site specific mutagenesis. 
     1. Transient Transfection and Production 
     The four T cell bispecifics were transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below. HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly). For transfection cells were centrifuged for 5 min by 210×g, supernatant was replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 μg DNA. After addition of 540 μl PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210×g, the solution is sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4° C. After production the supernatants were harvested and the antibody containing supernatants were filtered through 0.22 μm sterile filters and stored at 4° C. until purification. 
     2. Antibody Purification 
     All molecules were purified in two steps using standard procedures, such as protein A affinity purification (Äkta Explorer) and size exclusion chromatography. The supernatant obtained from transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to HiTrap PA HP (GE Healthcare, column volume (cv)=5 ml) equilibrated with 8 column volumes (cv) buffer A (20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M NaCl, 0.01% Tween-20, pH 7.5). After washing with 10 cv of buffer A, the protein was eluted using a pH gradient to buffer B (20 mM sodium citrate pH 2.5, 0.5 M NaCl, 0.01% Tween-20) over 20 cv. Fractions containing the protein of interest were pooled and the pH of the solution was gently adjusted to pH 6.0 (using 2 M Tris pH 8.0). Samples were concentrated to 1 ml using ultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius) and subsequently applied to a Superdex™ 200 10/300 GL (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween-20. The aggregate content of eluted fractions was analyzed by analytical size exclusion chromatography. Therefore, 30 μl of each fraction was applied to a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibrated in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN 3 , pH 6.7 running buffer at 25° C. Fractions containing less than 2% oligomers were pooled and concentrated to final concentration of 1-1.5 mg/ml using ultra concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius). The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Purified proteins were frozen in liquid N 2  and stored at −80° C. 
     3. Aggregation Temperature 
     Stability of the four constructs was tested on an Optim1000 (Avacta, PALL Corporation) by a gradient heating from 250 to 800 at 0.1° C./min. The temperature at onset of aggregation is recorded. 
     
       
         
           
               
             
               
                 TABLE 34 
               
             
            
               
                   
               
               
                 Yield, monomer content and aggregation 
               
               
                 temperature of four N-glycosylation site knock-out 
               
               
                 mutant of the 9D11 binder in the 2 + 1 inverted T-cell 
               
               
                 bispecific format. All four mutants behaved similarly 
               
               
                 to the wild-type 9D11 binder 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 Yield 
                 Monomer 
                 Aggregation 
               
               
                   
                 Clone 
                 Mutation 
                 [mg/L} 
                 [%] 
                 temperature 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 9D11 
                 T102N 
                 1.34 
                 97 
                 56° 
               
               
                   
                 9D11 
                 T102A 
                 1.29 
                 100 
                 56° 
               
               
                   
                 9D11 
                 N100Q 
                 2.5 
                 100 
                 56° 
               
               
                   
                 9D11 
                 N100S 
                 2.05 
                 100 
                 56° 
               
               
                   
                 9D11 
                 — 
                 2.6 
                 100 
                 57° 
               
               
                   
                   
               
            
           
         
       
     
     The following variants were generated: N100S (N95S); N100Q (N95Q), T102A (T97A) and T102N (T97N) (Kabat numbering indicated in parenthesis) and converted into the T-cell bispecific format. After transient production in HEK293 EBNA cells and purification the different variants were analyzed for target binding and cell killing activity in comparison to the original 9D11 clone. 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 primers used for removal 
               
               
                 of N-glycosylation 
               
               
                 site in CDR L3 of 9D11 
               
               
                 (sequences see 
               
               
                 below) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Amino acid 
                 Mutagenesis 
               
               
                   
                 # 
                 exchange 
                 primer 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 1 
                 N95S 
                 GAB-7735 
               
               
                   
                 2 
                 N95Q 
                 GAB-7734 
               
               
                   
                 3 
                 T97A 
                 GAB7736 
               
               
                   
                 4 
                 T97N 
                 GAB-7737 
               
               
                   
                   
               
            
           
         
       
     
     Example 18 
     Binding and T Cell Mediated Killing with 9D11 a-Glyco Variants 
     Due to a glycosylation site in the CDRs four different 9D11 variants were produced with a mutation removing the glycosylation site (Example 17). These four variants were tested in comparison to the original 9D11 for binding to FoIR1 on HeLa cells ( FIG. 10 ) and induction of tumor cell killing on SKOV3 and HT-29 ( FIGS. 11A-B , E-F). None of the variants showed differences in binding or induction of tumor cell killing. In parallel unspecific killing of the FoIR1 negative cell lines MKN-45 was addressed ( FIGS. 11C-D ). Also, no differences between the variants and the original binder could be observed. None of the constructs induced unspecific killing on FoLR1 negative tumor cells. 
     Example 19 
     FoIR1 Expression on Primary Epithelial Cells 
     FoIR1 is expressed at low levels on primary epithelial cells. Here we wanted to test if these levels are sufficient to induce T cell mediated killing in the presence of the FoIR1 TCBs. To test this we used primary human bronchial epithelial cells, primary human choroid plexus epithelial cell, primary human renal cortical epithelial cells and primary human retinal pigment epithelial cells. As positive control either FoIR1 positive SKOV3 cells or HT-29 cells were included. First we verified FoIR1 expression on the used primary cells and determined the amount of FoIR1 binding sites on these cells. Bronchial epithelial cells, renal cortical epithelial cells and retinal pigment epithelial cells express very low but significant levels of FoIR1 compared to the levels expressed on tumor cells. The choroid plexus epithelial cells do not express significant levels of FoIR1. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 FolR1 binding sites on 
               
               
                 primary epithelial cells 
               
            
           
           
               
               
               
            
               
                   
                 Cell line 
                 Binding sites 
               
               
                   
                   
               
               
                   
                 Bronchial epithelium 
                   492 
               
               
                   
                 Choroid plexus epithelium 
                   104 
               
               
                   
                 Renal cortical epithelium 
                   312 
               
               
                   
                 Retinal pigment epithelium 
                   822 
               
               
                   
                 Skov3 
                 69′890 
               
               
                   
                   
               
            
           
         
       
     
     The primary epithelial cells that demonstrated FoIR1 expression on the surface were used to address the question if these cells can be killed by T cells in the presence of FoLR1 TCBs. No significant levels of killing could be measured but induction of T cell activation in the presence of retinal pigment epithelial cells, bronchial epithelial cells and renal cortical cells resulting in upregulation of CD25 and CD69 was detected. The strongest activation is seen with retinal pigment epithelial cells resulting in upregulation of CD25 and CD69 both on CD4 +  T cells and CD8 +  T cells. In the presence of bronchial epithelial cells lower activation of T cells is induced with upregulation of CD69 on CD4 +  T cells and CD8 +  T cells but very low upregulation of CD25 only on CD4 +  T cells but not on CD8 +  T cells. The lowest activation of T cells is obtained in the presence of renal epithelial cells with no upregulation of CD25 on CD4 T +  cells and CD8 +  T cells and CD69 been only upregulated on CD8 +  T cells ( FIGS. 12A-X ). 
     Example 20 
     Comparison of Different TCB Formats Containing Either 16D5 or 9D11 Binder 
     To determine if the TCB 2+1 inverted format is the most active format with the selected FoIR1 binder, different formats containing either 16D5 or 9D11 were produced and compared in target cell binding, T cell mediated killing and T cell activation. The 16D5 binder was tested in the TCB 2+1 inverted ( FIG. 1A ), TCB 2+1 classical ( FIG. 1D ), TCB 1+1 classical ( FIG. 1C ) and TCB 1+1 head-to-tail ( FIG. 1B ) format; the 9D11 binder was tested in the TCB 2+1 inverted ( FIG. 1A ), TCB 1+1 classical ( FIG. 1C ) and TCB 1+1 head-to-tail ( FIG. 1B ) format. 
     All constructs were tested for binding to FoIR1 on HeLa cells. The molecules bivalent for binding to FoIR1 bind stronger compared to the monovalent constructs due to avidity. The difference between the bivalent vs. monovalent constructs is more pronounced for 16D5. The reason might be that due to the lower affinity of 16D5 the avidity effect for this binder is stronger. Between the two 1+1 TCBs there is no significant difference in binding but there is a difference between the two 2+1 constructs. The inverted 2+1 construct binds stronger to FoIR1 than the classical 2+1 construct. This indicates that in the classical 2+1 construct the binding to FoLR1 is influenced by the presence of the CD3 Fab whereas in the inverted construct binding is less influenced. 
     By testing T cell mediated killing with these constructs we could show that stronger binding of the 2+1 inverted TCB in converted into stronger tumor cell killing and T cell activation compared to the 2+1 classical TCB. The 16D5 FoIR1 TCB 2+1 classical is only a little bit more active than the respective 1+1 head-to-tail construct. The 1+1 head-to-tail construct is significantly more active than the 1+1 classical construct. This does not reflect the situation seen in binding and might be due to better crosslinking with the head-to-tail construct. Overall tumor cell killing and T cell activation is comparable with all tested constructs, the differences in potency seen with the differences are only in terms of EC50 values. In general it can be concluded that the FoIR1 TCB 2+1 inverted independent of the used binder is the preferred format to induce T cell mediated tumor cell killing and T cell activation (see  FIGS. 13A-C  and  FIGS. 14A-C ). 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 EC50 values of target cell binding and T cell 
               
               
                 mediated killing with different TCB formats 
               
            
           
           
               
               
               
               
            
               
                   
                 Binding EC50 
                 Killing 24 h 
                 Killing 48 h 
               
               
                 Construct 
                 (nM) 
                 (pM) 
                 (pM) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 16D5 FolR1 TCB 
                 11.03 
                 1.43 
                 0.18 
               
               
                 2 + 1 inverted 
                   
                   
                   
               
               
                 16D5 FolR1 TCB 
                 17.07 
                 5.60 
                 2.18 
               
               
                 2 + 1 classical 
                   
                   
                   
               
               
                 16D5 FolR1 TCB 
                 107.3 
                 n.d. 
                 n.d. 
               
               
                 1 + 1 classical 
                   
                   
                   
               
               
                 16D5 FoLR1 TCB 
                 102.6 
                 26.24 
                 6.06 
               
               
                 1 + 1 head-to-tail 
                   
                   
                   
               
               
                 9D11 FoLR1 TCB 
                 17.52 
                 0.74 
                 0.14 
               
               
                 2 + 1 inverted 
                   
                   
                   
               
               
                 9D11 FoLR1 TCB 
                 38.57 
                 20.92 
                 n.d. 
               
               
                 1 + 1 classical 
                   
                   
                   
               
               
                 9D11 FoLR1 TCB 
                 44.20 
                 4.73 
                 n.d. 
               
               
                 1 + 1 head-to-tail 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 EC50 values of T cell activation in the 
               
               
                 presence of SKOV3 cells with different TCB 
               
               
                 formats 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 CD4+ 
                 CD4+ 
                 CD8+ 
                 CD8+ 
               
               
                   
                   
                 CD25+ 
                 CD69+ 
                 CD25+ 
                 CD69+ 
               
               
                   
                 Construct 
                 (%) 
                 (%) 
                 (%) 
                 (%) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 16D5 FolR1 TCB 
                 1.96 
                 0.33 
                 2.10 
                 n.d. 
               
               
                   
                 2 + 1 inverted 
                   
                   
                   
                   
               
               
                   
                 16D5 FolR1 TCB 
                 13.83 
                 3.67 
                 12.88 
                 4.47 
               
               
                   
                 2 + 1 classical 
                   
                   
                   
                   
               
               
                   
                 16D5 FolR1 TCB 
                 38.54 
                 n.d. 
                 n.d. 
                 n.d. 
               
               
                   
                 1 + 1 classical 
                   
                   
                   
                   
               
               
                   
                 16D5 FolR1 TCB 
                 17.14 
                 7.47 
                 25.15 
                 n.d. 
               
               
                   
                 1 + 1 head-to-tail 
                   
                   
                   
                   
               
               
                   
                 9D11 FolR1 TCB 
                 1.41 
                 0.27 
                 1.24 
                 0.35 
               
               
                   
                 2 + 1 inverted 
                   
                   
                   
                   
               
               
                   
                 9D11 FolR1 TCB 
                 34.01 
                 n.d. 
                 34.39 
                 7.40 
               
               
                   
                 1 + 1 classical 
                   
                   
                   
                   
               
               
                   
                 9D11 FolR1 TCB 
                 3.73 
                 2.47 
                 4.98 
                 2.89 
               
               
                   
                 1 + 1 head-to-tail 
               
               
                   
                   
               
            
           
         
       
     
     Example 21 
     Tumor Cell Lines and Primary Cells 
     HeLa cells (CCL-2) were obtained from ATCC and cultured in DMEM with 10% FCS and 2 mM Glutamine, SKOV3 (HTB-77) were obtained from ATCC and cultured in RPMI with 10% FCS and 2 mM Glutamine, OVCAR5 were obtained from NCI and cultured in RPMI with 10% FCS and 2 mM Glutamine, HT-29 (ACC-299) were obtained from DSMZ and cultured in McCoy&#39;s 5A medium with 10% FCS and 2 mM Glutamine, MKN-45 (ACC-409) were obtained from DSMZ and cultured in RPMI with 10% FCS and 2 mM Glutamine. 
     All tested primary epithelial cells were obtained from ScienCell Research Laboratories. Human Bronchial Epithelium Cells (HBEpiC, Catalog Number 3210 were cultured in Bronchial Epithelial Cell Medium (BEpiCM, Cat. No. 3211, ScienCell). Human Colonic Epithelial Cells (HCoEpiC), Catalog Number 2950 were cultured in Colonic Epithelial Cell Medium (CoEpiCM, Cat. No. 2951, ScienCell). Human Retinal Pigment Epithelial Cells (HRPEpiC), Catalog Number 6540 were cultured in Epithelial Cell Medium (EpiCM, Cat. No. 4101, ScienCell). Human Renal Cortical Epithelial Cells (HRCEpiC), Catalog Number 4110, were cultured in Epithelial Cell Medium (EpiCM, Cat. No. 4101, ScienCell). Human Choroid Plexus Epithelial Cells (HCPEpiC), Catalog Number 1310 were cultured in Epithelial Cell Medium (EpiCM, Cat. No. 4101, ScienCell). 
     Example 22 
     Target Binding by Flow Cytometry 
     Target cells as indicated were harvested with Cell Dissociation Buffer, washed with PBS and resuspended in FACS buffer. The antibody staining was performed in a 96 well round bottom plate. Therefore 200,000 cells were seeded per well. The plate was centrifuged for 4 min at 400 g and the supernatant was removed. The test antibodies were diluted in FACS buffer and 20 μl of the antibody solution were added to the cells for 30 min at 4° C. To remove unbound antibody the cells were washed twice with FACS buffer before addition of the diluted secondary antibody (FITC conjugated AffiniPure F(ab′)2 fragment goat anti-human IgG, Fcg Fragment, Jackson ImmunoResearch #109-096-098 or PE-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgG Fcg Fragment Specific, Jackson ImmunoResearch #109-116-170. After 30 min incubation on 4° C. unbound secondary antibody was washed away. Before measurement the cells were resuspended in 200 μl FACS buffer and analyzed by flow cytometry using BD Canto II or BD Fortessa. 
     Example 23 
     Internalization 
     The cells were harvested and the viability was determined. The cells were re-suspended in fresh cold medium at 2 Mio cells per ml and the cell suspension was transferred in a 15 ml falcon tube for each antibody. The antibodies that should be tested for internalization were added with a final concentration of 20 μg per ml to the cells. The tubes were incubated for 45 min in the cold room on a shaker. After incubation the cells were washed three times with cold PBS to remove unbound antibodies. 0.2 Mio cells per well were transfer to the FACS plate as time point zero. The labeled cells were re-suspended in warm medium and incubated at 37° C. At the indicated time-points 0.2 Mio cells per well were transferred in cold PBS, washed in plated on the FACS plate. To detect the constructs that remain on the surface the cells were stained with PE-labeled anti-human Fc secondary antibody. Therefore 20 μl of the diluted antibody were added per well and the plate was incubated for 30 min at 4° C. Then the cells were washed twice to remove unbound antibodies and then fixed with 1% PFA to prevent any further internalization. The fluorescence was measured using BD FACS Cantoll. 
     Example 24 
     QIFIKIT® Analysis 
     QIFIKIT® contains a series of beads, 10 μm in diameter and coated with different, but well-defined quantities of mouse Mab molecules (high-affinity anti-human CD5, Clone CRIS-1, isotype IgG2a). The beads mimic cells with different antigen densities which have been labeled with a primary mouse Mab, isotype IgG. Briefly, cells were labeled with primary mouse monoclonal antibody directed against the antigen of interest. In a separate test well, cells were labeled with irrelevant mouse monoclonal antibody (isotype control). Then, cells, Set-Up Beads and Calibration Beads were labeled with a fluorescein-conjugated anti-mouse secondary antibody included in the kit. The primary antibody used for labeling of the cells has to be used at saturating concentration. The primary antibody may be of any mouse IgG isotype. Under these conditions, the number of bound primary antibody molecules corresponds to the number of antigenic sites present on the cell surface. The secondary antibody is also used at saturating concentration. Consequently, the fluorescence is correlated with the number of bound primary antibody molecules on the cells and on the beads. 
     Example 25 
     T Cell Mediated Tumor Cell Killing and T Cell Activation 
     Target cells were harvested with Trypsin/EDTA, counted and viability was checked. The cells were resuspended in their respective medium with a final concentration of 300,000 cells per ml. Then 100 μl of the target cell suspension was transferred into each well of a 96-flat bottom plate. The plate was incubated overnight at 37° C. in the incubator to allow adherence of the cells to the plate. On the next day PBMCs were isolated from whole blood from healthy donors. The blood was diluted 2:1 with PBS and overlayed on 15 ml Histopaque-1077 (#10771, Sigma-Aldrich) in Leucosep tubes and centrifuged for 30 min at 450 g without break. After centrifugation the band containing the cells was collected with a 10 ml pipette and transferred into 50 ml tubes. The tubes were filled up with PBS until 50 ml and centrifuged (400 g, 10 min, room temperature). The supernatant was removed and the pellet resuspended in PBS. After centrifugation (300 g, 10 min, room temperature), supernatants were discarded, 2 tubes were pooled and the washing step was repeated (this time centrifugation 350×g, 10 min, room temperature). Afterwards the cells were resuspended and the pellets pooled in 50 ml PBS for cell counting. After counting cells were centrifuged (350 g, 10 min, room temperature) and resuspended at 6 Mio cells per ml in RPMI with 2% FCS and 2 nM Glutamine. Medium was removed from plated target cells and the test antibodies diluted in RPMI with 2% FCS and 2 nM Glutamine were added as well as. 300,000 cells of the effector cell solution were transferred to each well resulting in a E:T ratio of 10:1. To determine the maximal release target cells were lysed with Triton X-100. LDH release was determined after 24 h and 48 h using Cytotoxicity Detection Kit (#1644793, Roche Applied Science). Activation marker upregulation on T cells after tumor cell killing was measured by flow cytometry. Briefly PBMCs were harvested, transferred into a 96 well round bottom plate and stained with CD4 PE-Cy7 (#3557852, BD Bioscience), CD8 FITC (#555634, BD Bioscience), CD25 APC (#555434, BD Bioscience), CD69 PE (#310906, BioLegend) antibodies diluted in FACS buffer. After 30 min incubation at 4° C. the cells were washed twice with FACS buffer. Before measuring the fluorescence using BD Canto II the cells were resuspended in 200 μl FACS buffer. 
     Example 26 
     T Cell Activation in Whole Blood 
     280 μl of fresh blood were added into a 96 well conical deep well plate. Then 20 μl of the diluted TCBs were added to the blood and mixed well by shaking the plate. After 24 h incubation at 37° C. in an incubator the blood was mixed and 35 μl were transferred to a 96 well round bottom plate. Then 20 μl of the antibody staining mix were added consisting of CD4 PE-Cy7 (#3557852, BD Bioscience), CD8 FITC (#555634, BD Bioscience), CD25 APC (#555434, BD Bioscience), CD69 PE (#310906, BioLegend) and CD45 V500 (#560777, BD Horizon) and incubated for 15 min in the dark at room temperature. Before measuring 200 μl of the freshly prepared BD FACS lysing solution (#349202, BD FCAS) was added to the blood. After 15 min incubation at room temperature the cells were measured with BD Fortessa. 
     Example 27 
     SDPK (Single Dose Pharmacokinetics) Study of Humanized FOLR1 TCB (Clone 16D5) in Immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) Mice 
     Female NOD/Shi-scid/IL-2Rγnull (NOG) mice, age 6-7 weeks at start of the experiment (bred at Taconic, Denmark) were maintained under specific-pathogen-free condition with daily cycles of 12 h light/12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by local government (P 2011/128). After arrival, animals were maintained for one week to get accustomed to the new environment and for observation. Continuous health monitoring was carried out on a regular basis. 
     Mice were injected i.v. with 10/1/0.1 μg/mouse of the FOLR1 TCB whereas 3 mice were bled per group and time point. All mice were injected with a total volume of 200 μl of the appropriate solution. To obtain the proper amount of the FOLR1 TCB per 200 μl, the stock solutions were diluted with PBS when necessary. Serum samples were collected 5 min, 1h, 3h, 8h, 24h, 48 h, 72h, 96h and 168h after therapy injection. 
       FIG. 15  shows that the 16D5 FOLR1 TCB shows typical and dose proportional IgG-like PK properties in NOG mice with slow clearance. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 21 
               
               
                   
               
               
                   
                   
                   
                 Concentration 
               
               
                 Compound 
                 Dose 
                 Formulation buffer 
                 (mg/mL) 
               
               
                   
               
             
            
               
                 FOLR1 TCB 
                 10 μg 
                 20 mM Histidine, 
                 5.43 
               
               
                 (16D5) 
                 (corresponding to 
                 140 mM NaCl, 
                 (= stock solution) 
               
               
                   
                 ca. 0.5 mg/kg) 
                 pH 6.0 
                   
               
               
                 FOLR1 TCB 
                 1 μg 
                 20 mM Histidine, 
                 5.43 
               
               
                 (16D5) 
                 (corresponding to 
                 140 mM NaCl, 
                 (= stock solution) 
               
               
                   
                 ca. 0.05 mg/kg) 
                 pH 6.0 
                   
               
               
                 FOLR1 TCB 
                 0.1 μg 
                 20 mM Histidine, 
                 5.43 
               
               
                 (16D5) 
                 (corresponding to 
                 140 mM NaCl, 
                 (= stock solution) 
               
               
                   
                 ca. 0.005 mg/kg) 
                 pH 6.0 
               
               
                   
               
            
           
         
       
     
     Example 28 
     In Vivo Efficacy of FOLR1 TCB (Clone 16D5) after Human PBMC Transfer in Skov3-Bearing NOG Mice 
     The FOLR1 TCB was tested in the human ovarian carcinoma cell line Skov3, injected s.c. into PBMC engrafted NOG mice. 
     The Skov3 ovarian carcinoma cells were obtained from ATCC (HTB-77). The tumor cell line was cultured in RPMI containing 10% FCS (Gibco) at 37° C. in a water-saturated atmosphere at 5% CO 2 . Passage 35 was used for transplantation, at a viability &gt;95%. 5×10 6  cells per animal were injected s.c. into the right flank of the animals in a total of 100 μl of RPMI cell culture medium (Gibco). 
     Female NOD/Shi-scid/IL-2Rγnull (NOG) mice, age 6-7 weeks at start of the experiment (bred at Taconic, Denmark) were maintained under specific-pathogen-free condition with daily cycles of 12 h light/12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by local government (P 2011/128). After arrival, animals were maintained for one week to get accustomed to the new environment and for observation. Continuous health monitoring was carried out on a regular basis. 
     According to the protocol ( FIG. 16 ), mice were injected s.c. on study day 0 with 5×10 6  of the Skov3. At study day 21, human PBMC of a healthy donor were isolated via the Ficoll method and 10×10 6  cells were injected i.p. into the tumor-bearing mice. Two days after, mice were randomized and equally distributed in five treatment groups (n=12) followed by i.v. injection with either 10/1/0.1 μg/mouse of the FOLR1 TCB or 10 μg/mouse of the DP47 control TCB once weekly for three weeks. All mice were injected i.v. with 200 μl of the appropriate solution. The mice in the vehicle group were injected with PBS. To obtain the proper amount of TCB per 200 μl, the stock solutions were diluted with PBS when necessary. Tumor growth was measured once weekly using a caliper ( FIG. 17 ) and tumor volume was calculated as followed: 
       T v : (W 2 /2)×L (W: Width, L: Length)
 
     The once weekly injection of the FOLR1 TCB resulted in a dose-dependent anti-tumoral effect. Whereas a dose of 10 μg/mouse and 1 μg/mouse induced tumor shrinkage and 0.1 μg/mouse a tumor stasis ( FIG. 17 , Table 22). Maximal tumor shrinkage was achieved at a dose of 10 μg/mouse as compared to a non-targeted control DP47 TCB. 
     
       
         
           
               
               
               
             
               
                 TABLE 22 
               
               
                   
               
               
                   
                   
                 Tumor growth 
               
               
                 Compound 
                 Dose 
                 inhibition 
               
               
                   
               
             
            
               
                 DP47 TCB 
                 10 μg 
                  7% 
               
               
                 control TCB 
                 (corresponding to 
                   
               
               
                   
                 ca. 0.5 mg/kg) 
                   
               
               
                 FOLR1 TCB 
                 10 μg 
                 90% 
               
               
                 (16D5) 
                 (corresponding to 
                   
               
               
                   
                 ca. 0.5 mg/kg) 
                   
               
               
                 FOLR1 TCB 
                 1 μg 
                 74% 
               
               
                 (16D5) 
                 (corresponding to 
                   
               
               
                   
                 ca. 0.05 mg/kg) 
                   
               
               
                 FOLR1 TCB 
                 0.1 μg 
                 56% 
               
               
                 (16D5) 
                 (corresponding to 
                   
               
               
                   
                 ca. 0.005 mg/kg) 
               
               
                   
               
            
           
         
       
     
     For PD read-outs, three mice per treatment group were sacrificed at study day 32, tumors were removed and single cell suspensions were prepared through an enzymatic digestion with Collagenase V, Dispase II and DNAse for subsequent FACS-analysis ( FIGS. 19 and 20 ). Single cells where either used directly for staining of extracellular antigens and activation markers or were re-stimulated using 5 ng/ml PMA and 500 ng/ml lonomycin in the presence of a protein transport inhibitor Monensin for 5h in normal culture medium. After re-stimulation, cells were stained for surface antigens, followed by a fixation and permeabilization step. Fix samples were then stained intracellulary for TNF-α, IFN-γ, IL-10 and IL-2 and analyzed by flow cytometry. Same procedure was used for the degranulation of cells, but an anti-CD107a antibody was added during the restimulation period and fixed samples were staining for intracellular perforin and granzyme-B contents. The FACS analysis revealed statistically higher number of infiltrating CD4 +  and CD8 +  T-cells in the tumor tissue upon treatment with FOLR1 TCB compared to vehicle and untargeted control TCB. Furthermore, higher numbers of TNF-1, IFN-Q and IL-2 producing as well as perforin + /granzym-B +  CD4 +  and CD8 +  T-cells were detected in FOLR1 TCB treated tumors. Tumor infiltrating T-cells treated with FOLR1 TCB also showed higher degranulation rates compared to control groups. 
     At study termination day 38, all animals were sacrificed; tumors were removed and weight ( FIG. 18 ). The weight of the tumors treated with 10 and 1 μg/mouse of the FOLR1 TCB showed a statistically significant difference compared to the control groups. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 23 
               
               
                   
                   
               
               
                   
                   
                   
                 Formulation 
                 Concentration 
               
               
                   
                 Compound 
                 Dose 
                 buffer 
                 (mg/mL) 
               
               
                   
                   
               
             
            
               
                   
                 PBS 
                   
                   
                   
               
               
                   
                 FOLR1 TCB 
                  10 μg 
                 20 mM Histidine, 
                 3.88 
               
               
                   
                 (16D5) 
                   
                 140 mM NaCl, 
                 (= stock solution) 
               
               
                   
                   
                   
                 pH 6.0 
                   
               
               
                   
                 FOLR1 TCB 
                   1 μg 
                 20 mM Histidine, 
                 3.88 
               
               
                   
                 (16D5) 
                   
                 140 mM NaCl, 
                 (= stock solution) 
               
               
                   
                   
                   
                 pH6.0 
                   
               
               
                   
                 FOLR1 TCB 
                 0.1 μg 
                 20 mM Histidine, 
                 3.88 
               
               
                   
                 (16D5) 
                   
                 140 mM NaCl, 
                 (= stock solution) 
               
               
                   
                   
                   
                 pH6.0 
                   
               
               
                   
                 DP47 TCB 
                  10 μg 
                 20 mM Histidine, 
                 4.35 
               
               
                   
                   
                   
                 140 mM NaCl, 
                 (= stock solution) 
               
               
                   
                   
                   
                 pH6.0 
               
               
                   
                   
               
            
           
         
       
     
     Example 29 
     Generation of a Bispecific FoIR1/CD3-kappa-lambda Antibody 
     To generate a bispecific antibody (monovalent for each antigen) that simultaneously can bind to human CD3 and human folate receptor alpha (FoIR1) without using any hetero-dimerization approach (e.g. knob-into-hole technology), a combination of a common light chain library with the so-called CrossMab technology was applied: The variable region of the humanized CD3 binder (CH2527_VL7_46/13) was fused to the CH1 domain of a standard human IgG1 antibody to form the VLVH crossed molecule (fused to Fc) which is common for both specificities. To generate the crossed counterparts (VHCL), a CD3 specific variable heavy chain domain (CH2527_VH_23/12) was fused to a constant human K light chain whereas a variable heavy chain domain specific for human FoIR1 (clone 16D5, isolated from common light chain library) was fused to a constant human k light chain. This enables the purification of the desired bispecific antibody by applying subsequent purification steps with KappaSelect and LambdaFabSelect columns (GE Healthcare) to remove undesired homodimeric antibodies. 
     All antibody expression vectors were generated using standard recombinant DNA technology as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Molecular biological reagents were used according the manufacturer&#39;s recommendations. Genes or gene fragments were either amplified by polymerase chain reaction (PCR) or generated from synthetic oligonucleotides at Geneart AG (Regensburg, Germany) by automated gene synthesis. PCR-amplified or subcloned DNA fragments were confirmed by DNA sequencing (Synergene GmbH, Switzerland). Plasmid DNA was transformed into and amplified in suitable  E. coli  host strains for preparation of transfection-grade plasmid DNA using standard Maxiprep kits (Qiagen). For production of the bispecific molecules HEK293 EBNA cells were transfected with plasmids encoding the respective genes using a standard polyethlenimine (PEI) based method. The used plasmid ratio of the three expression vectors was 1:1:1. Transfected cells were cultivated for 7 days before supernatants were harvested for purification. The bispecific FoIR1/CD3-kappa-lambda antibodies were produced and purified as follows. 
     1. Transient Transfection and Production 
     The kappa-lambda bispecific antibody was transiently produced in HEK293 EBNA cells using a PEI mediated transfection procedure for the required vectors as described below. HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection (for alternative scales all amounts were adjusted accordingly). For transfection cells were centrifuged for 5 min by 210×g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 μg DNA. After addition of 540 μl PEI solution is vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO 2  atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210×g, the solution is sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4° C. 
     2. Purification 
     The kappa-lambda bispecific antibody was purified in three steps, using an affinity step specific for kappa light chains, followed by an affinity step specific for lambda light chains and finally by a size exclusion chromatography step for removal of aggregates. The supernatant obtained from transient production was adjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to Capture Select kappa affinity matrix, or HiTrap KappaSelect, GE Healthcare, column volume (cv)=1 ml, equilibrated with 5 column volumes (cv) buffer A (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 8.0). After washing with 15 cv of buffer A, the protein was eluted using a pH gradient to buffer B (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 2.0) over 25 cv. Fractions containing the protein of interest were pooled and the pH of the solution was adjusted to pH 8.0 (using 2 M Tris pH 8.0). The neutralized pooled fractions were applied to Capture Select lambda affinity matrix (now: HiTrap LambdaFabSelect, GE Healthcare, column volume (cv)=1 ml) equilibrated with 5 column volumes (cv) buffer A (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 8.0). After washing with 15 cv of buffer A, the protein was eluted using a pH gradient to buffer B (50 mM Tris, 100 mM glycine, 150 mM NaCl, pH 2.0) over 25 cv. Fractions containing the protein of interest were pooled and the pH of the solution was adjusted to pH 8.0 (using 2 M Tris pH 8.0). This solution was concentrated using ultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius) and subsequently applied to a Superdex™ 200 10/300 GL (GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl, 0.01% Tween-20. The pooled fractions after size exclusion were again concentrated using ultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius). 
     The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of the constructs were analyzed by SDS capillary electrophoresis in the presence and absence of a reducing agent following the manufacturer instructions (instrument Caliper LabChipGX, Perkin Elmer). Only small amounts of protein could be purified with a final yield of 0.17 mg/L. 
     Example 30 
     T cell mediated killing with bispecific FoIR1/CD3-kappa-lambda antibody Activity of kappa lambda FoIR1 TCB was tested on SKOV3 cells in the presence of freshly isolated PBMCs. As negative control DP47 TCB was included. T cell mediated killing of SKOV3 cells was determined after 24 h and 48 h by LDH release. After 48 h the T cells were harvested and CD69 and CD25 upregulation on CD4 T cells and CD8 T cells was measured by flow cytometry. 
     The kappa lambda FoIR1 construct induces killing of SKOV3 cells in a concentration dependent manner which is accompanied by CD69 and CD25 upregulation both on CD4 T cells and on CD8 T cells. 
     SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 24 h and 48 h killing of tumor cells was determined by measuring LDH release ( FIG. 21 ). SKOV3 cells were incubated with PBMCs in the presence of either kappa lambda FoLR1 TCB or DP47 TCB. After 48 h CD25 and CD69 upregulation on CD4 T cells and CD8 T cells was measured by flow cytometry ( FIG. 22 ). 
     Example 31 
     Biochemical Characterization of 16D5 and 36F2 FoIR1 Binders by Surface Plasmon Resonance 
     Binding of anti-FoIR1 16D5 in different monovalent or bivalent T-cell bispecific formats and of anti-FoIR1 36F2 as IgG or as T-cell bispecific to recombinant human, cynomolgus and murine folate receptor 1 (all as Fc fusions) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, GE Healthcare). 
     1. Molecules Tested 
     The molecules used for affinity and avidity determination are described in Table 24. 
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 Name and description of the 6 
               
               
                 constructs used in SPR analysis 
               
            
           
           
               
               
               
            
               
                   
                 Name 
                 Description 
               
               
                   
                   
               
               
                   
                 16D5 TCB 
                 2 + 1 T-cell bispecific, inverted 
               
               
                   
                   
                 format (common light chain) 
               
               
                   
                 16D5 TCB classical 
                 2 + 1 T-cell bispecific, classical 
               
               
                   
                   
                 format (common light chain) 
               
               
                   
                 16D5 TCB 1 + 1 
                 1 + 1 T-cell bispecific (common 
               
               
                   
                   
                 light chain) 
               
               
                   
                 16D5 TCB 1 + 1 HT 
                 1 + 1 T-cell bispecific head-to-tail 
               
               
                   
                   
                 (common light chain) 
               
               
                   
                 36F2 IgG 
                 Human IgG1 with P329G LALA 
               
               
                   
                 36F2 TCB 
                 2 + 1 T-cell bispecific, inverted 
               
               
                   
                   
                 format, crossfab 
               
               
                   
                   
               
            
           
         
       
     
     2. Avidity to Folate Receptor 1 
     The avidity of the interaction between the anti-FoIR1 IgG or T cell bispecifics and the recombinant folate receptors was determined as described below (Table 25). 
     Recombinant biotinylated monomeric Fc fusions of human, cynomolgus and murine Folate Receptor 1 (FoIR1-Fc) were directly coupled on a SA chip using the standard coupling instruction (Biacore, GE Healthcare). The immobilization level was about 300-400 RU. The anti-FoIR1 IgGs or T cell bispecifics were passed at a concentration range from 3.7 to 900 nM with a flow of 30 μL/minutes through the flow cells over 180 seconds. The dissociation was monitored for 240 or 600 seconds. The chip surface was regenerated after every cycle using a double injection of 30 sec 10 mM Glycine-HCl pH 2. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell immobilized with recombinant biotinylated murine CD134 Fc fusion. The binding curves resulting from the bivalent binding of the IgG or T cell bispecifics were approximated to a 1:1 Langmuir binding (even though it is a 1:2 binding) and fitted with that model to get an apparent K D  representing the avidity of the bivalent binding. The apparent avidity constants for the interactions were derived from the rate constants of the fitting using the Bia Evaluation software (GE Healthcare). For the 1+1 T cell bispecifics format the interaction is a real 1:1 and the K D  represents affinity since there is only one FoIR1 binder in this construct. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Bivalent binding (avidity with apparent KD) 
               
               
                 of anti-FolR1 16D5 and 36F2 as IgG or as T-cell 
               
               
                 bispecifics (TCB) on human, cyno and murine FolR1. 
               
            
           
           
               
               
               
               
               
            
               
                 Analyte 
                 Ligand 
                 ka (1/Ms) 
                 kd (1/s) 
                 Apparent KD 
               
               
                   
               
               
                 36F2 IgG 
                 huFolR1 
                 2.07E+06 
                  1.3E−02 
                   6 nM 
               
               
                   
                 cyFolR1 
                 2.78E+06 
                  1.75E−02 
                   6 nM 
               
               
                   
                 muFolR1 
                 4.28E+05 
                  8.23E−04 
                   2 nM 
               
               
                 36F2 TCB 
                 huFolR1 
                 2.45E+06 
                 9.120E−03 
                   4 nM 
               
               
                   
                 cyFolR1 
                 4.31E+06 
                  1.45E−02 
                   3 nM 
               
               
                   
                 muFolR1 
                 6.97E+05 
                  9.51E−04 
                   1 nM 
               
               
                 16D5 TCB 
                 huFolR1 
                 1.57E+05 
                  3.92E−04 
                   3 nM 
               
               
                   
                 cyFolR1 
                 2.01E+05 
                  3.81E−04 
                   2 nM 
               
               
                 16D5 TCB 
                 huFolR1 
                 2.04E+05 
                  1.84E−04 
                 0.9 nM 
               
               
                 classical 
                 cyFolR1 
                 2.50E+05 
                  3.05E−04 
                   1 nM 
               
               
                 16D5 TCB 
                 huFolR1 
                 5.00E+04 
                  2.25E−03 
                  45 nM 
               
               
                 1 + 1 HT 
                 cyFolR1 
                 5.75E+04 
                  4.10E−03 
                  70 nM 
               
               
                 16D5 
                 huFolR1 
                 3.65E+04 
                  2.04E−03 
                  56 nM 
               
               
                 TCB 1 + 1 
                 cyFolR1 
                 4.09E+04 
                  3.60E−03 
                  90 nM 
               
               
                   
               
            
           
         
       
     
     3. Affinity to Folate Receptor 1 
     The affinity of the interaction between the anti-FoIR1 IgG or T cell bispecifics and the recombinant folate receptors was determined as described below (Table 26). 
     For affinity measurement, direct coupling of around 12000 resonance units (RU) of the anti-human Fab specific antibody (Fab capture kit, GE Healthcare) was performed on a CM5 chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). Anti-FoIR1 IgG or T cell bispecifics were captured at 20 nM with a flow rate of 10 μl/min for 40 sec, the reference flow cell was left without capture. Dilution series (12.3 to 3000 nM) of human, cyno or murine Folate Receptor 1 Fc fusion were passed on all flow cells at 30 μl/min for 240 sec to record the association phase. The dissociation phase was monitored for 300 s and triggered by switching from the sample solution to HBS-EP. The chip surface was regenerated after every cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 1.5. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell 1. The affinity constants for the interactions were derived from the rate constants by fitting to a 1:1 Langmuir binding using the Bia Evaluation software (GE Healthcare). 
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Monovalent binding (affinity) of anti-FolR1 
               
               
                 16D5 and 36F2 as IgG or as T-cell bispecifics (TCB) 
               
               
                 on human, cyno and murine FolR1. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Analyte 
                 Ligand 
                 ka (1/Ms) 
                 kd (1/s) 
                 KD 
               
               
                   
                   
               
               
                   
                 36F2 IgG 
                 huFolR1 
                 9.10E+04 
                 6.65E−02 
                  730 nM 
               
               
                   
                   
                 cyFolR1 
                 1.02E+05 
                 5.78E−02 
                  570 nM 
               
               
                   
                   
                 muFolR1 
                 8.32E+04 
                 1.78E−02 
                  210 nM 
               
               
                   
                 36F2 TCB 
                 huFolR1 
                 5.94E+04 
                 6.13E−02 
                 1000 nM 
               
               
                   
                   
                 cyFolR1 
                 6.29E+04 
                 5.42E−02 
                  860 nM 
               
               
                   
                   
                 muFolR1 
                 5.68E+04 
                 1.75E−02 
                  300 nM 
               
               
                   
                 16D5 TCB 
                 huFolR1 
                 2.23E+04 
                 7.33E−04 
                  33 nM 
               
               
                   
                   
                 cyFolR1 
                 1.57E+04 
                 1.60E−03 
                  100 nM 
               
               
                   
                 16D5 TCB 
                 huFolR1 
                 1.03E+04 
                 7.59E−04 
                  74 nM 
               
               
                   
                 classical 
                 cyFolR1 
                 9.18E+03 
                 1.61E−03 
                  175 nM 
               
               
                   
                 16D5 TCB 
                 huFolR1 
                 2.05E+04 
                 7.08E−04 
                  35 nM 
               
               
                   
                 1 + 1 HT 
                 cyFolR1 
                 1.67E+04 
                 1.53E−03 
                  92 nM 
               
               
                   
                 16D5 
                 huFolR1 
                 1.43E+04 
                 9.91E−04 
                  69 nM 
               
               
                   
                 TCB 1 + 1 
                 cyFolR1 
                 1.20E+04 
                 1.80E−03 
                  150 nM 
               
               
                   
                   
               
            
           
         
       
     
     The affinity (monovalent binding) to human and cyno FoIR1-Fc of 36F2 TCB is similar and around 1000 nM for both, whereas the affinity to murine FoIR1-Fc is slightly better and around 300 nM. The 36F2 can be used in murine and primate models, there is no need for a surrogate. 
     The avidity (apparent K D ) of 36F2 TCB to human FoIR1 is around 30 times lower than the affinity of the 16D5 TCB to human FoIR1. In the bivalent format, 36F2 TCB is in the low nanomolar range, whereas 16D5 TCB is in the low picomolar range (1000 fold difference). 
     FoIR1 is expressed on tumor cells overexpressed, at intermittent and high levels, on the surface of cancer cells in a spectrum of epithelial malignancies, including ovarian, breast, renal, colorectal, lung and other solid cancers and is also expressed on the apical surface of a limited subset of polarized epithelial cells in normal tissue. These non-tumorous, normal cells express FoIR1 only at low levels, and include, e.g., bronchiolal epithelial cells on alveolar surface, renal cortical luminal border of tubular cells, retinal pigment epithelium (basolateral membrane) and choroid plexus. 
     16D5 TCB binds to normal tissues cells expressing low amounts of FoIR1 which results in their T cell mediated killing. This might, at least in part, account for limited tolerance observed at 10 μg/kg in cynomolgus monkeys. The inventors wanted to determine if lowering the affinity of the T cell bispecific molecule could increase the differentiation between high and low target density tissues and, thereby, lower toxicity by making use of bivalent binding and avidity. Low affinity binders are ordinarily not selected as suitable candidates for further analysis because low affinity is often associated with low potency and efficacy. Nevertheless, the low affinity FoIR1 binder 36F2 was developed in several formats and characterized for its biological properties. For the 36F2 used in the bivalent T cell bispecific format the avidity effect (difference between monovalent and bivalent binding) is around 250 fold (1000 nM versus 4 nM). At low target density the affinity defined the interaction and with 1000 nM led to a low potency of the TCB. However, at high target density the molecule&#39;s avidity comes into play and with 4 nM led to a high activity of the TCB (see Example 32). 
     In an alternatively approach, the inventors generated monovalent formats of 16D5 and low affinity variant of 16D5 (affinity about 10-40 nM) in a bivalent format. The 16D5 binder used in a monovalent format (1+1) has an affinity of about 50 nM. The differentiation between high and low target density tissues can be better achieved by taking advantage of the avidity effect. 
     Example 32 
     T-Cell Killing of SKov-3 Cells Induced by 36F2 TCB, Mov19 TCB and 21A5 TCB 
     T-cell killing mediated by 36F2 TCB, Mov19 TCB and 21A5 TCB was assessed on SKov-3 cells (medium FoIR1). Human PBMCs were used as effectors and the killing was detected at 24 h and 48 h of incubation with the bispecific antibodies. Briefly, target cells were harvested with Trypsin/EDTA, washed, and plated at density of 25 000 cells/well using flat-bottom 96-well plates. Cells were left to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450×g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (400×g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350×g, 10 minutes). The resulting PBMC population was counted automatically (ViCell) and stored in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37° C., 5% CO2 in cell incubator until further use (no longer than 24 h). For the killing assay, the antibody was added at the indicated concentrations (range of 0.005 pM-5 nM in triplicates). PBMCs were added to target cells at final E:T ratio of 10:1. Target cell killing was assessed after 24 h and 48 h of incubation at 37° C., 5% CO2 by quantification of LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11 644 793 001). Maximal lysis of the target cells (=100%) was achieved by incubation of target cells with 1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells without bispecific construct. 
     The results show that the killing induced by 36F2 is strongly reduced in comparison to Mov19 TCB and 21A5 TCB ( FIGS. 23A-B ). The EC50 values related to killing assays, calculated using GraphPadPrism6 are summarized in Table 27. 
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 EC50 values (pM) for T-cell mediated 
               
               
                 killing of FolR1-expressing SKov-3 
               
               
                 cells induced by 36F2 
               
               
                 TCB, Mov19 TCB and 21A5 TCB. 
               
            
           
           
               
               
               
            
               
                   
                   
                 EC50 [pM] 
               
            
           
           
               
               
               
               
            
               
                   
                 Antibody 
                 24 h 
                 48 h 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 36F2 TCB 
                 1406.07* 
                 134.5 
               
               
                   
                 Mov19 TCB 
                 0.75 
                 0.05 
               
               
                   
                 21A5 TCB 
                 2.83 
                 0.10 
               
               
                   
                   
               
               
                   
                 *curve did not reach saturation, value is hypothetical 
               
            
           
         
       
     
     Example 33 
     T-Cell Killing Induced by 36F2 TCB and 16D5 TCB in Different Monovalent and Bivalent T-Cell Bispecific Formats 
     T-cell killing mediated by 36F2 TCB, 16D5 TCB, 16D5 TCB classical, 16D5 TCB 1+1 and 16D5 TCB HT antibodies of Hela (high FoIR1, about 2 million copies, Table 14,  FIG. 27 ), Skov-3 (medium FoIR1, about 70000-90000 copies, Table 14,  FIG. 27 ) and HT-29 (low FoIR1, about 10000, Table 14,  FIG. 27 ) human tumor cells was assessed. DP47 TCB antibody was included as negative control. Human PBMCs were used as effectors and the killing was detected at 24 h of incubation with the bispecific antibody. Briefly, target cells were harvested with Trypsin/EDTA, washed, and plated at density of 25 000 cells/well using flat-bottom 96-well plates. Cells were left to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450×g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (400×g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350×g, 10 minutes). The resulting PBMC population was counted automatically (ViCell) and stored in RPM11640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37° C., 5% CO2 in cell incubator until further use (no longer than 24 h). For the killing assay, the antibody was added at the indicated concentrations (range of 0.01 pM-100 nM in triplicates). PBMCs were added to target cells at final E:T ratio of 10:1. Target cell killing was assessed after 24 h of incubation at 37° C., 5% CO2 by quantification of LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11 644 793 001). Maximal lysis of the target cells (=100%) was achieved by incubation of target cells with 1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells without bispecific construct. 
     The results show that target-specific killing of all three FoIR1+ target cell lines induced by 36F2 TCB is much weaker compared to the killing induced by 16D5 TCB ( FIGS. 24A-C , Table 29). Target-specific killing induced by the monovalent 16D5 TCBs (16D5 HT and 16D5 1+1) is worse compared to the bivalent 16D5 TCBs (16D5 TCB and 16D5 TCB classical). The EC50 values related to killing assays, calculated using GraphPadPrism6, are summarized in Table 28. Importantly, this data shows that using the 36F2 FoIR1 binder in the bivalent 2+1 TCB format widens the therapeutic window compared to the 16D5 FOLR1 TCB ( FIGS. 24A-C ). Whereas the potency reduction between 16D5 and 36F2 FOLR1 TCB is approximately 45-fold for Hela cells (high FOLR1 expression, see Table 28: 16D5 TCB=0.8 versus 36F2 TCB 36.0) and approximately 297-fold for Skov3 cells (medium FOLR1 expression, see Table 28: 16D5 TCB=0.6 versus 36F2 TCB 178.4), this reduction is almost 7000-fold for HT29 with low FOLR1 expression (see Table 28: 16D5 TCB=5.7 versus 36F2 TCB 39573). Thus, the 36F2 FOLR1 TCB differentiates between high and low expressing cells which is of special importance to reduce toxicity as the cells of some normal, non-tumorous tissues express very low levels of FoIR1 (approximately less than 1000 copies per cell). Consistent with this observation, the results discussed in Example 35 below show that 36F2 TCB does not induce T-cell killing of primary cells ( FIGS. 26A-D ) whereas for 16D5 TCB some killing can be observed on HRCEpiC and HRPEpiC cells after 48 h of incubation ( FIGS. 26B  and C). This important characteristic of 36F2 TCB allows for dosing for the treatment of FoIR1-positive tumors so that it mediates potent killing of tumor tissues with high or medium FOLR1 expression, but not of normal tissues with low (partially polarized) expression. Notably, this characteristic appears to be mediated by the avidity of 36F2 TCB in the bivalent 2+1 inverted format, as it was not observed when using the 1+1 monovalent formats carrying the same low affinity 36F2 binder. 
     Stated another way, 36F2 TCB in the bivalent 2+1 format comprises FoIR1 binding moieties of relatively low affinity but it possesses an avidity effect which allows for differentiation between high and low FoIR1 expressing cells. Because tumor cells express FoIR1 at high or intermediate levels, this TCB selectively binds to tumor cells and not normal, non-cancerous cells that express FoIR1 at low levels or not at all. 
     In addition to the above advantageous characteristics, the 36F2 TCB in the bivalent 2+1 inverted format also has the advantage that it does not require chemical cross linking or other hybrid approach. This makes it suitable for manufacture of a medicament to treat patients, for example patients having FoIR1-positive cancerous tumors. The 36F2 TCB in the bivalent 2+1 inverted format can be produced using standard CHO processes with low aggregates. Further, the 36F2 TCB in the bivalent 2+1 comprises human and humanized sequences making it superior to molecules that employ rat and murine polypeptides that are highly immunogenic when administered to humans. Furthermore, the 36F2 TCB in the bivalent 2+1 format was engineered to abolish FcgR binding and, as such, does not cause FcgR crosslinking and infusion reactions, further enhancing its safety when administered to patients. 
     As demonstrated by the results described above, its head-to-tail geometry make the 36F2 TCB in the bivalent 2+1 inverted format a highly potent molecule that induces absolute target cell killing. Its bivalency enhance avidity and potency, but also allow for differentiation between high and low expressing cells. Its preference for high or medium target expressing cells due to its avidity affect reduce toxicity resulting from T cell mediated killing of normal cells that express FoIR1 at low levels. 
     A further advantage of the 36F2 TCB in the bivalent 2+1 format and other embodiments disclosed herein is that their clinical development does not require the use of surrogate molecules as they bind to human, cynomous and murine FoIR1. As such, the molecules disclosed herein recognize a different epitope than antibodies to FoIR1 previously described that do not recognize FoIR1 from all three species. 
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 EC50 values (pM) for T-cell mediated killing 
               
               
                 of FolR1-expressing tumor cells induced by 36F2 
               
               
                 TCB and 16D5 TCB in different monovalent 
               
               
                 and bivalent T-cell bispecific formats after 24 h of 
               
               
                 incubation. 
               
            
           
           
               
               
               
               
            
               
                   
                 Hela 
                 Skov-3 
                 HT-29 
               
               
                 Antibody 
                 (FolR1 high) 
                 (FolR1 medium) 
                 (FolR1 low) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 16D5 TCB 
                 0.8 
                 0.6 
                 5.7 
               
               
                 16D5 TCB 
                 4.6 
                 2.0 
                 13.0 
               
               
                 classical 
                   
                   
                   
               
               
                 16D5 TCB HT 
                 11.6 
                 12.3 
                 15.1 
               
               
                 16D5 TCB 
                 23.8 
                 48.9 
                 883.8* 
               
               
                 1 + 1 
                   
                   
                   
               
               
                 36F2 TCB 
                 36.0 
                 178.4 
                 39573.0* 
               
               
                   
               
               
                 *curve did not reach saturation, only hypothetical value 
               
            
           
         
       
     
     Table 29 shows a comparison of EC50 values of 16D5 TCB and 36F2 TCB on the different cell lines tested. Out of the obtained EC50 values the delta (EC50 of 16D5 TCB minus EC50 of 36F2 TCB) and the x-fold difference (EC50 of 16D5 TCB divided by the EC50 of 36F2 TCB) was calculated. 
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Comparison of EC50 values of 16D5 TCB and 36F2 TCB. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Hela 
                 Skov-3 
                 HT-29 
               
               
                   
                 Antibody 
                 (FoIR1 high) 
                 (FoIR1 medium) 
                 (FoIR1 low) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 16D5 TCB 
                 0.82 
                 0.63 
                 5.73 
               
               
                   
                 36F2 TCB 
                 35.99 
                 178.40 
                 39573.00* 
               
               
                   
                 Δ 
                 35.17 
                 177.77 
                 39567.27 
               
               
                   
                 x-fold 
                 43.83 
                 284.61 
                 6906.58 
               
               
                   
                   
               
               
                   
                 *curve did not reach saturation, only hypothetical value 
               
            
           
         
       
     
     The calculated EC50 values clearly show that the difference between 36F2 TCB and 16D5 TCB gets larger the lower the FoIR1 expression on the target cells is. 
     The same calculations as done for the comparison of the EC50 values of 16D5 TCB and 36F2 TCB were done for 16D5 TCB and the two monovalent 16D5 TCBs (16D5 TCB HT and 16D5 1+1). Tables 30 and 31 show the comparisons of the EC50 values of 16D5 TCB vs 16D5 TCB HT (Table 30) and 16D5 TCB vs 16D5 TCB 1+1 (Table 31) as well as the corresponding deltas (EC50 of 16D5 TCB minus EC50 of 16D5 TCB HT/1+1) and the x-fold differences (EC50 of 16D5 TCB divided by the EC50 of 16D5 TCB HT/1+1). 
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 Comparison of EC50 values of 16D5 TCB (2 + 1 inverted)  
               
               
                 and 16D5 TCB HT. 
               
            
           
           
               
               
               
               
            
               
                   
                 Hela 
                 Skov-3 
                 HT-29 
               
               
                 Antibody 
                 (FoIR1 high) 
                 (FoIR1 medium) 
                 (FoIR1 low) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 16D5 TCB 
                 0.82 
                 0.63 
                 5.73 
               
               
                 16D5 TCB HT 
                 11.61 
                 12.27 
                 15.11 
               
               
                 Δ 
                 10.79 
                 11.65 
                 9.38 
               
               
                 x-fold 
                 14.14 
                 19.58 
                 2.64 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 Comparison of EC50 values of 16D5 TCB and 16D5 TCB 1 + 1. 
               
            
           
           
               
               
               
               
            
               
                   
                 Hela 
                 Skov-3 
                 HT-29 
               
               
                 Antibody 
                 (FoIR1 high) 
                 (FoIR1 medium) 
                 (FoIR1 low) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 16D5 TCB 
                 0.82 
                 0.63 
                 5.73 
               
               
                 16D5 TCB 1 + 1 
                 23.84 
                 48.86 
                 883.78* 
               
               
                 Δ 
                 23.02 
                 48.24 
                 878.05 
               
               
                 x-fold 
                 29.03 
                 77.95 
                 154.24 
               
               
                   
               
               
                 *curve did not reach saturation, only hypothetical value 
               
            
           
         
       
     
     The comparison of the EC50 values of 16D5 TCB and 36F2 TCB (Table 29) shows that the difference in the EC50 values gets larger the lower the FoIR1 expression on the target cells is. This effect cannot be seen in the comparison of 16D5 TCB and the monovalent 16D5 TCBs (Table 29 and Table 30). For 16D5 TCB 1+1 (Table 31) there is also a slight increase in the difference between the EC50 of 16D5 TCB and 16D5 TCB 1+1 with decreasing FoIR1 expression but by far not as pronounced as can be seen in the comparison of 16D5 TCB vs 36F2 TCB. 
     Example 34 
     CD25 and CD69 Upregulation on CD8+ and CD4+ Effector Cells after T Cell-Killing of FoIR1-Expressing Tumor Cells Induced by 36F2 TCB and 16D5 TCB Antibody 
     Activation of CD8 +  and CD4 +  T cells after T-cell killing of FoIR1-expressing Hela, SKov-3 and HT-29 tumor cells mediated by 36F2 TCB and 16D5 TCB was assessed by FACS analysis using antibodies recognizing the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker). DP47 TCB was included as non-binding control. The antibody and the killing assay conditions were essentially as described above (Example 32) using the same antibody concentration range (0.01 pM-100 nM in triplicates), E:T ratio 10:1 and an incubation time of 48 h. 
     After the incubation, PBMCs were transferred to a round-bottom 96-well plate, centrifuged at 400×g for 4 min and washed twice with PBS containing 0.1% BSA. Surface staining for CD8 (PE anti-human CD8, BD #555635), CD4 (Brilliant Violet 421™ anti-human CD4, Biolegend #300532), CD69 (FITC anti-human CD69, BD #555530) and CD25 (APC anti-human CD25 BD #555434) was performed according to the manufacturer&#39;s instructions. Cells were washed twice with 150 μl/well PBS containing 0.1% BSA. After centrifugation, the samples were resuspended in 200 μl/well PBS 0.1% for the FACS measurement. Samples were analyzed at BD FACS Canto II. 
     36F2 TCB induced a target-specific up-regulation of activation markers (CD25, CD69) on CD8+ and CD4 +  T cells after killing of Hela ( FIG. 25A ) and SKov-3 ( FIG. 25B ) cells. In comparison to 16D5 TCB the up-regulation of CD25 and CD69 on CD8+ and CD4 +  T cells induced by 36F2 is much weaker. 
     On HT-29 (low FoIR1) an up-regulation of activation markers can only be seen at the highest concentration of 36F2 TCB. In contrast, with 16D5 TCB up-regulation of CD25 and CD69 can be seen already at much lower antibody concentrations ( FIG. 25C ). 
     As seen as well in the tumor lysis experiment, the analysis of activation markers (CD25 and CD69) on T cells (CD4+ and CD8+) after killing clearly shows that the difference between 16D5 TCB and 36F2 TCB becomes larger the lower the FoIR1 expression level on the target cells is. 
     Example 35 
     T-Cell Killing of Primary Cells Induced by 36F2 TCB and 16D5 TCB 
     T-cell killing mediated by 36F2 TCB and 16D5 TCB was assessed on primary cells (Human Renal Cortical Epithelial Cells (HRCEpiC) (ScienCell Research Laboratories; Cat No 4110) and Human Retinal Pigment Epithelial Cells (HRPEpiC) (ScienCell Research Laboratories; Cat No 6540)). HT-29 cells (low FoIR1) were included as control cell line. DP47 TCB served as non-binding control. Human PBMCs were used as effectors and the killing was detected at 24 h and 48 h of incubation with the bispecific antibodies. Briefly, target cells were harvested with Trypsin/EDTA, washed, and plated at density of 25 000 cells/well using flat-bottom 96-well plates. Cells were left to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450×g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (400×g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350×g, 10 minutes). The resulting PBMC population was counted automatically (ViCell) and stored in RPM11640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37° C., 5% CO 2  in cell incubator until further use (no longer than 24 h). For the killing assay, the antibody was added at the indicated concentrations (range of 0.01 pM-10 nM in triplicates). PBMCs were added to target cells at final E:T ratio of 10:1. Target cell killing was assessed after 24 h and 48 h of incubation at 37° C., 5% CO 2  by quantification of LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11 644 793 001). Maximal lysis of the target cells (=100%) was achieved by incubation of target cells with 1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells without bispecific construct. 
     The results show that 36F2 TCB does not induce T-cell killing of primary cells ( FIGS. 26A-D ) whereas for 16D5 TCB some killing can be observed on HRCEpiC and HRPEpiC cells after 48 h of incubation ( FIGS. 26B  and D). As described above, a strong difference in T-cell killing between of HT-29 cells was observed between 16D5 TCB and 36F2 TCB ( FIGS. 26E , F). 
     Example 36 
     Preparation of DP47 GS TCB (2+1 Crossfab-IgG P329G LALA Inverted=“Untargeted TCB”) 
     The “untargeted TCB” was used as a control in the above experiments. The bispecific antibody engages CD3e but does not bind to any other antigen and therefore cannot crosslink T cells to any target cells (and subsequently cannot induce any killing). It was therefore used as negative control in the assays to monitor any unspecific T cell activation. This untargeted TCB was prepared as described in WO2014/131712. In brief, the variable region of heavy and light chain DNA sequences have been subcloned in frame with either the constant heavy chain or the constant light chain pre-inserted into the respective recipient mammalian expression vector. The antibody expression was driven by an MPSV promoter and carries a synthetic polyA signal sequence at the 3′ end of the CDS. In addition each vector contains an EBV OriP sequence. 
     The molecule was produced by co-transfecting HEK293-EBNA cells with the mammalian expression vectors using polyethylenimine. The cells were transfected with the corresponding expression vectors in a 1:2:1:1 ratio (“vector heavy chain Fc(hole)”:“vector light chain”:“vector light chain Crossfab”:“vector heavy chain Fc(knob)-FabCrossfab”). 
     For transfection HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 min by 210×g, supernatant is replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 g DNA. After addition of 540 μl PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210×g, the solution was sterile filtered (0.22·m filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4° C. 
     The secreted protein was purified from cell culture supernatants by affinity chromatography using ProteinA. Supernatant was loaded on a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibrated with 40 ml 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein was removed by washing with at least 10 column volume 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Target protein was eluted during a gradient over 20 column volume from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 to 20 mM sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solution was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Target protein was concentrated and filtrated prior loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine, 140 mM sodium chloride solution of pH 6.0. 
     The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. 
     Purity and molecular weight of molecules were analyzed by CE-SDS analyses in the presence and absence of a reducing agent. The Caliper LabChip GXII system (Caliper lifescience) was used according to the manufacturer&#39;s instruction. 2 ug sample is used for analyses. 
     The aggregate content of antibody samples was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25° C. 
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 Summary production and purification of DP47 GS TCB. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Aggregate 
                   
                   
                   
               
               
                   
                   
                   
                 after 1 st   
                   
                   
                   
               
               
                   
                 Titer 
                 Yield 
                 purification 
                 HMW 
                 LMW 
                 Monomer 
               
               
                 Construct 
                 [mg/l] 
                 [mg/l] 
                 step [%] 
                 [%] 
                 [%] 
                 [%] 
               
               
                   
               
               
                 DP47 GS 
                 103.7 
                 8.04 
                 8 
                 2.3 
                 6.9 
                 91.8 
               
               
                 TCB 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 CE-SDS analyses of DP47 GS TCB. 
               
            
           
           
               
               
               
               
            
               
                   
                 Peak 
                 kDa 
                 Corresponding Chain 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 DP47 GS TCB  
                 1 
                 165.22 
                 Molecule with 2 missing 
               
               
                 non reduced (A) 
                   
                   
                 light chains 
               
               
                   
                 2 
                 181.35 
                 Molecule with 1 missing 
               
               
                   
                   
                   
                 light chain 
               
               
                   
                 3 
                 190.58 
                 Correct molecule without N- 
               
               
                   
                   
                   
                 linked glycosylation 
               
               
                   
                 4 
                 198.98 
                 Correct molecule 
               
               
                 DP47 GS TCB  
                 1 
                 27.86 
                 Light chain DP47 GS 
               
               
                 reduced (B) 
                 2 
                 35.74 
                 Light chain huCH2527 
               
               
                   
                 3 
                 63.57 
                 Fc(hole) 
               
               
                   
                 4 
                 93.02 
                 Fc(knob) 
               
               
                   
               
            
           
         
       
     
     Example 37 
     Binding of 16D5 TCB and 9D11 TCB and their Corresponding CD3 Deamidation Variants N100A and S100aA to CD3-Expressing Jurkat Cells 
     The binding of 16D5 TCB and the corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and the deamidation variants 9D11 TCB N100A and 9D11 TCB S100aA to human CD3 was assessed on a CD3-expressing immortalized T lymphocyte line (Jurkat). Briefly, cells were harvested, counted, checked for viability and resuspended at 2×10 6  cells/ml in FACS buffer (100 μl PBS 0.1% BSA). 100 μl of cell suspension (containing 0.2×10 6  cells) was incubated in round-bottom 96-well plates for 30 min at 4° C. with different concentrations of the bispecific antibodies (686 pM-500 nM). After two washing steps with cold PBS 0.1% BSA, samples were re-incubated for further 30 min at 4° C. with a PE-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgG Fcg Fragment Specific secondary antibody (Jackson Immuno Research Lab PE #109-116-170). After washing the samples twice with cold PBS 0.1% BSA they were immediately analyzed by FACS using a FACS Cantoll (Software FACS Diva). Binding curves were obtained using GraphPadPrism6 ( FIGS. 28A-B ). 
     The results show reduced binding of the deamidation variants N100A and S100aA to CD3 compared to the parental antibodies 16D5 TCB ( FIG. 28A ) and 9D11 TCB ( FIG. 28B ). 
     Example 38 
     T-Cell Killing of SKov-3 and HT-29 Cells Induced by 16D5 TCB and 9D11 TCB and their CD3 Deamidation Variants N100A and S100aA 
     T-cell killing mediated by 16D5 TCB and the corresponding CD3 deamidation variants 16D5 TCB N100A and 16D5 TCB S100aA and 9D11 TCB and the deamidation variants 9D11 TCB N100A and 9D11 TCB S100aA was assessed on SKov-3 (medium FoIR1) and HT-29 (low FoIR1) cells. Human PBMCs were used as effectors and the killing was detected at 24 h of incubation with the bispecific antibodies. Briefly, target cells were harvested with Trypsin/EDTA, washed, and plated at a density of 25 000 cells/well using flat-bottom 96-well plates. Cells were left to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of enriched lymphocyte preparations (buffy coats) obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered over Histopaque gradient (Sigma, #H8889). After centrifugation (450×g, 30 minutes, room temperature), the plasma above the PBMC-containing interphase was discarded and PBMCs transferred in a new falcon tube subsequently filled with 50 ml of PBS. The mixture was centrifuged (400×g, 10 minutes, room temperature), the supernatant discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps 350×g, 10 minutes). The resulting PBMC population was counted automatically (ViCell) and stored in RPM11640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37° C., 5% CO2 in cell incubator until further use (no longer than 24 h). For the killing assay, the antibody was added at the indicated concentrations (range of 0.01 pM-10 nM in triplicates). PBMCs were added to target cells at final E:T ratio of 10:1. Target cell killing was assessed after 24 h of incubation at 37° C., 5% CO 2  by quantification of LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11 644 793 001). Maximal lysis of the target cells (=100%) was achieved by incubation of target cells with 1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells without bispecific construct. 
     The results show that on SKov-3 cells the killing induced by the CD3 deamidation variants 16D5 TCB N100A and 16D5 S100aA is comparable to the one induced by 16D5 TCB ( FIG. 29A ). The same is true for 9D11 TCB and its variants 9D11 TCB N100A and 9D11 TCB S100aA ( FIG. 29B ). On FoIR1 low expressing HT-29 cells the S100aA variant shows an impaired killing efficiency which is the case for 16D5 TCB ( FIG. 30A ) as well as for 9D11 TCB ( FIG. 30B ). The EC50 values related to killing assays, calculated using GraphPadPrism6 are given in Table 35. 
     
       
         
           
               
             
               
                 TABLE 35 
               
             
            
               
                   
               
               
                 EC50 values (pM) for T-cell mediated killing of FoIR1-expressing  
               
               
                 SKov-3 and HT-29 cells induced by 16D5 TCB and 9D11 TCB  
               
               
                 and their deamidation variants N100A and A100aA. 
               
            
           
           
               
               
            
               
                   
                 EC50 [pM] 
               
            
           
           
               
               
               
            
               
                 Antibody 
                 SKov-3 
                 HT-29 
               
               
                   
               
            
           
           
               
               
               
            
               
                 16D5 TCB 
                 1.283 
                 56.67 
               
               
                 16D5 TCB 
                 1.886 
                 91.95 
               
               
                 N100A 
                   
                   
               
               
                 16D5 TCB 
                 1.939 
                 165.6 
               
               
                 S100aA 
                   
                   
               
               
                 9D11 TCB 
                 1.283 
                 2.827 
               
               
                 9D11 TCB 
                 1.886 
                 37.72 
               
               
                 N100A 
                   
                   
               
               
                 9D11 TCB 
                 1.939 
                 n.d.* 
               
               
                 S100aA 
               
               
                   
               
               
                 *not determined 
               
            
           
         
       
     
     Example 39 
     Generation of Mucin-1 T Cell Bi-Specific Constructs that Contain a Common Light Chain 
     Gene Synthesis 
     Desired gene segments were synthesized at Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. 
     Production and Purification of MUC1 Antigen 
     For the generation of common light chain (CLC) antibodies against the “sea urchin sperm protein, enterokinase and agrin” (SEA) domain of human Mucin-1 (MUC1), a DNA fragment encoding SEA domain was synthesized (Uniprot P15941, amino acids 1041-1151). In order to prevent autoproteolysis of the SEA between positions G1097 and S1098, a process described previously, by Ligtenber et al. (1992). Cell-associated episialin is a complex containing two proteins derived from a common precursor. J Biol Chem 267, 6171-7. Parry et al., Identification of MUC1 Proteolytic Cleavage Sites in Vivo. Biochem Biophys Res Commun 283, 715-20, four additional glycine residues were inserted between G1097 and S1098. This insertion results in the relief of the conformational stress and the protein remains intact. The DNA fragment was inserted into pETXX, an inducible bacterial expression vector. The resulting plasmid expresses the SEA domain with a C-terminal avi tag and a His6 tag (SEQ ID NO: 47). While the avi tag was used for BirA-mediated in vivo biotinylation, the His6 tag (SEQ ID NO: 47) was used for purification. 
     A 500 ml culture was inoculated with the bacterial strain BL21 D3, transformed with the corresponding plasmid and a plasmid expressing BirA, and induced with 1 mM IPTG at an OD 600  0.8. Afterwards, the cultures were incubated at 25° C. overnight and harvested by centrifugation. The bacterial pellet was resuspended with 25 ml BugBuster® Protein Extraction Reagent (Millipore) and incubated for 20 min at room temperature as described in the protocol. After centrifugation for 20 min at 16000×g, the supernatant was filtered and loaded on an IMAC column (His gravitrap, GE Healthcare). The column was washed with 40 ml washing buffer (500 mM NaCl, 20 mM Imidazole, 20 mM NaH 2 PO 4  pH 7.4). After the elution from the column (500 mM NaCl, 500 mM Imidazole, 20 mM NaH 2 PO 4  pH 7.4), the eluate was re-buffered using PD10 columns (GE Healthcare). 
     Selection of Anti-Human MUC1 SEA Domain Binders from the CLC Fab Libraries 
     Selections against the SEA domain of human MUC1 were carried out using  E. coli -derived and in vivo biotinylated MUC1. The antigens were enzymatically biotinylated by co-expression of the biotin ligase Bir A via an C-terminal avi-tag. Panning rounds were performed in solution according to the following pattern: 1. Binding of the phagemid particles of the CLC libraries to 100 nM biotinylated antigen protein for 0.5 h in a total volume of 1 ml, 2. capture of biotinylated antigen and attached specifically binding phage by addition of 5.4×10 7  streptavidin-coated magnetic beads for 10 min, 3. washing of beads using 5×1 ml PBS/Tween20 and 5×1 ml PBS, 4. elution of phage particles by addition of 1 ml 100 mM triethylamine (TEA) for 10 min and neutralization by addition of 500 ul 1 M Tris/HCl pH 7.4, 5. Re-infection of log-phase  E. coli  TG1 cells with the phage particles in the supernatant, infection with helperphage VCSM13 and subsequent PEG/NaCl precipitation of phagemid particles to be used in subsequent selection rounds. Selections were carried out over 3 rounds using either constant or decreasing (from 10 −7 M to 2×10 −9 M) antigen concentrations. In round 2, capture of antigen:phage complexes was performed using neutravidin plates instead of streptavidin beads. Specific binders were identified by ELISA as follows: 100 ul of 50 nM biotinylated antigen per well were coated on neutravidin plates. Fab-containing bacterial supernatants were added and binding Fabs were detected via their Flag-tags by using an anti-Flag/HRP secondary antibody. The VH domains of clones exhibiting significant signals over background were short-listed for sequencing (58D6 VH, 106D2 VH, 110A5 VH) and further analyses. All clones derive from the IGHV3-23 germline sequence ( FIG. 31 ). Of note, clone 58D6 and 110A5 originate from a library that was randomized only in CDR3, while clone 106D2 was identified from a library randomized in all 3 CDRs. Positions in CDR1 and 2 that deviate from the germline sequence are printed italic. All VH variants were expressed in combination with the same common light chain (common light chain VL). 
     Purification of Fabs 
     Fabs from bacterial cultures (protein sequence of variable heavy chains domains for 58D6 VH, 106D2 VH, 110A5 VH, all clones expressed the same CLC variable domain listed as SEQ ID NO: 31) were purified for the exact analysis of the kinetic parameters. For each clone, a 500 ml culture was inoculated with bacteria harboring the corresponding phagemid and induced with 1 mM IPTG at an OD 600  0.9. Afterwards, the cultures were incubated at 25° C. overnight and harvested by centrifugation. After the incubation of the resuspended pellet for 20 min in 25 ml PPB buffer (30 mM Tris-HCl pH8, 1 mM EDTA, 20% sucrose), bacteria were centrifuged again and the supernatant was harvested. This incubation step was repeated once with 25 ml of a 5 mM MgSO 4  solution. The supernatants of both incubation steps were pooled, filtered and loaded on an IMAC column (His gravitrap, GE Healthcare). Subsequently, the column was washed with 40 ml washing buffer (500 mM NaCl, 20 mM Imidazole, 20 mM NaH 2 PO 4  pH 7.4). After the elution (500 mM NaCl, 500 mM Imidazole, 20 mM NaH 2 PO 4  pH 7.4) the eluate was re-buffered using PD10 columns (GE Healthcare). The kinetic parameters of the purified Fabs were then studied by SPR-analysis (Proteon XPR36, Biorad) in a dilution row that ranged from 100 nM to 6.25 nM. 
     Affinity-Determination by SPR Using BioRad&#39;s ProteOn XPR36 Biosensor 
     Affinity (K D ) of selected Fab clones was measured by surface plasmon resonance using a ProteOn XPR36 instrument (Biorad) at 25° C. with biotinylated MUC1 antigen immobilized on NLC chips by neutravidin capture. Immobilization of recombinant antigens (ligand): Antigens were diluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween 20) to 10 μg/ml, then injected at 30 μl/minute at varying contact times, to achieve immobilization levels of about 200 response units (RU) in vertical orientation. Injection of analytes: For one-shot kinetics measurements, injection direction was changed to horizontal orientation, two-fold dilution series of purified Fab (varying concentration ranges between 100 and 6.25 nM) were injected simultaneously at 50 μl/min along separate channels 1-5, with association times of 250 or 300s, and dissociation times of 300s. Buffer (PBST) was injected along the sixth channel to provide an “in-line” blank for referencing. Association rate constants (k on ) and dissociation rate constants (k off ) were calculated using a simple one-to-one Langmuir binding model in ProteOn Manager v3.1 software by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K D ) was calculated as the ratio k off /k on . Regeneration was performed in horizontal orientation using 10 mM Glycine, pH 1.5 at a flow rate of 100 ul/min for a contact time of 30s. 3 clones bound specifically to MUC1 ( FIG. 32A ), but not to the “in-line” blank demonstrating the specificity of these binders. The kinetic and thermodynamic data of all measurements are summarized in  FIG. 32B . 
     Cloning, Expression and Characterization of the MUC1-Based 
     All Fabs demonstrating specific binding to MUC1 by SPR were converted into the T cell bi-specific (TCB) format. For this, the PCR-amplified DNA fragments of heavy and light chain VH-domains were inserted in frame into both Fc-containing Ig chains needed for the generation of a TCB ( FIG. 33 ). The antibody chain expression is driven by an MPSV promoter and transcription is terminated by a synthetic polyA signal sequence located downstream of the CDS. In addition to the expression cassette, each vector contains an EBV oriP sequence for autonomous replication in EBV-EBNA expressing cell lines. The resulting DNA constructs were co-expressed in combination with the CLC (clone 58D6; clone 110A5) and purified from mammalian-derived cell culture supernatant (clone 58D6: clone 110A5). A summary of the analytical data for two bi-specific antibodies is shown clones in  FIGS. 34A  and B. 
     Binding Analysis of the MUC1-Specific TCBs Using BioRad&#39;s ProteOn XPR36 Biosensor 
     Binding of the produced MUC1-specific TCBs was measured by surface plasmon resonance using a ProteOn XPR36 instrument (Biorad) at 25° C. Biotinylated MUC1 antigen and an unrelated biotinylated antigen were immobilized on NLC chips by neutravidin capture. Immobilization of the antigens was performed as described before. For one-shot kinetics measurements, injection direction was changed to horizontal orientation, two-fold dilution series of the purified constructs (varying concentration ranges between 30 and 1.88 nM or between 100 and 6.25 nM) were injected simultaneously at 50 μl/min along separate channels 1-5, with association times of 120s, and dissociation times of up to 600s. Buffer (PBST) was injected along the sixth channel to provide an “in-line” blank for referencing. 
     Regeneration was performed in horizontal orientation using 10 mM Glycine, pH 1.5 at a flow rate of 100 ul/min for a contact time of 30s. 3 clones specifically bound to MUC1 ( FIG. 35 ), but not to the “in-line” blank demonstrating the specificity of these binders. Due to the avidity effect of the bivalent TCB format, very strong binding to MUC1 was observed. In contrast, no binding to the unrelated antigen was detected indicating the specific binding of the TCB constructs. 
     Example 40 
     Generation of Anti-BCMA Antibodies 
     1.1: Production of Antigens and Tool Reagents 
     1.1.1: Recombinant, Soluble, Human BCMA Extracellular Domain 
     The extracellular domains of human, cynomolgus and murine BCMA that were used as antigens for phage display selections were transiently expressed as N-terminal monomeric Fc-fusion in HEK EBNA cells and in vivo site-specifically biotinylated via co-expression of BirA biotin ligase at the avi-tag recognition sequence located at the C-terminus of the Fc portion carrying the receptor chain (Fc knob chain). The extracellular domains of human, cynomolgus and murine BCMA comprised methionine 4 to asparagine 53, methionine 4 to asparagine 52, and alanine 2 to threonine 49, respectively. These were N-terminally fused to the hinge of a human IgG1 enabling heterodimerization with an unfused human IgG1 Fc portion (hole chain) by knobs-into-holes technology. 
     For recovering of the extracellular domain of BCMA the following primers were used: 
     AAGCTTGGATCCATGTTGCAGATGGCTGGGCAGTGCTCC-3 (SEQ ID NO: 48) incorporating a BamH1 site (bold, underlined) and 
                    reverse primer       (SEQ ID NO: 49)       5-GAATTCGCGGCCGCTCATCCTTTCACTGAATTGGTCACACTTGCATTA               C-3               primer       (SEQ ID NO: 50)       5-ACGTTAGATCTCCACTCAGTCCTGCATCTTGTTCCAGTTAAC-3       and               reverse primer       (SEQ ID NO: 51)       5-AACGTTGCGGCCGCTAGTTTCACAAACCCCAGG-3               (SEQ ID NO: 52)       GAATTCAAGCTTGCCACCATGTTGCAGATGGCTGGGCAGTGCTCC-3            
including a HindIII restriction site (bold, underlined) and Kozak consensus sequence and
 
     
       
         
           
               
            
               
                 reverse primer 
               
               
                 (SEQ ID NO: 53) 
               
               
                 5-GAATTCTCTAGATTACCTAGCAGAAATTGATTTCTCTATCTCCGTAG 
               
               
                   
               
               
                 C-3 
               
            
           
         
       
     
     Gene synthesis can be also used to obtain the extracellular domain of BCMA. 
     1.2: BCMA-Expressing Cells as Tools 
     1.2.1: Human Myeloma Cell Line Expressing BCMA on their Surface 
     BCMA expression was assessed on four human myeloma cell lines (NCI-H929, RPMI-8226, U266B1 and L-363) by flow cytometry. NCI-H929 cells ((H929) ATCC® CRL-9068™) were cultured in 80-90% RPMI 1640 with 10-20% heat-inactivated FCS and could contain 2 mM L-glutamine, 1 mM sodium pyruvate and 50 μM mercaptoethanol. RPMI-8226 cells ((RPMI) ATCC® CCL-155™) were cultured in a media containing 90% RPMI 1640 and 10% heat-inactivated FCS. U266B1 ((U266) ATCC® TIB-196™) cells were cultured in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate and 15% heat-inactivated FCS. L-363 cell line (Leibniz Institute DSMZ—German collection of microorganisms and cell cultures; DSMZ No. ACC 49) was cultured in 85% RPMI 1640 and 15% heat-inactivated FCS. Briefly, cells were harvested, washed, counted for viability, resuspended at 50,000 cells/well of a 96-well round bottom plate and incubated with anti-human BCMA antibody (Abcam, #ab54834, mouse IgG1) at 10 μg/ml for 30 min at 4° C. (to prevent internalization). A mouse IgG1 was used as isotype control (BD Biosciences, #554121). Cells were then centrifuged (5 min at 350×g), washed twice and incubated with the FITC-conjugated anti mouse secondary antibody for 30 min at 4° C. At the end of incubation time, cells were centrifuged (5 min at 350×g), washed twice with FACS buffer, resuspended in 100 ul FACS buffer and analyzed on a Cantoll device running FACS Diva software. The relative quantification of BCMA receptor number on the surface membrane of H929, U266B1, RPMI-8226 and L-363 myeloma cell lines was assessed by QIFIKIT analysis (Dako, #K0078, following manufacturer&#39;s instructions). H929 cells expressed human BCMA with the highest density, up to 3.8 to 27-fold higher than other myeloma cell lines. H929 is considered as a BCMA hi -expressing myeloma cell line as compared to U266 which is BCMA med/lo -expressing myeloma cells and RPMI-8226 and L363 which are BCMA lo -expressing myeloma cells. Table 36 summarizes the relative BCMA receptor number on the cell surface of human multiple myeloma cell lines. 
     
       
         
           
               
             
               
                 TABLE 36 
               
             
            
               
                   
               
               
                 Quantification of BCMA receptor number on cell 
               
               
                 surface of NCI-H929, U266B1, RPMI-8226 
               
               
                 and L-363 human myeloma cell lines 
               
            
           
           
               
               
               
            
               
                   
                   
                 Relative binding sites 
               
               
                   
                 Myeloma cell lines 
                 per cell 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 H929 
                 50000 
               
               
                   
                 U266 
                 13000 
               
               
                   
                 RPMI-8226 
                 2000 
               
               
                   
                 L363 
                 1800 
               
               
                   
                   
               
            
           
         
       
     
     1.3: Obtaining BCMA Binders Out of an In Vitro, Recombinant Library 
     1.3.1: Construction of Common Light Chain Fab-Libraries 
     Antibody libraries in the Fab-format are constructed on the basis of the humanized anti-CD3 antibody light chain were generated. Diversity was only introduced in the heavy chain. Six different heavy chain frameworks were used: VH1-46, VH1-69, VH3-15, VH3-23, VH4-59 and VH5-1. CDRs 1, 2 and 3 were randomized and each heavy chain library was combined with the non-randomized light chain. Libraries were generated as described by Nissim et al  EMBO J.  1994 Feb. 1; 13(3):692-8, and Silacci et al.  Proteomics.  2005 June; 5(9):2340-50. 
     1.3.2: Selection of Anti-BCMA Fab Clones 
     Anti-BCMA Fabs were established by phage display from synthetic Fab libraries consisting one constant VL and six different human VH sequences 
     
       
         
           
               
             
               
                 TABLE 37 
               
             
            
               
                   
               
               
                 Anti-BCMA  clones and respective VL/VH pairings 
               
            
           
           
               
               
               
            
               
                 Fab clone 
                 VL domain 
                 VH domain 
               
               
                   
               
               
                 pSCHLI372 
                 QAVVTQEPSLTVSPGGTVTLTCGS 
                 EVQLVESGGGLVKPGGSLRLSCAAS 
               
               
                   
                 STGAVTTSNYANWVQEKPGQAFRG 
                 GFTFSNSGMIWVRQAPGKGLEWVGH 
               
               
                   
                 LIGGTNKRAPGTPARFSGSLLGGK 
                 IRSKTDGGTTDYAAPVKGRFTISRD 
               
               
                   
                 AALTLSGAQPEDEAEYYCALWYSN 
                 DSKNTLYLQMNSLKTEDTAVYYCTT 
               
               
                   
                 LWVFGGGTKLTVL (SEQ ID 
                 GGSGSFDYWGQGTLVTVSS (SEQ 
               
               
                   
                 NO: 31) 
                 ID NO: 54) 
               
               
                   
               
               
                 pSCHLI373 
                 QAVVTQEPSLTVSPGGTVTLTCGS 
                 EVQLVESGGGLVKPGGSLRLSCAAS 
               
               
                   
                 STGAVTTSNYANWVQEKPGQAFRG 
                 GFSFSNSWMNWVRQAPGKGLEWVGT 
               
               
                   
                 LIGGTNKRAPGTPARFSGSLLGGK 
                 IRQKTYGGTTDYAAPVKGRFTISRD 
               
               
                   
                 AALTLSGAQPEDEAEYYCALWYSN 
                 DSKNTLYLQMNSLKTEDTAVYYCTT 
               
               
                   
                 LWVFGGGTKLTVL (SEQ ID 
                 GGLFGYWDYWGQGTLVTVSS (SEQ 
               
               
                   
                 NO: 31) 
                 ID NO: 55) 
               
               
                   
               
            
           
         
       
     
     Selection rounds (biopanning) were performed in solution according to the following pattern: 1) pre-clearing of ˜10 12  phagemid particles per library pool in immunotubes coated with 10 ug/ml of an unrelated human IgG to deplete the libraries of antibodies recognizing the Fc-portion of the antigens; 2) incubation of the non-Fc-binding phagemid particles with 100 nM biotinylated BCMA for 0.5 h in the presence of 100 nM unrelated non-biotinylated Fc knobs-into-holes construct for further depletion of Fc-binders in a total volume of 2 ml; 3) capture of biotinylated BCMA and specifically binding phage by splitting up and transferring the panning reaction into 16 wells on a neutravidin or streptavidin pre-coated microtiter plate for 20 min on a shaker; 4) washing of respective wells 10-30× with PBS/Tween20 and 10-30× with PBS using a plate washer; 5) optional competitive washing step by addition of 230 nM murine APRIL to displace Fab clones that recognize the binding site of the natural ligand thus selecting for APRIL-non-competing phage antibodies; 6) elution of phage particles by addition of 125 ul 100 mM TEA (triethylamine) per well for 5-10 min and neutralization by addition of an equal volume of 1 M Tris/HCl pH 7.4; 7) re-infection of log-phase  E. coli  TG1 cells with the eluted phage particles, infection with helperphage VCSM13, incubation on a shaker at 30° C. overnight and subsequent PEG/NaCl precipitation of phagemid particles to be used in the next selection round. 
     Selections were carried out over 3 to 5 rounds using constant antigen concentrations of 100 nM. Apart from selection campaigns during which only human BCMA was used as antigen, additional selection campaigns were carried out during which also cynomolgus or murine BCMA were used in an alternating fashion with human BCMA in order to select for cross-reactive antibodies. Moreover, as an alternative to streptavidin plate-based capture, capture of antigen:phage complexes was performed by addition of 5.4×10 7  streptavidin-coated magnetic beads to the panning reaction followed by washing steps using respective magnets under the conditions described above. 
     Specific binders were identified by surface plasmon resonance-screening of Fab-containing bacterial culture supernatants using BioRad&#39;s ProteOn XPR36 biosensor. In brief, after infection of log-phase  E. coli  TG1 cells with the eluted phage particles, single colony forming units (cfu) were plated and picked for inoculation of 1 ml expression cultures in 96-deep well plates. Fabs were captured from the supernatants on a ProteOn GLM chip that was derivatized with 8.000-10.000 RU of a goat anti-human IgG, F(ab′)2 fragment specific polyclonal antibody (Jackson ImmunoResearch, #109-005-006) in vertical orientation. Subsequently, human, cynomolgus and murine BCMA as well as an unrelated Fc knobs-into-holes construct were injected as analytes in horizontal orientation. Clones that exhibited significant binding responses to BCMA and did not bind the Fc-portion of the antigens, were bacterially expressed in a 0.5 liter culture volume, affinity purified and kinetically characterized by SPR-analysis using a one-shot-kinetics protocol on BioRad&#39;s ProteOn XPR36 biosensor. 
     Example 41 
     BCMA Binding Assays: Surface Plasmon Resonance 
     Assessment of binding of anti-BCMA antibodies to recombinant BCMA by surface plasmon resonance (SPR) as follow. All SPR experiments were performed on a Biacore T200 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany). The avidity of the interaction between anti-BCMA antibodies and recombinant BCMA Fc(kih) (human, cynomolgus and murine) was determined (Tables 38-40). Biotinylated recombinant human, cynomolgus and murine BCMA Fc(kih) were directly coupled on a SA chip following instructions (Biacore, Freiburg/Germany). The immobilization level ranged from 80 to 120 RU. The anti-BCMA antibodies were passed at a 4-fold concentration range (1.95 to 500 nM) with a flow of 30 μL/minutes through the flow cells over 120 seconds. The dissociation was monitored for 180 seconds. Bulk refractive index differences were corrected for by subtracting the response obtained on the reference flow cell. Here, the anti-BCMA antibodies were flown over an empty surface previously activated and deactivated as described in the standard amine coupling kit. Apparent kinetic constants were derived using the Biacore T200 Evaluation Software (v 1.0, Biacore AB, Uppsala/Sweden), to fit rate equations for 1:1 Langmuir binding by numerical integration, despite the bivalency of the interaction for comparison purposes. 
     
       
         
           
               
             
               
                 TABLE 38 
               
             
            
               
                   
               
               
                 Avidity values for comparison purposes only (Experiment 1) 
               
            
           
           
               
               
               
               
               
            
               
                 Analyte 
                 Ligand 
                 Kon[1/Ms] 
                 Koff[1/s] 
                 KD[nM] 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 pSCHLI333  
                 huBCMA Fc(kih) 
                 4.55E+06 
                 1.13E−02 
                 2.5 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI333  
                 cyBCMA Fc(kih) 
                 9.39E+06 
                 1.24E−02 
                 1.3 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI333  
                 muBCMA Fc(kih) 
                 8.26E+06 
                 1.62E−01 
                 19.6 
               
               
                 anti-BCMA IgG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 39 
               
             
            
               
                   
               
               
                 Avidity values for comparison purposes only (Experiment 2) 
               
            
           
           
               
               
               
               
               
            
               
                 Analyte 
                 Ligand 
                 Kon[1/Ms] 
                 Koff[1/s] 
                 KD[nM] 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 pSCHLI372 
                 huBCMA Fc(kih) 
                 3.13E+05 
                 2.66E−03 
                 8.5 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI372 
                 cyBCMA Fc(kih) 
                 7.85E+05 
                 3.50E−03 
                 4.5 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI372 
                 muBCMA Fc(kih) 
                 1.30E+05 
                 5.53E−01 
                 42.7 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI373 
                 huBCMA Fc(kih) 
                 1.40E+05 
                 3.23E−03 
                 23.0 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI373 
                 cyBCMA Fc(kih) 
                 9.36E+04 
                      9.28−04 
                 9.9 
               
               
                 anti-BCMA IgG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 40 
               
             
            
               
                   
               
               
                 Avidity values for comparison purposes only (Experiment 3) 
               
            
           
           
               
               
               
               
               
            
               
                 Analyte 
                 Ligand 
                 Kon[1/Ms] 
                 Koff[1/s] 
                 KD[nM] 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 pSCHLI372 
                 huBCMA Fc(kih) 
                 8.03E+05 
                 2.84E−03 
                 3.6 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI372 
                 cyBCMA Fc(kih) 
                 1.63E+06 
                 4.69E−03 
                 2.9 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI372 
                 muBCMA Fc(kih) 
                 4.69E+05 
                 1.21E−02 
                 25.7 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI373 
                 huBCMA Fc(kih) 
                 9.78E+04 
                 1.18E−03 
                 12.1 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI373 
                 cyBCMA Fc(kih) 
                 9.39E+04 
                 7.98E−04 
                 8.5 
               
               
                 anti-BCMA IgG 
               
               
                   
               
            
           
         
       
     
     The affinity of the interaction between anti-BCMA antibodies or BCMA-TCB CLC antibodies and recombinant human BCMA Fc(kih) was also determined. Anti-human Fab antibody (GE Healthcare) was directly coupled on a CM5 chip at pH 5.0 using the standard amine coupling kit (Biacore, Freiburg/Germany). The immobilization level was about 6500 RU. Anti-BCMA antibody or BCMA-TCB CLC antibody was captured for 90 seconds at 25 nM. Recombinant human BCMA Fc(kih) was passed at a 4-fold concentration range (1 0.95 to 500 nM) with a flow of 30 μL/minutes through the flow cells over 120 or 180 seconds. The dissociation was monitored for 120 or 400 seconds. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell. Here, recombinant BCMA was flown over a surface with immobilized anti-human Fab antibody but on which HBS-EP has been injected rather than anti-BCMA antibody or BCMA-TCB CLC antibody. Kinetic constants were derived using the Biacore T200 Evaluation Software (v 1.0, Biacore AB, Uppsala/Sweden), to fit rate equations for 1:1 Langmuir binding by numerical integration (Table 41). 
     
       
         
           
               
             
               
                 TABLE 41 
               
             
            
               
                   
               
               
                 Affinity constants determined by fitting rate  
               
               
                 equations for 1:1 Langmuir binding 
               
            
           
           
               
               
               
               
               
            
               
                 Ligand 
                 Analyte 
                 Kon[1/Ms] 
                 Koff[1/s] 
                 KD[nM] 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 pSCHLI372 
                 huBCMA Fc(kih) 
                 4.59E+04 
                 5.23E−03 
                 114 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI372 
                 cyBCMA Fc(kih) 
                 2.68E+04 
                 7.52E−03 
                 281 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI372 
                 muBCMA Fc(kih) 
                 9.92E+04 
                 1.30E−01 
                 1310 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI373 
                 huBCMA Fc(kih) 
                 4.22E+04 
                 5.70E−03 
                 135 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI373 
                 cyBCMA Fc(kih) 
                 2.10E+04 
                 1.12E−02 
                 535 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI373 
                 muBCMA Fc(kih) 
                 1.27E+05 
                 9.19E−02 
                 724 
               
               
                 anti-BCMA IgG 
                   
                   
                   
                   
               
               
                 pSCHLI333 
                 huBCMA Fc(kih) 
                 9.10E+03 
                 1.49E−03 
                 164 
               
               
                 BCMAxCD3 TCB 
                   
                   
                   
                   
               
               
                 pSCHLI333 
                 cyBCMA Fc(kih) 
                 1.44E+04 
                 2.63E−03 
                 183 
               
               
                 BCMAxCD3 TCB 
                   
                   
                   
                   
               
               
                 pSCHLI333 
                 muBCMA Fc(kih) 
                 4.83E+02 
                 3.47E−03 
                 7190 
               
               
                 BCMAxCD3 TCB 
                   
                   
                   
                   
               
               
                 pSCHLI372 
                 huBCMA Fc(kih) 
                 5.59E+04 
                 3.52E−03 
                 63 
               
               
                 BCMAxCD3 TCB 
                   
                   
                   
                   
               
               
                 pSCHLI372 
                 cyBCMA Fc(kih) 
                 4.58E+04 
                 7.57E−03 
                 165 
               
               
                 BCMAxCD3 TCB 
                   
                   
                   
                   
               
               
                 pSCHLI372 
                 muBCMA Fc(kih) 
                 1.42E+04 
                 4.87E−03 
                 344 
               
               
                 BCMAxCD3 TCB 
               
               
                   
               
            
           
         
       
     
     Example 42 
     Generation of Bivalent Anti-BCMA IgG Antibody with the Common Light Chain 
     To verify the hypothesis that the use of the common light chain could be applied to any BCMA antibodies, 83A10-CLC bivalent BCMA antibody was generated by substituting the native light chain of the bivalent BCMA antibody 83A10, previously described in WO/2014/122143, with the common light chain (CD3 LC (CLC)). The anti-BCMA antibody comprising two heavy chains of 83A10 and two common light chains were produced using the general techniques for generation of bivalent IgG antibodies as described in the Material and general methods section. Affinity of 83A10-CLC IgG antibody to human BCMA was measured by SPR with methods similar to the ones described in Example 41. Table 42 depicts the binding of recombinant human BCMA Fc(kih) to 83A10-CLC BCMA IgG antibody. 
     
       
         
           
               
             
               
                 TABLE 42 
               
             
            
               
                   
               
               
                 Affinity constants determined by fitting rate equations 
               
               
                 for 1:1 Langmuir binding: Binding of 
               
               
                 recombinant BCMA Fc(kih) to 83A10-CLC anti-BCMA antibody 
               
            
           
           
               
               
               
               
               
            
               
                 Ligand 
                 Analyte 
                 Kon[1/Ms] 
                 Koff[1/s] 
                 KD[nM] 
               
               
                   
               
               
                 83A10 CLC  
                 huBCMA Fc(kih) 
                 8.78E+04 
                 2.13E−3 
                 24.3 
               
               
                 anti-BCMA IgG 
               
               
                   
               
            
           
         
       
     
     Example 43 
     Specificity Test of Anti-BCMA IgG Antibodies to huTACI-R and huBAFF-R 
     As members of the TNF-TNF-R superfamily, TACI and BAFF receptors are related to BCMA receptor with respectively 22% and 18.5% homology in the extracellular domain. Therefore, surface plasmon resonance (SPR) binding experiments were performed to examine the specificity of anti-BCMA IgG antibodies. All SPR experiments were performed on a Biacore T200 (GE Healthcare) at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20). Fc fused huBCMA, huBAFF-R and huTACI-R were chemically immobilized with a high immobilization level (˜5000 RU) on different flow channels of a Biacore CM5 sensor chip at pH 5.0 using the standard amine coupling kit (GE Healthcare). Initially high concentrated solutions (3 μM, dissolved in HBS-EP) of anti-BCMA IgG pSCHLI372 as well as a-huTACI-R IgG or a-huBAFF-R IgG as positive controls were injected (association time: 80s, dissociation time: 600s, flow: 30 μl/min) to check if binding occurs. A positive binding event of the a-huTACI-R IgG to huTACI-R as well as for a-huBAFF-R IgG to huBAFF-R and anti-BCMA IgG antibodies to huBCMA indicated that all receptors were still recognized after immobilization. For anti-BCMA IgG antibodies binding with fast kinetic rate constants to huBAFF-R and/or huTACI-R, a careful examination of kinetic parameter with low immobilization levels (300 RU) was performed on a new CM5 sensor chip. Anti-BCMA IgG antibody dilutions at concentrations of 3000-93.75 nM (2-fold dilution in HBS-EP) are injected (association time: 80s, dissociation time: 300s, flow: 30 μl/min), and sample(s) were tested in duplicate. Regeneration was also performed when applicable i.e no fast and complete dissociation. Kinetic evaluation of the interaction between anti-BCMA IgG antibodies and huBAFF-R or huTACI-R was performed by global fitting of the data to a 1:1 interaction model that includes a term for mass transport (Biacore T200 evaluation Version 1.0). A steady state analysis was also performed. As depicted in  FIG. 37 , curve 1 corresponds to the signal on reference channel, curve 2 to the channel where the binding occurs (binding channel) and the curve 2-1 is the subtracted signal (binding channel—reference channel), meaning that this is the signal due to the binding event. As shown in  FIG. 37 , the SPR binding assay clearly demonstrated that pSCHLI372 IgG did not bind to human TACI receptor. As positive control for binding, another BCMA IgG known to slightly bind to TACI receptor but with very fast on- and off-rate was used (data not shown). 
     Example 44 
     Production and Purification of BCMA-TCB CLC Fc-Containing (2+1) Antibodies 
     For the production of the bispecific antibodies, bispecific antibodies were expressed by transient polymer-based cotransfection of the respective mammalian expression vectors in HEK293-EBNA cells, which were cultivated in suspension. One day prior to transfection the HEK293-EBNA cells were seeded at 1.5 Mio viable cells/mL in Ex-Cell medium, supplemented with 6 mM of L-Glutamine. For every mL of final production volume 2.0 Mio viable cells were centrifuged (5 minutes at 210×g). The supernatant was aspirated and the cells resuspended in 100 μL of CD CHO medium. The DNA for every mL of final production volume was prepared by mixing 1 μg of DNA (Ratio Bispecific Antibody Production:heavy chain:modified heavy chain:light chain:modified light chain=1:1:2:1; Ratio standard antibody:heavy chain:light chain=1:1) in 100 μL of CD CHO medium. After addition of 0.27 μL of PEI solution (1 mg/mL) the mixture was vortexed for 15 seconds and left at room temperature for 10 minutes. After 10 minutes, the resuspended cells and DNA/PEI mixture were put together and then transferred into an appropriate container which was placed in a shaking device (37° C., 5% CO 2 ). After a 3 hours incubation time 800 μL of Ex-Cell Medium, supplemented with 6 mM L-Glutamine, 1.25 mM valproic acid and 12.5% Pepsoy (50 g/L), was added for every mL of final Production volume. After 24 hours, 70 μL of feed solution was added for every mL of final production volume. After 7 days or when the cell viability was equal or lower than 70%, the cells were separated from the supernatant by centrifugation and sterile filtration. The antibodies were purified by an affinity step and one polishing step size exclusion chromatography. 
     For the affinity step the supernatant was loaded on a protein A column (HiTrap Protein A FF, 5 mL, GE Healthcare) equilibrated with 6 CV 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. After a washing step with the same buffer the antibody was eluted from the column by step elution with 20 mM sodium phosphate, 100 mM sodium chloride, 100 mM Glycine, pH 3.0. The fractions with the desired antibody were immediately neutralized by 0.5 M Sodium Phosphate, pH 8.0 (1:10), pooled and concentrated by centrifugation. The concentrate was sterile filtered and processed further by size exclusion chromatography. 
     For the size exclusion step the concentrated protein was injected in a XK16/60 HiLoad Superdex 200 column (GE Healthcare), and 20 mM Histidine, 140 mM Sodium Chloride, pH 6.0 with or without Tween20 as formulation buffer. The fractions containing the monomers were pooled, concentrated by centrifugation and sterile filtered into a sterile vial. 
     Determination of the antibody concentration was done by measurement of the absorbance at 280 nm, using the theoretical value of the absorbance of a 0.1% solution of the antibody. This value was based on the amino acid sequence and calculated by GPMAW software (Lighthouse data). 
     Purity and monomer content of the final protein preparation was determined by CE-SDS (Caliper LabChip GXII system (Caliper Life Sciences)) resp. HPLC (TSKgel G3000 SW XL analytical size exclusion column (Tosoh)) in a 25 mM potassium phosphate, 125 mM Sodium chloride, 200 mM L-arginine monohydrochloride, 0.02% (w/v) Sodium azide, pH 6.7 buffer. 
       FIGS. 38A-C  depicts the CE-SDS graphs (non-reduced (top panels) and reduced (bottom panels)) of the final protein preparations after Protein A (PA) affinity chromatography and size exclusion chromatographic (SEC) purification steps ( FIG. 38A ) pSCHLI333-TCB CLC, ( FIG. 38B ) pSCHLI372-TCB CLC, ( FIG. 38C ) pSCHLI373-TCB CLC. PA affinity chromatography and SEC purification steps applied to pSCHLI333-TCB CLC antibody resulted in a purity of 89.2% and 100% of monomer content ( FIG. 38A ), a purity of 88.5% and 100% of monomer content for pSCHLI372-TCB CLC antibody ( FIG. 38B ), and a purity of 91.8% and 100% of monomer content for pSCHLI372-TCB CLC antibody ( FIG. 38C ). Table 43 summarizes the properties of pSCHLI333-TCB CLC, pSCHLI372-TCB CLC, and pSCHLI373-TCB CLC antibodies following PA affinity chromatography and SEC purification steps. In all BCMA-TCB CLC antibodies, &gt;88% purity and 100% monomer content were consistently reached. The overall results clearly demonstrate that advantages in production/purification features could be achieved with using a common light chain (CLC) to TCB antibodies and that only two purification steps (i.e PA affinity chromatography and SEC) were required to achieve already high quality protein preparations with very good developability properties. 
     
       
         
           
               
             
               
                 TABLE 43 
               
             
            
               
                   
               
               
                 Production/purification profile of BCMA-TCB CLC 
               
               
                 antibodies following protein A affinity chromatography 
               
               
                 and size exclusion chromatography purification steps 
               
            
           
           
               
               
               
               
            
               
                   
                 pSCHLI333-TCB  
                 pSCHLI372-TCB  
                 pSCHLI373-TCB  
               
               
                   
                 CLC 
                 CLC 
                 CLC 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Purity (%) 
                 89.2 
                 88.5 
                 91.8 
               
               
                 Yield (mg/L) 
                 4.2 
                 4.1 
                 2.73 
               
               
                 Titer (mg/L) 
                 26.9 
                 21.2 
                 37.1 
               
               
                 Monomer (%) 
                 100 
                 100 
                 100 
               
               
                   
               
            
           
         
       
     
     Example 44 
     Binding of BCMA-TCB CLC Antibodies to BCMA-Positive Multiple Myeloma Cell Lines 
     BCMA-TCB CLC antibodies (pSCHLI333, pSCHLI372, pSCHLI373) were analyzed by flow cytometry for binding to human BCMA on BCMAhi-expressing H929 cells. MKN45 (human gastric adenocarcinoma cell line that does not express BCMA) was used as negative control. Briefly, cultured cells are harvested, counted and cell viability was evaluated using ViCell. Viable cells are then adjusted to 2×10 6  cells per ml in BSA-containing FACS Stain Buffer (BD Biosciences). 100 μl of this cell suspension were further aliquoted per well into a round-bottom 96-well plate and incubated with 30 μl of the BCMA-TCB CLC antibodies or corresponding TCB control for 30 min at 4° c. All BCMA-TCB CLC antibodies (and TCB controls) were titrated and analyzed in final concentration range between 0.12-500 nM. Cells were then centrifuged (5 min, 350×g), washed with 120 μl/well FACS Stain Buffer (BD Biosciences), resuspended and incubated for an additional 30 min at 4° C. with fluorochrome-conjugated PE-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgG Fc Fragment Specific (Jackson Immuno Research Lab; 109-116-170). Cells were then washed twice with Stain Buffer (BD Biosciences), fixed using 100 ul BD Fixation buffer per well (#BD Biosciences, 554655) at 4° C. for 20 min, resuspended in 120 μl FACS buffer and analyzed using BD FACS Cantoll. As depicted in  FIGS. 39A-B , the mean fluorescence intensity of BCMA-TCB CLC antibodies were plotted in function of antibody concentrations; ( FIG. 39A ) pSCHLI372-TCB CLC and pSCHLI373-TCB CLC on H929 cells; ( FIG. 39B ) pSCHLI372-TCB CLC and pSCHLI373-TCB CLC on MKN45 cells. BCMA-TCB CLC antibodies (pSCHLI333, pSCHLI372, pSCHLI373) antibodies did not bind to BCMA-negative and CD3-negative MKN45 cells at concentrations below 100 nM. When applicable, EC50 were calculated using Prism GraphPad (LaJolla, Calif., USA) and EC50 values denoting the antibody concentration required to reach 50% of the maximal binding for the binding of anti-BCMA/anti-CD3 TCB antibodies to H929 cells are summarized in Table 44. 
     
       
         
           
               
             
               
                 TABLE 44 
               
             
            
               
                   
               
               
                 EC50 values for binding of BCMA-TCB CLC antibodies to H929 cells 
               
            
           
           
               
               
               
            
               
                 Anti-BCMA/anti-CD3 TCB molecules 
                 EC50 (nM) 
                 EC50 (μg/ml) 
               
               
                   
               
               
                 pSCHLI372-TCB CLC 
                 344.8 
                 65.9 
               
               
                 pSCHLI373-TCB CLC 
                 no EC50 value 
                 no EC50 value 
               
               
                   
               
            
           
         
       
     
     Example 45 
     Binding of BCMA-TCB CLC Antibodies to CD3-Positive Jurkat T Cell Line (Flow Cytometry) 
     BCMA-TCB CLC antibodies (pSCHLI333, pSCHLI372, pSCHLI373) were also analyzed by flow cytometry for their binding properties to human CD3 expressed on human leukemic T cells Jurkat (ATCC TIB-152). Jurkat T cells were cultured in RPMI supplemented with 10% heat-inactivated FCS. Briefly, cultured cells were harvested, counted and cell viability was evaluated using ViCell. Viable cells were then adjusted to 2×10 6  cells per ml in FACS Stain Buffer (BD Biosciences) containing 0.1% BSA. 100 μl of this cell suspension were further aliquoted per well into a round-bottom 96-well plate. 30 μl of BCMA-TCB CLC antibodies or corresponding TCB control were added to the cell-containing wells to obtain final concentrations of 0.12 nM to 500 nM. BCMA-TCB CLC antibodies and control IgG were used at the same molarity. After incubation for 30 min at 4° C., cells were centrifuged (5 min, 350×g), washed twice with 150 μl/well BSA-containing FACS Stain Buffer (BD Biosciences), then cells were fixed using 100 ul BD Fixation buffer per well (#BD Biosciences, 554655) at 4° C. for 20 min, resuspended in 120 μl FACS buffer and analyzed using BD FACS Cantoll. Binding of BCMA-TCB CLC antibodies to T cells were evaluated and the median fluorescence intensity was determined gated on CD3-expressing Jurkat T cells and plotted in histograms or dot plots.  FIGS. 40A-B  show the median fluorescence intensity for BCMA-TCB CLC antibodies (pSCHLI333, pSCHLI372, pSCHLI373) binding to Jurkat T cells ( FIG. 40A ) or MKN45 cells ( FIG. 40B ) and plotted in function of antibody concentration. EC50 values and maximal binding of anti-BCMA/anti-CD3 TCB antibodies to CD3-positive Jurkat T cells were not reached. Isotype control antibody did not bind to Jurkat T cells and BCMA-TCB CLC antibodies (pSCHL1333, pSCHL1372, pSCHL1373) antibodies did not bind to BCMA-negative and CD3-negative MKN45 cells at concentrations below 100 nM. 
     Example 46 
     Activation of Human T Cells Upon Binding of BCMA-TCB CLC Antibodies to CD3-Positive T Cells and BCMA-Positive Multiple Myeloma Cell Lines 
     BCMA-TCB CLC antibodies (pSCHL1372, pSCHL1373) were analyzed by flow cytometry for their ability to induce T cell activation by evaluating the surface expression of the early activation marker CD69, or the late activation marker CD25 on CD4 +  and CD8 +  T cells in the presence or absence of human BCMA-expressing MM cells. Briefly, BCMA-positive H929 cells were harvested with Cell Dissociation buffer, counted and checked for viability. Cells were adjusted to 0.3×10 6  (viable) cells per ml in modified RPMI-1640 medium, 100 μl of this cell suspension were pipetted per well into a round-bottom 96-well plate (as indicated). 50 μl of the (diluted) BCMA-TCB CLC antibodies were added to the cell-containing wells to obtain a final concentration of 0.012 pM-100 nM. Human PBMC effector cells were isolated from fresh blood of a healthy donor and adjusted to 6×10 6  (viable) cells per ml in modified RPMI-1640 medium. 50 μl of this cell suspension was added per well of the assay plate to obtain a final E:T ratio of PBMC to myeloma tumor cells of 10:1. To analyze whether the BCMA-TCB CLC antibodies were able to activate T cells specifically in the presence of target cells expressing human BCMA, wells were included that contained 3 to 10 nM of the respective BCMA-TCB CLC antibodies molecules, as well as PBMCs, but no target cells. After 48 h incubation at 37° C., 5% CO 2 , cells were centrifuged (5 min, 350×g) and washed twice with 150 μl/well PBS containing 0.1% BSA. Surface staining for CD4 (mouse IgGI,K; clone RPA-T4), CD8 (mouse IgGI,K; clone HIT8a; BD #555635), CD69 (mouse IgGI; clone L78; BD #340560) and CD25 (mouse IgGI,K; clone M-A251; BD #555434) was performed at 4° C. for 30 min, according to the supplier&#39;s suggestions. Cells were washed twice with 150 μl/well PBS containing 0.1% BSA and fixed for 15 min at 4° C., using 100 μl/well fixation buffer (BD #554655). After centrifugation, the samples were resuspended in 200 μl/well PBS with 0.1% BSA and analyzed using a FACS Cantoll machine (Software FACS Diva).  FIG. 41  depicts the expression level of the early activation marker CD69 and the late activation marker CD25 on CD4 +  and CD8 +  T cells after 48 hours of incubation (representative results from two independent experiments). pSCHL1372-TCB CLC and pSCHL1373-TCB CLC antibodies induced an up-regulation of CD69 and CD25 activation markers in a concentration-dependent and specific manner in the presence of BCMA-positive target cells. No activation of CD4 +  and CD8+ T cells was observed when human PBMCs were treated with DP47-TCB control antibody, suggesting that despite binding to CD3 on the T cells T-cell activation does not occur when the TCB antibody does not bind to BCMA-positive target cells. 
     Example 47 
     Redirected T-Cell Cytotoxicity of BCMA hi -Expressing H929 Myeloma Cells Induced by Anti-BCMA/Anti-CD3 T Cell Bispecific Antibodies (Colorimetric LDH Release Assay) 
     BCMA-TCB CLC antibodies (pSCHLI372, pSCHLI373) were also analyzed for their potential to induce T cell-mediated apoptosis in BCMA-high expressing MM cells upon crosslinking of the construct via binding of the antigen binding moieties to BCMA on cells. Briefly, human BCMA-expressing H929 multiple myeloma target cells were harvested with Cell Dissociation Buffer, washed and resuspended in RPMI supplemented with 10% fetal bovine serum (Invitrogen). Approximately, 30,000 cells per well were plated in a round-bottom 96-well plate and the respective dilution of the construct was added for a desired final concentration (in triplicates); final concentrations ranging from 0.12 pM to 100 nM. For an appropriate comparison, all TCB constructs and controls were adjusted to the same molarity. Human total T cells (effector) were added into the wells to obtain a final E:T ratio of 5:1. When human PBMC were used as effector cells, a final E:T ratio of 10:1 was used. Negative control groups were represented by effector or target cells only. As a positive control for the activation of human pan T cells, 1 μg/ml PHA-M (Sigma #L8902) was used. For normalization, maximal lysis of the H929 MM target cells (=100%) was determined by incubation of the target cells with a final concentration of 1% Triton X-100, inducing cell death. Minimal lysis (=0%) was represented by target cells co-incubated with effector cells only, i.e. without any T cell bispecific antibody. After 20-24h or 48h incubation at 37° C., 5% CO 2 , LDH release from the apoptotic/necrotic MM target cells into the supernatant was then measured with the LDH detection kit (Roche Applied Science), following the manufacturer&#39;s instructions. The percentage of LDH release was plotted against the concentrations of BCMA-TCB CLC antibodies in concentration-response curves. The EC50 values were measured using Prism software (GraphPad) and determined as the TCB antibody concentration that results in 50% of maximum LDH release. As shown in  FIGS. 42A-B , BCMA-TCB CLC antibodies, pSCHLI372-TCB CLC ( FIGS. 42A , B) and pSCHLI373-TCB CLC ( FIG. 42B ) induced a concentration-dependent killing of BCMA-positive H929 myeloma cells as measured by LDH release. The killing of H929 cells was specific since DP47-TCB control antibody which does not bind to BCMA-positive target cells did not induce LDH release, even at the highest concentration tested of 100 nM. Table 45 summarizes the EC50 values for redirected T-cell killing of BCMA-positive H929 cells induced by BCMA-TCB CLC antibodies. 
     
       
         
           
               
             
               
                 TABLE 45 
               
             
            
               
                   
               
               
                 EC50 values for redirected T-cell killing of H929 
               
               
                 cells induced by BCMA-TCB CLC antibodies 
               
            
           
           
               
               
               
            
               
                 Anti-BCMA/anti-CD3 TCB molecules 
                 EC50 (pM) 
                 EC50 (μg/ml) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 pSCHLI333-TCB CLC 
                 980 
                 0.19 
               
               
                 pSCHLI372-TCB CLC (Experiment 1) 
                 450 
                 0.08 
               
               
                 pSCHLI373-TCB CLC 
                 1590 
                 0.31 
               
               
                 pSCHLI372-TCB CLC (Experiment 2) 
                 900 
                 0.17 
               
               
                   
               
            
           
         
       
     
     Example 48 
     Redirected T-Cell Cytotoxicity of BCMA med/lo -Expressing U266 Myeloma Cells Induced by BCMA-TCB CLC Antibodies (LDH Release Assay) 
     BCMA-TCB CLC antibodies (pSCHLI372, pSCHLI373) were analyzed for their ability to induce T cell-mediated apoptosis in BCMA med/lo -expressing MM cells upon crosslinking of the construct via binding of the antigen binding moieties to BCMA on cells. Briefly, human BCMA med/lo -expressing U266 multiple myeloma target cells were harvested with Cell Dissociation Buffer, washed and resuspended in RPMI supplemented with 10% fetal bovine serum (Invitrogen). Approximately, 30,000 cells per well are plated in a round-bottom 96-well plate and the respective dilution of the construct was added for a desired final concentration (in triplicates); final concentrations ranging from 0.12 pM to 100 nM. For an appropriate comparison, all TCB constructs and controls were adjusted to the same molarity. Human total T cells (effector) were added into the wells to obtain a final E:T ratio of 5:1. When human PBMC were used as effector cells, a final E:T ratio of 10:1 was used. Negative control groups were represented by effector or target cells only. As a positive control for the activation of human T cells, 1 μg/ml PHA-M (Sigma #L8902) was used. For normalization, maximal lysis of the MM target cells (=100%) was determined by incubation of the target cells with a final concentration of 1% Triton X-100, inducing cell death. Minimal lysis (=0%) was represented by target cells co-incubated with effector cells only, i.e. without any T cell bispecific antibody. After 20-24 h incubation at 37° C., 5% CO 2 , LDH release from the apoptotic/necrotic MM target cells into the supernatant was then measured with the LDH detection kit (Roche Applied Science), following the manufacturer&#39;s instructions. The percentage of LDH release was plotted against the concentrations of BCMA-TCB CLC antibodies in concentration-response curves. The EC50 values were measured using Prism software (GraphPad) and determined as the TCB antibody concentration that results in 50% of maximum LDH release. Table 46 summarizes the EC50 values for redirected T-cell killing of BCMA-positive U266 cells induced by BCMA-TCB CLC antibodies. 
     
       
         
           
               
             
               
                 TABLE 46 
               
             
            
               
                   
               
               
                 EC50 values for redirected T-cell killing of U266 cells 
               
               
                 induced by BCMA-TCB CLC antibodies 
               
            
           
           
               
               
               
            
               
                 Anti-BCMA/anti-CD3 TCB molecules 
                 EC50 (pM) 
                 EC50 (μg/ml) 
               
               
                   
               
               
                 pSCHLI372-TCB CLC 
                 5700 
                 1.1 
               
               
                   
               
            
           
         
       
     
     Amino Acid Sequences of Exemplary Embodiments 
     1) FoIR Binders Useful in Common Light Chain Format, Variable Heavy Chain 
       
     
       
         
           
               
            
               
                   
               
               
                 1) FoIR binders useful in common light chain format, variable heavy chain 
               
            
           
           
               
               
               
            
               
                 Description 
                 Sequence 
                 Seq ID No 
               
               
                   
               
            
           
           
               
               
               
            
               
                 16A3 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 1 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR NYYA   
                   
               
               
                   
                   GVTPFDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 18D3 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 2 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR NYYT   
                   
               
               
                   
                   GGSSAFDY WGQGTLVTVS 
                   
               
               
                   
               
               
                 15H7 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 3 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR NYYL   
                   
               
               
                   
                   FSTSFDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 15E36 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 4 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR NYYI   
                   
               
               
                   
                   GIVPFDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 21D1 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 5 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNYYV 
                   
               
               
                   
                 GVSPFDYWGQGTLVTVSS 
                   
               
               
                   
               
               
                 16F12 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 6 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR NFTV   
                   
               
               
                   
                   LRVPFDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 15A1 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 7 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR NYYI   
                   
               
               
                   
                   GVVTFDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 15A1_CDR1 
                 SYYMH 
                 8 
               
               
                   
               
               
                 15A1_CDR2 
                 IINPSGGSTSYAQKFQG 
                 9 
               
               
                   
               
               
                 15A1_CDR3 
                 NYYI GVVT FDY 
                 10 
               
               
                   
               
               
                 19E5 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 11 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GEWR   
                   
               
               
                   
                   RYTSFDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 19E5_CDR1 
                 SYYMH 
                 8 
               
               
                   
               
               
                 19E5_CDR2 
                 IINPSGGSTSYAQKFQG 
                 9 
               
               
                   
               
               
                 19E5_CDR3 
                 GEWRRYTSFDY 
                 12 
               
               
                   
               
               
                 19A4 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 13 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGWI   
                   
               
               
                   
                   RWEHFDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 19A4_CDR1 
                 SYYMH 
                 8 
               
               
                   
               
               
                 19A4_CDR2 
                 IINPSGGSTSYAQKFQG 
                 9 
               
               
                   
               
               
                 19A4_CDR3 
                 GGWIRWEHFDY 
                 14 
               
               
                   
               
               
                 16D5 
                 EVQLVESGGGLVKPGGSLRLSCAASGETFS NAWMS WVRQAPGKGLEWVG RI   
                 15 
               
               
                   
                   KSKTDGGTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT PW   
                   
               
               
                   
                   EWSWYDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 16D5_CDR1 
                 NAWMS 
                 16 
               
               
                   
               
               
                 16D5_CDR2 
                 RIKSKTDGGTTDYAAPVKG 
                 17 
               
               
                   
               
               
                 16D5_CDR3 
                 PWEWSWYDY 
                 18 
               
               
                   
               
               
                 15E12 
                 EVQLVESGGGLVKPGGSLRLSCAASGETFS NAWMS WVRQAPGKGLEWVG RI   
                 19 
               
               
                   
                   KSKTDGGTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT PW   
                   
               
               
                   
                   EWSYFDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 15E12_CDR1 
                 NAWMS 
                 16 
               
               
                   
               
               
                 15E12_CDR2 
                 RIKSKTDGGTTDYAAPVKG 
                 17 
               
               
                   
               
               
                 15E12_CDR3 
                 PWEWSYFDY 
                 20 
               
               
                   
               
               
                 21A5 
                 EVQLVESGGGLVKPGGSLRLSCAASGETFS NAWMS WVRQAPGKGLEWVG RI   
                 21 
               
               
                   
                   KSKTDGGTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT PW   
                   
               
               
                   
                   EWAWFDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 21A5_CDR1 
                 NAWMS 
                 16 
               
               
                   
               
               
                 21A5_CDR2 
                 RIKSKTDGGTTDYAAPVKG 
                 17 
               
               
                   
               
               
                 21A5_CDR3 
                 PWEWAWFDY 
                 22 
               
               
                   
               
               
                 21G8 
                 EVQLVESGGGLVKPGGSLRLSCAASGETFS NAWMS WVRQAPGKGLEWVG RI   
                 23 
               
               
                   
                   KSKTDGGTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT PW   
                   
               
               
                   
                   EWAYFDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 21G8_CDR1 
                 NAWMS 
                 16 
               
               
                   
               
               
                 21G8_CDR2 
                 RIKSKTDGGTTDYAAPVKG 
                 17 
               
               
                   
               
               
                 21G8_CDR3 
                 PWEWAYFDY 
                 24 
               
               
                   
               
               
                 19H3 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 25 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR TGWS   
                   
               
               
                   
                   RWGYMDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 19H3_CDR1 
                 SYYMH 
                 8 
               
               
                   
               
               
                 19H3_CDR2 
                 IINPSGGSTSYAQKFQG 
                 9 
               
               
                   
               
               
                 19H3_CDR3 
                 TGWSRWGYMDY 
                 26 
               
               
                   
               
               
                 20G6 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 27 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GEWI   
                   
               
               
                   
                   RYYHFDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 20G6_CDR1 
                 SYYMH 
                 8 
               
               
                   
               
               
                 20G6_CDR2 
                 IINPSGGSTSYAQKFQG 
                 9 
               
               
                   
               
               
                 20G6_CDR3 
                 GEWIRYYHFDY 
                 28 
               
               
                   
               
               
                 20H7 
                 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG II   
                 29 
               
               
                   
                   NPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR VGWY   
                   
               
               
                   
                   RWGYMDY WGQGTLVTVSS 
                   
               
               
                   
               
               
                 20H7_CDR1 
                 SYYMH 
                 8 
               
               
                   
               
               
                 20H7_CDR2 
                 IINPSGGSTSYAQKFQG 
                 9 
               
               
                   
               
               
                 20H7_CDR3 
                 VGWYRWGYMDY 
                 30 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 2) CD3 binder common light chain (CLC) 
               
            
           
           
               
               
               
            
               
                 Description 
                 Sequence 
                 Seq ID No 
               
               
                   
               
               
                 common 
                 QAVVTQEPSLTVSPGGTVTLTC GSSTGAVTTSNYAN WVQEKPGQAFRGLIG 
                 31 
               
               
                 CD3 light 
                   GTNKRAP GTPARFSGSLLGGKAALTLSGAQPEDEAEYYC ALWYSNLWV FGG 
                   
               
               
                 chain (VL) 
                 GTKLTVL 
                   
               
               
                   
               
               
                 common 
                 GSSTGAVTTSNYAN 
                 32 
               
               
                 CD3 light 
                   
                   
               
               
                 chain_CDR1 
                   
                   
               
               
                   
               
               
                 common 
                 GTNKRAP 
                 33 
               
               
                 CD3 light 
                   
                   
               
               
                 chain_CDR2 
                   
                   
               
               
                   
               
               
                 common 
                 ALWYSNLWV 
                 34 
               
               
                 CD3 light 
                   
                   
               
               
                 chain_CDR3 
                   
                   
               
               
                   
               
               
                 common 
                 QAVVTQEPSLTVSPGGTVTLTC GSSTGAVTTSNYAN WVQEKPGQAFRGLIG 
                 35 
               
               
                 CD3 light 
                   GTNKRAP GTPARFSGSLLGGKAALTLSGAQPEDEAEYYC ALWYSNLWV FGG 
                   
               
               
                 chain(VLCL) 
                 GTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA 
                   
               
               
                   
                 DSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGST 
                   
               
               
                   
                 VEKTVAPTECS 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 3) CD3 binder, heavy chain 
               
            
           
           
               
               
               
            
               
                 Description 
                 Sequence 
                 Seq ID No 
               
               
                   
               
               
                 CD3 variable 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSR 
                 36 
               
               
                 heavy chain 
                 IRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR 
                   
               
               
                 (VH) 
                 HGNFGNSYVSWFAYWGQGTLVTVSS 
                   
               
               
                   
               
               
                 CD3 heavy 
                 TYAMN 
                 37 
               
               
                 chain 
                   
                   
               
               
                 (VH)_CDR1 
                   
                   
               
               
                   
               
               
                 CD3 heavy 
                 RIRSKYNNYATYYADSVKG 
                 38 
               
               
                 chain 
                   
                   
               
               
                 (VH)_CDR2 
                   
                   
               
               
                   
               
               
                 CD3 heavy 
                 HGNFGNSYVSWFAY 
                 39 
               
               
                 chain 
                   
                   
               
               
                 (VH)_CDR3 
                   
                   
               
               
                   
               
               
                 CD3 full 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFS TYAMN WVRQAPGKGLEWVSR 
                 40 
               
               
                 heavy chain 
                   IRSKYNNYATYYADSVKG RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR 
                   
               
               
                 (VHCH1)_ 
                   HGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL 
                   
               
               
                   
                 GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS 
                   
               
               
                   
                 LGTQTYICNVNHKPSNTKVDKKVEPKSC 
                   
               
               
                   
               
               
                 CD3 
                 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV 
                 84 
               
               
                 constant 
                 HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP 
                   
               
               
                 heavy chain 
                 KSC 
                   
               
               
                 CH1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 4) MUC1 binders useful in common light chain format, variable heavy chain 
               
            
           
           
               
               
               
               
            
               
                 SEQ ID 
                   
                   
                 Amino Acid 
               
               
                 NOS 
                 Description 
                 Nucleotide 
                 Sequence 
               
               
                   
               
            
           
           
               
            
               
                 MUC1 Antigens 
               
            
           
           
               
               
               
               
            
               
                 57 and 
                 MUC1 SEA- 
                 ATGAGCTTTTTTTTCCTGAGCTTTCATATTAGCAACC 
                 MSFFFLSFHISNLQFN 
               
               
                 58 
                 avi-His 
                 TGCAGTTTAATAGCAGCCTGGAAGATCCGAGCACCGA 
                 SSLEDPSTDYYQELQR 
               
               
                   
                   
                 TTATTATCAAGAACTGCAGCGTGATATCAGCGAAATG 
                 DISEMFLQIYKQGGFL 
               
               
                   
                   
                 TTTCTGCAGATCTATAAACAGGGTGGTTTTCTGGGTC 
                 GLSNIKFRPGGGGGSV 
               
               
                   
                   
                 TGAGCAACATCAAATTTCGTCCGGGTGGTGGCGGTGG 
                 VVQLTLAFREGTINVH 
               
               
                   
                   
                 TTCAGTTGTTGTGCAGCTGACCCTGGCATTTCGTGAA 
                 DVETQFNQYKTEAASR 
               
               
                   
                   
                 GGCACCATTAATGTTCATGATGTGGAAACCCAGTTTA 
                 YNLTISDVSVSDVPFP 
               
               
                   
                   
                 ACCAGTATAAAACCGAAGCAGCAAGCCGTTATAATCT 
                 FSAQSVDGLNDIFEAQ 
               
               
                   
                   
                 GACCATTAGTGATGTTAGCGTTTCCGATGTTCCGTTT 
                 KIEWHELEHHHHHH 
               
               
                   
                   
                 CCGTTTAGCGCACAGAGTGTCGACGGTCTGAATGATA 
                   
               
               
                   
                   
                 TTTTTGAAGCCCAGAAAATCGAATGGCATGAACTCGA 
                   
               
               
                   
                   
                 GCACCACCACCACCACCAC 
                   
               
               
                   
               
            
           
           
               
            
               
                 MUC1 Binders 
               
            
           
           
               
               
               
               
            
               
                 59 and 
                 58D6 VH 
                 GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC 
                 EVQLLESGGGLVQPGG 
               
               
                 60 
                   
                 AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTC 
                 SLRLSCAASGFTFSSY 
               
               
                   
                   
                 CGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTC 
                 AMSWVRQAPGKGLEWV 
               
               
                   
                   
                 CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAG 
                 SAISGSGGSTYYADSV 
               
               
                   
                   
                 CTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGA 
                 KGRFTISRDNSKNTLY 
               
               
                   
                   
                 CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT 
                 LQMNSLRAEDTAVYYC 
               
               
                   
                   
                 TCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGA 
                 AKVEWSTLLYFDYWGQ 
               
               
                   
                   
                 GAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGT 
                 GTLVTVSS 
               
               
                   
                   
                 TGAATGGTCTACTCTGCTGTACTTCGACTACTGGGGC 
                   
               
               
                   
                   
                 CAAGGAACCCTGGTCACCGTCTCGAGT 
                   
               
               
                   
               
               
                 61 and 
                 106D2 VH 
                 GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC 
                 EVQLLESGGGLVQPGG 
               
               
                 62 
                   
                 AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTC 
                 SLRLSCAASGFTFRVY 
               
               
                   
                   
                 CGGATTCACCTTTCGGGTTTATGCAATGAGCTGGGTC 
                 AMSWVRQAPGKGLEWV 
               
               
                   
                   
                 CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAG 
                 SGISETGSYTYYADSV 
               
               
                   
                   
                 GTATTTCTGAAACTGGTTCTTACACATACTACGCAGA 
                 KGRFTISRDNSKNTLY 
               
               
                   
                   
                 CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT 
                 LQMNSLRAEDTAVYYC 
               
               
                   
                   
                 TCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGA 
                 ARYPYGFDYWGQGTLV 
               
               
                   
                   
                 GAGCCGAGGACACGGCCGTATATTACTGTGCGCGTTA 
                 TVSS 
               
               
                   
                   
                 CCCGTACGGTTTCGACTACTGGGGCCAAGGAACCCTG 
                   
               
               
                   
                   
                 GTCACCGTCTCGAGT 
                   
               
               
                   
               
               
                 63 and 
                 110A5 VH 
                 GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC 
                 EVQLLESGGGLVQPGG 
               
               
                 64 
                   
                 AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTC 
                 SLRLSCAASGFTFSSY 
               
               
                   
                   
                 CGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTC 
                 AMSWVRQAPGKGLEWV 
               
               
                   
                   
                 CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAG 
                 SAISGSGGSTYYADSV 
               
               
                   
                   
                 CTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGA 
                 KGRFTISRDNSKNTLY 
               
               
                   
                   
                 CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT 
                 LQMNSLRAEDTAVYYC 
               
               
                   
                   
                 TCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGA 
                 AKYSYRYVLAFDYWGQ 
               
               
                   
                   
                 GAGCCGAGGACACGGCCGTATATTACTGTGCGAAATA 
                 GTLVTVSS 
               
               
                   
                   
                 CTCTTACCGTTACGTTCTGGCTTTCGACTACTGGGGC 
                   
               
               
                   
                   
                 CAAGGAACCCTGGTCACCGTCTCGAGT 
                   
               
               
                   
               
               
                 65 and 
                 Common 
                 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGT 
                 QAVVTQEPSLTVSPGG 
               
               
                 66 
                 light chain 
                 CTCCTGGCGGCACCGTGACCCTGACATGTGGCAGTTC 
                 TVTLTCGSSTGAVTTS 
               
               
                   
                 VL 
                 TACAGGCGCCGTGACCACCAGCAACTACGCCAACTGG 
                 NYANWVQEKPGQAFRG 
               
               
                   
                   
                 GTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGA 
                 LIGGTNKRAPGTPARE 
               
               
                   
                   
                 TCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGC 
                 SGSLLGGKAALTLSGA 
               
               
                   
                   
                 CAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCC 
                 QPEDEAEYYCALWYSN 
               
               
                   
                   
                 CTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCG 
                 LWVFGGGTKLTVL 
               
               
                   
                   
                 AGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGT 
                   
               
               
                   
                   
                 GTTCGGCGGAGGCACCAAGCTGACAGTCCTA 
                   
               
               
                   
               
               
                 67 and 
                 Common 
                 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGT 
                 QAVVTQEPSLTVSPGG 
               
               
                 68 
                 light chain 
                 CTCCTGGCGGCACCGTGACCCTGACATGTGGCAGTTC 
                 TVTLTCGSSTGAVTTS 
               
               
                   
                   
                 TACAGGCGCCGTGACCACCAGCAACTACGCCAACTGG 
                 NYANWVQEKPGQAFRG 
               
               
                   
                   
                 GTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGA 
                 LIGGTNKRAPGTPARE 
               
               
                   
                   
                 TCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGC 
                 SGSLLGGKAALTLSGA 
               
               
                   
                   
                 CAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCC 
                 QPEDEAEYYCALWYSN 
               
               
                   
                   
                 CTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCG 
                 LWVFGGGTKLTVLRTQ 
               
               
                   
                   
                 AGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGT 
                 PKAAPSVTLFPPSSEE 
               
               
                   
                   
                 GTTCGGCGGAGGCACCAAGCTGACAGTGCTGCGTACG 
                 LQANKATLVCLISDFY 
               
               
                   
                   
                 CAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCC 
                 PGAVTVAWKADSSPVK 
               
               
                   
                   
                 CCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCT 
                 AGVETTTPSKQSNNKY 
               
               
                   
                   
                 GGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTG 
                 AASSYLSLTPEQWKSH 
               
               
                   
                   
                 ACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGG 
                 RSYSCQVTHEGSTVEK 
               
               
                   
                   
                 CCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAA 
                 TVAPTECS 
               
               
                   
                   
                 CAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACC 
                   
               
               
                   
                   
                 CCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCC 
                   
               
               
                   
                   
                 AGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGT 
                   
               
               
                   
                   
                 GGCCCCCACCGAGTGCTCC 
                   
               
               
                   
               
               
                 69 and 
                 58D6 knob 
                 GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC 
                 EVQLLESGGGLVQPGG 
               
               
                 70 
                 heavy chain 
                 AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTC 
                 SLRLSCAASGFTFSSY 
               
               
                   
                   
                 CGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTC 
                 AMSWVRQAPGKGLEWV 
               
               
                   
                   
                 CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAG 
                 SAISGSGGSTYYADSV 
               
               
                   
                   
                 CTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGA 
                 KGRFTISRDNSKNTLY 
               
               
                   
                   
                 CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT 
                 LQMNSLRAEDTAVYYC 
               
               
                   
                   
                 TCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGA 
                 AKVEWSTLLYFDYWGQ 
               
               
                   
                   
                 GAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGT 
                 GTLVTVSSASTKGPSV 
               
               
                   
                   
                 TGAATGGTCTACTCTGCTGTACTTCGACTACTGGGGC 
                 FPLAPSSKSTSGGTAA 
               
               
                   
                   
                 CAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACAA 
                 LGCLVKDYFPEPVTVS 
               
               
                   
                   
                 AGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAA 
                 WNSGALTSGVHTFPAV 
               
               
                   
                   
                 GAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTC 
                 LQSSGLYSLSSVVTVP 
               
               
                   
                   
                 GTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTT 
                 SSSLGTQTYICNVNHK 
               
               
                   
                   
                 GGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTT 
                 PSNTKVDKKVEPKSCD 
               
               
                   
                   
                 CCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTG 
                 GGGGSGGGGSEVQLLE 
               
               
                   
                   
                 AGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCA 
                 SGGGLVQPGGSLRLSC 
               
               
                   
                   
                 CCCAGACCTACATCTGCAACGTGAACCACAAGCCCAG 
                 AASGFTFSTYAMNWVR 
               
               
                   
                   
                 CAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGC 
                 QAPGKGLEWVSRIRSK 
               
               
                   
                   
                 TGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCG 
                 YNNYATYYADSVKGRF 
               
               
                   
                   
                 AGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCA 
                 TISRDDSKNTLYLQMN 
               
               
                   
                   
                 GCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGC 
                 SLRAEDTAVYYCVRHG 
               
               
                   
                   
                 GGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGC 
                 NFGNSYVSWFAYWGQG 
               
               
                   
                   
                 GCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCG 
                 TLVTVSSASTKGPSVF 
               
               
                   
                   
                 GATCAGAAGCAAGTACAACAACTACGCCACCTACTAC 
                 PLAPSSKSTSGGTAAL 
               
               
                   
                   
                 GCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGG 
                 GCLVKDYFPEPVTVSW 
               
               
                   
                   
                 ACGACAGCAAGAACACCCTGTACCTGCAGATGAACAG 
                 NSGALTSGVHTFPAVL 
               
               
                   
                   
                 CCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTG 
                 QSSGLYSLSSVVTVPS 
               
               
                   
                   
                 CGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGT 
                 SSLGTQTYICNVNHKP 
               
               
                   
                   
                 TTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTC 
                 SNTKVDKKVEPKSCDK 
               
               
                   
                   
                 AAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTG 
                 THTCPPCPAPEAAGGP 
               
               
                   
                   
                 GCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCG 
                 SVFLFPPKPKDTLMIS 
               
               
                   
                   
                 CTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCC 
                 RTPEVTCVVVDVSHED 
               
               
                   
                   
                 CGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGC 
                 PEVKFNWYVDGVEVHN 
               
               
                   
                   
                 GGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCG 
                 AKTKPREEQYNSTYRV 
               
               
                   
                   
                 GCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTC 
                 VSVLTVLHQDWLNGKE 
               
               
                   
                   
                 TAGCTCCCTGGGAACACAGACATATATCTGTAATGTC 
                 YKCKVSNKALGAPIEK 
               
               
                   
                   
                 AATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAG 
                 TISKAKGQPREPQVYT 
               
               
                   
                   
                 TCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCC 
                 LPPCRDELTKNQVSLW 
               
               
                   
                   
                 ACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCA 
                 CLVKGFYPSDIAVEWE 
               
               
                   
                   
                 GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA 
                 SNGQPENNYKTTPPVL 
               
               
                   
                   
                 TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT 
                 DSDGSFFLYSKLTVDK 
               
               
                   
                   
                 GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC 
                 SRWQQGNVFSCSVMHE 
               
               
                   
                   
                 TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGA 
                 ALHNHYTQKSLSLSPG 
               
               
                   
                   
                 CAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG 
                 K 
               
               
                   
                   
                 TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG 
                   
               
               
                   
                   
                 CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA 
                   
               
               
                   
                   
                 AAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAA 
                   
               
               
                   
                   
                 AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC 
                   
               
               
                   
                   
                 CTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGG 
                   
               
               
                   
                   
                 TCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAG 
                   
               
               
                   
                   
                 CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG 
                   
               
               
                   
                   
                 GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT 
                   
               
               
                   
                   
                 CCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT 
                   
               
               
                   
                   
                 GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA 
                   
               
               
                   
                   
                 TGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA 
                   
               
               
                   
                   
                 CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 71 and 
                 58D6 hole 
                 GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC 
                 EVQLLESGGGLVQPGG 
               
               
                 72 
                 heavy chain 
                 AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTC 
                 SLRLSCAASGFTFSSY 
               
               
                   
                   
                 CGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTC 
                 AMSWVRQAPGKGLEWV 
               
               
                   
                   
                 CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAG 
                 SAISGSGGSTYYADSV 
               
               
                   
                   
                 CTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGA 
                 KGRFTISRDNSKNTLY 
               
               
                   
                   
                 CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT 
                 LQMNSLRAEDTAVYYC 
               
               
                   
                   
                 TCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGA 
                 AKVEWSTLLYFDYWGQ 
               
               
                   
                   
                 GAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGT 
                 GTLVTVSSASTKGPSV 
               
               
                   
                   
                 TGAATGGTCTACTCTGCTGTACTTCGACTACTGGGGC 
                 FPLAPSSKSTSGGTAA 
               
               
                   
                   
                 CAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCA 
                 LGCLVKDYFPEPVTVS 
               
               
                   
                   
                 AGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAA 
                 WNSGALTSGVHTFPAV 
               
               
                   
                   
                 GAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTG 
                 LQSSGLYSLSSVVTVP 
               
               
                   
                   
                 GTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCT 
                 SSSLGTQTYICNVNHK 
               
               
                   
                   
                 GGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTT 
                 PSNTKVDKKVEPKSCD 
               
               
                   
                   
                 CCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTG 
                 KTHTCPPCPAPEAAGG 
               
               
                   
                   
                 AGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCA 
                 PSVFLFPPKPKDTLMI 
               
               
                   
                   
                 CCCAGACCTACATCTGCAACGTGAACCACAAGCCCAG 
                 SRTPEVTCVVVDVSHE 
               
               
                   
                   
                 CAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGC 
                 DPEVKFNWYVDGVEVH 
               
               
                   
                   
                 TGCGACAAAACTCACACATGCCCACCGTGCCCAGCAC 
                 NAKTKPREEQYNSTYR 
               
               
                   
                   
                 CTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCC 
                 VVSVLTVLHQDWLNGK 
               
               
                   
                   
                 CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC 
                 EYKCKVSNKALGAPIE 
               
               
                   
                   
                 CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG 
                 KTISKAKGQPREPQVC 
               
               
                   
                   
                 AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGG 
                 TLPPSRDELTKNQVSL 
               
               
                   
                   
                 CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG 
                 SCAVKGFYPSDIAVEW 
               
               
                   
                   
                 GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC 
                 ESNGQPENNYKTTPPV 
               
               
                   
                   
                 TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA 
                 LDSDGSFFLVSKLTVD 
               
               
                   
                   
                 GTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCC 
                 KSRWQQGNVFSCSVMH 
               
               
                   
                   
                 CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC 
                 EALHNHYTQKSLSLSP 
               
               
                   
                   
                 CCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCG 
                 GK 
               
               
                   
                   
                 GGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGC 
                   
               
               
                   
                   
                 GCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG 
                   
               
               
                   
                   
                 AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA 
                   
               
               
                   
                   
                 GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC 
                   
               
               
                   
                   
                 TTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGT 
                   
               
               
                   
                   
                 GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA 
                   
               
               
                   
                   
                 TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC 
                   
               
               
                   
                   
                 TCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 73 and 
                 110A5 knob 
                 GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC 
                 EVQLLESGGGLVQPGG 
               
               
                 74 
                 heavy chain 
                 AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTC 
                 SLRLSCAASGFTFSSY 
               
               
                   
                   
                 CGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTC 
                 AMSWVRQAPGKGLEWV 
               
               
                   
                   
                 CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAG 
                 SAISGSGGSTYYADSV 
               
               
                   
                   
                 CTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGA 
                 KGRFTISRDNSKNTLY 
               
               
                   
                   
                 CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT 
                 LQMNSLRAEDTAVYYC 
               
               
                   
                   
                 TCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGA 
                 AKYSYRYVLAFDYWGQ 
               
               
                   
                   
                 GAGCCGAGGACACGGCCGTATATTACTGTGCGAAATA 
                 GTLVTVSSASTKGPSV 
               
               
                   
                   
                 CTCTTACCGTTACGTTCTGGCTTTCGACTACTGGGGC 
                 FPLAPSSKSTSGGTAA 
               
               
                   
                   
                 CAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACAA 
                 LGCLVKDYFPEPVTVS 
               
               
                   
                   
                 AGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAA 
                 WNSGALTSGVHTFPAV 
               
               
                   
                   
                 GAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTC 
                 LQSSGLYSLSSVVTVP 
               
               
                   
                   
                 GTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTT 
                 SSSLGTQTYICNVNHK 
               
               
                   
                   
                 GGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTT 
                 PSNTKVDKKVEPKSCD 
               
               
                   
                   
                 CCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTG 
                 GGGGSGGGGSEVQLLE 
               
               
                   
                   
                 AGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCA 
                 SGGGLVQPGGSLRLSC 
               
               
                   
                   
                 CCCAGACCTACATCTGCAACGTGAACCACAAGCCCAG 
                 AASGFTFSTYAMNWVR 
               
               
                   
                   
                 CAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGC 
                 QAPGKGLEWVSRIRSK 
               
               
                   
                   
                 TGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCG 
                 YNNYATYYADSVKGRF 
               
               
                   
                   
                 AGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCA 
                 TISRDDSKNTLYLQMN 
               
               
                   
                   
                 GCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGC 
                 SLRAEDTAVYYCVRHG 
               
               
                   
                   
                 GGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGC 
                 NFGNSYVSWFAYWGQG 
               
               
                   
                   
                 GCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCG 
                 TLVTVSSASTKGPSVF 
               
               
                   
                   
                 GATCAGAAGCAAGTACAACAACTACGCCACCTACTAC 
                 PLAPSSKSTSGGTAAL 
               
               
                   
                   
                 GCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGG 
                 GCLVKDYFPEPVTVSW 
               
               
                   
                   
                 ACGACAGCAAGAACACCCTGTACCTGCAGATGAACAG 
                 NSGALTSGVHTFPAVL 
               
               
                   
                   
                 CCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTG 
                 QSSGLYSLSSVVTVPS 
               
               
                   
                   
                 CGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGT 
                 SSLGTQTYICNVNHKP 
               
               
                   
                   
                 TTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTC 
                 SNTKVDKKVEPKSCDK 
               
               
                   
                   
                 AAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTG 
                 THTCPPCPAPEAAGGP 
               
               
                   
                   
                 GCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCG 
                 SVFLFPPKPKDTLMIS 
               
               
                   
                   
                 CTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCC 
                 RTPEVTCVVVDVSHED 
               
               
                   
                   
                 CGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGC 
                 PEVKFNWYVDGVEVHN 
               
               
                   
                   
                 GGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCG 
                 AKTKPREEQYNSTYRV 
               
               
                   
                   
                 GCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTC 
                 VSVLTVLHQDWLNGKE 
               
               
                   
                   
                 TAGCTCCCTGGGAACACAGACATATATCTGTAATGTC 
                 YKCKVSNKALGAPIEK 
               
               
                   
                   
                 AATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAG 
                 TISKAKGQPREPQVYT 
               
               
                   
                   
                 TCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCC 
                 LPPCRDELTKNQVSLW 
               
               
                   
                   
                 ACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCA 
                 CLVKGFYPSDIAVEWE 
               
               
                   
                   
                 GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA 
                 SNGQPENNYKTTPPVL 
               
               
                   
                   
                 TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT 
                 DSDGSFFLYSKLTVDK 
               
               
                   
                   
                 GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC 
                 SRWQQGNVFSCSVMHE 
               
               
                   
                   
                 TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGA 
                 ALHNHYTQKSLSLSPG 
               
               
                   
                   
                 CAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG 
                 K 
               
               
                   
                   
                 TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG 
                   
               
               
                   
                   
                 CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA 
                   
               
               
                   
                   
                 AAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAA 
                   
               
               
                   
                   
                 AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC 
                   
               
               
                   
                   
                 CTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGG 
                   
               
               
                   
                   
                 TCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAG 
                   
               
               
                   
                   
                 CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG 
                   
               
               
                   
                   
                 GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT 
                   
               
               
                   
                   
                 CCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT 
                   
               
               
                   
                   
                 GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA 
                   
               
               
                   
                   
                 TGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA 
                   
               
               
                   
                   
                 CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 75 and 
                 110A5 hole 
                 GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC 
                 EVQLLESGGGLVQPGG 
               
               
                 76 
                 heavy chain 
                 AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTC 
                 SLRLSCAASGFTFSSY 
               
               
                   
                   
                 CGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTC 
                 AMSWVRQAPGKGLEWV 
               
               
                   
                   
                 CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAG 
                 SAISGSGGSTYYADSV 
               
               
                   
                   
                 CTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGA 
                 KGRFTISRDNSKNTLY 
               
               
                   
                   
                 CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT 
                 LQMNSLRAEDTAVYYC 
               
               
                   
                   
                 TCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGA 
                 AKYSYRYVLAFDYWGQ 
               
               
                   
                   
                 GAGCCGAGGACACGGCCGTATATTACTGTGCGAAATA 
                 GTLVTVSSASTKGPSV 
               
               
                   
                   
                 CTCTTACCGTTACGTTCTGGCTTTCGACTACTGGGGC 
                 FPLAPSSKSTSGGTAA 
               
               
                   
                   
                 CAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCA 
                 LGCLVKDYFPEPVTVS 
               
               
                   
                   
                 AGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAA 
                 WNSGALTSGVHTFPAV 
               
               
                   
                   
                 GAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTG 
                 LQSSGLYSLSSVVTVP 
               
               
                   
                   
                 GTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCT 
                 SSSLGTQTYICNVNHK 
               
               
                   
                   
                 GGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTT 
                 PSNTKVDKKVEPKSCD 
               
               
                   
                   
                 CCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTG 
                 KTHTCPPCPAPEAAGG 
               
               
                   
                   
                 AGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCA 
                 PSVFLFPPKPKDTLMI 
               
               
                   
                   
                 CCCAGACCTACATCTGCAACGTGAACCACAAGCCCAG 
                 SRTPEVTCVVVDVSHE 
               
               
                   
                   
                 CAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGC 
                 DPEVKFNWYVDGVEVH 
               
               
                   
                   
                 TGCGACAAAACTCACACATGCCCACCGTGCCCAGCAC 
                 NAKTKPREEQYNSTYR 
               
               
                   
                   
                 CTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCC 
                 VVSVLTVLHQDWLNGK 
               
               
                   
                   
                 CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC 
                 EYKCKVSNKALGAPIE 
               
               
                   
                   
                 CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG 
                 KTISKAKGQPREPQVC 
               
               
                   
                   
                 AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGG 
                 TLPPSRDELTKNQVSL 
               
               
                   
                   
                 CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG 
                 SCAVKGFYPSDIAVEW 
               
               
                   
                   
                 GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCC 
                 ESNGQPENNYKTTPPV 
               
               
                   
                   
                 TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA 
                 LDSDGSFFLVSKLTVD 
               
               
                   
                   
                 GTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCC 
                 KSRWQQGNVFSCSVMH 
               
               
                   
                   
                 CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC 
                 EALHNHYTQKSLSLSP 
               
               
                   
                   
                 CCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCG 
                 GK 
               
               
                   
                   
                 GGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGC 
                   
               
               
                   
                   
                 GCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG 
                   
               
               
                   
                   
                 AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA 
                   
               
               
                   
                   
                 GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC 
                   
               
               
                   
                   
                 TTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGT 
                   
               
               
                   
                   
                 GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA 
                   
               
               
                   
                   
                 TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC 
                   
               
               
                   
                   
                 TCCCTGTCTCCGGGTAAA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 5) BCMA  binders useful in common light chain format, variable heavy chain 
               
            
           
           
               
               
               
               
            
               
                 Amino Acid 
                   
                   
                   
               
               
                 Sequences 
                 pSCHLI333-TCB 
                 pSCHLI372-TCB 
                 pSCHLI373-TCB 
               
               
                   
               
               
                 BCMA VH 
                 QVQLVQSGAEVKKPGASV 
                 EVQLVESGGGLVKPGGSLRLS 
                 EVQLVESGGGLVKPGGSLRLSCAA 
               
               
                   
                 KVSCKASGYMFSSFGMSW 
                 CAASGFTFSNSGMIWVRQAPG 
                 SGFSFSNSWMNWVRQAPGKGLEWV 
               
               
                   
                 VRQAPGQGLEWMGWIYPV 
                 KGLEWVGHIRSKTDGGTTDYA 
                 GTIRQKTYGGTTDYAAPVKGRFTI 
               
               
                   
                 GQGTWYAQKFQGRVTMTR 
                 APVKGRFTISRDDSKNTLYLQ 
                 SRDDSKNTLYLQMNSLKTEDTAVY 
               
               
                   
                 DTSTSTVYMELSSLRSED 
                 MNSLKTEDTAVYYCTTGGSGS 
                 YCTTGGLFGYWDYWGQGTLVTVSS 
               
               
                   
                 TAVYYCARVSYPPSHFDY 
                 FDYWGQGTLVTVSS (SEQ 
                 (SEQ ID NO: 79) 
               
               
                   
                 WGQGTLVTVSS (SEQ 
                 ID NO: 78) 
                   
               
               
                   
                 ID NO: 77) 
                   
                   
               
               
                   
               
               
                 BCMA CDR1 
                 GYMFSSFGMS (SEQ ID 
                 GFTFSNSGMI (SEQ ID 
                 GFSFSNSWMN (SEQ ID NO: 
               
               
                 (VH) 
                 NO: 80) 
                 NO: 81) 
                 82) 
               
               
                   
               
               
                 BCMA CDR2 
                 WIYPVGQGTWYAQKFQG 
                 HIRSKTDGGTTDYAAPVKG 
                 TIRQKTYGGTTDYAAPVKG (SEQ 
               
               
                 (VH) 
                 (SEQ ID NO: 83) 
                 (SEQ ID NO: 85) 
                 ID NO: 86) 
               
               
                   
               
               
                 BCMA CDR3 
                 VSYPPSHFDY (SEQ ID 
                 GGSGSFDY (SEQ ID NO: 
                 GGLFGYWDY (SEQ ID NO: 
               
               
                 (VH) 
                 NO: 87) 
                 88) 
                 89) 
               
               
                   
               
               
                 CD3 VL 
                 QAVVTQEPSLTVSPGGTV 
                 QAVVTQEPSLTVSPGGTVTLT 
                 QAVVTQEPSLTVSPGGTVTLTCGS 
               
               
                   
                 TLTCGSSTGAVTTSNYAN 
                 CGSSTGAVTTSNYANWVQEKP 
                 STGAVTTSNYANWVQEKPGQAFRG 
               
               
                   
                 WVQEKPGQAFRGLIGGTN 
                 GQAFRGLIGGTNKRAPGTPAR 
                 LIGGTNKRAPGTPARFSGSLLGGK 
               
               
                   
                 KRAPGTPARFSGSLLGGK 
                 FSGSLLGGKAALTLSGAQPED 
                 AALTLSGAQPEDEAEYYCALWYSN 
               
               
                   
                 AALTLSGAQPEDEAEYYC 
                 EAEYYCALWYSNLWVFGGGTK 
                 LWVFGGGTKLTVL (SEQ ID 
               
               
                   
                 ALWYSNLWVFGGGTKLTV 
                 LTVL (SEQ ID NO: 91) 
                 NO: 92) 
               
               
                   
                 L (SEQ ID NO: 90) 
                   
                   
               
               
                   
               
               
                 CD3 CDR1 
                 GSSTGAVTTSNYAN 
                 GSSTGAVTTSNYAN (SEQ 
                 GSSTGAVTTSNYAN (SEQ ID 
               
               
                 (VL) 
                 (SEQ ID NO: 93) 
                 ID NO: 93) 
                 NO: 93) 
               
               
                   
               
               
                 CD3CDR2 
                 GTNKRAP (SEQ ID 
                 GTNKRAP (SEQ ID NO: 
                 GTNKRAP (SEQ ID NO: 94) 
               
               
                 (VL) 
                 NO: 94) 
                 94) 
                   
               
               
                   
               
               
                 CD3CDR3 
                 ALWYSNLWV (SEQ ID 
                 ALWYSNLWV (SEQ ID NO: 
                 ALWYSNLWV (SEQ ID NO: 
               
               
                 (VL) 
                 NO: 95) 
                 95) 
                 95) 
               
               
                   
               
               
                 CD3 VH 
                 EVQLLESGGGLVQPGGSL 
                 EVQLLESGGGLVQPGGSLRLS 
                 EVQLLESGGGLVQPGGSLRLSCAA 
               
               
                   
                 RLSCAASGFTFSTYAMNW 
                 CAASGFTFSTYAMNWVRQAPG 
                 SGFTFSTYAMNWVRQAPGKGLEWV 
               
               
                   
                 VRQAPGKGLEWVSRIRSK 
                 KGLEWVSRIRSKYNNYATYYA 
                 SRIRSKYNNYATYYADSVKGRFTI 
               
               
                   
                 YNNYATYYADSVKGRFTI 
                 DSVKGRFTISRDDSKNTLYLQ 
                 SRDDSKNTLYLQMNSLRAEDTAVY 
               
               
                   
                 SRDDSKNTLYLQMNSLRA 
                 MNSLRAEDTAVYYCVRHGNFG 
                 YCVRHGNFGNSYVSWFAYWGQGTL 
               
               
                   
                 EDTAVYYCVRHGNFGNSY 
                 NSYVSWFAYWGQGTLVTVSS 
                 VTVSS (SEQ ID NO: 96) 
               
               
                   
                 VSWFAYWGQGTLVTVSS 
                 (SEQ ID NO: 96) 
                   
               
               
                   
                 (SEQ ID NO: 96) 
                   
                   
               
               
                   
               
               
                 CD3 CDR1 
                 TYAMN (SEQ ID NO: 
                 TYAMN (SEQ ID NO: 98) 
                 TYAMN (SEQ ID NO: 98) 
               
               
                 (VH) 
                 98) 
                   
                   
               
               
                   
               
               
                 CD3CDR2 
                 RIRSKYNNYATYYADSVK 
                 RIRSKYNNYATYYADSVKG 
                 RIRSKYNNYATYYADSVKG (SEQ 
               
               
                 (VH) 
                 G (SEQ ID NO: 99) 
                 (SEQ ID NO: 99) 
                 ID NO: 99) 
               
               
                   
               
               
                 CD3CDR3 
                 HGNFGNSYVSWFAY 
                 HGNFGNSYVSWFAY (SEQ 
                 HGNFGNSYVSWFAY (SEQ ID 
               
               
                 (VH) 
                 (SEQ ID NO: 100) 
                 ID NO: 100) 
                 NO: 100) 
               
               
                   
               
               
                 CD3 LC 
                 QAVVTQEPSLTVSPGGTV 
                 QAVVTQEPSLTVSPGGTVTLT 
                 QAVVTQEPSLTVSPGGTVTLTCGS 
               
               
                 (CLC) 
                 TLTCGSSTGAVTTSNYAN 
                 CGSSTGAVTTSNYANWVQEKP 
                 STGAVTTSNYANWVQEKPGQAFRG 
               
               
                   
                 WVQEKPGQAFRGLIGGTN 
                 GQAFRGLIGGTNKRAPGTPAR 
                 LIGGTNKRAPGTPARFSGSLLGGK 
               
               
                   
                 KRAPGTPARFSGSLLGGK 
                 FSGSLLGGKAALTLSGAQPED 
                 AALTLSGAQPEDEAEYYCALWYSN 
               
               
                   
                 AALTLSGAQPEDEAEYYC 
                 EAEYYCALWYSNLWVEGGGTK 
                 LWVFGGGTKLTVLGQPKAAPSVTL 
               
               
                   
                 ALWYSNLWVFGGGTKLTV 
                 LTVLGQPKAAPSVTLFPPSSE 
                 FPPSSEELQANKATLVCLISDFYP 
               
               
                   
                 LGQPKAAPSVTLFPPSSE 
                 ELQANKATLVCLISDFYPGAV 
                 GAVTVAWKADSSPVKAGVETTTPS 
               
               
                   
                 ELQANKATLVCLISDFYP 
                 TVAWKADSSPVKAGVETTTPS 
                 KQSNNKYAASSYLSLTPEQWKSHR 
               
               
                   
                 GAVTVAWKADSSPVKAGV 
                 KQSNNKYAASSYLSLTPEQWK 
                 SYSCQVTHEGSTVEKTVAPTECS 
               
               
                   
                 ETTTPSKQSNNKYAASSY 
                 SHRSYSCQVTHEGSTVEKTVA 
                 (SEQ ID NO: 101) 
               
               
                   
                 LSLTPEQWKSHRSYSCQV 
                 PTECS (SEQ ID NO: 
                   
               
               
                   
                 THEGSTVEKTVAPTECS 
                 101) 
                   
               
               
                   
                 (SEQ ID NO: 101) 
                   
                   
               
               
                   
               
               
                 2 + 1 Fc 
                   
                   
                   
               
               
                 containing 
                   
                   
                   
               
               
                 TCB (full 
                   
                   
                   
               
               
                 molecule) 
                   
                   
                   
               
               
                   
               
               
                 BCMA  
                 QVQLVQSGAEVKKPGASV 
                 n/a 
                 n/a 
               
               
                 pSCHLI333 
                 KVSCKASGYMFSSFGMSW 
                   
                   
               
               
                 VH_CH1 x 
                 VRQAPGQGLEWMGWIYPV 
                   
                   
               
               
                 CD3 
                 GQGTWYAQKFQGRVTMTR 
                   
                   
               
               
                 VH_CH1 
                 DTSTSTVYMELSSLRSED 
                   
                   
               
               
                 Fcknob 
                 TAVYYCARVSYPPSHFDY 
                   
                   
               
               
                 LALAPG 
                 WGQGTLVTVSSASTKGPS 
                   
                   
               
               
                   
                 VFPLAPSSKSTSGGTAAL 
                   
                   
               
               
                   
                 GCLVKDYFPEPVTVSWNS 
                   
                   
               
               
                   
                 GALTSGVHTFPAVLQSSG 
                   
                   
               
               
                   
                 LYSLSSVVTVPSSSLGTQ 
                   
                   
               
               
                   
                 TYICNVNHKPSNTKVDKK 
                   
                   
               
               
                   
                 VEPKSCDGGGGSGGGGSE 
                   
                   
               
               
                   
                 VQLLESGGGLVQPGGSLR 
                   
                   
               
               
                   
                 LSCAASGFTFSTYAMNWV 
                   
                   
               
               
                   
                 RQAPGKGLEWVSRIRSKY 
                   
                   
               
               
                   
                 NNYATYYADSVKGRFTIS 
                   
                   
               
               
                   
                 RDDSKNTLYLQMNSLRAE 
                   
                   
               
               
                   
                 DTAVYYCVRHGNFGNSYV 
                   
                   
               
               
                   
                 SWFAYWGQGTLVTVSSAS 
                   
                   
               
               
                   
                 TKGPSVFPLAPSSKSTSG 
                   
                   
               
               
                   
                 GTAALGCLVKDYFPEPVT 
                   
                   
               
               
                   
                 VSWNSGALTSGVHTFPAV 
                   
                   
               
               
                   
                 LQSSGLYSLSSVVTVPSS 
                   
                   
               
               
                   
                 SLGTQTYICNVNHKPSNT 
                   
                   
               
               
                   
                 KVDKKVEPKSCDKTHTCP 
                   
                   
               
               
                   
                 PCPAPEAAGGPSVFLFPP 
                   
                   
               
               
                   
                 KPKDTLMISRTPEVTCVV 
                   
                   
               
               
                   
                 VDVSHEDPEVKFNWYVDG 
                   
                   
               
               
                   
                 VEVHNAKTKPREEQYNST 
                   
                   
               
               
                   
                 YRVVSVLTVLHQDWLNGK 
                   
                   
               
               
                   
                 EYKCKVSNKALGAPIEKT 
                   
                   
               
               
                   
                 ISKAKGQPREPQVYTLPP 
                   
                   
               
               
                   
                 CRDELTKNQVSLWCLVKG 
                   
                   
               
               
                   
                 FYPSDIAVEWESNGQPEN 
                   
                   
               
               
                   
                 NYKTTPPVLDSDGSFFLY 
                   
                   
               
               
                   
                 SKLTVDKSRWQQGNVFSC 
                   
                   
               
               
                   
                 SVMHEALHNHYTQKSLSL 
                   
                   
               
               
                   
                 SPGK (SEQ ID NO: 
                   
                   
               
               
                   
                 102) 
                   
                   
               
               
                   
               
               
                 BCMA  
                 QVQLVQSGAEVKKPGASV 
                 n/a 
                 n/a 
               
               
                 pSCHLI333 
                 KVSCKASGYMESSEGMSW 
                   
                   
               
               
                 HC hole 
                 VRQAPGQGLEWMGWIYPV 
                   
                   
               
               
                 LALAPG 
                 GQGTWYAQKFQGRVTMTR 
                   
                   
               
               
                   
                 DTSTSTVYMELSSLRSED 
                   
                   
               
               
                   
                 TAVYYCARVSYPPSHFDY 
                   
                   
               
               
                   
                 WGQGTLVTVSSASTKGPS 
                   
                   
               
               
                   
                 VFPLAPSSKSTSGGTAAL 
                   
                   
               
               
                   
                 GCLVKDYFPEPVTVSWNS 
                   
                   
               
               
                   
                 GALTSGVHTFPAVLQSSG 
                   
                   
               
               
                   
                 LYSLSSVVTVPSSSLGTQ 
                   
                   
               
               
                   
                 TYICNVNHKPSNTKVDKK 
                   
                   
               
               
                   
                 VEPKSCDKTHTCPPCPAP 
                   
                   
               
               
                   
                 EAAGGPSVFLFPPKPKDT 
                   
                   
               
               
                   
                 LMISRTPEVTCVVVDVSH 
                   
                   
               
               
                   
                 EDPEVKFNWYVDGVEVHN 
                   
                   
               
               
                   
                 AKTKPREEQYNSTYRVVS 
                   
                   
               
               
                   
                 VLTVLHQDWLNGKEYKCK 
                   
                   
               
               
                   
                 VSNKALGAPIEKTISKAK 
                   
                   
               
               
                   
                 GQPREPQVCTLPPSRDEL 
                   
                   
               
               
                   
                 TKNQVSLSCAVKGFYPSD 
                   
                   
               
               
                   
                 IAVEWESNGQPENNYKTT 
                   
                   
               
               
                   
                 PPVLDSDGSFFLVSKLTV 
                   
                   
               
               
                   
                 DKSRWQQGNVFSCSVMHE 
                   
                   
               
               
                   
                 ALHNHYTQKSLSLSPGK 
                   
                   
               
               
                   
                 (SEQ ID NO: 103) 
                   
                   
               
               
                   
               
               
                 BCMA  
                 n/a 
                 EVQLVESGGGLVKPGGSLRLS 
                 n/a 
               
               
                 pSCHLI372 
                   
                 CAASGETESNSGMIWVRQAPG 
                   
               
               
                 VH_CH1 x 
                   
                 KGLEWVGHIRSKTDGGTTDYA 
                   
               
               
                 CD3  
                   
                 APVKGRFTISRDDSKNTLYLQ 
                   
               
               
                 VH_CH1 
                   
                 MNSLKTEDTAVYYCTTGGSGS 
                   
               
               
                 Fcknob 
                   
                 FDYWGQGTLVTVSSASTKGPS 
                   
               
               
                 LALAPG 
                   
                 VFPLAPSSKSTSGGTAALGCL 
                   
               
               
                   
                   
                 VKDYFPEPVTVSWNSGALTSG 
                   
               
               
                   
                   
                 VHTFPAVLQSSGLYSLSSVVT 
                   
               
               
                   
                   
                 VPSSSLGTQTYICNVNHKPSN 
                   
               
               
                   
                   
                 TKVDKKVEPKSCDGGGGSGGG 
                   
               
               
                   
                   
                 GSEVQLLESGGGLVQPGGSLR 
                   
               
               
                   
                   
                 LSCAASGFTESTYAMNWVRQA 
                   
               
               
                   
                   
                 PGKGLEWVSRIRSKYNNYATY 
                   
               
               
                   
                   
                 YADSVKGRFTISRDDSKNTLY 
                   
               
               
                   
                   
                 LQMNSLRAEDTAVYYCVRHGN 
                   
               
               
                   
                   
                 FGNSYVSWFAYWGQGTLVTVS 
                   
               
               
                   
                   
                 SASTKGPSVFPLAPSSKSTSG 
                   
               
               
                   
                   
                 GTAALGCLVKDYFPEPVTVSW 
                   
               
               
                   
                   
                 NSGALTSGVHTFPAVLQSSGL 
                   
               
               
                   
                   
                 YSLSSVVTVPSSSLGTQTYIC 
                   
               
               
                   
                   
                 NVNHKPSNTKVDKKVEPKSCD 
                   
               
               
                   
                   
                 KTHTCPPCPAPEAAGGPSVFL 
                   
               
               
                   
                   
                 FPPKPKDTLMISRTPEVTCVV 
                   
               
               
                   
                   
                 VDVSHEDPEVKFNWYVDGVEV 
                   
               
               
                   
                   
                 HNAKTKPREEQYNSTYRVVSV 
                   
               
               
                   
                   
                 LTVLHQDWLNGKEYKCKVSNK 
                   
               
               
                   
                   
                 ALGAPIEKTISKAKGQPREPQ 
                   
               
               
                   
                   
                 VYTLPPCRDELTKNQVSLWCL 
                   
               
               
                   
                   
                 VKGFYPSDIAVEWESNGQPEN 
                   
               
               
                   
                   
                 NYKTTPPVLDSDGSFFLYSKL 
                   
               
               
                   
                   
                 TVDKSRWQQGNVFSCSVMHEA 
                   
               
               
                   
                   
                 LHNHYTQKSLSLSPGK (SEQ 
                   
               
               
                   
                   
                 ID NO: 110) 
                   
               
               
                   
               
               
                 BCMA  
                 n/a 
                 EVQLVESGGGLVKPGGSLRLS 
                 n/a 
               
               
                 pSCHLI372 
                   
                 CAASGETESNSGMIWVRQAPG 
                   
               
               
                 HC hole 
                   
                 KGLEWVGHIRSKTDGGTTDYA 
                   
               
               
                 LALAPG 
                   
                 APVKGRFTISRDDSKNTLYLQ 
                   
               
               
                   
                   
                 MNSLKTEDTAVYYCTTGGSGS 
                   
               
               
                   
                   
                 FDYWGQGTLVTVSSASTKGPS 
                   
               
               
                   
                   
                 VFPLAPSSKSTSGGTAALGCL 
                   
               
               
                   
                   
                 VKDYFPEPVTVSWNSGALTSG 
                   
               
               
                   
                   
                 VHTFPAVLQSSGLYSLSSVVT 
                   
               
               
                   
                   
                 VPSSSLGTQTYICNVNHKPSN 
                   
               
               
                   
                   
                 TKVDKKVEPKSCDKTHTCPPC 
                   
               
               
                   
                   
                 RAPEAAGGPSVFLFPPKPKDT 
                   
               
               
                   
                   
                 LMISRTPEVTCVVVDVSHEDP 
                   
               
               
                   
                   
                 EVKFNWYVDGVEVHNAKTKPR 
                   
               
               
                   
                   
                 EEQYNSTYRVVSVLTVLHQDW 
                   
               
               
                   
                   
                 LNGKEYKCKVSNKALGAPIEK 
                   
               
               
                   
                   
                 TISKAKGQPREPQVCTLPPSR 
                   
               
               
                   
                   
                 DELTKNQVSLSCAVKGFYPSD 
                   
               
               
                   
                   
                 IAVEWESNGQPENNYKTTPPV 
                   
               
               
                   
                   
                 LDSDGSFFLVSKLTVDKSRWQ 
                   
               
               
                   
                   
                 QGNVFSCSVMHEALHNHYTQK 
                   
               
               
                   
                   
                 SLSLSPGK (SEQ ID NO: 
                   
               
               
                   
                   
                 111) 
                   
               
               
                   
               
               
                 BCMA  
                 n/a 
                 n/a 
                 EVQLVESGGGLVKPGGSLRLSCAA 
               
               
                 pSCHLI373 
                   
                   
                 SGFSFSNSWMNWVRQAPGKGLEWV 
               
               
                 VH_CH1 x 
                   
                   
                 GTIRQKTYGGTTDYAAPVKGRFTI 
               
               
                 CD3  
                   
                   
                 SRDDSKNTLYLQMNSLKTEDTAVY 
               
               
                 VH_CH1 
                   
                   
                 YCTTGGLFGYWDYWGQGTLVTVSS 
               
               
                 Fcknob 
                   
                   
                 ASTKGPSVFPLAPSSKSTSGGTAA 
               
               
                 LALAPG 
                   
                   
                 LGCLVKDYFPEPVTVSWNSGALTS 
               
               
                   
                   
                   
                 GVHTFPAVLQSSGLYSLSSVVTVP 
               
               
                   
                   
                   
                 SSSLGTQTYICNVNHKPSNTKVDK 
               
               
                   
                   
                   
                 KVEPKSCDGGGGSGGGGSEVQLLE 
               
               
                   
                   
                   
                 SGGGLVQPGGSLRLSCAASGFTFS 
               
               
                   
                   
                   
                 TYAMNWVRQAPGKGLEWVSRIRSK 
               
               
                   
                   
                   
                 YNNYATYYADSVKGRFTISRDDSK 
               
               
                   
                   
                   
                 NTLYLQMNSLRAEDTAVYYCVRHG 
               
               
                   
                   
                   
                 NFGNSYVSWFAYWGQGTLVTVSSA 
               
               
                   
                   
                   
                 STKGPSVFPLAPSSKSTSGGTAAL 
               
               
                   
                   
                   
                 GCLVKDYFPEPVTVSWNSGALTSG 
               
               
                   
                   
                   
                 VHTFPAVLQSSGLYSLSSVVTVPS 
               
               
                   
                   
                   
                 SSLGTQTYICNVNHKPSNTKVDKK 
               
               
                   
                   
                   
                 VEPKSCDKTHTCPPCPAPEAAGGP 
               
               
                   
                   
                   
                 SVFLFPPKPKDTLMISRTPEVTCV 
               
               
                   
                   
                   
                 VVDVSHEDPEVKFNWYVDGVEVHN 
               
               
                   
                   
                   
                 AKTKPREEQYNSTYRVVSVLTVLH 
               
               
                   
                   
                   
                 QDWLNGKEYKCKVSNKALGAPIEK 
               
               
                   
                   
                   
                 TISKAKGQPREPQVYTLPPCRDEL 
               
               
                   
                   
                   
                 TKNQVSLWCLVKGFYPSDIAVEWE 
               
               
                   
                   
                   
                 SNGQPENNYKTTPPVLDSDGSFFL 
               
               
                   
                   
                   
                 YSKLTVDKSRWQQGNVFSCSVMHE 
               
               
                   
                   
                   
                 ALHNHYTQKSLSLSPGK (SEQ 
               
               
                   
                   
                   
                 ID NO: 113) 
               
               
                   
               
               
                 BCMA  
                 n/a 
                 n/a 
                 EVQLVESGGGLVKPGGSLRLSCAA 
               
               
                 pSCHLI373 
                   
                   
                 SGFSFSNSWMNWVRQAPGKGLEWV 
               
               
                 HC hole 
                   
                   
                 GTIRQKTYGGTTDYAAPVKGRFTI 
               
               
                 LALAPG 
                   
                   
                 SRDDSKNTLYLQMNSLKTEDTAVY 
               
               
                   
                   
                   
                 YCTTGGLFGYWDYWGQGTLVTVSS 
               
               
                   
                   
                   
                 ASTKGPSVFPLAPSSKSTSGGTAA 
               
               
                   
                   
                   
                 LGCLVKDYFPEPVTVSWNSGALTS 
               
               
                   
                   
                   
                 GVHTFPAVLQSSGLYSLSSVVTVP 
               
               
                   
                   
                   
                 SSSLGTQTYICNVNHKPSNTKVDK 
               
               
                   
                   
                   
                 KVEPKSCDKTHTCPPCPAPEAAGG 
               
               
                   
                   
                   
                 PSVFLFPPKPKDTLMISRTPEVTC 
               
               
                   
                   
                   
                 VVVDVSHEDPEVKFNWYVDGVEVH 
               
               
                   
                   
                   
                 NAKTKPREEQYNSTYRVVSVLTVL 
               
               
                   
                   
                   
                 HQDWLNGKEYKCKVSNKALGAPIE 
               
               
                   
                   
                   
                 KTISKAKGQPREPQVCTLPPSRDE 
               
               
                   
                   
                   
                 LTKNQVSLSCAVKGFYPSDIAVEW 
               
               
                   
                   
                   
                 ESNGQPENNYKTTPPVLDSDGSFF 
               
               
                   
                   
                   
                 LVSKLTVDKSRWQQGNVFSCSVMH 
               
               
                   
                   
                   
                 EALHNHYTQKSLSLSPGK (SEQ 
               
               
                   
                   
                   
                 ID NO: 114) 
               
               
                   
               
               
                 83A10 VH 
                 EVQLLESGGGLVQPGGSL 
                   
                   
               
               
                   
                 RLSCAASGFTFSSYAMSW 
                   
                   
               
               
                   
                 VRQAPGKGLEWVSAISGS 
                   
                   
               
               
                   
                 GGSTYYADSVKGRFTISR 
                   
                   
               
               
                   
                 DNSKNTLYLQMNSLRAED 
                   
                   
               
               
                   
                 TAVYYCAKVLGWFDYWGQ 
                   
                   
               
               
                   
                 GTLVTVSS (SEQ ID 
                   
                   
               
               
                   
                 NO: 115) 
                   
                   
               
               
                   
               
               
                 83A10 
                 SYAMS (SEQ ID NO: 
                   
                   
               
               
                 CDRH1 
                 116) 
                   
                   
               
               
                   
               
               
                 83A10 
                 AISGSGGSTYYADSVKG 
                   
                   
               
               
                 CDRH2 
                 (SEQ ID NO: 117) 
                   
                   
               
               
                   
               
               
                 83A10 
                 VLGWFDY (SEQ ID 
                   
                   
               
               
                 CDRH3 
                 NO: 118) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 6) Untargeted DP47 
               
            
           
           
               
               
               
            
               
                 Description 
                 Sequence 
                 Seq ID No 
               
               
                   
               
               
                 Light Chain 
                 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQ 
                 119 
               
               
                 DP47 GS 
                 KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR 
                   
               
               
                   
                 LEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVF 
                   
               
               
                   
                 IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ 
                   
               
               
                   
                 SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC 
                   
               
               
                   
                 EVTHQGLSSPVTKSFNRGEC 
                   
               
               
                   
               
               
                 Light Chain 
                 QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQ 
                 120 
               
               
                 humanized 
                 EKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLS 
                   
               
               
                 CD3 CH2527   
                 GAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGP 
                   
               
               
                 (Crossfab, 
                 SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA 
                   
               
               
                 VL-CH1) 
                 LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN 
                   
               
               
                   
                 VNHKPSNTKVDKKVEPKSC 
                   
               
               
                   
               
               
                 DP47 GS 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ 
                 121 
               
               
                 (VH-CH1)- 
                 APGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT 
                   
               
               
                 humanized 
                 LYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAS 
                   
               
               
                 CD3 CH2527   
                 TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW 
                   
               
               
                 (Crossfab 
                 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT 
                   
               
               
                 VH-Ck)- 
                 YICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLL 
                   
               
               
                 Fc(knob) 
                 ESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKG 
                   
               
               
                 P329GLALA 
                 LEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYL 
                   
               
               
                   
                 QMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVT 
                   
               
               
                   
                 VSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE 
                   
               
               
                   
                 AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS 
                   
               
               
                   
                 KADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCP 
                   
               
               
                   
                 PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV 
                   
               
               
                   
                 SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV 
                   
               
               
                   
                 LTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPR 
                   
               
               
                   
                 EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWES 
                   
               
               
                   
                 NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV 
                   
               
               
                   
                 FSCSVMHEALHNHYTQKSLSLSPGK 
                   
               
               
                   
               
               
                 DP47GS 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ 
                 122 
               
               
                 (VH-CH1)- 
                 APGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT 
                   
               
               
                 Fc(hole) 
                 LYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAS 
                   
               
               
                 P329GLALA 
                 TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW 
                   
               
               
                   
                 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT 
                   
               
               
                   
                 YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG 
                   
               
               
                   
                 GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF 
                   
               
               
                   
                 NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL 
                   
               
               
                   
                 NGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPP 
                   
               
               
                   
                 SRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYK 
                   
               
               
                   
                 TTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEA 
                   
               
               
                   
                 LHNHYTQKSLSLSPGK 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 7) Exemplary target sequences 
               
            
           
           
               
               
               
            
               
                 Description 
                 Sequence 
                 Seq ID No 
               
               
                   
               
               
                 Human CD3  
                   
                   
               
               
                   
               
               
                 Human FoIR1 
                 RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNA 
                 227 
               
               
                 (ECD 25-234) 
                 CCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDT 
                   
               
               
                   
                 CLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWE 
                   
               
               
                   
                 DCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPT 
                   
               
               
                   
                 PTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEE 
                   
               
               
                   
                 VARFYAAAM 
                   
               
               
                   
               
               
                 murine FoIR1 
                 TRARTELLNVCMDAKHHKEKPGPEDNLHDQCSPWKTNSCC 
                 230 
               
               
                 (ECD 25-232) 
                 STNTSQEAHKDISYLYRFNWNHCGTMTSECKRHFIQDTCL 
                   
               
               
                   
                 YECSPNLGPWIQQVDQSWRKERILDVPLCKEDCQQWWEDC 
                   
               
               
                   
                 QSSFTCKSNWHKGWNWSSGHNECPVGASCHPFTFYFPTSA 
                   
               
               
                   
                 ALCEEIWSHSYKLSNYSRGSGRCIQMWFDPAQGNPNEEVA 
                   
               
               
                   
                 RFYAEAMS 
                   
               
               
                   
               
               
                 cynomolgus 
                 EAQTRTARARTELLNVCMNAKHHKEKPGPEDKLHEQCRPW 
                 231 
               
               
                 FoIR1 
                 KKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHF 
                   
               
               
                 (ECD 25-234) 
                 IQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCE 
                   
               
               
                   
                 RWWEDCRTSYCKSNWHKGWNWTSGFNKCPVGAACQPFHFY 
                   
               
               
                   
                 FPTPTVLCNEIWTYSYKVSNYSRGSGRCIQMWFDPAQGNP 
                   
               
               
                   
                 NEEVARFYAAAMS 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 8) Nucleotide sequences of exemplary embodiments 
               
            
           
           
               
               
               
            
               
                   
                 Sequence 
                 Seq ID No 
               
               
                   
               
               
                 Description 
                   
                   
               
               
                 16A3 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 151 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTAC 
                   
               
               
                   
                 TACGCTGGTGTTACTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCT 
                   
               
               
                   
               
               
                 15A1 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 152 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTAC 
                   
               
               
                   
                 TACATCGGTGTTGTTACTTTCGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCT 
                   
               
               
                   
               
               
                 18D3 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 153 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTAC 
                   
               
               
                   
                 TACACTGGTGGTTCTTCTGCTTTCGACTATTGGGGTCAAGGCACCCTCGT 
                   
               
               
                   
                 AACGGTTTCTTCT 
                   
               
               
                   
               
               
                 19E5 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGNTTC 
                 154 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAA 
                   
               
               
                   
                 TGGCGTCGTTACACTTCTTTCGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCT 
                   
               
               
                   
               
               
                 19A4 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 155 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGGT 
                   
               
               
                   
                 TGGATCCGTTGGGAACATTTCGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCT 
                   
               
               
                   
               
               
                 15H7 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 156 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTAC 
                   
               
               
                   
                 TACCTGTTCTCTACTTCTTTCGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCT 
                   
               
               
                   
               
               
                 15B6 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAGGTAAAAAAACCGGGCGCTTC 
                 157 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTAC 
                   
               
               
                   
                 TACATCGGTATCGTTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCT 
                   
               
               
                   
               
               
                 16D5 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 158 
               
               
                   
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                   
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                   
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                   
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                   
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                   
                 CCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCC 
                   
               
               
                   
               
               
                 15E12 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 159 
               
               
                   
                 CCNGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                   
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                   
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                   
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                   
                 AGATGAACTCTCTGAAAACCGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                   
                 CCGTGGGAATGGTCTTACTTCGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCC 
                   
               
               
                   
               
               
                 21D1 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 160 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTAC 
                   
               
               
                   
                 TACGTTGGTGTTTCTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCT 
                   
               
               
                   
               
               
                 16F12 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGNTTC 
                 161 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCNTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTTC 
                   
               
               
                   
                 ACTGTTCTGCGTGTTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCT 
                   
               
               
                   
               
               
                 21A5 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 162 
               
               
                   
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                   
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                   
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                   
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                   
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                   
                 CCGTGGGAATGGGCTTGGTTCGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCC 
                   
               
               
                   
               
               
                 21G8 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 163 
               
               
                   
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                   
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                   
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                   
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                   
                 AGATGAACTCTCTGAAAACCGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                   
                 CCTTGGGAATGGGCTTACTTCGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCC 
                   
               
               
                   
               
               
                 19H3 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 164 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCACTGGT 
                   
               
               
                   
                 TGGTCTCGTTGGGGTTACATGGACTATTGGGGCCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCT 
                   
               
               
                   
               
               
                 20G6 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 165 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAA 
                   
               
               
                   
                 TGGATCCGTTACTACCATTTCGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCT 
                   
               
               
                   
               
               
                 20H7 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 166 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGTTGGT 
                   
               
               
                   
                 TGGTACCGTTGGGGTTACATGGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCT 
                   
               
               
                   
               
               
                 11F8_VH 
                 CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTC 
                 167 
               
               
                   
                 GGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTA 
                   
               
               
                   
                 TAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGG 
                   
               
               
                   
                 ATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAG 
                   
               
               
                   
                 GGTAACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGA 
                   
               
               
                   
                 GCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGCTGTT 
                   
               
               
                   
                 TTCTACCGTGCTTGGTACTCTTTCGACTACTGGGGCCAAGGGACCACCGT 
                   
               
               
                   
                 GACCGTCTCCTCA 
                   
               
               
                   
               
               
                 11F8_VL 
                 GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGA 
                 168 
               
               
                   
                 CCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGG 
                   
               
               
                   
                 CCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAT 
                   
               
               
                   
                 GCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATC 
                   
               
               
                   
                 CGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTG 
                   
               
               
                   
                 CAACTTATTACTGCCAACAGTATACCAGCCCACCACCAACGTTTGGCCAG 
                   
               
               
                   
                 GGCACCAAAGTCGAGATCAAG 
                   
               
               
                   
               
               
                 36F2_VH 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 169 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTC 
                   
               
               
                   
                 TTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGT 
                   
               
               
                   
                 TTCTTCT 
                   
               
               
                   
               
               
                 36F2_VL 
                 GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA 
                 170 
               
               
                   
                 AAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACT 
                   
               
               
                   
                 TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT 
                   
               
               
                   
                 GGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG 
                   
               
               
                   
                 ATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT 
                   
               
               
                   
                 TTGCAGTGTATTACTGTCAGCAGTATACCAACGAACATTATTATACGTTC 
                   
               
               
                   
                 GGCCAGGGGACCAAAGTGGAAATCAAA 
                   
               
               
                   
               
               
                 9D11_VH 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 171 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAC 
                   
               
               
                   
                 TTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTC 
                   
               
               
                   
                 T 
                   
               
               
                   
               
               
                 9D11_VL 
                 GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGA 
                 172 
               
               
                   
                 ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACG 
                   
               
               
                   
                 GCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAG 
                   
               
               
                   
                 CTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTT 
                   
               
               
                   
                 CAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTG 
                   
               
               
                   
                 AAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAAC 
                   
               
               
                   
                 CGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAA 
                   
               
               
                   
               
               
                 9D11_VL 
                 GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGA 
                 173 
               
               
                 N95S 
                 ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACG 
                   
               
               
                   
                 GCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAG 
                   
               
               
                   
                 CTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTT 
                   
               
               
                   
                 CAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTG 
                   
               
               
                   
                 AAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAGC 
                   
               
               
                   
                 CGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAA 
                   
               
               
                   
               
               
                 9D11_VL 
                 GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGA 
                 174 
               
               
                 N95Q 
                 ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACG 
                   
               
               
                   
                 GCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAG 
                   
               
               
                   
                 CTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTT 
                   
               
               
                   
                 CAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTG 
                   
               
               
                   
                 AAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGCAG 
                   
               
               
                   
                 CGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAA 
                   
               
               
                   
               
               
                 9D11_VL 
                 GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGA 
                 175 
               
               
                 T97A 
                 ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACG 
                   
               
               
                   
                 GCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAG 
                   
               
               
                   
                 CTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTT 
                   
               
               
                   
                 CAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTG 
                   
               
               
                   
                 AAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAAC 
                   
               
               
                   
                 CGGGCTTTTGGTCAAGGCACCAAGGTCGAAATTAAA 
                   
               
               
                   
               
               
                 9D11_VL 
                 GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGA 
                 176 
               
               
                 T97N 
                 ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACG 
                   
               
               
                   
                 GCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAG 
                   
               
               
                   
                 CTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTT 
                   
               
               
                   
                 CAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTG 
                   
               
               
                   
                 AAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAAC 
                   
               
               
                   
                 CGGAATTTTGGTCAAGGCACCAAGGTCGAAATTAAA 
                   
               
               
                   
               
               
                 5D9_VH 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 177 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTAC 
                   
               
               
                   
                 ATCGACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 
                   
               
               
                   
               
               
                 5D9_VL 
                 GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA 
                 178 
               
               
                   
                 AAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACT 
                   
               
               
                   
                 TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT 
                   
               
               
                   
                 GGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG 
                   
               
               
                   
                 ATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT 
                   
               
               
                   
                 TTGCAGTGTATTACTGTCAGCAGGATAACTGGAGCCCAACGTTCGGCCAG 
                   
               
               
                   
                 GGGACCAAAGTGGAAATCAAA 
                   
               
               
                   
               
               
                 6B6_VH 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 179 
               
               
                   
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTAC 
                   
               
               
                   
                 GTTGACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 
                   
               
               
                   
               
               
                 6B6_VL 
                 GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA 
                 180 
               
               
                   
                 AAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACC 
                   
               
               
                   
                 TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT 
                   
               
               
                   
                 GGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG 
                   
               
               
                   
                 ATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT 
                   
               
               
                   
                 TTGCAGTGTATTACTGTCAGCAGGATATTTGGAGCCCAACGTTCGGCCAG 
                   
               
               
                   
                 GGGACCAAAGTGGAAATCAAA 
                   
               
               
                   
               
               
                 14E4_VH 
                 GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC 
                 181 
               
               
                   
                 CCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCA 
                   
               
               
                   
                 TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT 
                   
               
               
                   
                 ATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCG 
                   
               
               
                   
                 GTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGA 
                   
               
               
                   
                 ACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACTCT 
                   
               
               
                   
                 TCTTACGTTGAATGGTACGCTTTCGACTACTGGGGCCAAGGAACCCTGGT 
                   
               
               
                   
                 CACCGTCTCGAGT 
                   
               
               
                   
               
               
                 14E4_VL 
                 GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA 
                 182 
               
               
                   
                 AAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACT 
                   
               
               
                   
                 TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT 
                   
               
               
                   
                 GGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG 
                   
               
               
                   
                 ATCCGGGACAGACTCCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT 
                   
               
               
                   
                 TTGCAGTGTATTACTGTCAGCAGCCAACCAGCAGCCCAATTACGTTCGGC 
                   
               
               
                   
                 CAGGGGACCAAAGTGGAAATCAAA 
                   
               
               
                   
               
               
                 CD3 heavy 
                 GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATC 
                 183 
               
               
                 chain 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCA 
                   
               
               
                 (VHCH1) 
                 TGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGG 
                   
               
               
                   
                 ATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAA 
                   
               
               
                   
                 GGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                   
                 AGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGG 
                   
               
               
                   
                 CACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCA 
                   
               
               
                   
                 GGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGT 
                   
               
               
                   
                 TCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTG 
                   
               
               
                   
                 GGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAA 
                   
               
               
                   
                 CTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGA 
                   
               
               
                   
                 GCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCC 
                   
               
               
                   
                 CTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACAC 
                   
               
               
                   
                 CAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGC 
                   
               
               
                   
               
               
                 Crossed CD3 
                 GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATC 
                 184 
               
               
                 heavy chain 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCA 
                   
               
               
                 (VHCκ) 
                 TGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGG 
                   
               
               
                   
                 ATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAA 
                   
               
               
                   
                 GGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                   
                 AGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGG 
                   
               
               
                   
                 CACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCA 
                   
               
               
                   
                 GGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGT 
                   
               
               
                   
                 TTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTC 
                   
               
               
                   
                 GTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAA 
                   
               
               
                   
                 AGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAAC 
                   
               
               
                   
                 AGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCT 
                   
               
               
                   
                 AAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA 
                   
               
               
                   
                 GGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 
                   
               
               
                   
               
               
                 Mutagenesis 
                 GCAGGCAAGCATTATGCAGCGGACTTTTGGTCAAGG 
                 185 
               
               
                 primer 
                   
                   
               
               
                 GAB7734 
                   
                   
               
               
                 N95Q 
                   
                   
               
               
                   
               
               
                 Mutagenesis 
                 CAGGCAAGCATTATGAGCCGGACTTTTGGTCAAGG 
                 186 
               
               
                 primer 
                   
                   
               
               
                 GAB7735 
                   
                   
               
               
                 N95S 
                   
                   
               
               
                   
               
               
                 Mutagenesis 
                 CATTATGAACCGGGCTTTTGGTCAAGGCACCAAGGTC 
                 187 
               
               
                 primer 
                   
                   
               
               
                 GAB7736 
                   
                   
               
               
                 T97A 
                   
                   
               
               
                   
               
               
                 Mutagenesis 
                 CATTATGAACCGGAATTTTGGTCAAGGCACCAAGGTC 
                 188 
               
               
                 primer 
                   
                   
               
               
                 GAB7737 
                   
                   
               
               
                 T97N 
                   
                   
               
               
                   
               
               
                 VHCH1[16D5]_ 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 189 
               
               
                 VHCH1[CD3]_ 
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                 Fcknob_PGLALA 
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                 pCON999 
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                 (Inverted TCB 
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                 with 16D5 
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                 2 + 1: 
                 CCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                 pCON999 + 
                 GGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCA 
                   
               
               
                 pCON983 + 
                 GCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAG 
                   
               
               
                 pETR13197) 
                 GACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGAC 
                   
               
               
                   
                 AAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACT 
                   
               
               
                   
                 CCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACC 
                   
               
               
                   
                 TACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAA 
                   
               
               
                   
                 GGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGAT 
                   
               
               
                   
                 CCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGA 
                   
               
               
                   
                 TCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGC 
                   
               
               
                   
                 CATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCC 
                   
               
               
                   
                 GGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTG 
                   
               
               
                   
                 AAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCT 
                   
               
               
                   
                 GCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGC 
                   
               
               
                   
                 GGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGC 
                   
               
               
                   
                 CAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGT 
                   
               
               
                   
                 GTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTC 
                   
               
               
                   
                 TGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGG 
                   
               
               
                   
                 AACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCA 
                   
               
               
                   
                 GAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCT 
                   
               
               
                   
                 CCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAAC 
                   
               
               
                   
                 ACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACAC 
                   
               
               
                   
                 ATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCC 
                   
               
               
                   
                 TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG 
                   
               
               
                   
                 GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT 
                   
               
               
                   
                 CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC 
                   
               
               
                   
                 GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC 
                   
               
               
                   
                 CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA 
                   
               
               
                   
                 CAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC 
                   
               
               
                   
                 AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTG 
                   
               
               
                   
                 ACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAG 
                   
               
               
                   
                 CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA 
                   
               
               
                   
                 AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC 
                   
               
               
                   
                 AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG 
                   
               
               
                   
                 CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT 
                   
               
               
                   
                 CCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 VHCH1[16D5]_ 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 190 
               
               
                 Fchole_PGLALA_ 
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                 HYRF 
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                 pCON983 
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                   
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                   
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                   
                 CCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCA 
                   
               
               
                   
                 GCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAG 
                   
               
               
                   
                 GACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGAC 
                   
               
               
                   
                 CTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATA 
                   
               
               
                   
                 GCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACC 
                   
               
               
                   
                 TACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAA 
                   
               
               
                   
                 GGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAG 
                   
               
               
                   
                 CACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC 
                   
               
               
                   
                 AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT 
                   
               
               
                   
                 GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG 
                   
               
               
                   
                 GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC 
                   
               
               
                   
                 AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT 
                   
               
               
                   
                 GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCC 
                   
               
               
                   
                 CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG 
                   
               
               
                   
                 GTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG 
                   
               
               
                   
                 CCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT 
                   
               
               
                   
                 GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG 
                   
               
               
                   
                 CTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAA 
                   
               
               
                   
                 GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG 
                   
               
               
                   
                 CTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 CD3_common 
                 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCAC 
                 191 
               
               
                 light chain 
                 CGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACT 
                   
               
               
                 pETR13197 
                 ACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATC 
                   
               
               
                   
                 GGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATC 
                   
               
               
                   
                 TCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAG 
                   
               
               
                   
                 ATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTC 
                   
               
               
                   
                 GGCGGAGGCACCAAGCTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAG 
                   
               
               
                   
                 CGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCA 
                   
               
               
                   
                 CCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCC 
                   
               
               
                   
                 TGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCC 
                   
               
               
                   
                 CAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGA 
                   
               
               
                   
                 CCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCAC 
                   
               
               
                   
                 GAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC 
                   
               
               
                   
               
               
                 VHCH1[CD3]_ 
                 GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATC 
                 192 
               
               
                 VHCH1[16D5]_ 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCA 
                   
               
               
                 Fcknob_PGLALA 
                 TGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGG 
                   
               
               
                 pETR13932 
                 ATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAA 
                   
               
               
                 (Classical 
                 GGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                 TCB with 
                 AGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGG 
                   
               
               
                 16D5; 2 + 1: 
                 CACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCA 
                   
               
               
                 pETR13932 + 
                 GGGCACCCTCGTGACCGTGTCATCTGCTAGCACAAAGGGCCCTAGCGTGT 
                   
               
               
                 pCON983 + 
                 TCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTG 
                   
               
               
                 pETR13197) 
                 GGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAA 
                   
               
               
                   
                 CAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGA 
                   
               
               
                   
                 GCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGC 
                   
               
               
                   
                 CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACAC 
                   
               
               
                   
                 CAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGT 
                   
               
               
                   
                 CCGGAGGCGGAGGATCCGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTG 
                   
               
               
                   
                 GTAAAACCGGGCGGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCAC 
                   
               
               
                   
                 CTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCC 
                   
               
               
                   
                 TCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGAT 
                   
               
               
                   
                 TACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAA 
                   
               
               
                   
                 AAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAG 
                   
               
               
                   
                 TCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGC 
                   
               
               
                   
                 CAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACCAAGGGCCCCAGCGT 
                   
               
               
                   
                 GTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTC 
                   
               
               
                   
                 TGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGG 
                   
               
               
                   
                 AACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCA 
                   
               
               
                   
                 GAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCT 
                   
               
               
                   
                 CCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAAC 
                   
               
               
                   
                 ACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACAC 
                   
               
               
                   
                 ATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCC 
                   
               
               
                   
                 TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG 
                   
               
               
                   
                 GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT 
                   
               
               
                   
                 CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC 
                   
               
               
                   
                 GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC 
                   
               
               
                   
                 CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA 
                   
               
               
                   
                 CAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC 
                   
               
               
                   
                 AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTG 
                   
               
               
                   
                 ACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAG 
                   
               
               
                   
                 CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA 
                   
               
               
                   
                 AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC 
                   
               
               
                   
                 AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG 
                   
               
               
                   
                 CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT 
                   
               
               
                   
                 CCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 VHCH1[CD3]_ 
                 GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATC 
                 193 
               
               
                 Fcknob_PGLALA 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCA 
                   
               
               
                 pETR13719 
                 TGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGG 
                   
               
               
                 (16D5 IgG 
                 ATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAA 
                   
               
               
                 format, 1 + 1: 
                 GGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                 pETR13719 + 
                 AGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGG 
                   
               
               
                 pCON983 + 
                 CACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCA 
                   
               
               
                 pETR13197) 
                 GGGCACCCTCGTGACCGTGTCATCTGCTAGCACCAAGGGCCCATCGGTCT 
                   
               
               
                   
                 TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTG 
                   
               
               
                   
                 GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAA 
                   
               
               
                   
                 CTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGT 
                   
               
               
                   
                 CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC 
                   
               
               
                   
                 TTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACAC 
                   
               
               
                   
                 CAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT 
                   
               
               
                   
                 GCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTC 
                   
               
               
                   
                 TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT 
                   
               
               
                   
                 CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA 
                   
               
               
                   
                 ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG 
                   
               
               
                   
                 GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT 
                   
               
               
                   
                 GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA 
                   
               
               
                   
                 AAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG 
                   
               
               
                   
                 CCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGAC 
                   
               
               
                   
                 CAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCG 
                   
               
               
                   
                 ACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG 
                   
               
               
                   
                 ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAA 
                   
               
               
                   
                 GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT 
                   
               
               
                   
                 CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC 
                   
               
               
                   
                 CTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 Fc_hole_PGLALA_ 
                 GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG 
                 194 
               
               
                 HYRF 
                 ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT 
                   
               
               
                 pETR10755 
                 CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC 
                   
               
               
                 (16D5 Head- 
                 CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC 
                   
               
               
                 to-tail, 1 + 1: 
                 CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA 
                   
               
               
                 pCON999 + 
                 GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG 
                   
               
               
                 pETR10755 + 
                 TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTC 
                   
               
               
                 pETR13197) 
                 CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCAT 
                   
               
               
                   
                 CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAA 
                   
               
               
                   
                 GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC 
                   
               
               
                   
                 GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT 
                   
               
               
                   
                 TCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG 
                   
               
               
                   
                 AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCAC 
                   
               
               
                   
                 GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 VHCH1[9D11]_ 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 195 
               
               
                 VHCL[CD3]_ 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                 Fcknob_PGLALA 
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                 pCON1057 
                 ATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                 (9D11 
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                 inverted 
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAC 
                   
               
               
                 format, 2 + 1: 
                 TTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTC 
                   
               
               
                 pCON1057 + 
                 TGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGA 
                   
               
               
                 pCON1051 + 
                 GCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTT 
                   
               
               
                 pCON1063 + 
                 CCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGT 
                   
               
               
                 pETR12940) 
                 GCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCA 
                   
               
               
                   
                 GCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGC 
                   
               
               
                   
                 AACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCC 
                   
               
               
                   
                 CAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGC 
                   
               
               
                   
                 AGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGA 
                   
               
               
                   
                 CTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTG 
                   
               
               
                   
                 GGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAA 
                   
               
               
                   
                 GCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGG 
                   
               
               
                   
                 TTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAA 
                   
               
               
                   
                 CAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCA 
                   
               
               
                   
                 ACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACC 
                   
               
               
                   
                 CTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTT 
                   
               
               
                   
                 TCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTC 
                   
               
               
                   
                 TGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGAT 
                   
               
               
                   
                 AACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTC 
                   
               
               
                   
                 CAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTG 
                   
               
               
                   
                 ATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG 
                   
               
               
                   
                 AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCA 
                   
               
               
                   
                 CACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGT 
                   
               
               
                   
                 TCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCC 
                   
               
               
                   
                 GAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAA 
                   
               
               
                   
                 GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGC 
                   
               
               
                   
                 CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC 
                   
               
               
                   
                 GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC 
                   
               
               
                   
                 CAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG 
                   
               
               
                   
                 GGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAG 
                   
               
               
                   
                 CTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCC 
                   
               
               
                   
                 CAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT 
                   
               
               
                   
                 ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC 
                   
               
               
                   
                 AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC 
                   
               
               
                   
                 ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC 
                   
               
               
                   
                 TCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 9D11_Fchole_ 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 196 
               
               
                 PGLALA_HYRF 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                 pCON1051 
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAC 
                   
               
               
                   
                 TTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTC 
                   
               
               
                   
                 TGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGA 
                   
               
               
                   
                 GCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTC 
                   
               
               
                   
                 CCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGT 
                   
               
               
                   
                 GCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCA 
                   
               
               
                   
                 GCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGC 
                   
               
               
                   
                 AACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCC 
                   
               
               
                   
                 CAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAG 
                   
               
               
                   
                 CTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC 
                   
               
               
                   
                 CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG 
                   
               
               
                   
                 CCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGG 
                   
               
               
                   
                 TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC 
                   
               
               
                   
                 CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA 
                   
               
               
                   
                 GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGA 
                   
               
               
                   
                 AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACC 
                   
               
               
                   
                 CTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTG 
                   
               
               
                   
                 CGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA 
                   
               
               
                   
                 ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC 
                   
               
               
                   
                 GACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTG 
                   
               
               
                   
                 GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA 
                   
               
               
                   
                 ACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 9D11_LC 
                 GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGA 
                 197 
               
               
                 pCON1063 
                 ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACG 
                   
               
               
                   
                 GCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAG 
                   
               
               
                   
                 CTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTT 
                   
               
               
                   
                 CAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTG 
                   
               
               
                   
                 AAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAAC 
                   
               
               
                   
                 CGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACGGTGGCTGC 
                   
               
               
                   
                 ACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA 
                   
               
               
                   
                 CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA 
                   
               
               
                   
                 GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAG 
                   
               
               
                   
                 TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCC 
                   
               
               
                   
                 TGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA 
                   
               
               
                   
                 GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG 
                   
               
               
                   
                 AGAGTGT 
                   
               
               
                   
               
               
                 VLCH1[CD3] 
                 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCAC 
                 198 
               
               
                 pETR12940 
                 CGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACT 
                   
               
               
                   
                 ACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATC 
                   
               
               
                   
                 GGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATC 
                   
               
               
                   
                 TCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAG 
                   
               
               
                   
                 ATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTC 
                   
               
               
                   
                 GGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAAGGCCC 
                   
               
               
                   
                 TTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCG 
                   
               
               
                   
                 CTGCTCTCGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTG 
                   
               
               
                   
                 TCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGT 
                   
               
               
                   
                 GCTGCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCA 
                   
               
               
                   
                 GCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCC 
                   
               
               
                   
                 AGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCTTGT 
                   
               
               
                   
               
               
                 VHCL[CD3]_ 
                 GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATC 
                 199 
               
               
                 Fcknob_PGLALA 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCA 
                   
               
               
                 pETR13378 
                 TGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGG 
                   
               
               
                 (9D11 
                 ATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAA 
                   
               
               
                 CrossMab 
                 GGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                 format, 1 + 1: 
                 AGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGG 
                   
               
               
                 pETR13378 + 
                 CACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCA 
                   
               
               
                 pCON1051 + 
                 GGGCACCCTCGTGACCGTGTCATCTGCTAGCGTGGCCGCTCCCTCCGTGT 
                   
               
               
                 pCON1063 + 
                 TTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTC 
                   
               
               
                 pETR12940) 
                 GTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAA 
                   
               
               
                   
                 AGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAAC 
                   
               
               
                   
                 AGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCT 
                   
               
               
                   
                 AAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA 
                   
               
               
                   
                 GGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACA 
                   
               
               
                   
                 AGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCT 
                   
               
               
                   
                 TCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCG 
                   
               
               
                   
                 GACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTG 
                   
               
               
                   
                 AAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAG 
                   
               
               
                   
                 ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT 
                   
               
               
                   
                 CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA 
                   
               
               
                   
                 AGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAA 
                   
               
               
                   
                 GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCG 
                   
               
               
                   
                 GGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCT 
                   
               
               
                   
                 TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG 
                   
               
               
                   
                 AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT 
                   
               
               
                   
                 CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG 
                   
               
               
                   
                 TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG 
                   
               
               
                   
                 AAGAGCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 16D5 inverted 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 200 
               
               
                 2 + 1 with 
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                 N100A in CDRH3 
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                 pETR14096 
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                 (pETR14096 + 
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                 pCON983 + 
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                 pETR13197) 
                 CCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCA 
                   
               
               
                   
                 GCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAG 
                   
               
               
                   
                 GACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGAC 
                   
               
               
                   
                 AAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACT 
                   
               
               
                   
                 CCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACC 
                   
               
               
                   
                 TACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAA 
                   
               
               
                   
                 GGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGAT 
                   
               
               
                   
                 CCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGA 
                   
               
               
                   
                 TCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGC 
                   
               
               
                   
                 CATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCC 
                   
               
               
                   
                 GGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTG 
                   
               
               
                   
                 AAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCT 
                   
               
               
                   
                 GCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGC 
                   
               
               
                   
                 GGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGC 
                   
               
               
                   
                 CAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGT 
                   
               
               
                   
                 GTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTC 
                   
               
               
                   
                 TGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGG 
                   
               
               
                   
                 AACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCA 
                   
               
               
                   
                 GAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCT 
                   
               
               
                   
                 CCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAAC 
                   
               
               
                   
                 ACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACAC 
                   
               
               
                   
                 ATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCC 
                   
               
               
                   
                 TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG 
                   
               
               
                   
                 GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT 
                   
               
               
                   
                 CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC 
                   
               
               
                   
                 GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC 
                   
               
               
                   
                 CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA 
                   
               
               
                   
                 CAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC 
                   
               
               
                   
                 AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTG 
                   
               
               
                   
                 ACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAG 
                   
               
               
                   
                 CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA 
                   
               
               
                   
                 AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC 
                   
               
               
                   
                 AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG 
                   
               
               
                   
                 CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT 
                   
               
               
                   
                 CCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 16D5 inverted 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 201 
               
               
                 2 + 1 with 
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                 S100aA in 
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                 CDR H3 
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                 pETR14097 
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                 (pETR14097 + 
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                 pCON983 + 
                 CCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                 pETR13197) 
                 GGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCA 
                   
               
               
                   
                 GCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAG 
                   
               
               
                   
                 GACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGAC 
                   
               
               
                   
                 AAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACT 
                   
               
               
                   
                 CCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACC 
                   
               
               
                   
                 TACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAA 
                   
               
               
                   
                 GGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGAT 
                   
               
               
                   
                 CCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGA 
                   
               
               
                   
                 TCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGC 
                   
               
               
                   
                 CATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCC 
                   
               
               
                   
                 GGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTG 
                   
               
               
                   
                 AAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCT 
                   
               
               
                   
                 GCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGC 
                   
               
               
                   
                 GGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGC 
                   
               
               
                   
                 CAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGT 
                   
               
               
                   
                 GTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTC 
                   
               
               
                   
                 TGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGG 
                   
               
               
                   
                 AACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCA 
                   
               
               
                   
                 GAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCT 
                   
               
               
                   
                 CCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAAC 
                   
               
               
                   
                 ACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACAC 
                   
               
               
                   
                 ATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCC 
                   
               
               
                   
                 TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG 
                   
               
               
                   
                 GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT 
                   
               
               
                   
                 CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC 
                   
               
               
                   
                 GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC 
                   
               
               
                   
                 CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA 
                   
               
               
                   
                 CAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC 
                   
               
               
                   
                 AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTG 
                   
               
               
                   
                 ACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAG 
                   
               
               
                   
                 CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA 
                   
               
               
                   
                 AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC 
                   
               
               
                   
                 AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG 
                   
               
               
                   
                 CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT 
                   
               
               
                   
                 CCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 CD3 light 
                 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCAC 
                 202 
               
               
                 chain fused to 
                 CGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACT 
                   
               
               
                 CH1; 
                 ACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATC 
                   
               
               
                 Fc_PGLALA; 
                 GGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATC 
                   
               
               
                 pETR13862 
                 TCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAG 
                   
               
               
                 (Kappa- 
                 ATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTC 
                   
               
               
                 lambda 
                 GGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTAGCACCAAGGGCCC 
                   
               
               
                 antibody with 
                 ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAG 
                   
               
               
                 CD3 common 
                 CGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTG 
                   
               
               
                 light chain 
                 TCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGT 
                   
               
               
                 fused to CH1 + 
                 CCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCT 
                   
               
               
                 Fc_PGLALA. 
                 CCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCC 
                   
               
               
                 VHs fused to 
                 AGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAAC 
                   
               
               
                 kappa or 
                 TCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAG 
                   
               
               
                 lambda 
                 TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC 
                   
               
               
                 constant chain 
                 CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGT 
                   
               
               
                 pETR13859 + 
                 CAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAA 
                   
               
               
                 pETR13860 + 
                 AGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC 
                   
               
               
                 pETR13862) 
                 ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT 
                   
               
               
                   
                 CTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCA 
                   
               
               
                   
                 AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAT 
                   
               
               
                   
                 GAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA 
                   
               
               
                   
                 TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA 
                   
               
               
                   
                 ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC 
                   
               
               
                   
                 TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT 
                   
               
               
                   
                 CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGA 
                   
               
               
                   
                 GCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 16D5 VH 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 203 
               
               
                 fused to 
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                 constant 
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                 kappa chain; 
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                 pETR13859 
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                   
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                   
                 CCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCCGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTT 
                   
               
               
                   
                 CCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAAC 
                   
               
               
                   
                 AACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCT 
                   
               
               
                   
                 GCAGTCCGGCAACAGCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACA 
                   
               
               
                   
                 GCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAG 
                   
               
               
                   
                 AAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCC 
                   
               
               
                   
                 CGTGACCAAGTCTTTCAACCGGGGCGAGTGC 
                   
               
               
                   
               
               
                 CD3 VH fused 
                 GAAGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGATC 
                 204 
               
               
                 to constant 
                 TCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCACCTACGCCA 
                   
               
               
                 lambda chain; 
                 TGAACTGGGTGCGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGTCCCGG 
                   
               
               
                 pETR13860 
                 ATCAGATCCAAGTACAACAACTACGCCACCTACTACGCCGACTCCGTGAA 
                   
               
               
                   
                 GGGCCGGTTCACCATCTCTCGGGACGACTCCAAGAACACCCTGTACCTGC 
                   
               
               
                   
                 AGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGG 
                   
               
               
                   
                 CACGGCAACTTCGGCAACTCCTATGTGTCTTGGTTTGCCTACTGGGGCCA 
                   
               
               
                   
                 GGGCACCCTCGTGACCGTGTCATCTGCTAGCCCCAAGGCTGCCCCCAGCG 
                   
               
               
                   
                 TGACCCTGTTTCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACC 
                   
               
               
                   
                 CTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTG 
                   
               
               
                   
                 GAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCA 
                   
               
               
                   
                 GCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACC 
                   
               
               
                   
                 CCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGA 
                   
               
               
                   
                 GGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC 
                   
               
               
                   
               
               
                 VHCH1[36F2]_ 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 246 
               
               
                 VHCL[CD3]_ 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                 Fcknob_PGLALA 
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                 pCON1056 
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTC 
                   
               
               
                   
                 TTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGT 
                   
               
               
                   
                 TTCTTCTGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCA 
                   
               
               
                   
                 GCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGAC 
                   
               
               
                   
                 TACTTTCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAG 
                   
               
               
                   
                 CGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTC 
                   
               
               
                   
                 TGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTAC 
                   
               
               
                   
                 ATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGT 
                   
               
               
                   
                 GGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCG 
                   
               
               
                   
                 AGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCT 
                   
               
               
                   
                 CTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCAT 
                   
               
               
                   
                 GAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGA 
                   
               
               
                   
                 TCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAG 
                   
               
               
                   
                 GGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCA 
                   
               
               
                   
                 GATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGC 
                   
               
               
                   
                 ACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAG 
                   
               
               
                   
                 GGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTT 
                   
               
               
                   
                 TATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCG 
                   
               
               
                   
                 TGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAA 
                   
               
               
                   
                 GTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACA 
                   
               
               
                   
                 GGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTA 
                   
               
               
                   
                 AGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG 
                   
               
               
                   
                 GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAA 
                   
               
               
                   
                 GACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTT 
                   
               
               
                   
                 CTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGG 
                   
               
               
                   
                 ACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGA 
                   
               
               
                   
                 AGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGA 
                   
               
               
                   
                 CAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC 
                   
               
               
                   
                 CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA 
                   
               
               
                   
                 GGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAG 
                   
               
               
                   
                 CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGG 
                   
               
               
                   
                 GATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTT 
                   
               
               
                   
                 CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA 
                   
               
               
                   
                 ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC 
                   
               
               
                   
                 CTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT 
                   
               
               
                   
                 CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA 
                   
               
               
                   
                 AGAGCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 36F2-Fchole 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 247 
               
               
                 PGLALA 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                 pCON1050 
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTC 
                   
               
               
                   
                 TTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGT 
                   
               
               
                   
                 TTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCA 
                   
               
               
                   
                 GCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGAC 
                   
               
               
                   
                 TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTC 
                   
               
               
                   
                 CGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCC 
                   
               
               
                   
                 TGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTAC 
                   
               
               
                   
                 ATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGT 
                   
               
               
                   
                 GGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCAC 
                   
               
               
                   
                 CTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG 
                   
               
               
                   
                 GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGA 
                   
               
               
                   
                 CGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG 
                   
               
               
                   
                 TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC 
                   
               
               
                   
                 ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA 
                   
               
               
                   
                 TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCA 
                   
               
               
                   
                 TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG 
                   
               
               
                   
                 TGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT 
                   
               
               
                   
                 CTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG 
                   
               
               
                   
                 AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG 
                   
               
               
                   
                 GACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAG 
                   
               
               
                   
                 CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC 
                   
               
               
                   
                 TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 36F2 LC 
                 GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA 
                 97 
               
               
                 pCON1062 
                 AAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACT 
                   
               
               
                   
                 TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT 
                   
               
               
                   
                 GGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG 
                   
               
               
                   
                 ATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT 
                   
               
               
                   
                 TTGCAGTGTATTACTGTCAGCAGTATACCAACGAACATTATTATACGTTC 
                   
               
               
                   
                 GGCCAGGGGACCAAAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGT 
                   
               
               
                   
                 CTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTG 
                   
               
               
                   
                 TTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG 
                   
               
               
                   
                 AAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA 
                   
               
               
                   
                 GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA 
                   
               
               
                   
                 GCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT 
                   
               
               
                   
                 CANGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 
                   
               
               
                   
               
               
                 CD3 VLCH1 
                 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCAC 
                 198 
               
               
                 pETR12940 
                 CGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACT 
                   
               
               
                   
                 ACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATC 
                   
               
               
                   
                 GGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATC 
                   
               
               
                   
                 TCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAG 
                   
               
               
                   
                 ATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTC 
                   
               
               
                   
                 GGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAAGGCCC 
                   
               
               
                   
                 TTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCG 
                   
               
               
                   
                 CTGCTCTCGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTG 
                   
               
               
                   
                 TCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGT 
                   
               
               
                   
                 GCTGCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCA 
                   
               
               
                   
                 GCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCC 
                   
               
               
                   
                 AGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCTTGT 
                   
               
               
                   
               
               
                 Name 
                   
                   
               
               
                 K53A 
                 CAGACCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCAC 
                 205 
               
               
                 nt 
                 CGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACT 
                   
               
               
                   
                 ACGCCAACTGGGTGCAGCAGAAGCCAGGCCAGGCTCCCAGAGGACTGATC 
                   
               
               
                   
                 GGCGGCACCAACGCCAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATC 
                   
               
               
                   
                 TCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGTGCAGCCTGAAG 
                   
               
               
                   
                 ATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTC 
                   
               
               
                   
                 GGCGGAGGCACCAAGCTGACAGTCCTA 
                   
               
               
                   
               
               
                 S93A 
                 CAGACCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCAC 
                 206 
               
               
                 nt 
                 CGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACT 
                   
               
               
                   
                 ACGCCAACTGGGTGCAGCAGAAGCCAGGCCAGGCTCCCAGAGGACTGATC 
                   
               
               
                   
                 GGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATC 
                   
               
               
                   
                 TCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGTGCAGCCTGAAG 
                   
               
               
                   
                 ATGAGGCCGAGTACTACTGCGCCCTGTGGTACGCCAACCTGTGGGTGTTC 
                   
               
               
                   
                 GGCGGAGGCACCAAGCTGACAGTCCTA 
                   
               
               
                   
               
               
                 S35H 
                 GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATC 
                 207 
               
               
                 nt 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGA 
                   
               
               
                   
                 TGCACTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGG 
                   
               
               
                   
                 ATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAA 
                   
               
               
                   
                 GGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                   
                 AGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACC 
                   
               
               
                   
                 CCCTGGGAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGAC 
                   
               
               
                   
                 CGTGTCCTCTGCTAGC 
                   
               
               
                   
               
               
                 G49S 
                 GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATC 
                 208 
               
               
                 nt 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGA 
                   
               
               
                   
                 TGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGTCCCGG 
                   
               
               
                   
                 ATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAA 
                   
               
               
                   
                 GGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                   
                 AGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACC 
                   
               
               
                   
                 CCCTGGGAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGAC 
                   
               
               
                   
                 CGTGTCCTCTGCTAGC 
                   
               
               
                   
               
               
                 R50S 
                 GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATC 
                 209 
               
               
                 nt 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGA 
                   
               
               
                   
                 TGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGATCT 
                   
               
               
                   
                 ATCAAGAGCAAGACCGACGGCGGCACCACCGACTATGCCGCCCCTGTGAA 
                   
               
               
                   
                 GGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                   
                 AGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACC 
                   
               
               
                   
                 CCCTGGGAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGAC 
                   
               
               
                   
                 CGTGTCCTCTGCTAGC 
                   
               
               
                   
               
               
                 W96Y 
                 GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATC 
                 210 
               
               
                 nt 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGA 
                   
               
               
                   
                 TGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGG 
                   
               
               
                   
                 ATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAA 
                   
               
               
                   
                 GGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                   
                 AGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACC 
                   
               
               
                   
                 CCCTACGAGTGGTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGAC 
                   
               
               
                   
                 CGTGTCATCTGCTAGC 
                   
               
               
                   
               
               
                 W98Y 
                 GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATC 
                 211 
               
               
                 nt 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGA 
                   
               
               
                   
                 TGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGG 
                   
               
               
                   
                 ATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAA 
                   
               
               
                   
                 GGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                   
                 AGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACC 
                   
               
               
                   
                 CCCTGGGAGTACTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGAC 
                   
               
               
                   
                 CGTGTCATCTGCTAGC 
                   
               
               
                   
               
               
                 90D7 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 212 
               
               
                 nt 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTAC 
                   
               
               
                   
                 ACTATCGTTGTTTCTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCTGCTAGC 
                   
               
               
                   
               
               
                 90C1 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 213 
               
               
                 nt 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTAC 
                   
               
               
                   
                 TTCATCGGTTCTGTTGCTATGGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCTGCTAGC 
                   
               
               
                   
               
               
                 5E8 VH 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 214 
               
               
                 nt 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTCTG 
                   
               
               
                   
                 ACTTACTCTATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTC 
                   
               
               
                   
                 TGCTAGC 
                   
               
               
                   
               
               
                 5E8 VL 
                 GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGA 
                 215 
               
               
                 nt 
                 ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACG 
                   
               
               
                   
                 GCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAG 
                   
               
               
                   
                 CTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTT 
                   
               
               
                   
                 CAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTG 
                   
               
               
                   
                 AAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGATTCCA 
                   
               
               
                   
                 AACACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 
                   
               
               
                   
               
               
                 12A4 VH 
                 GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC 
                 216 
               
               
                 nt 
                 CCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCA 
                   
               
               
                   
                 TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT 
                   
               
               
                   
                 ATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCG 
                   
               
               
                   
                 GTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGA 
                   
               
               
                   
                 ACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATACGCT 
                   
               
               
                   
                 TACGCTCTGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGC 
                   
               
               
                   
                 TAGC 
                   
               
               
                   
               
               
                 12A4 VL 
                 GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA 
                 217 
               
               
                 nt 
                 AAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACT 
                   
               
               
                   
                 TAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT 
                   
               
               
                   
                 GGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGG 
                   
               
               
                   
                 ATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATT 
                   
               
               
                   
                 TTGCAGTGTATTACTGTCAGCAGCATGGCAGCAGCAGCACGTTCGGCCAG 
                   
               
               
                   
                 GGGACCAAAGTGGAAATCAAACGTACG 
                   
               
               
                   
               
               
                 7A3 VH 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 218 
               
               
                 nt 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAC 
                   
               
               
                   
                 TTCTCTGCTGGTCGTCTGATGGACTATTGGGGTCAAGGCACCCTCGTAAC 
                   
               
               
                   
                 GGTTTCTTCTGCTAGC 
                   
               
               
                   
               
               
                 7A3 VL 
                 GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGA 
                 219 
               
               
                 nt 
                 ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACG 
                   
               
               
                   
                 GCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAG 
                   
               
               
                   
                 CTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTT 
                   
               
               
                   
                 CAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTG 
                   
               
               
                   
                 AAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGACCCCA 
                   
               
               
                   
                 CCAATTACCTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 
                   
               
               
                   
               
               
                 6E10 VH 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 220 
               
               
                 nt 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAC 
                   
               
               
                   
                 TACAACGCTTTCGACTATTGGGGTCACGGCACCCTCGTAACGGTTTCTTC 
                   
               
               
                   
                 TGCTAGC 
                   
               
               
                   
               
               
                 6E10 VL 
                 GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGA 
                 221 
               
               
                 nt 
                 ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACG 
                   
               
               
                   
                 GCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAG 
                   
               
               
                   
                 CTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTT 
                   
               
               
                   
                 CAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTG 
                   
               
               
                   
                 AAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCATGGCATAGCCCA 
                   
               
               
                   
                 ACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 
                   
               
               
                   
               
               
                 12F9 VH 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 222 
               
               
                 nt 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGCT 
                   
               
               
                   
                 ACTTACACTATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTC 
                   
               
               
                   
                 TGCTAGC 
                   
               
               
                   
               
               
                 12F9 VL 
                 GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGA 
                 223 
               
               
                 nt 
                 ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACG 
                   
               
               
                   
                 GCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAG 
                   
               
               
                   
                 CTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTT 
                   
               
               
                   
                 CAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTG 
                   
               
               
                   
                 AAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGACCCCA 
                   
               
               
                   
                 ATTACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 
                   
               
               
                   
               
               
                 pETR11646 
                 CAGGTGCAGCTGCAGCAGTCTGGCGCCGAGCTCGTGAAACCTGGCGCCTC 
                 224 
               
               
                 Mov19 VH- 
                 CGTGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACTTCA 
                   
               
               
                 CH1-Fchole 
                 TGAACTGGGTCAAGCAGAGCCACGGCAAGAGCCTGGAATGGATCGGCAGA 
                   
               
               
                 PG/LALA 
                 ATCCACCCCTACGACGGCGACACCTTCTACAACCAGAACTTCAAGGACAA 
                   
               
               
                   
                 GGCCACCCTGACCGTGGACAAGAGCAGCAACACCGCCCACATGGAACTGC 
                   
               
               
                   
                 TGAGCCTGACCAGCGAGGACTTCGCCGTGTACTACTGCACCAGATACGAC 
                   
               
               
                   
                 GGCAGCCGGGCCATGGATTATTGGGGCCAGGGCACCACCGTGACAGTGTC 
                   
               
               
                   
                 CAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCA 
                   
               
               
                   
                 AGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTAC 
                   
               
               
                   
                 TTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGG 
                   
               
               
                   
                 CGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGA 
                   
               
               
                   
                 GCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATC 
                   
               
               
                   
                 TGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGA 
                   
               
               
                   
                 GCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG 
                   
               
               
                   
                 AAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGAC 
                   
               
               
                   
                 ACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGT 
                   
               
               
                   
                 GAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGG 
                   
               
               
                   
                 AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG 
                   
               
               
                   
                 TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG 
                   
               
               
                   
                 CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCG 
                   
               
               
                   
                 AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGC 
                   
               
               
                   
                 ACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTC 
                   
               
               
                   
                 GTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA 
                   
               
               
                   
                 GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC 
                   
               
               
                   
                 TCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAG 
                   
               
               
                   
                 GTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC 
                   
               
               
                   
                 ACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 pETR11647 
                 CAGGTGCAGCTGCAGCAGTCTGGCGCCGAGCTCGTGAAACCTGGCGCCTC 
                 225 
               
               
                 Mov19 VH- 
                 CGTGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACTTCA 
                   
               
               
                 CH1-CD3 
                 TGAACTGGGTCAAGCAGAGCCACGGCAAGAGCCTGGAATGGATCGGCAGA 
                   
               
               
                 VH-CL- 
                 ATCCACCCCTACGACGGCGACACCTTCTACAACCAGAACTTCAAGGACAA 
                   
               
               
                 Fcknob 
                 GGCCACCCTGACCGTGGACAAGAGCAGCAACACCGCCCACATGGAACTGC 
                   
               
               
                 PG/LALA 
                 TGAGCCTGACCAGCGAGGACTTCGCCGTGTACTACTGCACCAGATACGAC 
                   
               
               
                   
                 GGCAGCCGGGCCATGGATTATTGGGGCCAGGGCACCACCGTGACAGTGTC 
                   
               
               
                   
                 CAGCGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCA 
                   
               
               
                   
                 AGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTAC 
                   
               
               
                   
                 TTTCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGG 
                   
               
               
                   
                 CGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGA 
                   
               
               
                   
                 GCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATC 
                   
               
               
                   
                 TGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGA 
                   
               
               
                   
                 GCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAAG 
                   
               
               
                   
                 TGCAGCTGGTGGAAAGCGGCGGAGGCCTGGTGCAGCCTAAGGGCTCTCTG 
                   
               
               
                   
                 AAGCTGAGCTGTGCCGCCAGCGGCTTCACCTTCAACACCTACGCCATGAA 
                   
               
               
                   
                 CTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGGCCCGGATCA 
                   
               
               
                   
                 GAAGCAAGTACAACAATTACGCCACCTACTACGCCGACAGCGTGAAGGAC 
                   
               
               
                   
                 CGGTTCACCATCAGCCGGGACGACAGCCAGAGCATCCTGTACCTGCAGAT 
                   
               
               
                   
                 GAACAACCTGAAAACCGAGGACACCGCCATGTACTACTGCGTGCGGCACG 
                   
               
               
                   
                 GCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGC 
                   
               
               
                   
                 ACCCTCGTGACAGTGTCTGCTGCTAGCGTGGCCGCTCCCTCCGTGTTTAT 
                   
               
               
                   
                 CTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGT 
                   
               
               
                   
                 GTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTG 
                   
               
               
                   
                 GATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGA 
                   
               
               
                   
                 CTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGG 
                   
               
               
                   
                 CTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC 
                   
               
               
                   
                 CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGAC 
                   
               
               
                   
                 CCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTG 
                   
               
               
                   
                 TGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACC 
                   
               
               
                   
                 CCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGT 
                   
               
               
                   
                 GAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAA 
                   
               
               
                   
                 AGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC 
                   
               
               
                   
                 ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT 
                   
               
               
                   
                 CTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCA 
                   
               
               
                   
                 AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGAT 
                   
               
               
                   
                 GAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTA 
                   
               
               
                   
                 TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA 
                   
               
               
                   
                 ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC 
                   
               
               
                   
                 TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT 
                   
               
               
                   
                 CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGA 
                   
               
               
                   
                 GCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 pETR11644 
                 GACATCGAGCTGACCCAGAGCCCTGCCTCTCTGGCCGTGTCTCTGGGACA 
                 226 
               
               
                 Mov19 LC 
                 GAGAGCCATCATCAGCTGCAAGGCCAGCCAGAGCGTGTCCTTTGCCGGCA 
                   
               
               
                   
                 CCTCTCTGATGCACTGGTATCACCAGAAGCCCGGCCAGCAGCCCAAGCTG 
                   
               
               
                   
                 CTGATCTACAGAGCCAGCAACCTGGAAGCCGGCGTGCCCACAAGATTTTC 
                   
               
               
                   
                 CGGCAGCGGCAGCAAGACCGACTTCACCCTGAACATCCACCCCGTGGAAG 
                   
               
               
                   
                 AAGAGGACGCCGCCACCTACTACTGCCAGCAGAGCAGAGAGTACCCCTAC 
                   
               
               
                   
                 ACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGTACGGTGGCTGCACC 
                   
               
               
                   
                 ATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG 
                   
               
               
                   
                 CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA 
                   
               
               
                   
                 CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGT 
                   
               
               
                   
                 CACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGA 
                   
               
               
                   
                 CGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC 
                   
               
               
                   
                 ACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA 
                   
               
               
                   
                 GTGT 
                   
               
               
                   
               
               
                 Variant 
                   
                   
               
               
                 16D5 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 261 
               
               
                 VH_D52dE 
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                   
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                   
                 ATCAAGTCTAAAACTGAGGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                   
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                   
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                   
                 CCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCC 
                   
               
               
                   
               
               
                 16D5 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 262 
               
               
                 VH_D52dQ 
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                   
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                   
                 ATCAAGTCTAAAACTCAGGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                   
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                   
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                   
                 CCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCC 
                   
               
               
                   
               
               
                 CD3_VH 
                 GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATC 
                 263 
               
               
                 N100A 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCA 
                   
               
               
                   
                 TGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGG 
                   
               
               
                   
                 ATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAA 
                   
               
               
                   
                 GGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                   
                 AGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGG 
                   
               
               
                   
                 CACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCA 
                   
               
               
                   
                 GGGCACCCTCGTGACCGTGTCAAGC 
                   
               
               
                   
               
               
                 CD3_VH 
                 GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATC 
                 264 
               
               
                 S100aA 
                 TCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCA 
                   
               
               
                   
                 TGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGG 
                   
               
               
                   
                 ATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAA 
                   
               
               
                   
                 GGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGC 
                   
               
               
                   
                 AGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGG 
                   
               
               
                   
                 CACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCA 
                   
               
               
                   
                 GGGCACCCTCGTGACCGTGTCAAGC 
                   
               
               
                   
               
               
                 16D5 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 265 
               
               
                 [VHCH1]- 
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                 CD3[VHCH1- 
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                 N100A]- 
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                 Fcknob_PGLALA 
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                   
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                   
                 CCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCA 
                   
               
               
                   
                 GCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAG 
                   
               
               
                   
                 GACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGAC 
                   
               
               
                   
                 AAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACT 
                   
               
               
                   
                 CCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACC 
                   
               
               
                   
                 TACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAA 
                   
               
               
                   
                 GGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGAT 
                   
               
               
                   
                 CCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGA 
                   
               
               
                   
                 TCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGC 
                   
               
               
                   
                 CATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCC 
                   
               
               
                   
                 GGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTG 
                   
               
               
                   
                 AAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCT 
                   
               
               
                   
                 GCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGC 
                   
               
               
                   
                 GGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGC 
                   
               
               
                   
                 CAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGT 
                   
               
               
                   
                 GTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTC 
                   
               
               
                   
                 TGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGG 
                   
               
               
                   
                 AACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCA 
                   
               
               
                   
                 GAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCT 
                   
               
               
                   
                 CCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAAC 
                   
               
               
                   
                 ACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACAC 
                   
               
               
                   
                 ATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCC 
                   
               
               
                   
                 TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG 
                   
               
               
                   
                 GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT 
                   
               
               
                   
                 CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC 
                   
               
               
                   
                 GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC 
                   
               
               
                   
                 CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA 
                   
               
               
                   
                 CAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC 
                   
               
               
                   
                 AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTG 
                   
               
               
                   
                 ACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAG 
                   
               
               
                   
                 CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA 
                   
               
               
                   
                 AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC 
                   
               
               
                   
                 AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG 
                   
               
               
                   
                 CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT 
                   
               
               
                   
                 CCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 16D5- 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 266 
               
               
                 Fchole- 
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                 PGLALA 
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                   
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                   
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                   
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                   
                 CCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCA 
                   
               
               
                   
                 GCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAG 
                   
               
               
                   
                 GACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGAC 
                   
               
               
                   
                 CTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATA 
                   
               
               
                   
                 GCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACC 
                   
               
               
                   
                 TACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAA 
                   
               
               
                   
                 GGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAG 
                   
               
               
                   
                 CACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC 
                   
               
               
                   
                 AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT 
                   
               
               
                   
                 GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG 
                   
               
               
                   
                 GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC 
                   
               
               
                   
                 AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT 
                   
               
               
                   
                 GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCC 
                   
               
               
                   
                 CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG 
                   
               
               
                   
                 GTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG 
                   
               
               
                   
                 CCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT 
                   
               
               
                   
                 GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG 
                   
               
               
                   
                 CTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAA 
                   
               
               
                   
                 GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG 
                   
               
               
                   
                 CTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 CD3-CLC 
                 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCAC 
                 267 
               
               
                   
                 CGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACT 
                   
               
               
                   
                 ACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATC 
                   
               
               
                   
                 GGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATC 
                   
               
               
                   
                 TCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAG 
                   
               
               
                   
                 ATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTC 
                   
               
               
                   
                 GGCGGAGGCACCAAGCTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAG 
                   
               
               
                   
                 CGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCA 
                   
               
               
                   
                 CCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCC 
                   
               
               
                   
                 TGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCC 
                   
               
               
                   
                 CAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGA 
                   
               
               
                   
                 CCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCAC 
                   
               
               
                   
                 GAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC 
                   
               
               
                   
               
               
                 16D5 
                 GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC 
                 268 
               
               
                 [VHCH1]- 
                 CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGA 
                   
               
               
                 CD3[VHCH1- 
                 TGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGT 
                   
               
               
                 S100aA]- 
                 ATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAA 
                   
               
               
                 Fcknob_PGLALA 
                 AGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGC 
                   
               
               
                   
                 AGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACC 
                   
               
               
                   
                 CCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTAC 
                   
               
               
                   
                 GGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCA 
                   
               
               
                   
                 GCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAG 
                   
               
               
                   
                 GACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGAC 
                   
               
               
                   
                 AAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACT 
                   
               
               
                   
                 CCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACC 
                   
               
               
                   
                 TACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAA 
                   
               
               
                   
                 GGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGAT 
                   
               
               
                   
                 CCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGA 
                   
               
               
                   
                 TCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGC 
                   
               
               
                   
                 CATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCC 
                   
               
               
                   
                 GGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTG 
                   
               
               
                   
                 AAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCT 
                   
               
               
                   
                 GCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGC 
                   
               
               
                   
                 GGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGC 
                   
               
               
                   
                 CAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGT 
                   
               
               
                   
                 GTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTC 
                   
               
               
                   
                 TGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGG 
                   
               
               
                   
                 AACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCA 
                   
               
               
                   
                 GAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCT 
                   
               
               
                   
                 CCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAAC 
                   
               
               
                   
                 ACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACAC 
                   
               
               
                   
                 ATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCC 
                   
               
               
                   
                 TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG 
                   
               
               
                   
                 GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT 
                   
               
               
                   
                 CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC 
                   
               
               
                   
                 GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC 
                   
               
               
                   
                 CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA 
                   
               
               
                   
                 CAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC 
                   
               
               
                   
                 AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTG 
                   
               
               
                   
                 ACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAG 
                   
               
               
                   
                 CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA 
                   
               
               
                   
                 AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC 
                   
               
               
                   
                 AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG 
                   
               
               
                   
                 CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT 
                   
               
               
                   
                 CCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 9D11 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 269 
               
               
                 [VHCH1]- 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                 CD3[VHCL- 
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                 N100A]- 
                 ATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                 Fcknob_PGLALA 
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAC 
                   
               
               
                   
                 TTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTC 
                   
               
               
                   
                 TGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGA 
                   
               
               
                   
                 GCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTT 
                   
               
               
                   
                 CCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGT 
                   
               
               
                   
                 GCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCA 
                   
               
               
                   
                 GCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGC 
                   
               
               
                   
                 AACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCC 
                   
               
               
                   
                 CAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGC 
                   
               
               
                   
                 AGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGA 
                   
               
               
                   
                 CTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTG 
                   
               
               
                   
                 GGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAA 
                   
               
               
                   
                 GCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGG 
                   
               
               
                   
                 TTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAA 
                   
               
               
                   
                 CAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCA 
                   
               
               
                   
                 ACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACC 
                   
               
               
                   
                 CTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTT 
                   
               
               
                   
                 TCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTC 
                   
               
               
                   
                 TGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGAT 
                   
               
               
                   
                 AACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTC 
                   
               
               
                   
                 CAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTG 
                   
               
               
                   
                 ATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG 
                   
               
               
                   
                 AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCA 
                   
               
               
                   
                 CACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGT 
                   
               
               
                   
                 TCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCC 
                   
               
               
                   
                 GAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAA 
                   
               
               
                   
                 GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGC 
                   
               
               
                   
                 CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC 
                   
               
               
                   
                 GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC 
                   
               
               
                   
                 CAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG 
                   
               
               
                   
                 GGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAG 
                   
               
               
                   
                 CTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCC 
                   
               
               
                   
                 CAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT 
                   
               
               
                   
                 ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC 
                   
               
               
                   
                 AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC 
                   
               
               
                   
                 ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC 
                   
               
               
                   
                 TCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 9D11- 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 270 
               
               
                 Fchole 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                   
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                   
                 ATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                   
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAC 
                   
               
               
                   
                 TTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTC 
                   
               
               
                   
                 TGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGA 
                   
               
               
                   
                 GCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTC 
                   
               
               
                   
                 CCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGT 
                   
               
               
                   
                 GCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCA 
                   
               
               
                   
                 GCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGC 
                   
               
               
                   
                 AACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCC 
                   
               
               
                   
                 CAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAG 
                   
               
               
                   
                 CTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC 
                   
               
               
                   
                 CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG 
                   
               
               
                   
                 CCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGG 
                   
               
               
                   
                 TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC 
                   
               
               
                   
                 CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA 
                   
               
               
                   
                 GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGA 
                   
               
               
                   
                 AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACC 
                   
               
               
                   
                 CTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTG 
                   
               
               
                   
                 CGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA 
                   
               
               
                   
                 ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC 
                   
               
               
                   
                 GACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTG 
                   
               
               
                   
                 GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA 
                   
               
               
                   
                 ACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 
                   
               
               
                   
               
               
                 9D11_LC 
                 GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGA 
                 271 
               
               
                 [N95Q] 
                 ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACG 
                   
               
               
                   
                 GCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAG 
                   
               
               
                   
                 CTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTT 
                   
               
               
                   
                 CAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTG 
                   
               
               
                   
                 AAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGCAG 
                   
               
               
                   
                 CGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACGGTGGCTGC 
                   
               
               
                   
                 ACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA 
                   
               
               
                   
                 CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA 
                   
               
               
                   
                 GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAG 
                   
               
               
                   
                 TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCC 
                   
               
               
                   
                 TGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA 
                   
               
               
                   
                 GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG 
                   
               
               
                   
                 AGAGTGT 
                   
               
               
                   
               
               
                 CD3_VLCH1 
                 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCAC 
                 272 
               
               
                   
                 CGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACT 
                   
               
               
                   
                 ACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATC 
                   
               
               
                   
                 GGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATC 
                   
               
               
                   
                 TCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAG 
                   
               
               
                   
                 ATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTC 
                   
               
               
                   
                 GGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAAGGCCC 
                   
               
               
                   
                 TTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCG 
                   
               
               
                   
                 CTGCTCTCGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTG 
                   
               
               
                   
                 TCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGT 
                   
               
               
                   
                 GCTGCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCA 
                   
               
               
                   
                 GCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCC 
                   
               
               
                   
                 AGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCTTGT 
                   
               
               
                   
               
               
                 9D11 
                 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTC 
                 273 
               
               
                 [VHCH1]- 
                 CGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACA 
                   
               
               
                 CD3[VHCH1- 
                 TGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATC 
                   
               
               
                 S100aA]- 
                 ATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCG 
                   
               
               
                 Fcknob_PGLALA 
                 CGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGT 
                   
               
               
                   
                 CCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAC 
                   
               
               
                   
                 TTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTC 
                   
               
               
                   
                 TGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGA 
                   
               
               
                   
                 GCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTT 
                   
               
               
                   
                 CCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGT 
                   
               
               
                   
                 GCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCA 
                   
               
               
                   
                 GCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGC 
                   
               
               
                   
                 AACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCC 
                   
               
               
                   
                 CAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGC 
                   
               
               
                   
                 AGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGA 
                   
               
               
                   
                 CTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTG 
                   
               
               
                   
                 GGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAA 
                   
               
               
                   
                 GCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGG 
                   
               
               
                   
                 TTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAA 
                   
               
               
                   
                 CAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCA 
                   
               
               
                   
                 ACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACC 
                   
               
               
                   
                 CTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTT 
                   
               
               
                   
                 TCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTC 
                   
               
               
                   
                 TGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGAT 
                   
               
               
                   
                 AACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTC 
                   
               
               
                   
                 CAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTG 
                   
               
               
                   
                 ATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG 
                   
               
               
                   
                 AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCA 
                   
               
               
                   
                 CACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGT 
                   
               
               
                   
                 TCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCC 
                   
               
               
                   
                 GAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAA 
                   
               
               
                   
                 GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGC 
                   
               
               
                   
                 CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC 
                   
               
               
                   
                 GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC 
                   
               
               
                   
                 CAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG 
                   
               
               
                   
                 GGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAG 
                   
               
               
                   
                 CTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCC 
                   
               
               
                   
                 CAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT 
                   
               
               
                   
                 ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC 
                   
               
               
                   
                 AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC 
                   
               
               
                   
                 ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC 
                   
               
               
                   
                 TCTCCCTGTCTCCGGGTAAA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 9) Additonal Common Light Chain Protein and Nucleotide Sequences 
               
            
           
           
               
               
            
               
                 CH2527 (VL_7-46(13)/VH_3-23(12) 
                 SEQ ID NO. 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Heavy chain “CH2527 (VH_3-23(12))” 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGK GLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMN SLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 
                 123 
               
               
                   
               
               
                 Light chain “CH2527 (VL_7-46(13))” 
                 QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPG QAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEA EYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQ ANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 
                 124 
               
               
                   
               
               
                 VH “CH2527 (VH_3-23(12))” 
                 EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGK GLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMN SLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 
                 36 
               
               
                   
               
               
                 VH CDR H1 “CH2527 (VH_3-23(12))” 
                 TYAMN 
                 37 
               
               
                   
               
               
                 VH CDR H2 “CH2527 (VH_3-23(12))” 
                 RIRSKYNNYATYYADSVKG 
                 38 
               
               
                   
               
               
                 VH CDR H3 “CH2527 (VH_3-23(12))” 
                 HGNFGNSYVSWFAY 
                 39 
               
               
                   
               
               
                 VL “CH2527 (VL_7-46(13))” 
                 QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPG QAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEA EYYCALWYSNLWVFGGGTKLTVL 
                 125 
               
               
                   
               
               
                 VL CDR L1 “CH2527 (VL_7-46(13))” 
                 GSSTGAVTTSNYAN 
                 32 
               
               
                   
               
               
                 VL CDR L2 “CH2527 (VL_7-46(13))” 
                 GTNKRAP 
                 33 
               
               
                   
               
               
                 VL CDR L3 “CH2527 (VL_7-46(13))” 
                 ALWYSNLWV 
                 34 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 10) DNA sequences humanized CD3 CH2527  (CDR/VH/VL) 
               
            
           
           
               
               
            
               
                 CH2527 (VL_7-46(13)/VH_3-23(12) 
                 SEQ ID NO. 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Heavy chain “CH2527 (VH_3-23(12))” 
                 ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA GCTACCGGTGTGCATTCCGAGGTGCAGCTGCTGGAATCT GGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTG AGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCC ATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA TGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCC ACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATC AGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATG AACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGT GTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGG TTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCA TCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCA CCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTG GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC ACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGC AACACCAAGGTGGAGAAGAAAGTTGAGCCCAAATCTTGT GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC CTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTC AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAATGA 
                 126 
               
               
                   
               
               
                 Light chain “CH2527 (VL_7-46(13))” 
                 ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA GCTACCGGTGTGCATTCTCAGGCCGTCGTGACCCAGGAA CCCAGCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTG ACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAAC TACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTC AGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGC ACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAG GCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAG GCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGG GTGTTCGGCGGAGGCACCAAGCTGACAGTCCTAGGTCAA CCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGC AGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGC CTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCC TGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAG ACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCC GCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAG AGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGC AGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC TGA 
                 127 
               
               
                   
               
               
                 VH “CH2527 (VH_3-23(12))” 
                 ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA GCTACCGGTGTGCATTCCGAGGTGCAGCTGCTGGAATCT GGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTG AGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCC ATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA TGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCC ACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATC AGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATG AACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGT GTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGG TTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCA TCT 
                 128 
               
               
                   
               
               
                 VH CDR H1 “CH2527 (VH_3-23(12))” 
                 ACCTACGCCATGAAC 
                 129 
               
               
                   
               
               
                 VH CDR H2 “CH2527 (VH_3-23(12))” 
                 CGGATCAGAAGCAAGTACAACAACTACGCCACCTACTAC GCCGACAGCGTGAAGGGC 
                 130 
               
               
                   
               
               
                 VH CDR H3 “CH2527 (VH_3-23(12))” 
                 CACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCC TAC 
                 131 
               
               
                   
               
               
                 VL “CH2527 (VL_7-46(13))” 
                 ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA GCTACCGGTGTGCATTCTCAGGCCGTCGTGACCCAGGAA CCCAGCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTG ACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAAC TACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTC AGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGC ACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAG GCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAG GCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGG GTGTTCGGCGGAGGCACCAAGCTGACAGTCCTA 
                 132 
               
               
                   
               
               
                 VL CDR L1 “CH2527 (VL_7-46(13))” 
                 GGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCC AAC 
                 133 
               
               
                   
               
               
                 VL CDR L2 “CH2527 (VL_7-46(13))” 
                 GGCACCAACAAGAGAGCCCCT 
                 134 
               
               
                   
               
               
                 VL CDR L3 “CH2527 (VL_7-46(13))” 
                 GCCCTGTGGTACAGCAACCTGTGGGTG 
                 135 
               
               
                   
               
            
           
         
       
     
     Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.