# Patent

## Title
Therapeutic binding molecules

## Abstract

A molecule comprising at least one antigen binding site, comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence Asn-Tyr-Ile-Ile-His (NYIIH), said CDR2 having the amino acid sequence Tyr-Phe-Asn-Pro-Tyr-Asn-His-Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (YFNPYNHGTKYNEKFKG) and said CDR3 having the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (SGPYAWFDT); e.g. further comprising in sequence the hypervariable regions CDR1′, CDR2′ and CDR3′, CDR1′ having the amino acid sequence Arg-Ala-Ser-Gln-Asn-Ile-Gly-Thr-Ser-Ile-Gln (RASQNIGTSIQ), CDR2′ having the amino acid sequence Ser-Ser-Ser-Glu-Ser-Ile-Ser (SSSESIS) and CDR3′ having the amino acid sequence Gln-Gln-Ser-Asn-Thr-Trp-Pro-Phe-Thr (QQSNTWPFT), e.g. a chimeric or humanised antibody, useful as a pharmaceutical.

## Background
FIELD OF THE INVENTION
The present invention relates to organic compounds, such as to binding molecules against CD45 antigen isoforms, such as for example monoclonal antibodies (mAbs).
BACKGROUND OF THE INVENTION
One approach in the treatment of a variety of diseases is to achieve the elimination or the inactivation of pathogenic leukocytes and the potential for induction of tolerance to inactivate pathological immune responses.
Organ, cell and tissue transplant rejection and the various autoimmune diseases are thought to be primarily the result of T-cell mediated immune response triggered by helper T-cells which are capable of recognizing specific antigens which are captured, processed and presented to the helper T cells by antigen presenting cell (APC) such as macrophages and dendritic cells, in the form of an antigen-MHC complex, i.e. the helper T-cell when recognizing specific antigens is stimulated to produce cytokines such as IL-2 and to express or upregulate some cytokine receptors and other activation molecules and to proliferate. Some of these activated helper T-cells may act directly or indirectly, i.e. assisting effector cytotoxic T-cells or B cells, to destroy cells or tissues expressing the selected antigen. After the termination of the immune response some of the mature clonally selected cells remain as memory helper and memory cytotoxic T-cells, which circulate in the body and rapidly recognize the antigen if appearing again. If the antigen triggering this response is an innocuous environmental antigen the result is allergy, if the antigen is not a foreign antigen, but a self antigen, it can result is autoimmune disease; if the antigen is an antigen from a transplanted organ, the result can be graft rejection.
The immune system has developed to recognize self from non-self. This property enables an organism to survive in an environment exposed to the daily challenges of pathogens. This specificity for non-self and tolerance towards self arises during the development of the T cell repertoire in the thymus through processes of positive and negative selection, which also comprise the recognition and elimination of autoreactive T cells. This type of tolerance is referred to as central tolerance. However, some of these autoreactive cells escape this selective mechanism and pose a potential hazard for the development of autoimmune diseases. To control the autoreactive T cells that have escaped to the periphery, the immune system has peripheral regulatory mechanisms that provide protection against autoimmunity. These mechanisms are a basis for peripheral tolerance.
Cell surface antigens recognized by specific mAbs are generally designated by a CD (Cluster of Differentiation) number assigned by successive International Leukocyte Typing workshops and the term CD45 applied herein refers to the cell surface leukocyte common antigen CD45; and an mAb to that antigen is designated herein as “anti-CD45”.
Antibodies against the leukocyte common antigen (LCA) or CD45 are a major component of anti-lymphocyte globulin (ALG). CD45 belongs to the family of transmembrane tyrosine phosphatases and is both a positive and negative regulator of cell activation, depending upon receptor interaction. The phosphatase activity of CD45 appears to be required for activation of Src-family kinases associated with antigen receptor of B and T lymphocytes (Trowbridge I S et al, Annu Rev Immunol. 1994; 12:85-116). Thus, in T cell activation, CD45 is essential for signal 1 and CD45-deficient cells have profound defects in TCR-mediated activation events.
The CD45 antigen exists in different isoforms comprising a family of transmembrane glycoproteins. Distinct isoforms of CD45 differ in their extracellular domain structure which arise from alternative splicing of 3 variable exons coding for part of the CD45 extracellular region (Streuli M F. et al, J. Exp. Med. 1987; 166:1548-1566). The various isoforms of CD45 have different extra-cellular domains, but have the same transmembrane and cytoplasmic segments having two homologous, highly conserved phosphatase domains of approximately 300 residues. Different isoform combinations are differentially expressed on subpopulations of T and B lymphocytes (Thomas M L. et al, Immunol. Today 1988; 9:320-325). Some monoclonal antibodies recognize an epitope common to all the different isoforms, while other mAbs have a restricted (CD45R) specificity, dependent on which of the alternatively spliced exons (A, B or C) they recognize. For example, monoclonal antibodies recognizing the product of exon A are consequently designated CD45RA, those recognizing the various isoforms containing exon B have been designated CD45RB (Beverley PCL et al, Immunol. Supp. 1988; 1:3-5). Antibodies such as UCHL1 selectively bind to the 180 kDa isoform CD45RO (without any of the variable exons A, B or C) which appears to be restricted to a subset of activated T cells, memory cells and cortical thymocytes and is not detected on B cells (Terry L A et al, Immunol. 1988; 64:331-336).



DESCRIPTION OF THE FIGURES
FIG. 1 shows that the inhibition of primary MLR by the “candidate mAb” is dose-dependent in the range of 0.001 and 10 μg/ml. “Concentration” is concentration of the “candidate mAb”.
FIG. 2 shows the plasmid map of the expression vector HCMV-G1 HuAb-VHQ comprising the heavy chain having the nucleotide sequence SEQ ID NO:12 (3921-4274) in the complete expression vector nucleotide sequence SEQ ID NO:15.
FIG. 3 shows the plasmid map of the expression vector HCMV-G1 HuAb-VHE comprising the heavy chain having the nucleotide sequence SEQ ID NO:11 (3921-4274) in the complete expression vector nucleotide sequence SEQ ID NO:16.
FIG. 4 shows the plasmid map of the expression vector HCMV-K HuAb-humV1 comprising the light chain having the nucleotide sequence SEQ ID NO:14 (3964-4284) in the complete expression vector nucleotide sequence SEQ ID NO:17.
FIG. 5 shows the plasmid map of the expression vector HCMV-K HuAb-humV2 comprising the light chain having the nucleotide sequence SEQ ID NO:13 (3926-4246) in the complete expression vector nucleotide sequence SEQ ID NO:18.



DESCRIPTION OF THE INVENTION
We have now found a binding molecule which comprises a polypeptide sequence which binds to CD45RO and CD45RB, hereinafter also designated as a “CD45RO/RB binding molecule”. These binding molecule according to the invention may induce immunosuppression, inhibit primary T cell responses and induce T cell tolerance. Furthermore, the binding molecules of the invention inhibit primary mixed lymphocyte responses (MLR). Cells derived from cultures treated with CD45RO/RB binding molecules preferredly also have impaired proliferative responses in secondary MLR even in the absence of CD45RO/RB binding molecules in the secondary MLR. Such impaired proliferative responses in secondary MLR are an indication of the ability of binding molecules of the invention to induce tolerance.
Furthermore, it is found that in vivo administration of CD45RO/RB binding molecule to severe combined immunodeficiency (SCID) mice undergoing xeno-GVHD following injection with human PBMC may prolong mice survival, compared to control treated mice, even though circulating human T cells may still be detected in CD45RO/RB binding molecule treated mice. CD45RB/RO binding molecule may also suppress the inflammatory process that mediates human allograft skin rejection.
By “CD45RO/RB binding molecule” is meant any molecule capable of binding specifically to the CD45RB and CD45RO isoforms of the CD45 antigen, either alone or associated with other molecules. The binding reaction may be shown by standard methods (qualitative assay) including for example any kind of binding assay such as direct or indirect immunofluorescence together with fluorescence microscopy or cytofluorimetric (FACS) analysis, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay in which binding of the molecule to cells expressing a particular CD45 isoform can be visualized. In addition, the binding of this molecule may result in the alteration of the function of the cells expressing these isoforms. For example inhibition of primary or secondary mixed lymphocyte response (MLR) may be determined, such as an in vitro assay or a bioassay for determining the inhibition of primary or secondary MLR in the presence and in the absence of a CD46RO/RB binding molecule and determining the differences in primary MLR inhibition.
Alternatively, the in vitro functional modulatory effects can also be determined by measuring the PBMC or T cells or CD4+ T cells proliferation, production of cytokines, change in the expression of cell surface molecules e.g. following cell activation in MLR, or following stimulation with specific antigen such as tetanus toxoid or other antigens, or with polyclonal stimulators such as phytohemagglutinin (PHA) or anti-CD3 and anti-CD28 antibodies or phorbol esters and Ca2+ ionophores. The cultures are set up in a similar manner as described for MLR except that instead of allogeneic cells as stimulators soluble antigen or polyclonal stimulators such as those mentioned above are used. T cell proliferation is measured preferably as described above by 3H-thymidine incorporation.
Cytokine production is measured preferably by sandwich ELISA where a cytokine capture antibody is coated on the surface of a 96-well plate, the supernatants from the cultures are added and incubated for 1 hr at room temperature and a detecting antibody specific for the particular cytokine is then added, following a second-step antibody conjugated to an enzyme such as Horseradish peroxidase followed by the corresponding substrate and the absorbance is measured in a plate reader. The change in cell surface molecules may be preferably measured by direct or indirect immunofluorescence after staining the target cells with antibodies specific for a particular cell surface molecule. The antibody can be either directly labeled with flourochrome or a fluorescently labeled second step antibody specific for the first antibody can be used, and the cells are analysed with a cytofluorimeter.
The binding molecule of the invention has a binding specificity for both CD45RO and CD45RB (“CD45RB/RO binding molecule”).
Preferably the binding molecule binds to CD45RO isoforms with a dissociation constant (Kd)<20 nM, preferably with a Kd<15 nM or <10 nM, more preferably with a Kd<5 nM. Preferably the binding molecule binds to CD45RB isoforms with a Kd<50 nM, preferably with a Kd<15 nM or <10 nM, more preferably with a Kd<5 nM.
In a further preferred embodiment the binding molecule of the invention binds those CD45 isoforms which
1) include the A and B epitopes but not the C epitope of the CD45 molecule; and/or
2) include the B epitope but not the A and not the C epitope of the CD45 molecule; and/or
3) do not include any of the A, B or C epitopes of the CD45 molecule.
In yet a further preferred embodiment the binding molecule of the invention does not bind CD45 isoforms which include
1) all of the A, B and C epitopes of the CD45 molecule; and/or
2) both the B and C epitopes but not the A epitope of the CD45 molecule.
In further preferred embodiments the binding molecule of the invention further
1) recognises memory and in vivo alloactivated T cells; and/or
2) binds to its target on human T cells, such as for example PEER cells; wherein said binding preferably is with a Kd<15 nM, more preferably with a Kd<10 nM, most preferably with a Kd<5 nM; and/or
3) inhibits in vitro alloreactive T cell function, preferably with an IC50 of about less than 100 nM, preferably less than 50 nM or 30 nM, more preferably with an IC50 of about 10 or 5 nM, most preferably with an IC50 of about 0.5 nM or even 0.1 nM; and/or
4) induces cell death through apoptosis in human T lymphocytes; and/or
5) induces alloantigen-specific T cell tolerance in vitro; and/or
6) prevents lethal xenogeneic graft versus host disease (GvHD) induced in SCID mice by injection of human PBMC when administered in an effective amount; and/or
7) binds to T lymphocytes, monocytes, stem cells, natural killer cells and/or granulocytes, but not to platelets or B lymphocytes; and/or
8) supports the differentiation of T cells with a characteristic T regulatory cell (Treg) phenotype; and/or
9) induces T regulatory cells capable of suppressing naïve T cell activation; and/or
10) suppresses the inflammatory process that mediates human allograft skin rejection, in particular, suppresses the inflammatory process that mediates human allograft skin rejection in vivo in SCID mice transplanted with human skin and engrafted with mononuclear splenocytes.
In a further preferred embodiment the binding molecule of the invention binds to the same epitope as the monoclonal antibody “A6” as described by Aversa et al., Cellular Immunology 158, 314-328 (1994).
Due to the above-described binding properties and biological activities, such binding molecules of the invention are particularly useful in medicine for therapy and/or prophylaxis. Diseases in which binding molecules of the invention are particularly useful include autoimmune diseases, transplant rejection, psoriasis, inflammatory bowel disease and allergies, as will be further set out below.
We have found that a molecule comprising a polypeptide of SEQ ID NO: 1 and a polypeptide of SEQ ID NO: 2 is a CD45RO/RB binding molecule. We also have found the hypervariable regions CDR1′, CDR2′ and CDR3′ in a CD45RO/RB binding molecule of SEQ ID NO:1, CDR1′ having the amino acid sequence Arg-Ala-Ser-Gln-Asn-Ile-Gly-Thr-Ser-Ile-Gln (RASQNIGTSIQ), CDR2′ having the amino acid sequence Ser-Ser-Ser-Glu-Ser-Ile-Ser (SSSESIS) and CDR3′ having the amino acid sequence Gln-Gln-Ser-Asn-Thr-Trp-Pro-Phe-Thr (QQSNTWPFT).
We also have found the hypervariable regions CDR1, CDR2 and CDR3 in a CD45RO/RB binding molecule of SEQ ID NO:2, CDR1 having the amino acid sequence Asn-Tyr-Ile-Ile-His (NYIIH), CDR2 having the amino acid sequence Tyr-Phe-Asn-Pro-Tyr-Asn-His-Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (YFNPYNHGTKYNEKFKG) and CDR3 having the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (SGPYAWFDT).
CDRs are 3 specific complementary determining regions which are also called hypervariable regions which essentially determine the antigen binding characteristics. These CDRs are part of the variable region, e.g. of SEQ ID NO: 1 or SEQ ID NO: 2, respectively, wherein these CDRs alternate with framework regions (FR's) e.g. constant regions. A SEQ ID NO: 1 is part of a light chain, e.g. of SEQ ID NO: 3, and a SEQ ID NO:2 is part of a heavy chain, e.g. of SEQ ID NO: 4, in a chimeric antibody according to the present invention. The CDRs of a heavy chain together with the CDRs of an associated light chain essentially constitute the antigen binding site of a molecule of the present invention. It is known that the contribution made by a light chain variable region to the energetics of binding is small compared to that made by the associated heavy chain variable region and that isolated heavy chain variable regions have an antigen binding activity on their own. Such molecules are commonly referred to as single domain antibodies.
In one aspect the present invention provides a molecule comprising at least one antigen binding site, e.g. a CD45RO/RB binding molecule, comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence Asn-Tyr-Ile-Ile-His (NYIIH), said CDR2 having the amino acid sequence Tyr-Phe-Ash-Pro-Tyr-Asn-His-Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (YFNPYNHGTKYNEKFKG) and said CDR3 having the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (SGPYAWFDT); e.g. and direct equivalents thereof.
In another aspect the present invention provides a molecule comprising at least one antigen binding site, e.g. a CD45RO/RB binding molecule, comprising

    
    
        a) a first domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence Asn-Tyr-Ile-Ile-His (NYIIH), said CDR2 having the amino acid sequence Tyr-Phe-Asn-Pro-Tyr-Asn-His-Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (YFNPYNHGTKYNEKFKG) and said CDR3 having the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (SGPYAWFDT); and
        b) a second domain comprising in sequence the hypervariable regions CDR1′, CDR2′ and CDR3′, CDR1′ having the amino acid sequence Arg-Ala-Ser-Gln-Asn-Ile-Gly-Thr-Ser-Ile-Gln (RASQNIGTSIQ), CDR2′ having the amino acid sequence Ser-Ser-Ser-Glu-Ser-Ile-Ser (SSSESIS) and CDR3′ having the amino acid sequence Gln-Gln-Ser-Asn-Thr-Trp-Pro-Phe-Thr (QQSNTWPFT),

e.g. and direct equivalents thereof.

    
    


In a preferred embodiment the first domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3 is an immunoglobulin heavy chain, and the second domain comprising in sequence the hypervariable regions CDR1′, CDR2′ and CDR3′ is an immunoglobulin light chain.
In another aspect the present invention provides a molecule, e.g. a CD45RO/RB binding molecule, comprising a polypeptide of SEQ ID NO: 1 and/or a polypeptide of SEQ ID NO: 2, preferably comprising in one domain a polypeptide of SEQ ID NO: 1 and in another domain a polypeptide of SEQ ID NO: 2, e.g. a chimeric monoclonal antibody, and in another aspect A molecule, e.g. a CD45RO/RB binding molecule, comprising a polypeptide of SEQ ID NO: 3 and/or a polypeptide of SEQ ID NO: 4, preferably comprising in one domain a polypeptide of SEQ ID NO: 3 and in another domain a polypeptide of SEQ ID NO: 4, e.g. a chimeric monoclonal antibody.
When the antigen binding site comprises both the first and second domains or a polypeptide of SEQ ID NO: 1 or SEQ ID NO:3, respectively, and a polypeptide of SEQ ID NO: 2 or of SEQ ID NO:4, respectively, these may be located on the same polypeptide, or, preferably each domain may be on a different chain, e.g. the first domain being part of an heavy chain, e.g. immunoglobulin heavy chain, or fragment thereof and the second domain being part of a light chain, e.g. an immunoglobulin light chain or fragment thereof.
We have further found that a CD45RO/RB binding molecule according to the present invention is a CD45RO/RB binding molecule in mammalian, e.g. human, body environment. A CD45RO/RB binding molecule according to the present invention can thus be designated as a monoclonal antibody (mAb), wherein the binding activity is determined mainly by the CDR regions as described above, e.g. said CDR regions being associated with other molecules without binding specificity, such as framework, e.g. constant regions, which are substantially of human origin.
In another aspect the present invention provides a CD45RO/RB binding molecule which is not the monoclonal antibody “A6” as described by Aversa et al., Cellular Immunology 158, 314-328 (1994), which is incorporated by reference for the passages characterizing A6.
In another aspect the present invention provides a CD45RO/RB binding molecule according to the present invention which is a chimeric, a humanised or a fully human monoclonal antibody.
Examples of a CD45RO/RB binding molecules include chimeric or humanised antibodies e.g. derived from antibodies as produced by B-cells or hybridomas and or any fragment thereof, e.g. F(ab′)2 and Fab fragments, as well as single chain or single domain antibodies. A single chain antibody consists of the variable regions of antibody heavy and light chains covalently bound by a peptide linker, usually consisting of from 10 to 30 amino acids, preferably from 15 to 25 amino acids. Therefore, such a structure does not include the constant part of the heavy and light chains and it is believed that the small peptide spacer should be less antigenic than a whole constant part. By a chimeric antibody is meant an antibody in which the constant regions of heavy and light chains or both are of human origin while the variable domains of both heavy and light chains are of non-human (e.g. murine) origin. By a humanised antibody is meant an antibody in which the hypervariable regions (CDRs) are of non-human (e.g. murine) origin while all or substantially all the other part, e.g. the constant regions and the highly conserved parts of the variable regions are of human origins. A humanised antibody may however retain a few amino acids of the murine sequence in the parts of the variable regions adjacent to the hypervariable regions.
Hypervariable regions, i.e. CDR's according to the present invention may be associated with any kind of framework regions, e.g. constant parts of the light and heavy chains, of human origin. Suitable framework regions are e.g. described in “Sequences of proteins of immunological interest”, Kabat, E. A. et al, US department of health and human services, Public health service, National Institute of health. Preferably the constant part of a human heavy chain may be of the IgG1 type, including subtypes, preferably the constant part of a human light chain may be of the κ or λ type, more preferably of the κ type. A preferred constant part of a heavy chain is a polypeptide of SEQ ID NO: 4 (without the CDR1′, CDR2′ and CDR3′ sequence parts which are specified above) and a preferred constant part of a light chain is a polypeptide of SEQ ID NO: 3 (without the CDR1, CDR2 and CDR3 sequence parts which are specified above).
We also have found a humanised antibody comprising a light chain variable region of amino acid SEQ ID NO:7 or of amino acid SEQ ID NO:8, which comprises CDR1′, CDR2′ and CDR3′ according to the present invention and a heavy chain variable region of SEQ:ID NO:9 or of SEQ:ID NO:10, which comprises CDR1, CDR2 and CDR3 according to the present invention.
In another aspect the present invention provides a humanised antibody comprising a polypeptide of SEQ ID NO:9 or of SEQ ID NO:10 and a polypeptide of SEQ ID NO:7 or of SEQ ID NO:8.
In another aspect the present invention provides a humanised antibody comprising

    
    
        a polypeptide of SEQ ID NO:9 and a polypeptide of SEQ ID NO:7,
        a polypeptide of SEQ ID NO:9 and a polypeptide of SEQ ID NO:8,
        a polypeptide of SEQ ID NO:10 and a polypeptide of SEQ ID NO:7, or
        a polypeptide of SEQ ID NO:10 and a polypeptide of SEQ ID NO:8.
    
    


A polypeptide according to the present invention, e.g. of a herein specified sequence, e.g. of CDR1, CDR2, CDR3, CDR1′, CDR2′, CDR3′, or of a SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 includes direct equivalents of said (poly)peptide (sequence); e.g. including a functional derivative of said polypeptide. Said functional derivative may include covalent modifications of a specified sequence, and/or said functional derivative may include amino acid sequence variants of a specified sequence.
“Polypeptide”, if not otherwise specified herein, includes any peptide or protein comprising amino acids joined to each other by peptide bonds, having an amino acid sequence starting at the N-terminal extremity and ending at the C-terminal extremity. Preferably the polypeptide of the present invention is a monoclonal antibody, more preferred is a chimeric (V-grafted) or humanised (CDR-grafted) monoclonal antibody. The humanised (CDR-grafted) monoclonal antibody may or may not include further mutations introduced into the framework (FR) sequences of the acceptor antibody.
A functional derivative of a polypeptide as used herein includes a molecule having a qualitative biological activity in common with a polypeptide to the present invention, i.e. having the ability to bind to CD45RO and CD45RB. A functional derivative includes fragments and peptide analogs of a polypeptide according to the present invention. Fragments comprise regions within the sequence of a polypeptide according to the present invention, e.g. of a specified sequence. The term “derivative” is used to define amino acid sequence variants, and covalent modifications of a polypeptide according to the present invention. e.g. of a specified sequence. The functional derivatives of a polypeptide according to the present invention, e.g. of a specified sequence, preferably have at least about 65%, more preferably at least about 75%, even more preferably at least about 85%, most preferably at least about 95% overall sequence homology with the amino acid sequence of a polypeptide according to the present invention, e.g. of a specified sequence, and substantially retain the ability to bind to CD45RO and CD45RB.
The term “covalent modification” includes modifications of a polypeptide according to the present invention, e.g. of a specified sequence; or a fragment thereof with an organic proteinaceous or non-proteinaceous derivatizing agent, fusions to heterologous polypeptide sequences, and post-translational modifications. Covalent modified polypeptides, e.g. of a specified sequence, still have the ability bind to CD45RO and CD45RB by crosslinking. Covalent modifications are traditionally introduced by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected sides or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deaminated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deaminated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, tyrosine or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains, see e.g. T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983). Covalent modifications e.g. include fusion proteins comprising a polypeptide according to the present invention, e.g. of a specified sequence and their amino acid sequence variants, such as immunoadhesins, and N-terminal fusions to heterologous signal sequences.
“Homology” with respect to a native polypeptide and its functional derivative is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known.
“Amino acid(s)” refer to all naturally occurring L-α-amino acids, e.g. and including D-amino acids. The amino acids are identified by either the well known single-letter or three-letter designations.
The term “amino acid sequence variant” refers to molecules with some differences in their amino acid sequences as compared to a polypeptide according to the present invention, e.g. of a specified sequence. Amino acid sequence variants of a polypeptide according to the present invention, e.g. of a specified sequence, still have the ability to bind to CD45RO and CD45RB. Substitutional variants are those that have at least one amino acid residue removed and a different amino acid inserted in its place at the same position in a polypeptide according to the present invention, e.g. of a specified sequence. These substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. Insertional variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a polypeptide according to the present invention, e.g. of a specified sequence. Immediately adjacent to an amino acid means connected to either the α-carboxy or α-amino functional group of the amino acid. Deletional variants are those with one or more amino acids in a polypeptide according to the present invention, e.g. of a specified sequence, removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule.
We also have found the polynucleotide sequences of

    
    
        GGCCAGTCAGAACATTGGCACAAGCATACAGTG, encoding the amino acid sequence of CDR1,
        TTCTTCTGAGTCTATCTCTGG; encoding the amino acid sequence of CDR 2,
        ACAAAGTAATACCTGGCCATTCACGTT encoding the amino acid sequence of CDR 3,
        TTATATTATCCACTG, encoding the amino acid sequence of CDR1′,
        TTTTAATCCTTACAATCATGGTACTAAGTACAATGAGAAGTTCAAAGGCAG encoding the amino acid sequence of CDR2′, AGGACCCTATGCCTGGTTTGACACCTG encoding the amino acid sequence of CDR3′,
        SEQ ID NO:5 encoding a polypeptide of SEQ ID NO: 1, i.e. the variable region of a light chain of an mAb according to the present invention;
        SEQ ID NO:6 encoding a polypeptide of SEQ ID NO:2, i.e. the variable region of the heavy chain of an mAb according to the present invention;
        SEQ ID NO:11 encoding a polypeptide of SEQ ID NO:9. i.e. a heavy chain variable region including CDR1, CDR2 and CDR3 according to the present invention;
        SEQ ID NO:12 encoding a polypeptide of SEQ ID NO:10, i.e. a heavy chain variable region including CDR1, CDR2 and CDR3 according to the present invention;
        SEQ ID NO:13 encoding a polypeptide of SEQ ID NO:7, i.e. a light chain variable region including CDR1′, CDR2′ and CDR3′ according to the present invention; and
        SEQ ID NO:14 encoding a polypeptide of SEQ ID NO:8, i.e. a light chain variable region including CDR1′, CDR2′ and CDR3′ according to the present invention.
    
    


In another aspect the present invention provides isolated polynucleotides comprising polynucleotides encoding a CD45RO/RB-biding molecule, e.g. encoding the amino acid sequence of CDR1, CDR2 and CDR3 according to the present invention and/or, preferably and, polynucleotides encoding the amino acid sequence of CDR1′, CDR2′ and CDR3′ according to the present invention; and
Polynucleotides comprising a polynucleotide of SEQ ID NO: 5 and/or, preferably and, a polynucleotide of SEQ ID NO: 6; and
Polynucleotides comprising polynucleotides encoding a polypeptide of SEQ ID NO:7 or SEQ ID NO:8 and a polypeptide of SEQ ID NO:9 or SEQ ID NO:10; e.g. encoding

    
    
        a polypeptide of SEQ ID NO:7 and a polypeptide of SEQ ID NO:9,
        a polypeptide of SEQ ID NO:7 and a polypeptide of SEQ ID NO:10,
        a polypeptide of SEQ ID NO:8 and a polypeptide of SEQ ID NO:9, or
        a polypeptide of SEQ ID NO:8 and a polypeptide of SEQ ID NO:10; and

Polynucleotides comprising a polynucleotide of SEQ ID NO:11 or of SEQ ID NO:12 and a polynucleotide of SEQ ID NO:13 or a polynucleotide of SEQ ID NO:14, preferably comprising

        a polynucleotide of SEQ ID NO:11 and a polynucleotide of SEQ ID NO:13,
        a polynucleotide of SEQ ID NO:11 and a polynucleotide of SEQ ID NO:14,
        a polynucleotide of SEQ ID NO:12 and a polynucleotide of SEQ ID NO:13, or
        a polynucleotide of SEQ ID NO:12 and a polynucleotide of SEQ ID NO:14.
    
    


“Polynucleotide”, if not otherwise specified herein, includes any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA, including without limitation single and double stranded RNA, and RNA that is a mixture of single- and double-stranded regions.
A polynucleotide according to the present invention, e.g. a polynucleotide encoding the amino acid sequence CDR1, CDR2, CDR3, CDR1′, CDR2′, CDR3′, or of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, respectively, such as a polynucleotide of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, respectively, includes allelic variants thereof and/or their complements; e.g. including a polynucleotide that hybridizes to the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, respectively; e.g. encoding a polypeptide having at least 80% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, respectively, e.g. including a functional derivative of said polypeptide, e.g. said functional derivative having at least 65% homology with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, respectively, e.g. said functional derivative including covalent modifications of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, respectively, e.g. said functional derivative including amino acid sequence variants of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, respectively; e.g. a SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, respectively includes a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes a polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, respectively, or encodes a polypeptide with an amino acid sequence which has at least 80% identity with the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, respectively.
A CD45RO/RB binding molecule, e.g. which is a chimeric or humanised antibody, may be produced by recombinant DNA techniques. Thus, one or more DNA molecules encoding the CD45RO/RB may be constructed, placed under appropriate control sequences and transferred into a suitable host (organism) for expression by an appropriate vector.
In another aspect the present invention provides a polynucleotide which encodes a single, heavy and/or a light chain of a CD45RO/RB binding molecule according to the present invention; and the use of a polynucleotide according to the present invention for the production of a CD45RO/RB binding molecule according to the present invention by recombinant means.
A CD45RO/RB binding molecule may be obtained according, e.g. analogously, to a method as conventional together with the information provided herein, e.g. with the knowledge of the amino acid sequence of the hypervariable or variable regions and the polynucleotide sequences encoding these regions. A method for constructing a variable domain gene is e.g. described in EP 239 400 and may be briefly summarized as follows: A gene encoding a variable region of a mAb of whatever specificity may be cloned. The DNA segments encoding the frame work and hypervariable regions are determined and the DNA segments encoding the hypervariable regions are removed. Double stranded synthetic CDR cassettes are prepared by DNA synthesis according to the CDR and CDR′ sequences as specified herein. These cassettes are provided with sticky ends so that they can be ligated at junctions of a desired framework of human origin. Polynucleotides encoding single chain antibodies may also be prepared according to, e.g. analogously, to a method as conventional. A polynucleotide according to the present invention thus prepared may be conveniently transferred into an appropriate expression vector.
Appropriate cell lines may be found according, e.g. analogously, to a method as conventional. Expression vectors, e.g. comprising suitable promoter(s) and genes encoding heavy and light chain constant parts are known e.g. and are commercially available. Appropriate hosts are known or may be found according, e.g. analogously, to a method as conventional and include cell culture or transgenic animals.
In another aspect the present invention provides an expression vector comprising a polynucleotide encoding a CD45RO/RB binding molecule according to the present invention, e.g. of sequence SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18.
In another aspect the present invention provides

    
    
        An expression system comprising a polynucleotide according to the present invention wherein said expression system or part thereof is capable of producing a CD45RO/RB binding molecule according to the present invention, when said expression system or part thereof is present in a compatible host cell; and
        An isolated host cell which comprises an expression system as defined above.
    
    


We have further found that a CD45RO/RB binding molecule according to the present invention inhibit primary alloimmune responses in a dose-dependent fashion as determined by in vitro MLR. The results indicate that the cells which had been alloactivated in the presence of a CD45RO/RB binding molecule according to the present invention are impaired in their responses to alloantigen. This confirms the indication that a CD45RO/RB binding molecule according to the present invention can act directly on the effector alloreactive T cells and modulate their function. In addition, the functional properties of T cells derived from the primary MLR were further studied in restimulation experiments in secondary MLR, using specific stimulator cells or third-party stimulators to assess the specificity of the observed functional effects. We have found that the cells derived from primary MLRs in which a CD45RO/RB binding molecule according to the present invention is present, were impaired in their ability to respond to subsequent optimal stimulation with specific stimulator cells, although there was no antibody added to the secondary cultures. The specificity of the inhibition was demonstrated by the ability of cells treated with a CD45RO/RB binding molecule according to the present invention to respond normally to stimulator cells from unrelated third-party donors. Restimulation experiments using T cells derived from primary MLR cultures thus indicate that the cells which had been alloactivated a CD45RO/RB binding molecule according to the present invention are hyporesponsive, i.e. tolerant, to the original alloantigen. Further biological activities are described in examples 7, and 9 to 12.
Furthermore we have found that cell proliferation in cells pre-treated with a CD45RO/RB binding molecule according to the present invention could be rescued by exogenous IL-2. This indicates that treatment of alloreactive T cells with a CD45RO/RB binding molecule according to the present invention induces a state of tolerance. Indeed, the reduced proliferative responses observed in cells treated with a CD45RO/RB binding molecule according to the present invention, was due to impairment of T cell function, and these cells were able to respond to exogenous IL-2, indicating that these cells are in an anergic, true unresponsive state. The specificity of this response was shown by the ability of cells treated with a CD45RO/RB binding molecule according to the present invention to proliferate normally to unrelated donor cells to the level of the control treated cells.
In addition experiments indicate that the binding of a CD45RO/RB binding molecule according to the present invention to CD45RO and CD45RB may inhibit the memory responses of peripheral blood mononuclear cells (PBMC) from immunized donors to specific recall antigen. Binding of a CD45RO/RB binding molecule according to the present invention to CD45RO and CD45RB thus is also effective in inhibiting memory responses to soluble Ag. The ability of a CD45RO/RB binding molecule according to the present invention to inhibit recall responses to tetanus in PBMC from immunized donors indicate that a CD45RO/RB binding molecule according to the present invention is able to target and modulate the activation of memory T cells. E.g. these data indicate that a CD45RO/RB binding molecule according to the present invention in addition to recognizing alloreactive and activated T cells is able to modulate their function, resulting in induction of T cell anergy. This property may be important in treatment of ongoing immune responses to autoantigens and allergens and possibly to alloantigens as seen in autoimmune diseases, allergy and chronic rejection, and diseases, such as psoriasis, inflammatory bowel disease, where memory responses play a role in the maintenance of disease state. It is believed to be an important feature in a disease situation, such as in autoimmune diseases in which memory responses to autoantigens may play a major role for the disease maintenance.
We have also found that a CD45RO/RB binding molecule according to the present invention may modulate T cell proliferative responses in a mixed lymphocyte response (MLR) in vivo, i.e. a CD45RO/RB binding molecule according to the present invention was found to have corresponding inhibitory properties in vivo testing.
A CD45RO/RB binding molecule according to the present invention may thus have immunosuppressive and tolerogenic properties and may be useful for in vivo and ex-vivo tolerance induction to alloantigens, autoantigens, allergens and bacterial flora antigens, e.g. a CD45RO/RB binding molecule according to the present invention may be useful in the treatment and prophylaxis of diseases e.g. including autoimmune diseases, such as, but not limited to, rheumatoid arthritis, autoimmune thyroditis, Graves disease, type I and type II diabetes, multiple sclerosis, systemic lupus erythematosus, Sjögren syndrome, scleroderma, autoimmune gastritis, glomerulonephritis, transplant rejection, e.g. organ and tissue allograft and xenograft rejection, graft versus host disease (GVHD), and also psoriasis, inflammatory bowel disease and allergies.
In another aspect the present invention provides the use of a CD45RO/RB binding molecule according to the present invention as a pharmaceutical, e.g. in the treatment and prophylaxis of autoimmune diseases, transplant rejection, psoriasis, inflammatory bowel disease and allergies.
In another aspect the present invention provides a CD45RO/RB binding molecule according to the present invention for the production of a medicament in the treatment and prophylaxis of diseases associated with autoimmune diseases, transplant rejection, psoriasis, inflammatory bowel disease and allergies.
In another aspect the present invention provides a pharmaceutical composition comprising a CD45RO/RB binding molecule according to the present invention in association with at least one pharmaceutically acceptable carrier or diluent.
A pharmaceutical composition may comprise further, e.g. active, ingredients, e.g. other immunomodulatory antibodies such as, but not confined to anti-ICOS, anti-CD154, anti-CD134L or recombinant proteins such as, but not confined to rCTLA4 (CD152), rOX40 (CD134), or immunomodulatory compounds such as, but not confined to cyclosporin A, FTY720, RAD, rapamycin, FK506, 15-deoxyspergualin, steroids.
In another aspect the present invention provides a method of treatment and/or prophylaxis of diseases associated with autoimmune diseases, transplant rejection, psoriasis, inflammatory bowel disease and allergies comprising administering to a subject in need of such treatment and/or prophylaxis an effective amount of a CD45RO/RB binding molecule according to the present invention, e.g. in the form of a pharmaceutical composition according to the present invention.
Autoimmune diseases to be treated with binding molecule of the present invention further include, but are not limited to, rheumatoid arthritis, autoimmune thyroditis, Graves disease, type I and type II diabetes, multiple sclerosis, systemic lupus erythematosus, Sjögren syndrome, scleroderma, autoimmune gastritis, glomerulonephritis; transplant rejection, e.g. organ and tissue allograft and xenograft rejection and graft-versus-host disease (GVHD).
EXAMPLES
The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. In the following examples all temperatures are in degree Celsius.
The “candidate mAb” or “chimeric antibody” is a CD45RO/RB binding molecule according to the present invention comprising light chain of SEQ ID NO:3 and heavy chain of SEQ ID NO:4.
The “humanised antibody” is a CD45RO/RB binding molecule according to the present invention comprising a polypeptide of SEQ ID NO:8 and polypeptide of SEQ ID NO:9 or a polypeptide of SEQ ID NO:8 and a polypeptide of SEQ ID NO:10.
The following abbreviations are used:
















APC
antigen presenting cell



c.p.m.
counts per minute



ELISA
enzyme linked immuno-sorbant assay



FACS
fluorescence activated cell sorting



Fc
fragment crystallizable



F(ab′)2
fragment antigen-binding; bivalent



FITC
fluorescein isothiocyanate



FBS
foetal bovine serum



GVHD
graft-vs-host disease



HCMV
human cytomegalovirus promoter



IFN-γ
interferon gamma



IgE
immunoglobulin isotype E



lgG
immunoglobulin isotype G



IL-2
interleukin-2



IU
international units



MLR
mixed lymphocyte reaction



MLC
mixed lymphocyte culture



MP1
matrix protein 1 from hemophilus influenza



PBS
phosphate-buffered saline



PBL
peripheral blood leukocytes



PBMC
peripheral blood mononuclear cells



PCR
polymerase chain reaction



SCID
severe combined immunodeficiency



Treg
T regulatory cells



xGVHD
xeno-graft-vs-host disease










Example 1
Primary Mixed Lymphocyte Response (MLR)
Cells
Blood samples are obtained from healthy human donors. Peripheral blood mononuclear cells (PBMC) are isolated by centrifugation over Ficoll-Hypaque (Pharmacia LKB) from leukocytes from whole peripheral blood, leukopheresis or buffy coats with known blood type, but unknown HLA type. In some MLR experiments, PBMC are directly used as the stimulator cells after the irradiation at 40 Gy. In the other experiments, T cells were depleted from PBMC by using CD2 or CD3 Dynabeads (Dynal, Oslo, Norway). Beads and contaminating cells are removed by magnetic field. T cell-depleted PBMC are used as simulator cells after the irradiation.
PBMC, CD3+ T cells or CD4+ T cells are used as the responder cells in MLR. Cells are prepared from different donors to stimulator cells. CD3+ T cells are purified by negative selection using anti-CD16 mAb (Zymed, Calif.), goat anti-mouse IgG Dynabeads, anti-CD14 Dynabeads, CD19 Dynabeads. In addition anti-CD8 Dynabeads are used to purify CD4+ T cells. The cells obtained are analyzed by FACScan or FACSCalibur (Becton Dickinson & Co., Calif.) and the purity of the cells obtained was >75%. Cells are suspended in RPMI1640 medium, supplemented with 10% heat-inactivated FBS, penicillin, streptomycin and L-glutamine.
Reagents
The chimeric anti-CD45R0/RB mAb “candidate mAb” and an isotype matched control chimeric antibody is also generated. Mouse (Human) control IgG1 antibody specific for KLH (keyhole limpet hemocyanin) or recombinant human IL-10 is purchased from BD Pharmingen (San Diego, Calif.). Anti-human CD154 mAb 5c8 is according to Lederman et al 1992.
Primary Mixed Lymphocyte Response (MLR)
Aliquots of 1×105 PBMC or 5×104 of CD3+ or CD4+ cells are mixed with 1×105 irradiated PBMC or 5×104 T cells-depleted irradiated (50 Gy) PBMC in the each well of 96-well culture plates (Costar, Cambridge, Mass.) in the presence of the indicated mAb or absence of Ab. In some experiments, F(ab′)2 fragment of goat anti-mouse Ig or goat anti-human Ig specific for Fc portion (Jackson ImmunoResearch, West Grove, Pa.) is added at 10 μg/ml in addition to the candidate mAb To ensure optimal in vitro cross-linking of the target CD45 molecules. The mixed cells are cultured for 4 or 5 days at 37° C. in 5% CO2 and proliferation is determined by pulsing the cells with 3H-thymidine for the last 16-20 hours of culture. Other experiments are similar to those described above, but with the following exceptions: 1) Medium used is EX-VIVO (Bio-Whittaker) containing 10% FBS and 1% human plasma; 2) Anti-mouse total IgG (5 μg/ml) is used as secondary cross-linking step; 3) Irradiation of stimulator cells is 60 Gy.
Primary MLR is performed in the presence of the “candidate mAb” or control chimeric IgG1 (10 μg/ml) both with a second step reagent, F(ab′)2 fragment of goat anti-human Ig specific for Fc portion (10 μg/ml). Percentage inhibition by the “candidate mAb” is calculated in comparison with the cell proliferation in the presence of control IgG1. Results are shown in TABLE 1 below:







TABLE 1







Inhibition of primary MLR by 10 μg/ml of a candidate


mAb according to the present invention









Responder
Stimulator (Irr. PBMC)
% of Inhibition





#211 CD4
#219 CD3
63.51


#220 CD4
#219 CD3 depl.
63.07


#227 CD4
#220 CD3 depl.
65.96


#229 CD4
#219 CD3 depl.
50.76










Average ± SD
60.83 ± 6.83*







*Significantly different from control value (P < 0.001)






A candidate mAb according to the present invention inhibits primary MLR as can be seen from TABLE 1. The average inhibitory effect is 60.83±6.83% in four different donors-derived CD4+ T cells and statistically significant.
The inhibition of primary MLR by the “candidate mAb” is shown to be dose-dependent in the range of 0.001 and 10 μg/ml of the “candidate mAb” as shown in FIG. 1.
The IC50 for the inhibition of primary MLR by a “candidate mAb” is determined from the results of three separate MLR experiments using one donor PBMC as responder cells. Thus, responder CD4+ T cells from Donor #229 and #219 and irradiated PBMC depleted of T cells as stimulators are mixed in the presence of a “candidate mAb” or control chimeric Ab with 10 μg/ml of F(ab′)2 fragment of goat anti-human Ig. Experiments are repeated 3 times and percentage of proliferation in the presence of a “candidate mAb” is calculated in comparison with the T cell proliferation in the presence of control Ab. IC50 value is determined using Origin (V. 6.0®). The cellular activity IC50 value is calculated to be 0.87±0.35 nM (0.13±0.052 μg/ml).
Example 2
Secondary MLR
In order to assess whether a “candidate mAb” induces unresponsiveness of CD4+ T cells to specific alloantigens, secondary MLR is performed in the absence of any antibodies after the primary MLC. CD4+ T cells are cultured with irradiated allogeneic stimulator cells (T cells-depleted PBMC) in the presence of the indicated antibody in 96-well culture plates for 10 days (primary MLC). Then, cells are collected, layered on a Ficoll-Hypaque gradient to remove dead cells, washed twice with RPMI, and restimulated with the same stimulator, 3rd party stimulator cells or IL-2 (50 U/ml). The cells are cultured for 3 days and the proliferative response is determined by pulsing the cells with 3H-thymidine for the last 16-20 hours of culture.
Specifically, CD4+ T cells are cultured with irradiated allogeneic stimulator cells (T cells-depleted PBMC taken from other donors) in the presence of 10 μg/ml of the “candidate mAb”, control IgG1 chimeric Ab and F(ab′)2 fragment of goat anti-human Ig. Primary MLR proliferation is determined on day 5. For secondary MLR, the responder and stimulator cells are cultured for 10 days in the presence of the “candidate mAb”, then the cells are harvested, washed twice in RPMI1640 and restimulated with specific stimulator, third-party stimulators or IL-2 (50 U/ml) in the absence of any Ab. Cell proliferation is determined on day
3. Results set out in TABLE 2:










TABLE 2







Responder CD4+ T cells Donor #
% Inhibition of 2ry MLR









#211
49.90*



#220
59.33*



#227
58.68*







*Significantly different from control value (p =< 0.001 determined by t-test, SigmaStat V.2.03).



#p =< 0.046






In order to test whether the impaired proliferation is due to unresponsiveness as a consequence of the treatment with a “candidate mAb”, the cells derived from primary MLR are cultured in the presence of IL-2 (50 U/ml). Addition of IL-2 results in the rescue of proliferative responses of the T cells which had been treated with a “candidate mAb” in primary MLR, to levels similar to those observed in the presence of IgG1 control Ab. These data indicate that the impaired secondary response in T cells treated with a “candidate mAb” is due to functional alteration of the responder T cells which become unresponsive to the specific stimulator cells.
Percentage inhibition is calculated according to the following formula:




  
    
      
        
          c
          .
          p
          .
          m
          .
          
            
          
          ⁢
          with
        
        ⁢
        
          
        
        ⁢
        control
        ⁢
        
          
        
        ⁢
        Ab
      
      -
      
        
          c
          .
          p
          .
          m
          .
          
            
          
          ⁢
          with
        
        ⁢
        
          
        
        ⁢
        
           
          
            “
            
              candidate
              ⁢
              
                
              
              ⁢
              mAb
            
          
        
      
    
    
      
        c
        .
        p
        .
        m
        .
        
          
        
        ⁢
        with
      
      ⁢
      
        
      
      ⁢
      control
      ⁢
      
        
      
      ⁢
      Ab
    
  
  ×
  100




Statistical analysis is performed using SigmaStat (Vers. 2.03).
The data is analyzed by two-way ANOVA followed by Dunnett method. In all test procedures probabilities<0.05 are considered as significant. In some experiments t-test is used (SigmaStat V.2.03).
Example 3
In Vivo Survival Studies in SCID-Mice
Engraftment of hu-PBL in SCID Mice
Human peripheral blood mononuclear cells (PBMC) are injected intraperitoneally into SCID mice C.B 17/GbmsTac-Prkdcscid Lysebg mice (Taconic, Germantown, N.Y.) in an amount sufficient to induce a lethal xenogeneic graft-versus-host disease (xGvHD) in >90% of the mice within 4 weeks after cell transfer. Such treated SCID mice are hereinafter designated as hu-PBL-SCID mice
Mab-Treatment of hu-PBL-SCID Mice
Hu-PBL-SCID mice are treated with a “candidate mAb” or mouse or chimeric isotype matched mAb controls at day 0, immediately after PBMC injection, at day 3, day 7 and at weekly intervals thereafter. Mabs are delivered subcutaneously in 100 μl PBS at a final concentration of 5 mg/kg body weight. The treatment was stopped when all control mice were dead.
Evaluation of Treatment Results
The main criterion to assess the efficacy of a “candidate mAb” in this study was the survival of the hu-PBL-SCID mice. The significance of the results is evaluated by the statistical method of survival analysis using the Log-rank test (Mantel method) with the help of the Systat v9.01 software. The method of survival analysis is a non-parametric test, which not only consider whether a particular mouse is still alive but also whether if it was sacrificed for reasons irrelevant to the treatment/disease such as the requirement of perform in vitro analysis with its organs/cells. Biopsies of liver, lung, kidney and spleen are obtained from dead mice for further evaluation. In addition, hu-PBL-SCID mice are weighed at the beginning (before cell transfer) and throughout (every two days) the experiment as an indirect estimation of their health status. Linear regression lines were generated using the body weight versus days post-PBMC transfer values obtained from each mouse and subsequently, their slopes (control versus anti-CD45 treated mice) were compared using the non-parametric Mann-Whitney test.
Results
All hu-PBL-SCID mice treated with mouse mAb controls had infiltrated human leukocytes in the lung, liver and spleen and died (4/4) within ca. 2 to 3 weeks after cell transfer. Death is a likely consequence of xGvHD. Control mAb-treated mice furthermore lost weight in a linear manner, ca. 10% and more within 3 weeks.
All hu-PBL-SCID mice treated with a “candidate mAb” survived (4/4) without any apparent sign of disease more than 4 weeks, even although “candidate mAb”-treatment was stopped after 3 weeks. “Candidate mAb”-treated mice increased weight in a linear manner, up to ca. 5% within 4 weeks.
Example 4
Expression of Antibodies of the Invention
Expression of Humanised Antibody Comprising a SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10
Expression vectors according to the plasmid map shown in FIGS. 2 to 5 are constructed, comprising the corresponding nucleotides encoding the amino acid sequence of humanised light chain variable region humV1 (SEQ ID NO:7), humanised light chain variable region humV2 (SEQ ID NO:8), humanised heavy chain variable region VHE (SEQ ID NO:9), or humanised heavy chain variable region VHQ (SEQ ID NO:10), respectively. These expression vectors have the DNA (nucleotide) sequences SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, or SEQ ID NO 18, respectively.
Construction of Humanised Antibody Heavy and Light Chain Expression Vectors
Human kappa light chain expression vectors for versions VLh and VLm
In order to construct the final expression vector encoding for the complete humanised light chain of human kappa isotype, DNA fragments encoding the complete light chain variable regions (VLh and VLm) were excised from the VLh and VLm containing PCR-Script cloning vectors (Stratagene) (VLm region) using HindIII and BglII. The gel-purified fragments were then subcloned into the HindIII and BamHI sites of C21-HCMV Kappa expression vector which was created during construction of the humanised anti-IgE antibody TESC-21 (Kolbinger et al 1993) and which originally received from M. Bendig (MRC Collaborative Centre, London, UK) (Maeda et al. 1991). The ligation products were purified by phenol/chloroform extraction, and electroporated into electrocoporation-competent Epicurian Coli® XL1-Blue strain (Cat. No #200228, Stratagene). After plating on LB/amp agar plates overnight at 37° C., each 12 colonies were picked to prepare plasmid DNA from a 3 ml culture using the BioRobot 9600 (Qiagen). This yielded the light chain expression vectors for the humanised antibody versions VLh and VLm, respectively, as further described in the Figures.
Human Gamma-1 Heavy Chain Expression Vectors for VHQ
For the construction of the VHQ expression vector, a step-wise approach was taken. First, the complete variable region of VHQ was assembled by PCR according to the methology as described in Kolbinger et al 1993 (Protein Eng. November 1993; 6(8):971-80) and subcloned into the C21-HCMV-gamma-1 expression from which the C21 insert had been removed using the same enzymes. A HindIII/BamHI fragment of PCRScript clone VHQ containing the complete variable region was then subcloned into expression vector C21-HCMV-gamma-1 cleaved with the same enzymes. This yielded the final expression vector for the humanised antibody version VHQ.
Human Gamma-1 Heavy Chain Expression Vectors for VHE
The construction of the final VHE expression vector encoding for the complete humanised heavy chain of human gamma-1 isotype was achieved by directly ligating a HindIII and BamHI restricted PCR fragment encoding the variable region into the HindIII and BamHI sites of C21-HCMV gamma-1 expression vector which was created during construction of the humanised anti-IgE antibody TESC-21 (Kolbinger et al 1993) and which was also originally received from M. Bendig (MRC Collaborative Centre, London, UK) (Maeda et al. 1991).
Transient Expression in COS Cells
The following transfection protocol is adapted for adherent COS cells in 150 mm cell culture dishes, using SuperFectTM Transfection Reagent (Cat. No 301305, Qiagen). The four different expression vectors described above are used for transient transfection of cells. For expression of humanised antibody, each of two clones containing heavy chain inserts (VHE or VHQ, respectively) are co-transfected into cells with each of the two clones encoding for the light chains (humV1 or humV2, respectively), in total 4 different combinations of heavy and light chain expression vectors (VHE/humV1, VHE/humV2, VHQ/humV1 and VHQ/humV2). Before transfection, the plasmids are linearized with the restriction endonuclease PvuI which cleaves in the region encoding the resistance gene for ampicillin. The day before transfection, 4×106 COS cells in 30 ml of fresh culture medium are seeded in 150 mm cell culture dishes. Seeding at this cell density generally yielded 80% confluency after 24 hours. On the day of transfection, four different combinations of linearized heavy- and light-chain DNA expression vectors (15 μg each) are diluted in a total volume of 900 μl of fresh medium without serum and antibiotics. 180 μl of SuperFect Transfection Reagent is then mixed thoroughly with the DNA solution. The DNA mixture is incubated for 10 min at room temperature to allow complex formation. While complex formation takes place, the growth medium is removed from COS cell cultures, and cells are washed once with PBS. 9 ml of fresh culture medium (containing 10% FBS and antibiotics) are then added to each reaction tube containing the transfection complexes and well mixed. The final preparation is immediately transferred to each of 4 cultures to be transfected and gently mixed. Cell cultures are then incubated with the DNA complexes for 3 hours at 37° C. and 5% CO2. After incubation, the medium containing transfection complexes is removed and replaced with 30 ml of fresh culture medium. At 48 hr post transfection, the culture supernatants are harvested.
Concentration of Culture Supernatants
For ELISA and FACS analysis, the culture supernatants collected from COS cells transfected with heavy- and light-chain plasmids are concentrated as follows. 10 ml of each supernatant are added to Centriprep YM-50 Centrifugal Filter Devices (Cat. No 4310, Millipore) as described by the manufacturer. The Centriprep filters are centrifuged for 10 min at 3000 rpm at room temperature. The centrifugation step is then repeated again with the remaining 20 ml of supernatant using only 5 min of centrifugation and supervising the concentration evolution. The intermediate 500 μl of concentrated supernatant is recovered, transferred to new Microcon Centrifugal Filter Devices (Cat. No 42412, Microcon) and further concentrated following the manufacturer's protocol. The concentrated supernatants are centrifuged four times for 24 min at 3000 rpm at room temperature, one time for 10 min at 6000 rpm and then, three times for 5 min, always supervising the concentration evolution. The final volume of concentrated conditioned medium achieved is 100-120 μl corresponding to a 250 to 300-fold concentration of original culture medium and is stored at 4° C. until use. For comparison and control, culture medium from untransfected cells is similarly concentrated, using the same centrifugation protocol described above.
Generation of Stable Sp2/0 Myeloma Transfectants Secreting Humanised Anti-CD45RO/RB Antibodies
The mouse myeloma cell line Sp2/0 (ATCC, CRL-1581) is electroporated with vectors encoding heavy (VHE or VHQ) and light (humV1 or humV2) chain of the CD45RO/RB binding humanised antibodies. Four different combinations of heavy and light chain expression vectors (VHE/humV1, VHE/humV2, VHQ/humV1 and VHQ/humV2) are transfected according to the following protocol: 20 μg supercoiled DNA of each plasmid is mixed in an electroporation cuvette (0.4 cm gap) with 8×106 live Sp2/0 cells suspended in DMEM/10% FCS culture medium. Electroporation settings are 1500 V, 25 μF using a BioRad GenePulser instrument. After electroporation, cells are cultured for 20 h in culture medium (DMEM supplemented with 10% FCS penicillin, streptomycin and L-glutamine). On day two the selection drug G418 (Cat. No 10131-019, Gibco) is added to a final concentration of 1 mg active drug/ml and the cells are distributed into one 96-well plate, 200 μl each well with approx. 105 cells per well. Ten to 15 days later, G418-surviving clones are expanded in G418-containing medium. Secretion of humanised mAbs from these transfectants is assessed by ELISA, using a coating antibody goat anti-human IgG/Fcγ (Cat. No 109-005-098, Jackson Labs) and a peroxidase-coupled antibody against human kappa light chain (Cat. No A-7164, Sigma). Transfectants, which score positive in this assay are selected for a comparison of productivity on a per cell per day basis, again using ELISA (see below). The best clone of each transfectant is selected for immediate subcloning by limiting dilution, using a seeding density of 1 cell per well. Productivity of G418-surviving subclones is again determined as described above. Subclones are expanded in G418-containing selection medium, until the culture volume reaches 150 ml, at which stage the culture is continued without G418 in flasks destined to feed roller bottles.
After the first transfection and selection, stable transfectants grow out of the 96-well plates at a frequency of 20.8% for VHE/humV1, 11.5% for VHQ/humV1, 18.8% for VHE/humV2 and 7.3% for VHQ/humV2. After two rounds of subcloning the best two producers are clone 1.33.25 (3.87 pg/cell/day) and clone 1.33.26 (3.43 pg/cell/day) for VHE/humV1 and clone 12.1.4 (1.19 pg/cell/day) and clone 12.1.20 (1.05 pg/cell/day) for VHQ/humV1. The stable Sp2/0 transfectants for VHE/humV1 and VHQ/humV1 are subsequently expanded for antibody production and purification.
The antibodies are purified from supernatants of stably transfected SP2/0 myeloma cell lines containing 10% FCS by a combination of affinity chromatography using an immobilized anti-human IgGFc matrix and size-exclusion chromatography. If required, endotoxin is removed using an Acticlean Etox column (Sterogene Bioseparations).
Example 5
Determination of Recombinant Human IgG Expression by ELISA
To determine IgG concentrations of recombinant human antibody expressed in the culture supernatants, a sandwich ELISA protocol has been developed and optimized using human IgG as standard. Flat bottom 96-well microtiter plates (Cat. No 4-39454, Nunc Immunoplate Maxisorp) are coated overnight at 4° C. with 100 μl of goat anti-human IgG (whole molecule, Cat. No I1011, SIGMA) at the final concentration of 0.5 pg/ml in PBS. Wells are then washed 3 times with washing buffer (PBS containing 0.05% Tween 20) and blocked for 1.5 hours at 37° C. with blocking buffer (0.5% BSA in PBS). After 3 washing cycles, the antibody samples and the standard human IgG (Cat. No. 14506, SIGMA) are prepared by serial 1.5-fold dilution in blocking buffer. 100 ∥l of diluted samples or standard are transferred in duplicate to the coated plate and incubated for 1 hour at room temperature. After incubation, the plates are washed 3 times with washing buffer and subsequently incubated for 1 hour with 100 μl of horseradish peroxidase-conjugated goat anti-human IgG kappa-light chain (Cat. No A-7164, SIGMA) diluted at 1/4000 in blocking buffer. Control wells received 100 μl of blocking buffer or concentrated normal culture medium. After washing, the colorimetric quantification of bound peroxidase in the sample and standard wells is performed, using a TMB Peroxidase EIA Substrate Kit (Cat. No 172-1067, Bio-Rad) according to the manufacturer's instructions. The peroxidase mixture is added at 100 μl per well and incubated for 30 min at room temperature in the dark. The colorimetric reaction is stopped by addition of 100 μl of 1 M sulfuric acid and the absorbance in each well is read at 450 nm, using an ELISA plate reader (Model 3350-UV, BioRad).
With a correlation coefficient of 0.998 for the IgG standard curve, the following concentrations are determined for the four different culture concentrates (ca. 250-300 fold concentrated) obtained from transfected COS cells:

    VHE/humV1 supernatant=8.26 μg/ml
    VHE/humV2 supernatant=6.27 μg/ml
    VHQ/humV1 supernatant=5.3 μg/ml
    VHQ/humV2 supernatant=5.56 μg/ml


Example 6
FACS Competition Analysis
Binding Affinity
The human T-cell line PEER is chosen as the target cell for FACS analysis because it expressed the CD45 antigen on its cell surface. To analyze the binding affinity of humanised antibody supernatants, competition experiments using FITC-labeled chimeric antibody as a reference are performed and compared with the inhibition of purified mouse antibody and of chimeric antibody. PEER cell cultures are centrifuged for 10 seconds at 3000 rpm and the medium is removed. Cells are resuspended in FACS buffer (PBS containing 1% FBS and 0.1% sodium azide) and seeded into 96-well round-bottom microtiter plate at a cell density of 1×105 cells per well. The plate is centrifuged and the supernatant is discarded. For blocking studies, 25 μl of concentrated untransfected medium or isotype matched control antibody (negative controls), unlabeled mouse antibody or chimeric antibody (positive controls) as well as concentrated supernatant containing the various combinations of humanised antibody (samples), is first added in each well at the indicated concentrations in the text. After 1 hour of incubation at 4° C., PEER cells are washed with 200 μl of FACS buffer by centrifugation. Cells are subsequently incubated for 1 hour at 4° C. with chimeric antibody conjugated with FITC in 25 μl of FACS buffer at the final concentration of 20 μg/ml. Cells are washed and resuspended in 300 μl of FACS buffer containing 2 μg/ml propidium iodide which allows gating of viable cells. The cell preparations are analyzed on a flow cytometer (FACSCalibur, Becton Dickinson).
FACS analyses indicate a dose-dependent blockade of fluorochrome-labeled chimeric antibody by the concentrated humanised antibody culture supernatants. No dose-dependent blockade of chimeric antibody binding is seen with the isotype matched control antibody, indicating that the blocking effect by the different humanised antibody combinations is epitope specific and that epitope specificity appears to be retained after the humanisation process.
Undiluted supernatant from the above mentioned SP2/0 transfectants or chimeric antibody (positive controls) or isotype matched control antibody (negative controls) at 2 μg/ml in culture medium are incubated with 1.5×105 PEER cells in 100 μl for 30 min at 4° C. Then, 100 μl PBS containing FITC-labeled chimeric antibody is added to each sample and incubation at 4° C. continues for another 30 minutes. After washing, cells are resuspended in FACS-PBS containing 1 μg/ml 7-Amino-Actinomycin D and analyzed by flow cytometry using a Becton Dickinson FACSCalibur instrument and the CellQuest Pro Software. Gating was on live cells, i.e. 7-Amino-Actinomycin D—negative events.
FACS analyses show that unlabeled humanised CD45RB/RO binding molecules, e.g. VHE/humV1 and VHQ/humV1 but not the isotype matched control antibody compete with FITC-labeled chimeric antibody for binding to the human CD45-positive T cell line PEER.
Example 7
Biological Activities of CD45RB/RO Binding Molecules
In this study, we have addressed whether CD45RB/RO binding chimeric antibody, when present in cultures of polyclonally activated primary human T cells (i) supports the differentiation of T cells with a characteristic Treg phenotype, (ii) prevents or enhances apoptosis following T cell activation, and (iii) affects expression of subset-specific antigens and receptors after restimulation.
CD45RB/RO Binding Chimeric Antibody Enhances Cell Death in Polyclonally Activated T Cells
Primary T cells (mixture of CD4+ and CD8+ T subsets) were subjected to activation by anti-CD3 plus anti-CD28 mAb (200 ng/ml each) in the presence or absence (=control) of CD45RB/RO binding chimeric antibody. Excess antibodies were removed by washing on day 2. 7-amino-actinomycin D (7-MD) as a DNA-staining dye taken up by apoptotic and necrotic cells was used to measure cell death following activation. The results show that activation of T cells in the presence of CD45RB/RO binding chimeric antibody increased the fraction of 7-MD positive cells than two-fold on day 2 after activation. On day 7, the portion of 7-MD positive cells was again similar in CD45RB/RO binding chimeric antibody-treated and control cultures.
CD45RB/RO Binding Chimeric Antibody but not Control mAb Treated T Cells Display a T Regulatory Cell (Treg) Phenotype
Increased expression of CD25 and the negative regulatory protein CTLA-4 (CD152) is a marker of Treg cells. Functional suppression of primary and secondary T cell responses by CD45RB/RO binding chimeric antibody may be due to the induction of Treg cells. To address this issue, T cells were activated by anti-CD3+CD28 mAbs and cultured in the presence of CD45RB/RO binding chimeric antibody or anti-LPS control mAb. The time course of CTLA-4 and CD25 expression reveals marked differences between controls and CD45RB/RO binding chimeric antibody-treated T cells on days 1 and 3 after secondary stimulation, indicating a Treg phenotype.
Intracellular CTLA-4 Expression is Sustained in the Presence of CD45RB/RO Binding Chimeric Antibody
It has been reported that substantial amounts of CTLA-4 can also be found intracellularly. Therefore, in parallel to surface CTLA-4 staining, intracellular CTLA-4 expression was analyzed. Moderate differences between T cell cultures were seen on day 4 after stimulation. After prolonged culture, however, high levels of intracellular CTLA-4 were sustained only in CD45RB/RO binding chimeric antibody-treated but not in control T cells.
CD45RB/RO Binding Chimeric Antibody-Treated T Cells Become Double Positive for CD4 and CD8
Following stimulation, T cells induce and upregulate the expression of several surface receptors, such as CD25, CD152 (CTLA-4), CD154 (CD40-Ligand) and others. In contrast, the level of expression of CD4 or CD8 is thought to stay relatively constant. We reproducibly observed a strong increase of both CD4 and CD8 antigens on CD45RB/RO binding chimeric antibody-treated but not on control Ab-treated T cells after activation. The emergence of a CD4/CD8 double-positive T cell population seems to be due to the upregulation of CD4 on the CD8+ subset and conversely, CD8 on the CD4+ subset. This contrasts with a moderately low percentage of double positive T cells in control cultures.
High IL-2 Receptor Alpha-Chain, but Very Low Beta-Chain Expression by CD45RB/RO Binding Chimeric Antibody-Treated T Cells
Treg cells are known to be constitutively positive for CD25, the IL-2 receptor alpha-chain. The regulation of other subunits of the trimeric IL-2 receptor on Treg cells is not known. Recently we have compared the expression of the beta-chain of IL-2 receptor, e.g. CD122, on T cells activated and propagated in the presence or absence of CD45RB/RO binding chimeric antibody. The results show that CD45RB/RO binding chimeric antibody-treated T cells have about ten-fold lower CD122 expression as compared to T cells in control cultures. This difference may indicate that Treg cells require factors other than IL-2 to proliferate.
Example 8
Sequences of the Invention
CDR Sequences of the Invention are Underlined
SEQ ID NO:1
Part of the Amino Acid Sequence of Chimeric Light Chain








DILLTQSPAILSVSPGERVSFSCRASQNIGTSIQWYQQRTNGSPRLLIR



 


SSSESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNTWPFTFG


 


SGTKLEIK






SEQ ID NO:2

Part of the Amino Acid Sequence of Chimeric Heavy Chain









EVQLQQSGPELVKPGASVKMSCKASGYTFTNYIIHWVKQEPGQGLEWIG



 


YFNPYNHGTKYNEKFKGRATLTADKSSNTAYMDLSSLTSEDSAIYYCAR


 


SGPYAWFDTWGQGTTVTVSS






SEQ ID NO:3

Amino Acid Sequence of Chimeric Light Chain









DILLTQSPAILSVSPGERVSFSCRASQNIGTSIQWYQQRTNGSPRLLIR



 


SSSESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNTWPFT


 


FGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ


 


WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT


 


HQGLSSPVTKSFNRGEC






SEQ ID NO:4

Amino Acid Sequence of Chimeric Heavy Chain









EVQLQQSGPELVKPGASVKMSCKASGYTFTNYIIHWVKQEPGQGLEWIG



 


YFNPYNHGTKYNEKFKGRATLTADKSSNTAYMDLSSLTSEDSAIYYCAR


 


SGPYAWFDTWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV


 


KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ


 


TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK


 


PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY


 


NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP


 


QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP


 


VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS


 


PGK






SEQ ID NO:5

Nucleotide Sequence Encoding a Polypeptide of SEQ ID NO:1









GACATTCTGCTGACCCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGA



 


AAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATAC


 


AGTGGTATCAACAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAGGTCT


 


TCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATC


 


AGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTG


 


CAGATTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTCGGCTCG


 


GGGACCAAGCTTGAAATCAAA






SEQ ID NO:6

Nucleotide Sequence Encoding a Polypeptide of SEQ ID NO:2









GAGGTGCAGCTGCAGCAGTCAGGACCTGAACTGGTAAAGCCTGGGGCTTC



 


AGTGAAGATGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTA


 


TCCACTGGGTGAAGCAGGAGCCTGGTCAGGGCCTTGAATGGATTGGATAT


 


TTTAATCCTTACAATCATGGTACTAAGTACAATGAGAAGTTCAAAGGCAG


 


GGCCACACTAACTGCAGACAAATCCTCCAACACAGCCTACATGGACCTCA


 


GCAGCCTGACCTCTGAGGACTCTGCGATCTACTACTGTGCAAGATCAGGA


 


CCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTC


 


CTCA






SEQ ID NO:7

Part of Amino Acid Sequence of Humanised Light Chain Designated humV2 (humV2=VLm)











DILLTQSPAT LSLSPGERAT FSCRASQNIG TSIQWYQQKT




 



NGAPRLLIRS SSESISGIPS RFSGSGSGTD FTLTISSLEP



 



EDFAVYYCQQ SNTWPFTFGQ GTKLEIK






SEQ ID NO:8

Part of Amino Acid Sequence of Humanised Light Chain Designated humV1 (humV1=VLh)











DILLTQSPAT LSLSPGERAT LSCRASQNIG TSIQWYQQKP




 



GQAPRLLIRS SSESISGIPS RFSGSGSGTD FTLTISSLEP



 



EDFAVYYCQQ SNTWPFTFGQ GTKLEIK






SEQ ID NO:9

Part of Amino Acid Sequence of Humanised Heavy Chain Designated VHE











EVQLVESGAE VKKPGASVKV SCKASGYTFT NYIIHWVKQE




 



PGQGLEWIGY FNPYNHGTKY NEKFKGRATL TANKSISTAY



 



MELSSLRSED TAVYYCARSG PYAWFDTWGQ GTTVTVSS






SEQ ID NO:10

Part of Amino Acid Sequence of Humanised Heavy Chain Designated VHQ











QVQLVESGAE VKKPGASVKV SCKASGYTFT NYIIHWVKQE




 



PGQGLEWIGY FNPYNHGTKY NEKFKGRATL TANKSISTAY



 



MELSSLRSED TAVYYCARSG PYAWFDTWGQ GTTVTVSS






SEQ ID NO:11

Nucleotide Sequence Encoding Amino Acid Sequence SEQ ID NO:9









GAGGTGCAGCTGGTGGAGTCAGGAGCCGAAGTGAAAAAGCCTGGGGCTTCAGTGAAG



 


GTGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCA


 


GGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTA


 


AGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAAACAAATCCATCAGCACA


 


GCCTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACTGCGGTCTACTACTGTGCAA


 


GATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTC


 


CTCA






SEQ ID NO:12

Nucleotide Sequence Encoding Amino Acid Sequence SEQ ID NO:10









CAGGTGCAGCTGGTGGAGTCAGGAGCCGAAGTGAAAAAGCCTGGGGCTTCAGTGAAG



 


GTGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCA


 


GGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTA


 


AGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAAACAAATCCATCAGCACA


 


GCCTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACTGCGGTCTACTACTGTGCAA


 


GATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTC


 


CTCA






SEQ ID NO:13

Nucleotide Sequence Encoding Amino Acid Sequence SEQ ID NO:7









GACATTCTGCTGACCCAGTCTCCAGCCACCCTGTCTCTGAGTCCAGGAGAAAGAGCCA



 


CTTTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAAA


 


ACAAATGGTGCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCC


 


TTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTACCATCAGCAGTCTGG


 


AGCCTGAAGATTTTGCAGTGTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTC


 


GGCCAGGGGACCAAGCTGGAGATCAAA






SEQ ID NO:14

Nucleotide Sequence Encoding Amino Acid Sequence SEQ ID NO:8









GACATTCTGCTGACCCAGTCTCCAGCCACCCTGTCTCTGAGTCCAGGAGAAAGAGCCA



 


CTCTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAAA


 


CCAGGTCAGGCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCC


 


TTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTACCATCAGCAGTCTGG


 


AGCCTGAAGATTTTGCAGTGTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTC


 


GGCCAGGGGACCAAGCTGGAGATCAAA






SEQ ID NO:15

Nucleotide Sequence of the Expression Vector HCMV-G1 HuAb-VHQ (Complete DNA Sequence of a Humanised Heavy Chain Expression Vector Comprising SEQ ID NO:12 (VHQ) from 3921-4274)










1
AGCTTTTTGC AAAAGCCTAG GCCTCCAAAA AAGCCTCCTC




ACTACTTCTG


 


51
GAATAGCTCA GAGGCCGAGG CGGCCTCGGC CTCTGCATAA



ATAAAAAAAA


 


101
TTAGTCAGCC ATGGGGCGGA GAATGGGCGG AACTGGGCGG



AGTTAGGGGC


 


151
GGGATGGGCG GAGTTAGGGG CGGGACTATG GTTGCTGACT



AATTGAGATG


 


201
CATGCTTTGC ATACTTCTGC CTGCTGGGGA GCCTGGTTGC



TGACTAATTG


 


251
AGATGCATGC TTTGCATACT TCTGCCTGCT GGGGAGCCTG



GGGACTTTCC


 


301
ACACCCTAAC TGACACACAT TCCACAGCTG CCTCGCGCGT



TTCGGTGATG


 


351
ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT



CACAGCTTGT


 


401
CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG



CGTCAGCGGG


 


451
TGTTGGCGGG TGTCGGGGCG CAGCCATGAC CCAGTCACGT



AGCGATAGCG


 


501
GAGTGTATAC TGGCTTAACT ATGCGGCATC AGAGCAGATT



GTACTGAGAG


 


551
TGCACCATAT GCGGTGTGAA ATACCGCACA GATGCGTAAG



GAGAAAATAC


 


601
CGCATCAGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC



TGCGCTCGGT


 


651
CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG



GTAATACGGT


 


701
TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG



AGCAAAAGGC


 


751
CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG



CGTTTTTCCA


 


801
TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC



TCAAGTCAGA


 


851
GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT



TCCCCCTGGA


 


901
AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA



CCGGATACCT


 


951
GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT



AGCTCACGCT


 


1001
GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT



GGGCTGTGTG


 


1051
CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG



GTAACTATCG


 


1101
TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG



GCAGCAGCCA


 


1151
CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC



TACAGAGTTC


 


1201
TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG



TATTTGGTAT


 


1251
CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT



GGTAGCTCTT


 


1301
GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT



TGTTTGCAAG


 


1351
CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC



CTTTGATCTT


 


1401
TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT



TAAGGGATTT


 


1451
TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT



TTTAAATTAA


 


1501
AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA



CTTGGTCTGA


 


1551
CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG



ATCTGTCTAT


 


1601
TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT



AACTACGATA


 


1651
CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC



CGCGAGACCC


 


1701
ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA



GCCGGAAGGG


 


1751
CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT



CCAGTCTATT


 


1801
AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA



ATAGTTTGCG


 


1851
CAACGTTGTT GCCATTGCTG CAGGCATCGT GGTGTCACGC



TCGTCGTTTG


 


1901
GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG



AGTTACATGA


 


1951
TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC



CTCCGATCGT


 


2001
TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT



ATGGCAGCAC


 


2051
TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT



TTCTGTGACT


 


2101
GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC



GGCGACCGAG


 


2151
TTGCTCTTGC CCGGCGTCAA CACGGGATAA TACCGCGCCA



CATAGCAGAA


 


2201
CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG



AAAACTCTCA


 


2251
AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA



CTCGTGCACC


 


2301
CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT



GGGTGAGCAA


 


2351
AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC



GACACGGAAA


 


2401
TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA



GCATTTATCA


 


2451
GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT



TAGAAAAATA


 


2501
AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC



ACCTGACGTC


 


2551
TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA



GGCGTATCAC


 


2601
GAGGCCCTTT CGTCTTCAAG AATTCAGCTT GGCTGCAGTG



AATAATAAAA


 


2651
TGTGTGTTTG TCCGAAATAC GCGTTTTGAG ATTTCTGTCG



CCGACTAAAT


 


2701
TCATGTCGCG CGATAGTGGT GTTTATCGCC GATAGAGATG



GCGATATTGG


 


2751
AAAAATCGAT ATTTGAAAAT ATGGCATATT GAAAATGTCG



CCGATGTGAG


 


2801
TTTCTGTGTA ACTGATATCG CCATTTTTCC AAAAGTGATT



TTTGGGCATA


 


2851
CGCGATATCT GGCGATAGCG CTTATATCGT TTACGGGGGA



TGGCGATAGA


 


2901
CGACTTTGGT GACTTGGGCG ATTCTGTGTG TCGCAAATAT



CGCAGTTTCG


 


2951
ATATAGGTGA CAGACGATAT GAGGCTATAT CGCCGATAGA



GGCGACATCA


 


3001
AGCTGGCACA TGGCCAATGC ATATCGATCT ATACATTGAA



TCAATATTGG


 


3051
CCATTAGCCA TATTATTCAT TGGTTATATA GCATAAATCA



ATATTGGCTA


 


3101
TTGGCCATTG CATACGTTGT ATCCATATCA TAATATGTAC



ATTTATATTG


 


3151
GCTCATGTCC AACATTACCG CCATGTTGAC ATTGATTATT



GACTAGTTAT


 


3201
TAATAGTAAT CAATTACGGG GTCATTAGTT CATAGCCCAT



ATATGGAGTT


 


3251
CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA



CCGCCCAACG


 


3301
ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT



AGTAACGCCA


 


3351
ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC



GGTAAACTGC


 


3401
CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG



CCCCCTATTG


 


3451
ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA



GTACATGACC


 


3501
TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG



TCATCGCTAT


 


3551
TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT



GGATAGCGGT


 


3601
TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT



CAATGGGAGT


 


3651
TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC



GTAACAACTC


 


3701
CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG



GAGGTCTATA


 


3751
TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG



ACGCCATCCA


 


3801
CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA



GCCTCCGCAA


 


3851
GCTTGCCGCC ACCATGGACT GGACCTGGAG GGTGTTCTGC



CTGCTGGCCG


 


3901
TGGCCCCCGG CGCCCACAGC CAGGTGCAGC TGGTGGAGTC



AGGAGCCGAA


 


3951
GTGAAAAAGC CTGGGGCTTC AGTGAAGGTG TCCTGCAAGG



CCTCTGGATA


 


4001
CACATTCACT AATTATATTA TCCACTGGGT GAAGCAGGAG



CCTGGTCAGG


 


4051
GCCTTGAATG GATTGGATAT TTTAATCCTT ACAATCATGG



TACTAAGTAC


 


4101
AATGAGAAGT TCAAAGGCAG GGCCACACTA ACTGCAAACA



AATCCATCAG


 


4151
CACAGCCTAC ATGGAGCTCA GCAGCCTGCG CTCTGAGGAC



ACTGCGGTCT


 


4201
ACTACTGTGC AAGATCAGGA CCCTATGCCT GGTTTGACAC



CTGGGGCCAA


 


4251
GGGACCACGG TCACCGTCTC CTCAGGTGAG TTCTAGAAGG



ATCCCAAGCT


 


4301
AGCTTTCTGG GGCAGGCCAG GCCTGACCTT GGCTTTGGGG



CAGGGAGGGG


 


4351
GCTAAGGTGA GGCAGGTGGC GCCAGCCAGG TGCACACCCA



ATGCCCATGA


 


4401
GCCCAGACAC TGGACGCTGA ACCTCGCGGA CAGTTAAGAA



CCCAGGGGCC


 


4451
TCTGCGCCCT GGGCCCAGCT CTGTCCCACA CCGCGGTCAC



ATGGCACCAC


 


4501
CTCTCTTGCA GCCTCCACCA AGGGCCCATC GGTCTTCCCC



CTGGCACCCT


 


4551
CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG



CCTGGTCAAG


 


4601
GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG



GCGCCCTGAC


 


4651
CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA



GGACTCTACT


 


4701
CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG



CACCCAGACC


 


4751
TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG



TGGACAAGAA


 


4801
AGTTGGTGAG AGGCCAGCAC AGGGAGGGAG GGTGTCTGCT



GGAAGCCAGG


 


4851
CTCAGCGCTC CTGCCTGGAC GCATCCCGGC TATGCAGCCC



CAGTCCAGGG


 


4901
CAGCAAGGCA GGCCCCGTCT GCCTCTTCAC CCGGAGGCCT



CTGCCCGCCC


 


4951
CACTCATGCT CAGGGAGAGG GTCTTCTGGC TTTTTCCCCA



GGCTCTGGGC


 


5001
AGGCACAGGC TAGGTGCCCC TAACCCAGGC CCTGCACACA



AAGGGGCAGG


 


5051
TGCTGGGCTC AGACCTGCCA AGAGCCATAT CCGGGAGGAC



CCTGCCCCTG


 


5101
ACCTAAGCCC ACCCCAAAGG CCAAACTCTC CACTCCCTCA



GCTCGGACAC


 


5151
CTTCTCTCCT CCCAGATTCC AGTAACTCCC AATCTTCTCT



CTGCAGAGCC


 


5201
CAAATCTTGT GACAAAACTC ACACATGCCC ACCGTGCCCA



GGTAAGCCAG


 


5251
CCCAGGCCTC GCCCTCCAGC TCAAGGCGGG ACAGGTGCCC



TAGAGTAGCC


 


5301
TGCATCCAGG GACAGGCCCC AGCCGGGTGC TGACACGTCC



ACCTCCATCT


 


5351
CTTCCTCAGC ACCTGAACTC CTGGGGGGAC CGTCAGTCTT



CCTCTTCCCC


 


5401
CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG



AGGTCACATG


 


5451
CGTGGTGGTG GACGTGAGCC ACGAAGACCC TGAGGTCAAG



TTCAACTGGT


 


5501
ACGTGGACGG CGTGGAGGTG CATAATGCCA AGACAAAGCC



GCGGGAGGAG


 


5551
CAGTACAACA GCACGTACCG TGTGGTCAGC GTCCTCACCG



TCCTGCACCA


 


5601
GGACTGGCTG AATGGCAAGG AGTACAAGTG CAAGGTCTCC



AACAAAGCCC


 


5651
TCCCAGCCCC CATCGAGAAA ACCATCTCCA AAGCCAAAGG



TGGGACCCGT


 


5701
GGGGTGCGAG GGCCACATGG ACAGAGGCCG GCTCGGCCCA



CCCTCTGCCC


 


5751
TGAGAGTGAC CGCTGTACCA ACCTCTGTCC CTACAGGGCA



GCCCCGAGAA


 


5801
CCACAGGTGT ACACCCTGCC CCCATCCCGG GATGAGCTGA



CCAAGAACCA


 


5851
GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC



GACATCGCCG


 


5901
TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA



GACCACGCCT


 


5951
CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA



AGCTCACCGT


 


6001
GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC



TCCGTGATGC


 


6051
ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC



CCTGTCTCCG


 


6101
GGTAAATGAG TGCGACGGCC GGCAAGCCCC CGCTCCCCGG



GCTCTCGCGG


 


6151
TCGCACGAGG ATGCTTGGCA CGTACCCCCT GTACATACTT



CCCGGGCGCC


 


6201
CAGCATGGAA ATAAAGCACC CAGCGCTGCC CTGGGCCCCT



GCGAGACTGT


 


6251
GATGGTTCTT TCCACGGGTC AGGCCGAGTC TGAGGCCTGA



GTGGCATGAG


 


6301
ATCTGATATC ATCGATGAAT TCGAGCTCGG TACCCGGGGA



TCGATCCAGA


 


6351
CATGATAAGA TACATTGATG AGTTTGGACA AACCACAACT



AGAATGCAGT


 


6401
GAAAAAAATG CTTTATTTGT GAAATTTGTG ATGCTATTGC



TTTATTTGTA


 


6451
ACCATTATAA GCTGCAATAA ACAAGTTAAC AACAACAATT



GCATTCATTT


 


6501
TATGTTTCAG GTTCAGGGGG AGGTGTGGGA GGTTTTTTAA



AGCAAGTAAA


 


6551
ACCTCTACAA ATGTGGTATG GCTGATTATG ATCTCTAGTC



AAGGCACTAT


 


6601
ACATCAAATA TTCCTTATTA ACCCCTTTAC AAATTAAAAA



GCTAAAGGTA


 


6651
CACAATTTTT GAGCATAGTT ATTAATAGCA GACACTCTAT



GCCTGTGTGG


 


6701
AGTAAGAAAA AACAGTATGT TATGATTATA ACTGTTATGC



CTACTTATAA


 


6751
AGGTTACAGA ATATTTTTCC ATAATTTTCT TGTATAGCAG



TGCAGCTTTT


 


6801
TCCTTTGTGG TGTAAATAGC AAAGCAAGCA AGAGTTCTAT



TACTAAACAC


 


6851
AGCATGACTC AAAAAACTTA GCAATTCTGA AGGAAAGTCC



TTGGGGTCTT


 


6901
CTACCTTTCT CTTCTTTTTT GGAGGAGTAG AATGTTGAGA



GTCAGCAGTA


 


6951
GCCTCATCAT CACTAGATGG CATTTCTTCT GAGCAAAACA



GGTTTTCCTC


 


7001
ATTAAAGGCA TTCCACCACT GCTCCCATTC ATCAGTTCCA



TAGGTTGGAA


 


7051
TCTAAAATAC ACAAACAATT AGAATCAGTA GTTTAACACA



TTATACACTT


 


7101
AAAAATTTTA TATTTACCTT AGAGCTTTAA ATCTCTGTAG



GTAGTTTGTC


 


7151
CAATTATGTC ACACCACAGA AGTAAGGTTC CTTCACAAAG



ATCCGGGACC


 


7201
AAAGCGGCCA TCGTGCCTCC CCACTCCTGC AGTTCGGGGG



CATGGATGCG


 


7251
CGGATAGCCG CTGCTGGTTT CCTGGATGCC GACGGATTTG



CACTGCCGGT


 


7301
AGAACTCCGC GAGGTCGTCC AGCCTCAGGC AGCAGCTGAA



CCAACTCGCG


 


7351
AGGGGATCGA GCCCGGGGTG GGCGAAGAAC TCCAGCATGA



GATCCCCGCG


 


7401
CTGGAGGATC ATCCAGCCGG CGTCCCGGAA AACGATTCCG



AAGCCCAACC


 


7451
TTTCATAGAA GGCGGCGGTG GAATCGAAAT CTCGTGATGG



CAGGTTGGGC


 


7501
GTCGCTTGGT CGGTCATTTC GAACCCCAGA GTCCCGCTCA



GAAGAACTCG


 


7551
TCAAGAAGGC GATAGAAGGC GATGCGCTGC GAATCGGGAG



CGGCGATACC


 


7601
GTAAAGCACG AGGAAGCGGT CAGCCCATTC GCCGCCAAGC



TCTTCAGCAA


 


7651
TATCACGGGT AGCCAACGCT ATGTCCTGAT AGCGGTCCGC



CACACCCAGC


 


7701
CGGCCACAGT CGATGAATCC AGAAAAGCGG CCATTTTCCA



CCATGATATT


 


7751
CGGCAAGCAG GCATCGCCAT GGGTCACGAC GAGATCCTCG



CCGTCGGGCA


 


7801
TGCGCGCCTT GAGCCTGGCG AACAGTTCGG CTGGCGCGAG



CCCCTGATGC


 


7851
TCTTCGTCCA GATCATCCTG ATCGACAAGA CCGGCTTCCA



TCCGAGTACG


 


7901
TGCTCGCTCG ATGCGATGTT TCGCTTGGTG GTCGAATGGG



CAGGTAGCCG


 


7951
GATCAAGCGT ATGCAGCCGC CGCATTGCAT CAGCCATGAT



GGATACTTTC


 


8001
TCGGCAGGAG CAAGGTGAGA TGACAGGAGA TCCTGCCCCG



GCACTTCGCC


 


8051
CAATAGCAGC CAGTCCCTTC CCGCTTCAGT GACAACGTCG



AGCACAGCTG


 


8101
CGCAAGGAAC GCCCGTCGTG GCCAGCCACG ATAGCCGCGC



TGCCTCGTCC


 


8151
TGCAGTTCAT TCAGGGCACC GGACAGGTCG GTCTTGACAA



AAAGAACCGG


 


8201
GCGCCCCTGC GCTGACAGCC GGAACACGGC GGCATCAGAG



CAGCCGATTG


 


8251
TCTGTTGTGC CCAGTCATAG CCGAATAGCC TCTCCACCCA



AGCGGCCGGA


 


8301
GAACCTGCGT GCAATCCATC TTGTTCAATC ATGCGAAACG



ATCCTCATCC


 


8351
TGTCTCTTGA TCAGATCTTG ATCCCCTGCG CCATCAGATC



CTTGGCGGCA


 


8401
AGAAAGCCAT CCAGTTTACT TTGCAGGGCT TCCCAACCTT



ACCAGAGGGC


 


8451
GCCCCAGCTG GCAATTCCGG TTCGCTTGCT GTCCATAAAA



CCGCCCAGTC


 


8501
TAGCTATCGC CATGTAAGCC CACTGCAAGC TACCTGCTTT



CTCTTTGCGC


 


8551
TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACATT



CATCCGGGGT


 


8601
CAGCACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT



TCCTTTAGCA


 


8651
GCCCTTGCGC CCTGAGTGCT TGCGGCAGCG TGAAGCT






SEQ ID NO:16

Nucleotide Sequence of the Expression Vector HCMV-G1 HuAb-VHE (Complete DNA Sequence of a Humanised Heavy Chain Expression Vector Comprising SEQ ID NO: 11 (VHE) from 3921-4274)










1
AGCTTTTTGC AAAAGCCTAG GCCTCCAAAA AAGCCTCCTC




ACTACTTCTG


 


51
GAATAGCTCA GAGGCCGAGG CGGCCTCGGC CTCTGCATAA



ATAAAAAAAA


 


101
TTAGTCAGCC ATGGGGCGGA GAATGGGCGG AACTGGGCGG



AGTTAGGGGC


 


151
GGGATGGGCG GAGTTAGGGG CGGGACTATG GTTGCTGACT



AATTGAGATG


 


201
CATGCTTTGC ATACTTCTGC CTGCTGGGGA GCCTGGTTGC



TGACTAATTG


 


251
AGATGCATGC TTTGCATACT TCTGCCTGCT GGGGAGCCTG



GGGACTTTCC


 


301
ACACCCTAAC TGACACACAT TCCACAGCTG CCTCGCGCGT



TTCGGTGATG


 


351
ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT



CACAGCTTGT


 


401
CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG



CGTCAGCGGG


 


451
TGTTGGCGGG TGTCGGGGCG CAGCCATGAC CCAGTCACGT



AGCGATAGCG


 


501
GAGTGTATAC TGGCTTAACT ATGCGGCATC AGAGCAGATT



GTACTGAGAG


 


551
TGCACCATAT GCGGTGTGAA ATACCGCACA GATGCGTAAG



GAGAAAATAC


 


601
CGCATCAGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC



TGCGCTCGGT


 


651
CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG



GTAATACGGT


 


701
TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG



AGCAAAAGGC


 


751
CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG



CGTTTTTCCA


 


801
TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC



TCAAGTCAGA


 


851
GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT



TCCCCCTGGA


 


901
AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA



CCGGATACCT


 


951
GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT



AGCTCACGCT


 


1001
GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT



GGGCTGTGTG


 


1051
CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG



GTAACTATCG


 


1101
TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG



GCAGCAGCCA


 


1151
CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC



TACAGAGTTC


 


1201
TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG



TATTTGGTAT


 


1251
CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT



GGTAGCTCTT


 


1301
GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT



TGTTTGCAAG


 


1351
CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC



CTTTGATCTT


 


1401
TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT



TAAGGGATTT


 


1451
TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT



TTTAAATTAA


 


1501
AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA



CTTGGTCTGA


 


1551
CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG



ATCTGTCTAT


 


1601
TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT



AACTACGATA


 


1651
CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC



CGCGAGACCC


 


1701
ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA



GCCGGAAGGG


 


1751
CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT



CCAGTCTATT


 


1801
AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA



ATAGTTTGCG


 


1851
CAACGTTGTT GCCATTGCTG CAGGCATCGT GGTGTCACGC



TCGTCGTTTG


 


1901
GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG



AGTTACATGA


 


1951
TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC



CTCCGATCGT


 


2001
TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT



ATGGCAGCAC


 


2051
TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT



TTCTGTGACT


 


2101
GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC



GGCGACCGAG


 


2151
TTGCTCTTGC CCGGCGTCAA CACGGGATAA TACCGCGCCA



CATAGCAGAA


 


2201
CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG



AAAACTCTCA


 


2251
AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA



CTCGTGCACC


 


2301
CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT



GGGTGAGCAA


 


2351
AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC



GACACGGAAA


 


2401
TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA



GCATTTATCA


 


2451
GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT



TAGAAAAATA


 


2501
AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC



ACCTGACGTC


 


2551
TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA



GGCGTATCAC


 


2601
GAGGCCCTTT CGTCTTCAAG AATTCAGCTT GGCTGCAGTG



AATAATAAAA


 


2651
TGTGTGTTTG TCCGAAATAC GCGTTTTGAG ATTTCTGTCG



CCGACTAAAT


 


2701
TCATGTCGCG CGATAGTGGT GTTTATCGCC GATAGAGATG



GCGATATTGG


 


2751
AAAAATCGAT ATTTGAAAAT ATGGCATATT GAAAATGTCG



CCGATGTGAG


 


2801
TTTCTGTGTA ACTGATATCG CCATTTTTCC AAAAGTGATT



TTTGGGCATA


 


2851
CGCGATATCT GGCGATAGCG CTTATATCGT TTACGGGGGA



TGGCGATAGA


 


2901
CGACTTTGGT GACTTGGGCG ATTCTGTGTG TCGCAAATAT



CGCAGTTTCG


 


2951
ATATAGGTGA CAGACGATAT GAGGCTATAT CGCCGATAGA



GGCGACATCA


 


3001
AGCTGGCACA TGGCCAATGC ATATCGATCT ATACATTGAA



TCAATATTGG


 


3051
CCATTAGCCA TATTATTCAT TGGTTATATA GCATAAATCA



ATATTGGCTA


 


3101
TTGGCCATTG CATACGTTGT ATCCATATCA TAATATGTAC



ATTTATATTG


 


3151
GCTCATGTCC AACATTACCG CCATGTTGAC ATTGATTATT



GACTAGTTAT


 


3201
TAATAGTAAT CAATTACGGG GTCATTAGTT CATAGCCCAT



ATATGGAGTT


 


3251
CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA



CCGCCCAACG


 


3301
ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT



AGTAACGCCA


 


3351
ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC



GGTAAACTGC


 


3401
CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG



CCCCCTATTG


 


3451
ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA



GTACATGACC


 


3501
TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG



TCATCGCTAT


 


3551
TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT



GGATAGCGGT


 


3601
TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT



CAATGGGAGT


 


3651
TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC



GTAACAACTC


 


3701
CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG



GAGGTCTATA


 


3751
TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG



ACGCCATCCA


 


3801
CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA



GCCTCCGCAA


 


3851
GCTTGCCGCC ACCATGGACT GGACCTGGAG GGTGTTCTGC



CTGCTGGCCG


 


3901
TGGCCCCCGG CGCCCACAGC GAGGTGCAGC TGGTGGAGTC



AGGAGCCGAA


 


3951
GTGAAAAAGC CTGGGGCTTC AGTGAAGGTG TCCTGCAAGG



CCTCTGGATA


 


4001
CACATTCACT AATTATATTA TCCACTGGGT GAAGCAGGAG



CCTGGTCAGG


 


4051
GCCTTGAATG GATTGGATAT TTTAATCCTT ACAATCATGG



TACTAAGTAC


 


4101
AATGAGAAGT TCAAAGGCAG GGCCACACTA ACTGCAAACA



AATCCATCAG


 


4151
CACAGCCTAC ATGGAGCTCA GCAGCCTGCG CTCTGAGGAC



ACTGCGGTCT


 


4201
ACTACTGTGC AAGATCAGGA CCCTATGCCT GGTTTGACAC



CTGGGGCCAA


 


4251
GGGACCACGG TCACCGTCTC CTCAGGTGAG TTCTAGAAGG



ATCCCAAGCT


 


4301
AGCTTTCTGG GGCAGGCCAG GCCTGACCTT GGCTTTGGGG



CAGGGAGGGG


 


4351
GCTAAGGTGA GGCAGGTGGC GCCAGCCAGG TGCACACCCA



ATGCCCATGA


 


4401
GCCCAGACAC TGGACGCTGA ACCTCGCGGA CAGTTAAGAA



CCCAGGGGCC


 


4451
TCTGCGCCCT GGGCCCAGCT CTGTCCCACA CCGCGGTCAC



ATGGCACCAC


 


4501
CTCTCTTGCA GCCTCCACCA AGGGCCCATC GGTCTTCCCC



CTGGCACCCT


 


4551
CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG



CCTGGTCAAG


 


4601
GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG



GCGCCCTGAC


 


4651
CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA



GGACTCTACT


 


4701
CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG



CACCCAGACC


 


4751
TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG



TGGACAAGAA


 


4801
AGTTGGTGAG AGGCCAGCAC AGGGAGGGAG GGTGTCTGCT



GGAAGCCAGG


 


4851
CTCAGCGCTC CTGCCTGGAC GCATCCCGGC TATGCAGCCC



CAGTCCAGGG


 


4901
CAGCAAGGCA GGCCCCGTCT GCCTCTTCAC CCGGAGGCCT



CTGCCCGCCC


 


4951
CACTCATGCT CAGGGAGAGG GTCTTCTGGC TTTTTCCCCA



GGCTCTGGGC


 


5001
AGGCACAGGC TAGGTGCCCC TAACCCAGGC CCTGCACACA



AAGGGGCAGG


 


5051
TGCTGGGCTC AGACCTGCCA AGAGCCATAT CCGGGAGGAC



CCTGCCCCTG


 


5101
ACCTAAGCCC ACCCCAAAGG CCAAACTCTC CACTCCCTCA



GCTCGGACAC


 


5151
CTTCTCTCCT CCCAGATTCC AGTAACTCCC AATCTTCTCT



CTGCAGAGCC


 


5201
CAAATCTTGT GACAAAACTC ACACATGCCC ACCGTGCCCA



GGTAAGCCAG


 


5251
CCCAGGCCTC GCCCTCCAGC TCAAGGCGGG ACAGGTGCCC



TAGAGTAGCC


 


5301
TGCATCCAGG GACAGGCCCC AGCCGGGTGC TGACACGTCC



ACCTCCATCT


 


5351
CTTCCTCAGC ACCTGAACTC CTGGGGGGAC CGTCAGTCTT



CCTCTTCCCC


 


5401
CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG



AGGTCACATG


 


5451
CGTGGTGGTG GACGTGAGCC ACGAAGACCC TGAGGTCAAG



TTCAACTGGT


 


5501
ACGTGGACGG CGTGGAGGTG CATAATGCCA AGACAAAGCC



GCGGGAGGAG


 


5551
CAGTACAACA GCACGTACCG TGTGGTCAGC GTCCTCACCG



TCCTGCACCA


 


5601
GGACTGGCTG AATGGCAAGG AGTACAAGTG CAAGGTCTCC



AACAAAGCCC


 


5651
TCCCAGCCCC CATCGAGAAA ACCATCTCCA AAGCCAAAGG



TGGGACCCGT


 


5701
GGGGTGCGAG GGCCACATGG ACAGAGGCCG GCTCGGCCCA



CCCTCTGCCC


 


5751
TGAGAGTGAC CGCTGTACCA ACCTCTGTCC CTACAGGGCA



GCCCCGAGAA


 


5801
CCACAGGTGT ACACCCTGCC CCCATCCCGG GATGAGCTGA



CCAAGAACCA


 


5851
GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC



GACATCGCCG


 


5901
TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA



GACCACGCCT


 


5951
CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA



AGCTCACCGT


 


6001
GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC



TCCGTGATGC


 


6051
ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC



CCTGTCTCCG


 


6101
GGTAAATGAG TGCGACGGCC GGCAAGCCCC CGCTCCCCGG



GCTCTCGCGG


 


6151
TCGCACGAGG ATGCTTGGCA CGTACCCCCT GTACATACTT



CCCGGGCGCC


 


6201
CAGCATGGAA ATAAAGCACC CAGCGCTGCC CTGGGCCCCT



GCGAGACTGT


 


6251
GATGGTTCTT TCCACGGGTC AGGCCGAGTC TGAGGCCTGA



GTGGCATGAG


 


6301
ATCTGATATC ATCGATGAAT TCGAGCTCGG TACCCGGGGA



TCGATCCAGA


 


6351
CATGATAAGA TACATTGATG AGTTTGGACA AACCACAACT



AGAATGCAGT


 


6401
GAAAAAAATG CTTTATTTGT GAAATTTGTG ATGCTATTGC



TTTATTTGTA


 


6451
ACCATTATAA GCTGCAATAA ACAAGTTAAC AACAACAATT



GCATTCATTT


 


6501
TATGTTTCAG GTTCAGGGGG AGGTGTGGGA GGTTTTTTAA



AGCAAGTAAA


 


6551
ACCTCTACAA ATGTGGTATG GCTGATTATG ATCTCTAGTC



AAGGCACTAT


 


6601
ACATCAAATA TTCCTTATTA ACCCCTTTAC AAATTAAAAA



GCTAAAGGTA


 


6651
CACAATTTTT GAGCATAGTT ATTAATAGCA GACACTCTAT



GCCTGTGTGG


 


6701
AGTAAGAAAA AACAGTATGT TATGATTATA ACTGTTATGC



CTACTTATAA


 


6751
AGGTTACAGA ATATTTTTCC ATAATTTTCT TGTATAGCAG



TGCAGCTTTT


 


6801
TCCTTTGTGG TGTAAATAGC AAAGCAAGCA AGAGTTCTAT



TACTAAACAC


 


6851
AGCATGACTC AAAAAACTTA GCAATTCTGA AGGAAAGTCC



TTGGGGTCTT


 


6901
CTACCTTTCT CTTCTTTTTT GGAGGAGTAG AATGTTGAGA



GTCAGCAGTA


 


6951
GCCTCATCAT CACTAGATGG CATTTCTTCT GAGCAAAACA



GGTTTTCCTC


 


7001
ATTAAAGGCA TTCCACCACT GCTCCCATTC ATCAGTTCCA



TAGGTTGGAA


 


7051
TCTAAAATAC ACAAACAATT AGAATCAGTA GTTTAACACA



TTATACACTT


 


7101
AAAAATTTTA TATTTACCTT AGAGCTTTAA ATCTCTGTAG



GTAGTTTGTC


 


7151
CAATTATGTC ACACCACAGA AGTAAGGTTC CTTCACAAAG



ATCCGGGACC


 


7201
AAAGCGGCCA TCGTGCCTCC CCACTCCTGC AGTTCGGGGG



CATGGATGCG


 


7251
CGGATAGCCG CTGCTGGTTT CCTGGATGCC GACGGATTTG



CACTGCCGGT


 


7301
AGAACTCCGC GAGGTCGTCC AGCCTCAGGC AGCAGCTGAA



CCAACTCGCG


 


7351
AGGGGATCGA GCCCGGGGTG GGCGAAGAAC TCCAGCATGA



GATCCCCGCG


 


7401
CTGGAGGATC ATCCAGCCGG CGTCCCGGAA AACGATTCCG



AAGCCCAACC


 


7451
TTTCATAGAA GGCGGCGGTG GAATCGAAAT CTCGTGATGG



CAGGTTGGGC


 


7501
GTCGCTTGGT CGGTCATTTC GAACCCCAGA GTCCCGCTCA



GAAGAACTCG


 


7551
TCAAGAAGGC GATAGAAGGC GATGCGCTGC GAATCGGGAG



CGGCGATACC


 


7601
GTAAAGCACG AGGAAGCGGT CAGCCCATTC GCCGCCAAGC



TCTTCAGCAA


 


7651
TATCACGGGT AGCCAACGCT ATGTCCTGAT AGCGGTCCGC



CACACCCAGC


 


7701
CGGCCACAGT CGATGAATCC AGAAAAGCGG CCATTTTCCA



CCATGATATT


 


7751
CGGCAAGCAG GCATCGCCAT GGGTCACGAC GAGATCCTCG



CCGTCGGGCA


 


7801
TGCGCGCCTT GAGCCTGGCG AACAGTTCGG CTGGCGCGAG



CCCCTGATGC


 


7851
TCTTCGTCCA GATCATCCTG ATCGACAAGA CCGGCTTCCA



TCCGAGTACG


 


7901
TGCTCGCTCG ATGCGATGTT TCGCTTGGTG GTCGAATGGG



CAGGTAGCCG


 


7951
GATCAAGCGT ATGCAGCCGC CGCATTGCAT CAGCCATGAT



GGATACTTTC


 


8001
TCGGCAGGAG CAAGGTGAGA TGACAGGAGA TCCTGCCCCG



GCACTTCGCC


 


8051
CAATAGCAGC CAGTCCCTTC CCGCTTCAGT GACAACGTCG



AGCACAGCTG


 


8101
CGCAAGGAAC GCCCGTCGTG GCCAGCCACG ATAGCCGCGC



TGCCTCGTCC


 


8151
TGCAGTTCAT TCAGGGCACC GGACAGGTCG GTCTTGACAA



AAAGAACCGG


 


8201
GCGCCCCTGC GCTGACAGCC GGAACACGGC GGCATCAGAG



CAGCCGATTG


 


8251
TCTGTTGTGC CCAGTCATAG CCGAATAGCC TCTCCACCCA



AGCGGCCGGA


 


8301
GAACCTGCGT GCAATCCATC TTGTTCAATC ATGCGAAACG



ATCCTCATCC


 


8351
TGTCTCTTGA TCAGATCTTG ATCCCCTGCG CCATCAGATC



CTTGGCGGCA


 


8401
AGAAAGCCAT CCAGTTTACT TTGCAGGGCT TCCCAACCTT



ACCAGAGGGC


 


8451
GCCCCAGCTG GCAATTCCGG TTCGCTTGCT GTCCATAAAA



CCGCCCAGTC


 


8501
TAGCTATCGC CATGTAAGCC CACTGCAAGC TACCTGCTTT



CTCTTTGCGC


 


8551
TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACATT



CATCCGGGGT


 


8601
CAGCACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT



TCCTTTAGCA


 


8651
GCCCTTGCGC CCTGAGTGCT TGCGGCAGCG TGAAGCT






SEQ ID NO:17

Nucleotide Sequence of the Expression Vector HCMV-K HuAb-VL1 hum V1 (Complete DNA Sequence of a Humanised Light Chain Expression Vector Comprising SEQ ID NO: 14 (humV1=VLh) from 3964-4284)










1
CTAGCTTTTT GCAAAAGCCT AGGCCTCCAA AAAAGCCTCC




TCACTACTTC


 


51
TGGAATAGCT CAGAGGCCGA GGCGGCCTCG GCCTCTGCAT



AAATAAAAAA


 


101
AATTAGTCAG CCATGGGGCG GAGAATGGGC GGAACTGGGC



GGAGTTAGGG


 


151
GCGGGATGGG CGGAGTTAGG GGCGGGACTA TGGTTGCTGA



CTAATTGAGA


 


201
TGCATGCTTT GCATACTTCT GCCTGCTGGG GAGCCTGGTT



GCTGACTAAT


 


251
TGAGATGCAT GCTTTGCATA CTTCTGCCTG CTGGGGAGCC



TGGGGACTTT


 


301
CCACACCCTA ACTGACACAC ATTCCACAGC TGCCTCGCGC



GTTTCGGTGA


 


351
TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG



GTCACAGCTT


 


401
GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG



CGCGTCAGCG


 


451
GGTGTTGGCG GGTGTCGGGG CGCAGCCATG ACCCAGTCAC



GTAGCGATAG


 


501
CGGAGTGTAT ACTGGCTTAA CTATGCGGCA TCAGAGCAGA



TTGTACTGAG


 


551
AGTGCACCAT ATGCGGTGTG AAATACCGCA CAGATGCGTA



AGGAGAAAAT


 


601
ACCGCATCAG GCGCTCTTCC GCTTCCTCGC TCACTGACTC



GCTGCGCTCG


 


651
GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG



CGGTAATACG


 


701
GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG



TGAGCAAAAG


 


751
GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT



GGCGTTTTTC


 


801
CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC



GCTCAAGTCA


 


851
GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG



TTTCCCCCTG


 


901
GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT



TACCGGATAC


 


951
CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC



ATAGCTCACG


 


1001
CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG



CTGGGCTGTG


 


1051
TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC



CGGTAACTAT


 


1101
CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC



TGGCAGCAGC


 


1151
CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT



GCTACAGAGT


 


1201
TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC



AGTATTTGGT


 


1251
ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG



TTGGTAGCTC


 


1301
TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT



TTTGTTTGCA


 


1351
AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA



TCCTTTGATC


 


1401
TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC



GTTAAGGGAT


 


1451
TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC



CTTTTAAATT


 


1501
AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA



AACTTGGTCT


 


1551
GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG



CGATCTGTCT


 


1601
ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG



ATAACTACGA


 


1651
TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT



ACCGCGAGAC


 


1701
CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC



CAGCCGGAAG


 


1751
GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC



ATCCAGTCTA


 


1801
TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT



TAATAGTTTG


 


1851
CGCAACGTTG TTGCCATTGC TGCAGGCATC GTGGTGTCAC



GCTCGTCGTT


 


1901
TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG



CGAGTTACAT


 


1951
GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG



TCCTCCGATC


 


2001
GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG



TTATGGCAGC


 


2051
ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC



TTTTCTGTGA


 


2101
CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT



GCGGCGACCG


 


2151
AGTTGCTCTT GCCCGGCGTC AACACGGGAT AATACCGCGC



CACATAGCAG


 


2201
AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG



CGAAAACTCT


 


2251
CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC



CACTCGTGCA


 


2301
CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT



CTGGGTGAGC


 


2351
AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG



GCGACACGGA


 


2401
AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG



AAGCATTTAT


 


2451
CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA



TTTAGAAAAA


 


2501
TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG



CCACCTGACG


 


2551
TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA



TAGGCGTATC


 


2601
ACGAGGCCCT TTCGTCTTCA AGAATTCAGC TTGGCTGCAG



TGAATAATAA


 


2651
AATGTGTGTT TGTCCGAAAT ACGCGTTTTG AGATTTCTGT



CGCCGACTAA


 


2701
ATTCATGTCG CGCGATAGTG GTGTTTATCG CCGATAGAGA



TGGCGATATT


 


2751
GGAAAAATCG ATATTTGAAA ATATGGCATA TTGAAAATGT



CGCCGATGTG


 


2801
AGTTTCTGTG TAACTGATAT CGCCATTTTT CCAAAAGTGA



TTTTTGGGCA


 


2851
TACGCGATAT CTGGCGATAG CGCTTATATC GTTTACGGGG



GATGGCGATA


 


2901
GACGACTTTG GTGACTTGGG CGATTCTGTG TGTCGCAAAT



ATCGCAGTTT


 


2951
CGATATAGGT GACAGACGAT ATGAGGCTAT ATCGCCGATA



GAGGCGACAT


 


3001
CAAGCTGGCA CATGGCCAAT GCATATCGAT CTATACATTG



AATCAATATT


 


3051
GGCCATTAGC CATATTATTC ATTGGTTATA TAGCATAAAT



CAATATTGGC


 


3101
TATTGGCCAT TGCATACGTT GTATCCATAT CATAATATGT



ACATTTATAT


 


3151
TGGCTCATGT CCAACATTAC CGCCATGTTG ACATTGATTA



TTGACTAGTT


 


3201
ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC



ATATATGGAG


 


3251
TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT



GACCGCCCAA


 


3301
CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC



ATAGTAACGC


 


3351
CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT



ACGGTAAACT


 


3401
GCCCACTTGG CAGTACATCA AGTGTATCAT ATGCCAAGTA



CGCCCCCTAT


 


3451
TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC



CAGTACATGA


 


3501
CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT



AGTCATCGCT


 


3551
ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC



GTGGATAGCG


 


3601
GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC



GTCAATGGGA


 


3651
GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG



TCGTAACAAC


 


3701
TCCGCCCCAT TGACGCAAAT GGGCGGTAGG CGTGTACGGT



GGGAGGTCTA


 


3751
TATAAGCAGA GCTCGTTTAG TGAACCGTCA GATCGCCTGG



AGACGCCATC


 


3801
CACGCTGTTT TGACCTCCAT AGAAGACACC GGGACCGATC



CAGCCTCCGC


 


3851
AAGCTTGATA TCGAATTCCT GCAGCCCGGG GGATCCGCCC



GCTTGCCGCC


 


3901
ACCATGGAGA CCCCCGCCCA GCTGCTGTTC CTGCTGCTGC



TGTGGCTGCC


 


3951
CGACACCACC GGCGACATTC TGCTGACCCA GTCTCCAGCC



ACCCTGTCTC


 


4001
TGAGTCCAGG AGAAAGAGCC ACTCTCTCCT GCAGGGCCAG



TCAGAACATT


 


4051
GGCACAAGCA TACAGTGGTA TCAACAAAAA CCAGGTCAGG



CTCCAAGGCT


 


4101
TCTCATAAGG TCTTCTTCTG AGTCTATCTC TGGGATCCCT



TCCAGGTTTA


 


4151
GTGGCAGTGG ATCAGGGACA GATTTTACTC TTACCATCAG



CAGTCTGGAG


 


4201
CCTGAAGATT TTGCAGTGTA TTACTGTCAA CAAAGTAATA



CCTGGCCATT


 


4251
CACGTTCGGC CAGGGGACCA AGCTGGAGAT CAAACGTGAG



TATTCTAGAA


 


4301
AGATCCTAGA ATTCTAAACT CTGAGGGGGT CGGATGACGT



GGCCATTCTT


 


4351
TGCCTAAAGC ATTGAGTTTA CTGCAAGGTC AGAAAAGCAT



GCAAAGCCCT


 


4401
CAGAATGGCT GCAAAGAGCT CCAACAAAAC AATTTAGAAC



TTTATTAAGG


 


4451
AATAGGGGGA AGCTAGGAAG AAACTCAAAA CATCAAGATT



TTAAATACGC


 


4501
TTCTTGGTCT CCTTGCTATA ATTATCTGGG ATAAGCATGC



TGTTTTCTGT


 


4551
CTGTCCCTAA CATGCCCTGT GATTATCCGC AAACAACACA



CCCAAGGGCA


 


4601
GAACTTTGTT ACTTAAACAC CATCCTGTTT GCTTCTTTCC



TCAGGAACTG


 


4651
TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA



GCAGTTGAAA


 


4701
TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT



ATCCCAGAGA


 


4751
GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG



GGTAACTCCC


 


4801
AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA



CAGCCTCAGC


 


4851
AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA



AAGTCTACGC


 


4901
CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA



AAGAGCTTCA


 


4951
ACAGGGGAGA GTGTTAGAGG GAGAAGTGCC CCCACCTGCT



CCTCAGTTCC


 


5001
AGCCTGACCC CCTCCCATCC TTTGGCCTCT GACCCTTTTT



CCACAGGGGA


 


5051
CCTACCCCTA TTGCGGTCCT CCAGCTCATC TTTCACCTCA



CCCCCCTCCT


 


5101
CCTCCTTGGC TTTAATTATG CTAATGTTGG AGGAGAATGA



ATAAATAAAG


 


5151
TGAATCTTTG CACCTGTGGT TTCTCTCTTT CCTCATTTAA



TAATTATTAT


 


5201
CTGTTGTTTA CCAACTACTC AATTTCTCTT ATAAGGGACT



AAATATGTAG


 


5251
TCATCCTAAG GCGCATAACC ATTTATAAAA ATCATCCTTC



ATTCTATTTT


 


5301
ACCCTATCAT CCTCTGCAAG ACAGTCCTCC CTCAAACCCA



CAAGCCTTCT


 


5351
GTCCTCACAG TCCCCTGGGC CATGGTAGGA GAGACTTGCT



TCCTTGTTTT


 


5401
CCCCTCCTCA GCAAGCCCTC ATAGTCCTTT TTAAGGGTGA



CAGGTCTTAC


 


5451
AGTCATATAT CCTTTGATTC AATTCCCTGA GAATCAACCA



AAGCAAATTT


 


5501
TTCAAAAGAA GAAACCTGCT ATAAAGAGAA TCATTCATTG



CAACATGATA


 


5551
TAAAATAACA ACACAATAAA AGCAATTAAA TAAACAAACA



ATAGGGAAAT


 


5601
GTTTAAGTTC ATCATGGTAC TTAGACTTAA TGGAATGTCA



TGCCTTATTT


 


5651
ACATTTTTAA ACAGGTACTG AGGGACTCCT GTCTGCCAAG



GGCCGTATTG


 


5701
AGTACTTTCC ACAACCTAAT TTAATCCACA CTATACTGTG



AGATTAAAAA


 


5751
CATTCATTAA AATGTTGCAA AGGTTCTATA AAGCTGAGAG



ACAAATATAT


 


5801
TCTATAACTC AGCAATCCCA CTTCTAGATG ACTGAGTGTC



CCCACCCACC


 


5851
AAAAAACTAT GCAAGAATGT TCAAAGCAGC TTTATTTACA



AAAGCCAAAA


 


5901
ATTGGAAATA GCCCGATTGT CCAACAATAG AATGAGTTAT



TAAACTGTGG


 


5951
TATGTTTATA CATTAGAATA CCCAATGAGG AGAATTAACA



AGCTACAACT


 


6001
ATACCTACTC ACACAGATGA ATCTCATAAA AATAATGTTA



CATAAGAGAA


 


6051
ACTCAATGCA AAAGATATGT TCTGTATGTT TTCATCCATA



TAAAGTTCAA


 


6101
AACCAGGTAA AAATAAAGTT AGAAATTTGG ATGGAAATTA



CTCTTAGCTG


 


6151
GGGGTGGGCG AGTTAGTGCC TGGGAGAAGA CAAGAAGGGG



CTTCTGGGGT


 


6201
CTTGGTAATG TTCTGTTCCT CGTGTGGGGT TGTGCAGTTA



TGATCTGTGC


 


6251
ACTGTTCTGT ATACACATTA TGCTTCAAAA TAACTTCACA



TAAAGAACAT


 


6301
CTTATACCCA GTTAATAGAT AGAAGAGGAA TAAGTAATAG



GTCAAGACCA


 


6351
CGCAGCTGGT AAGTGGGGGG GCCTGGGATC AAATAGCTAC



CTGCCTAATC


 


6401
CTGCCCTCTT GAGCCCTGAA TGAGTCTGCC TTCCAGGGCT



CAAGGTGCTC


 


6451
AACAAAACAA CAGGCCTGCT ATTTTCCTGG CATCTGTGCC



CTGTTTGGCT


 


6501
AGCTAGGAGC ACACATACAT AGAAATTAAA TGAAACAGAC



CTTCAGCAAG


 


6551
GGGACAGAGG ACAGAATTAA CCTTGCCCAG ACACTGGAAA



CCCATGTATG


 


6601
AACACTCACA TGTTTGGGAA GGGGGAAGGG CACATGTAAA



TGAGGACTCT


 


6651
TCCTCATTCT ATGGGGCACT CTGGCCCTGC CCCTCTCAGC



TACTCATCCA


 


6701
TCCAACACAC CTTTCTAAGT ACCTCTCTCT GCCTACACTC



TGAAGGGGTT


 


6751
CAGGAGTAAC TAACACAGCA TCCCTTCCCT CAAATGACTG



ACAATCCCTT


 


6801
TGTCCTGCTT TGTTTTTCTT TCCAGTCAGT ACTGGGAAAG



TGGGGAAGGA


 


6851
CAGTCATGGA GAAACTACAT AAGGAAGCAC CTTGCCCTTC



TGCCTCTTGA


 


6901
GAATGTTGAT GAGTATCAAA TCTTTCAAAC TTTGGAGGTT



TGAGTAGGGG


 


6951
TGAGACTCAG TAATGTCCCT TCCAATGACA TGAACTTGCT



CACTCATCCC


 


7001
TGGGGGCCAA ATTGAACAAT CAAAGGCAGG CATAATCCAG



CTATGAATTC


 


7051
TAGGATCGAT CCAGACATGA TAAGATACAT TGATGAGTTT



GGACAAACCA


 


7101
CAACTAGAAT GCAGTGAAAA AAATGCTTTA TTTGTGAAAT



TTGTGATGCT


 


7151
ATTGCTTTAT TTGTAACCAT TATAAGCTGC AATAAACAAG



TTAACAACAA


 


7201
CAATTGCATT CATTTTATGT TTCAGGTTCA GGGGGAGGTG



TGGGAGGTTT


 


7251
TTTAAAGCAA GTAAAACCTC TACAAATGTG GTATGGCTGA



TTATGATCTC


 


7301
TAGTCAAGGC ACTATACATC AAATATTCCT TATTAACCCC



TTTACAAATT


 


7351
AAAAAGCTAA AGGTACACAA TTTTTGAGCA TAGTTATTAA



TAGCAGACAC


 


7401
TCTATGCCTG TGTGGAGTAA GAAAAAACAG TATGTTATGA



TTATAACTGT


 


7451
TATGCCTACT TATAAAGGTT ACAGAATATT TTTCCATAAT



TTTCTTGTAT


 


7501
AGCAGTGCAG CTTTTTCCTT TGTGGTGTAA ATAGCAAAGC



AAGCAAGAGT


 


7551
TCTATTACTA AACACAGCAT GACTCAAAAA ACTTAGCAAT



TCTGAAGGAA


 


7601
AGTCCTTGGG GTCTTCTACC TTTCTCTTCT TTTTTGGAGG



AGTAGAATGT


 


7651
TGAGAGTCAG CAGTAGCCTC ATCATCACTA GATGGCATTT



CTTCTGAGCA


 


7701
AAACAGGTTT TCCTCATTAA AGGCATTCCA CCACTGCTCC



CATTCATCAG


 


7751
TTCCATAGGT TGGAATCTAA AATACACAAA CAATTAGAAT



CAGTAGTTTA


 


7801
ACACATTATA CACTTAAAAA TTTTATATTT ACCTTAGAGC



TTTAAATCTC


 


7851
TGTAGGTAGT TTGTCCAATT ATGTCACACC ACAGAAGTAA



GGTTCCTTCA


 


7901
CAAAGATCCG GGACCAAAGC GGCCATCGTG CCTCCCCACT



CCTGCAGTTC


 


7951
GGGGGCATGG ATGCGCGGAT AGCCGCTGCT GGTTTCCTGG



ATGCCGACGG


 


8001
ATTTGCACTG CCGGTAGAAC TCCGCGAGGT CGTCCAGCCT



CAGGCAGCAG


 


8051
CTGAACCAAC TCGCGAGGGG ATCGAGCCCG GGGTGGGCGA



AGAACTCCAG


 


8101
CATGAGATCC CCGCGCTGGA GGATCATCCA GCCGGCGTCC



CGGAAAACGA


 


8151
TTCCGAAGCC CAACCTTTCA TAGAAGGCGG CGGTGGAATC



GAAATCTCGT


 


8201
GATGGCAGGT TGGGCGTCGC TTGGTCGGTC ATTTCGAACC



CCAGAGTCCC


 


8251
GCTCAGAAGA ACTCGTCAAG AAGGCGATAG AAGGCGATGC



GCTGCGAATC


 


8301
GGGAGCGGCG ATACCGTAAA GCACGAGGAA GCGGTCAGCC



CATTCGCCGC


 


8351
CAAGCTCTTC AGCAATATCA CGGGTAGCCA ACGCTATGTC



CTGATAGCGG


 


8401
TCCGCCACAC CCAGCCGGCC ACAGTCGATG AATCCAGAAA



AGCGGCCATT


 


8451
TTCCACCATG ATATTCGGCA AGCAGGCATC GCCATGGGTC



ACGACGAGAT


 


8501
CCTCGCCGTC GGGCATGCGC GCCTTGAGCC TGGCGAACAG



TTCGGCTGGC


 


8551
GCGAGCCCCT GATGCTCTTC GTCCAGATCA TCCTGATCGA



CAAGACCGGC


 


8601
TTCCATCCGA GTACGTGCTC GCTCGATGCG ATGTTTCGCT



TGGTGGTCGA


 


8651
ATGGGCAGGT AGCCGGATCA AGCGTATGCA GCCGCCGCAT



TGCATCAGCC


 


8701
ATGATGGATA CTTTCTCGGC AGGAGCAAGG TGAGATGACA



GGAGATCCTG


 


8751
CCCCGGCACT TCGCCCAATA GCAGCCAGTC CCTTCCCGCT



TCAGTGACAA


 


8801
CGTCGAGCAC AGCTGCGCAA GGAACGCCCG TCGTGGCCAG



CCACGATAGC


 


8851
CGCGCTGCCT CGTCCTGCAG TTCATTCAGG GCACCGGACA



GGTCGGTCTT


 


8901
GACAAAAAGA ACCGGGCGCC CCTGCGCTGA CAGCCGGAAC



ACGGCGGCAT


 


8951
CAGAGCAGCC GATTGTCTGT TGTGCCCAGT CATAGCCGAA



TAGCCTCTCC


 


9001
ACCCAAGCGG CCGGAGAACC TGCGTGCAAT CCATCTTGTT



CAATCATGCG


 


9051
AAACGATCCT CATCCTGTCT CTTGATCAGA TCTTGATCCC



CTGCGCCATC


 


9101
AGATCCTTGG CGGCAAGAAA GCCATCCAGT TTACTTTGCA



GGGCTTCCCA


 


9151
ACCTTACCAG AGGGCGCCCC AGCTGGCAAT TCCGGTTCGC



TTGCTGTCCA


 


9201
TAAAACCGCC CAGTCTAGCT ATCGCCATGT AAGCCCACTG



CAAGCTACCT


 


9251
GCTTTCTCTT TGCGCTTGCG TTTTCCCTTG TCCAGATAGC



CCAGTAGCTG


 


9301
ACATTCATCC GGGGTCAGCA CCGTTTCTGC GGACTGGCTT



TCTACGTGTT


 


9351
CCGCTTCCTT TAGCAGCCCT TGCGCCCTGA GTGCTTGCGG



CAGCGTGAAG






SEQ ID NO:18

Nucleotide Sequence of the Expression Vector HCMV-K HuAb-VL1 hum V2 (Complete DNA Sequence of a Humanised Light Chain Expression Vector Comprising SEQ ID NO: 13 (humV2=VLm) from 3926-4246)










1
CTAGCTTTTT GCAAAAGCCT AGGCCTCCAA AAAAGCCTCC




TCACTACTTC


 


51
TGGAATAGCT CAGAGGCCGA GGCGGCCTCG GCCTCTGCAT



AAATAAAAAA


 


101
AATTAGTCAG CCATGGGGCG GAGAATGGGC GGAACTGGGC



GGAGTTAGGG


 


151
GCGGGATGGG CGGAGTTAGG GGCGGGACTA TGGTTGCTGA



CTAATTGAGA


 


201
TGCATGCTTT GCATACTTCT GCCTGCTGGG GAGCCTGGTT



GCTGACTAAT


 


251
TGAGATGCAT GCTTTGCATA CTTCTGCCTG CTGGGGAGCC



TGGGGACTTT


 


301
CCACACCCTA ACTGACACAC ATTCCACAGC TGCCTCGCGC



GTTTCGGTGA


 


351
TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG



GTCACAGCTT


 


401
GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG



CGCGTCAGCG


 


451
GGTGTTGGCG GGTGTCGGGG CGCAGCCATG ACCCAGTCAC



GTAGCGATAG


 


501
CGGAGTGTAT ACTGGCTTAA CTATGCGGCA TCAGAGCAGA



TTGTACTGAG


 


551
AGTGCACCAT ATGCGGTGTG AAATACCGCA CAGATGCGTA



AGGAGAAAAT


 


601
ACCGCATCAG GCGCTCTTCC GCTTCCTCGC TCACTGACTC



GCTGCGCTCG


 


651
GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG



CGGTAATACG


 


701
GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG



TGAGCAAAAG


 


751
GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT



GGCGTTTTTC


 


801
CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC



GCTCAAGTCA


 


851
GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG



TTTCCCCCTG


 


901
GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT



TACCGGATAC


 


951
CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC



ATAGCTCACG


 


1001
CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG



CTGGGCTGTG


 


1051
TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC



CGGTAACTAT


 


1101
CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC



TGGCAGCAGC


 


1151
CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT



GCTACAGAGT


 


1201
TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC



AGTATTTGGT


 


1251
ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG



TTGGTAGCTC


 


1301
TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT



TTTGTTTGCA


 


1351
AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA



TCCTTTGATC


 


1401
TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC



GTTAAGGGAT


 


1451
TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC



CTTTTAAATT


 


1501
AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA



AACTTGGTCT


 


1551
GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG



CGATCTGTCT


 


1601
ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG



ATAACTACGA


 


1651
TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT



ACCGCGAGAC


 


1701
CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC



CAGCCGGAAG


 


1751
GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC



ATCCAGTCTA


 


1801
TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT



TAATAGTTTG


 


1851
CGCAACGTTG TTGCCATTGC TGCAGGCATC GTGGTGTCAC



GCTCGTCGTT


 


1901
TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG



CGAGTTACAT


 


1951
GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG



TCCTCCGATC


 


2001
GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG



TTATGGCAGC


 


2051
ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC



TTTTCTGTGA


 


2101
CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT



GCGGCGACCG


 


2151
AGTTGCTCTT GCCCGGCGTC AACACGGGAT AATACCGCGC



CACATAGCAG


 


2201
AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG



CGAAAACTCT


 


2251
CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC



CACTCGTGCA


 


2301
CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT



CTGGGTGAGC


 


2351
AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG



GCGACACGGA


 


2401
AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG



AAGCATTTAT


 


2451
CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA



TTTAGAAAAA


 


2501
TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG



CCACCTGACG


 


2551
TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA



TAGGCGTATC


 


2601
ACGAGGCCCT TTCGTCTTCA AGAATTCAGC TTGGCTGCAG



TGAATAATAA


 


2651
AATGTGTGTT TGTCCGAAAT ACGCGTTTTG AGATTTCTGT



CGCCGACTAA


 


2701
ATTCATGTCG CGCGATAGTG GTGTTTATCG CCGATAGAGA



TGGCGATATT


 


2751
GGAAAAATCG ATATTTGAAA ATATGGCATA TTGAAAATGT



CGCCGATGTG


 


2801
AGTTTCTGTG TAACTGATAT CGCCATTTTT CCAAAAGTGA



TTTTTGGGCA


 


2851
TACGCGATAT CTGGCGATAG CGCTTATATC GTTTACGGGG



GATGGCGATA


 


2901
GACGACTTTG GTGACTTGGG CGATTCTGTG TGTCGCAAAT



ATCGCAGTTT


 


2951
CGATATAGGT GACAGACGAT ATGAGGCTAT ATCGCCGATA



GAGGCGACAT


 


3001
CAAGCTGGCA CATGGCCAAT GCATATCGAT CTATACATTG



AATCAATATT


 


3051
GGCCATTAGC CATATTATTC ATTGGTTATA TAGCATAAAT



CAATATTGGC


 


3101
TATTGGCCAT TGCATACGTT GTATCCATAT CATAATATGT



ACATTTATAT


 


3151
TGGCTCATGT CCAACATTAC CGCCATGTTG ACATTGATTA



TTGACTAGTT


 


3201
ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC



ATATATGGAG


 


3251
TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT



GACCGCCCAA


 


3301
CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC



ATAGTAACGC


 


3351
CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT



ACGGTAAACT


 


3401
GCCCACTTGG CAGTACATCA AGTGTATCAT ATGCCAAGTA



CGCCCCCTAT


 


3451
TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC



CAGTACATGA


 


3501
CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT



AGTCATCGCT


 


3551
ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC



GTGGATAGCG


 


3601
GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC



GTCAATGGGA


 


3651
GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG



TCGTAACAAC


 


3701
TCCGCCCCAT TGACGCAAAT GGGCGGTAGG CGTGTACGGT



GGGAGGTCTA


 


3751
TATAAGCAGA GCTCGTTTAG TGAACCGTCA GATCGCCTGG



AGACGCCATC


 


3801
CACGCTGTTT TGACCTCCAT AGAAGACACC GGGACCGATC



CAGCCTCCGC


 


3851
AAGCTTGCCG CCACCATGGA GACCCCCGCC CAGCTGCTGT



TCCTGCTGCT


 


3901
GCTGTGGCTG CCCGACACCA CCGGCGACAT TCTGCTGACC



CAGTCTCCAG


 


3951
CCACCCTGTC TCTGAGTCCA GGAGAAAGAG CCACTTTCTC



CTGCAGGGCC


 


4001
AGTCAGAACA TTGGCACAAG CATACAGTGG TATCAACAAA



AAACAAATGG


 


4051
TGCTCCAAGG CTTCTCATAA GGTCTTCTTC TGAGTCTATC



TCTGGGATCC


 


4101
CTTCCAGGTT TAGTGGCAGT GGATCAGGGA CAGATTTTAC



TCTTACCATC


 


4151
AGCAGTCTGG AGCCTGAAGA TTTTGCAGTG TATTACTGTC



AACAAAGTAA


 


4201
TACCTGGCCA TTCACGTTCG GCCAGGGGAC CAAGCTGGAG



ATCAAACGTG


 


4251
AGTATTCTAG AAAGATCCTA GAATTCTAAA CTCTGAGGGG



GTCGGATGAC


 


4301
GTGGCCATTC TTTGCCTAAA GCATTGAGTT TACTGCAAGG



TCAGAAAAGC


 


4351
ATGCAAAGCC CTCAGAATGG CTGCAAAGAG CTCCAACAAA



ACAATTTAGA


 


4401
ACTTTATTAA GGAATAGGGG GAAGCTAGGA AGAAACTCAA



AACATCAAGA


 


4451
TTTTAAATAC GCTTCTTGGT CTCCTTGCTA TAATTATCTG



GGATAAGCAT


 


4501
GCTGTTTTCT GTCTGTCCCT AACATGCCCT GTGATTATCC



GCAAACAACA


 


4551
CACCCAAGGG CAGAACTTTG TTACTTAAAC ACCATCCTGT



TTGCTTCTTT


 


4601
CCTCAGGAAC TGTGGCTGCA CCATCTGTCT TCATCTTCCC



GCCATCTGAT


 


4651
GAGCAGTTGA AATCTGGAAC TGCCTCTGTT GTGTGCCTGC



TGAATAACTT


 


4701
CTATCCCAGA GAGGCCAAAG TACAGTGGAA GGTGGATAAC



GCCCTCCAAT


 


4751
CGGGTAACTC CCAGGAGAGT GTCACAGAGC AGGACAGCAA



GGACAGCACC


 


4801
TACAGCCTCA GCAGCACCCT GACGCTGAGC AAAGCAGACT



ACGAGAAACA


 


4851
CAAAGTCTAC GCCTGCGAAG TCACCCATCA GGGCCTGAGC



TCGCCCGTCA


 


4901
CAAAGAGCTT CAACAGGGGA GAGTGTTAGA GGGAGAAGTG



CCCCCACCTG


 


4951
CTCCTCAGTT CCAGCCTGAC CCCCTCCCAT CCTTTGGCCT



CTGACCCTTT


 


5001
TTCCACAGGG GACCTACCCC TATTGCGGTC CTCCAGCTCA



TCTTTCACCT


 


5051
CACCCCCCTC CTCCTCCTTG GCTTTAATTA TGCTAATGTT



GGAGGAGAAT


 


5101
GAATAAATAA AGTGAATCTT TGCACCTGTG GTTTCTCTCT



TTCCTCATTT


 


5151
AATAATTATT ATCTGTTGTT TACCAACTAC TCAATTTCTC



TTATAAGGGA


 


5201
CTAAATATGT AGTCATCCTA AGGCGCATAA CCATTTATAA



AAATCATCCT


 


5251
TCATTCTATT TTACCCTATC ATCCTCTGCA AGACAGTCCT



CCCTCAAACC


 


5301
CACAAGCCTT CTGTCCTCAC AGTCCCCTGG GCCATGGTAG



GAGAGACTTG


 


5351
CTTCCTTGTT TTCCCCTCCT CAGCAAGCCC TCATAGTCCT



TTTTAAGGGT


 


5401
GACAGGTCTT ACAGTCATAT ATCCTTTGAT TCAATTCCCT



GAGAATCAAC


 


5451
CAAAGCAAAT TTTTCAAAAG AAGAAACCTG CTATAAAGAG



AATCATTCAT


 


5501
TGCAACATGA TATAAAATAA CAACACAATA AAAGCAATTA



AATAAACAAA


 


5551
CAATAGGGAA ATGTTTAAGT TCATCATGGT ACTTAGACTT



AATGGAATGT


 


5601
CATGCCTTAT TTACATTTTT AAACAGGTAC TGAGGGACTC



CTGTCTGCCA


 


5651
AGGGCCGTAT TGAGTACTTT CCACAACCTA ATTTAATCCA



CACTATACTG


 


5701
TGAGATTAAA AACATTCATT AAAATGTTGC AAAGGTTCTA



TAAAGCTGAG


 


5751
AGACAAATAT ATTCTATAAC TCAGCAATCC CACTTCTAGA



TGACTGAGTG


 


5801
TCCCCACCCA CCAAAAAACT ATGCAAGAAT GTTCAAAGCA



GCTTTATTTA


 


5851
CAAAAGCCAA AAATTGGAAA TAGCCCGATT GTCCAACAAT



AGAATGAGTT


 


5901
ATTAAACTGT GGTATGTTTA TACATTAGAA TACCCAATGA



GGAGAATTAA


 


5951
CAAGCTACAA CTATACCTAC TCACACAGAT GAATCTCATA



AAAATAATGT


 


6001
TACATAAGAG AAACTCAATG CAAAAGATAT GTTCTGTATG



TTTTCATCCA


 


6051
TATAAAGTTC AAAACCAGGT AAAAATAAAG TTAGAAATTT



GGATGGAAAT


 


6101
TACTCTTAGC TGGGGGTGGG CGAGTTAGTG CCTGGGAGAA



GACAAGAAGG


 


6151
GGCTTCTGGG GTCTTGGTAA TGTTCTGTTC CTCGTGTGGG



GTTGTGCAGT


 


6201
TATGATCTGT GCACTGTTCT GTATACACAT TATGCTTCAA



AATAACTTCA


 


6251
CATAAAGAAC ATCTTATACC CAGTTAATAG ATAGAAGAGG



AATAAGTAAT


 


6301
AGGTCAAGAC CACGCAGCTG GTAAGTGGGG GGGCCTGGGA



TCAAATAGCT


 


6351
ACCTGCCTAA TCCTGCCCTC TTGAGCCCTG AATGAGTCTG



CCTTCCAGGG


 


6401
CTCAAGGTGC TCAACAAAAC AACAGGCCTG CTATTTTCCT



GGCATCTGTG


 


6451
CCCTGTTTGG CTAGCTAGGA GCACACATAC ATAGAAATTA



AATGAAACAG


 


6501
ACCTTCAGCA AGGGGACAGA GGACAGAATT AACCTTGCCC



AGACACTGGA


 


6551
AACCCATGTA TGAACACTCA CATGTTTGGG AAGGGGGAAG



GGCACATGTA


 


6601
AATGAGGACT CTTCCTCATT CTATGGGGCA CTCTGGCCCT



GCCCCTCTCA


 


6651
GCTACTCATC CATCCAACAC ACCTTTCTAA GTACCTCTCT



CTGCCTACAC


 


6701
TCTGAAGGGG TTCAGGAGTA ACTAACACAG CATCCCTTCC



CTCAAATGAC


 


6751
TGACAATCCC TTTGTCCTGC TTTGTTTTTC TTTCCAGTCA



GTACTGGGAA


 


6801
AGTGGGGAAG GACAGTCATG GAGAAACTAC ATAAGGAAGC



ACCTTGCCCT


 


6851
TCTGCCTCTT GAGAATGTTG ATGAGTATCA AATCTTTCAA



ACTTTGGAGG


 


6901
TTTGAGTAGG GGTGAGACTC AGTAATGTCC CTTCCAATGA



CATGAACTTG


 


6951
CTCACTCATC CCTGGGGGCC AAATTGAACA ATCAAAGGCA



GGCATAATCC


 


7001
AGCTATGAAT TCTAGGATCG ATCCAGACAT GATAAGATAC



ATTGATGAGT


 


7051
TTGGACAAAC CACAACTAGA ATGCAGTGAA AAAAATGCTT



TATTTGTGAA


 


7101
ATTTGTGATG CTATTGCTTT ATTTGTAACC ATTATAAGCT



GCAATAAACA


 


7151
AGTTAACAAC AACAATTGCA TTCATTTTAT GTTTCAGGTT



CAGGGGGAGG


 


7201
TGTGGGAGGT TTTTTAAAGC AAGTAAAACC TCTACAAATG



TGGTATGGCT


 


7251
GATTATGATC TCTAGTCAAG GCACTATACA TCAAATATTC



CTTATTAACC


 


7301
CCTTTACAAA TTAAAAAGCT AAAGGTACAC AATTTTTGAG



CATAGTTATT


 


7351
AATAGCAGAC ACTCTATGCC TGTGTGGAGT AAGAAAAAAC



AGTATGTTAT


 


7401
GATTATAACT GTTATGCCTA CTTATAAAGG TTACAGAATA



TTTTTCCATA


 


7451
ATTTTCTTGT ATAGCAGTGC AGCTTTTTCC TTTGTGGTGT



AAATAGCAAA


 


7501
GCAAGCAAGA GTTCTATTAC TAAACACAGC ATGACTCAAA



AAACTTAGCA


 


7551
ATTCTGAAGG AAAGTCCTTG GGGTCTTCTA CCTTTCTCTT



CTTTTTTGGA


 


7601
GGAGTAGAAT GTTGAGAGTC AGCAGTAGCC TCATCATCAC



TAGATGGCAT


 


7651
TTCTTCTGAG CAAAACAGGT TTTCCTCATT AAAGGCATTC



CACCACTGCT


 


7701
CCCATTCATC AGTTCCATAG GTTGGAATCT AAAATACACA



AACAATTAGA


 


7751
ATCAGTAGTT TAACACATTA TACACTTAAA AATTTTATAT



TTACCTTAGA


 


7801
GCTTTAAATC TCTGTAGGTA GTTTGTCCAA TTATGTCACA



CCACAGAAGT


 


7851
AAGGTTCCTT CACAAAGATC CGGGACCAAA GCGGCCATCG



TGCCTCCCCA


 


7901
CTCCTGCAGT TCGGGGGCAT GGATGCGCGG ATAGCCGCTG



CTGGTTTCCT


 


7951
GGATGCCGAC GGATTTGCAC TGCCGGTAGA ACTCCGCGAG



GTCGTCCAGC


 


8001
CTCAGGCAGC AGCTGAACCA ACTCGCGAGG GGATCGAGCC



CGGGGTGGGC


 


8051
GAAGAACTCC AGCATGAGAT CCCCGCGCTG GAGGATCATC



CAGCCGGCGT


 


8101
CCCGGAAAAC GATTCCGAAG CCCAACCTTT CATAGAAGGC



GGCGGTGGAA


 


8151
TCGAAATCTC GTGATGGCAG GTTGGGCGTC GCTTGGTCGG



TCATTTCGAA


 


8201
CCCCAGAGTC CCGCTCAGAA GAACTCGTCA AGAAGGCGAT



AGAAGGCGAT


 


8251
GCGCTGCGAA TCGGGAGCGG CGATACCGTA AAGCACGAGG



AAGCGGTCAG


 


8301
CCCATTCGCC GCCAAGCTCT TCAGCAATAT CACGGGTAGC



CAACGCTATG


 


8351
TCCTGATAGC GGTCCGCCAC ACCCAGCCGG CCACAGTCGA



TGAATCCAGA


 


8401
AAAGCGGCCA TTTTCCACCA TGATATTCGG CAAGCAGGCA



TCGCCATGGG


 


8451
TCACGACGAG ATCCTCGCCG TCGGGCATGC GCGCCTTGAG



CCTGGCGAAC


 


8501
AGTTCGGCTG GCGCGAGCCC CTGATGCTCT TCGTCCAGAT



CATCCTGATC


 


8551
GACAAGACCG GCTTCCATCC GAGTACGTGC TCGCTCGATG



CGATGTTTCG


 


8601
CTTGGTGGTC GAATGGGCAG GTAGCCGGAT CAAGCGTATG



CAGCCGCCGC


 


8651
ATTGCATCAG CCATGATGGA TACTTTCTCG GCAGGAGCAA



GGTGAGATGA


 


8701
CAGGAGATCC TGCCCCGGCA CTTCGCCCAA TAGCAGCCAG



TCCCTTCCCG


 


8751
CTTCAGTGAC AACGTCGAGC ACAGCTGCGC AAGGAACGCC



CGTCGTGGCC


 


8801
AGCCACGATA GCCGCGCTGC CTCGTCCTGC AGTTCATTCA



GGGCACCGGA


 


8851
CAGGTCGGTC TTGACAAAAA GAACCGGGCG CCCCTGCGCT



GACAGCCGGA


 


8901
ACACGGCGGC ATCAGAGCAG CCGATTGTCT GTTGTGCCCA



GTCATAGCCG


 


8951
AATAGCCTCT CCACCCAAGC GGCCGGAGAA CCTGCGTGCA



ATCCATCTTG


 


9001
TTCAATCATG CGAAACGATC CTCATCCTGT CTCTTGATCA



GATCTTGATC


 


9051
CCCTGCGCCA TCAGATCCTT GGCGGCAAGA AAGCCATCCA



GTTTACTTTG


 


9101
CAGGGCTTCC CAACCTTACC AGAGGGCGCC CCAGCTGGCA



ATTCCGGTTC


 


9151
GCTTGCTGTC CATAAAACCG CCCAGTCTAG CTATCGCCAT



GTAAGCCCAC


 


9201
TGCAAGCTAC CTGCTTTCTC TTTGCGCTTG CGTTTTCCCT



TGTCCAGATA


 


9251
GCCCAGTAGC TGACATTCAT CCGGGGTCAG CACCGTTTCT



GCGGACTGGC


 


9301
TTTCTACGTG TTCCGCTTCC TTTAGCAGCC CTTGCGCCCT



GAGTGCTTGC


 


9351
GGCAGCGTGA AG






Example 9
In vitro Efficacy of CD45RO/RB Binding Humanised Antibodies
To determine the efficacy of the CD45RO/RB binding humanised antibodies VHE/humV1 and VHQ/humV1 in comparison to the chimeric antibody the ability to induce apoptosis in human T cells and also the ability to inhibit human T cell proliferation is analysed.
Cells and Reagents
Peripheral blood mononuclear cells (PBMC) are isolated from leukopheresis samples of healthy human donors with known blood type, but unknown HLA type by centrifugation over Ficoll-Hypaque (Pharmacia LKB). PBMC used as stimulators are first depleted of T and NK cells by using CD3-coated ferromagnetic beads (Miltenyi). Beads and contaminating cells are removed by magnetic field. T cell-depleted PBMC are used as stimulator cells after irradiation (50 Gy). CD4+ T cells are used as responder cells in MLR and are isolated from PBMC with a CD4 T cell negative selection kit (Miltenyi).
The obtained cells are analyzed by FACScan or FACSCalibur (Becton Dickinson & Co., Calif.) and the purity of the obtained cells is >75%. Cells are suspended in RPMI1640 medium supplemented with 10% heat-inactivated FCS, penicillin, streptomycin and L-glutamine.
Apoptosis Assays
Human PBMC of three healthy voluntary donors are cultured in growth medium (RPMI1640+10% FCS) overnight (<16 h) in the presence of CD45RO/RB binding chimeric mAb, humanized antibodies (VHE/humV1 and VHQ/humV1) or anti-LPS control mAb. If indicated, a cross-linking reagent, F(ab′)2-fragment of goat anti-human IgG (Cat. No. 109-006-098, JacksonLab) is included at a μg/ml concentration being twice as high as the sample's anti-CD45 antibodies concentration. The PBS-concentration in all wells introduced by the antibody reagents is kept constant among all samples, namely at 20% (v/v) for samples without cross-linker or at 40% (v/v) for samples with cross-linker. Earlier experiments demonstrate that the amount of PBS does not affect the readout.
After overnight culture in the presence of the antibodies, the samples are subjected to flow cytometry analyses and stained with the apoptosis marker AnnexinV-FITC (Cat. No. 556419, BD/Pharmingen) and the T cell marker CD2-PE (Cat. No. 556609, BD/Pharmingen). The samples are run in a Becton Dickinson FACSCalibur instrument and the data are analyzed using the CellQuest Pro Software.
From the data collected, curves are fitted using the software Origin v7.0300 The equation used for fitting is




A1: final value (for fitting sessions set to “shared” and “floating”)
A2: initial value (for fitting sessions set to “shared” and “floating”)
p: power
X0: ED50; IC50 (see below).
In the absence of cross-linker, VHE/humV1 is most effective, with an ED50 value of 148±71 nM, followed by VHQ/humV1 with 377±219 nM. CD45RO/RB binding chimeric antibody is less effective with an ED50 value of 2440±1205 nM.
In the presence of a cross-linking antiserum, the ED50 values are shifted dramatically towards higher efficacy by at least two orders of magnitude. In addition, the presence of cross-linker permitted higher levels of apoptosis at very high antibody concentrations, now reaching up to 80%, whereas the absence of cross-linker only allowed for up to 50% of apoptosis. In the presence of cross-linker, the curves (antibody concentration/% apoptosis) are bi-modal with two plateaus: the first plateau is reached at low antibody concentrations ( ̃5 nM), where the apoptosis level corresponds to the maximum level obtained in the absence of cross-linker. The second plateau is reached at high antibody concentrations ( ̃500 nM) and apoptosis is observed within 70-80% of the T cell population.
Both CD45R0/RB binding humanised mAb are equally effective and better or equal compared to CD45R0/RB binding chimeric mAb with respect to their ability to induce apoptosis in primary human T cells.
Mixed Lymphocyte Reaction Assays
One ×105 PBMC or 5×104 of CD4+ cells are mixed with 1× or 5×104 T cells-depleted irradiated (50 Gy) PBMC in each well of 96-well culture plates in the presence or absence of the different concentrations of mAb.
The mixed cells are cultured for 5 days and proliferation is determined by pulsing the cells with 3H-thymidine for the last 16-20 hours of culture. MLR inhibition at each antibody concentration is expressed as percentage inhibition as described in Example 2.
The effect of increasing concentrations of VHE/humV1 and VHQ/humV1 on MLR is evaluated in three responder:stimulator combinations. All antibodies inhibit the MLR in a dose-dependent manner. The IC50 values (see above) are similar for the humanized Ab VHE/humV1 (7±7 nM) and VHQ/humV1 (39±54 nM). Both humanised antibodies are more potent in inhibiting MLR than the parental chimeric antibody (IC50 of 347±434 nM). As usually seen with MLR experiments, donor variability is high in these experiments.
Example 10
Specificity of CD45RB/RO Binding Molecule
The CD45 molecule is expressed on all leukocytes. However, different CD45 isoforms are expressed by the various leukocyte subsets. In order to determine the leukocyte subset reactivity of CD45RB/RO binding chimeric antibody molecule immunofluorescent labeling of human leukocytes with subset-specific markers and simultaneous immunofluorescent labeling with a dye-conjugated CD45RB/RO binding chimeric antibody is performed, followed by flow cytometry analysis. Briefly, specific subsets of a freshly isolated preparation of human peripheral blood mononuclear cells (PBMC), human platelets, human peripheral blood neutrophils or human bone-marrow derived hematopoietic stem cells are identified by incubation with phycoerythrin-coupled antibodies against CD2 (T lymphocytes), CD14 (monocytes), CD19 (B lymphocytes), CD34 (stem cells), CD42a (platelets), CD56 (natural killer cells) or CD66b (granulocytes). Simultaneous binding of a FITC-labeled chimeric CD45RB/RO binding molecule is detected on T lymphocytes, monocytes, stem cells, natural killer cells and granulocytes, but not on platelets or B lymphocytes.
Example 11
In Vitro Induction of Suppressor T Cells (T Regulatory Cells) and of Functionally Paralyzed T Cells
To demonstrate the ability of a CD45RO/RB binding chimeric antibody to induce suppressor T cells, the antibody is included at various concentrations during the generation of CD8+ T cell lines reactive with the antigen matrix protein 1 (MP1) of hemophilus influenza. These lines are generated through repeated co-culture of CD8+ human lymphocytes with CD14+ human monocytes pulsed with the antigen. Later on, CD14+ monocytes can be replaced with a human leukocyte antigen-2 positive cell line as an MP1 antigen-presenting cell (APC). If such MP1-specific CD8+ T cells from a culture including CD45RO/RB binding chimeric antibody are mixed with freshly isolated human CD8+ T cells and this mixture of cells is stimulated with the MP1 antigen on APC, the addition of CD8+ T cells from the culture in the presence of CD45RO/RB binding molecule is able to reduce the IFN-γ production in an antibody-dose-dependent fashion. No CD45RO/RB binding chimeric antibody is present during this IFN-γ assay culture, indicating that the pre-treatment with the CD45RO/RB mAb has induced CD8+ T cells capable of suppressing the activation of freshly isolated T cells. Because of this induction of suppressor T regulatory cells by the CD45RO/RB binding chimeric antibody, the antibody may be useful in diseases, where a dysregulated and/or activated T cell population is thought to contribute to the pathology. Examples of such diseases include autoimmune diseases, transplant rejection, psoriasis, inflammatory bowel disease and allergies.
To demonstrate the ability of a chimeric CD45RO/RB binding molecule to render T cells hyporesponsive (anergic) to further stimulation, i.e. to functionally paralyze T cells, the antibody is included during the generation of CD8+ T cell lines reactive with the antigen matrix protein 1 (MP1) of hemophilus influenza as outlined above. Paralysis is assessed by activating the T cells (exposed prior to CD45RO/RB binding chimeric antibody) with MP1 antigen presented by APC. No CD45RO/RB binding molecule is present in this culture. CD8+ T cells not exposed to CD45RO/RB binding chimeric antibody previously produce IFN-γ upon the mentioned stimulus. In contrast, CD8+ T cells pre-treated with CD45RO/RB binding chimeric antibody show a markedly reduced to inexistent production of this cytokine in response to the antigen-stimulus, demonstrating the CD45RO/RB binding chimeric antibody's ability to functionally paralyze human T cells. Because of this induction of functional T cell hyporesponsiveness by the CD45RO/RB binding molecule, the antibody may be used in diseases, such autoimmune diseases, transplant rejection, psoriasis, inflammatory bowel disease or allergies, where an activated T cell population is thought to contribute to the pathology.
Example 12
In Vivo Studies in SCID-hu Skin Mice
In this study, the utility of the CD45RB/RO binding chimeric antibody in a Psoriasis model system is tested. Human skin from normal individuals is transplanted to SCID (SCID-hu Skin) mice and the inflammatory process is mimicked by transferring mononuclear cells of unrelated donors into the SCID-hu Skin mice.
Transplantation of Human Adult Skin in SCID Mice (SCID-hu Skin Mice)
Two small pieces (1 cm2) of human adult skin (obtained from the West Hungarian Regional Tissue Bank; WHRTB, Gyor) consisting of the entire epidermis, the papillary dermis and part of the reticular dermis, are transplanted at the right and left upper-back sides of SCID mice C.B 17/GbmsTac-Prkdcscid Lystbg mice (Taconic, Germantown, N.Y.) in replacement of mouse skin. The quality of the grafts is monitored during 5-6 weeks following transplantation and successfully transplanted mice (SCID-hu Skin mice, generally >85%) are selected for in vivo testing of CD45RB/RO binding chimeric antibody.
Engraftment of Human Mononuclear Cells in SCID Mice
Mononuclear splenocytes (Spl) are isolated from human adult spleen biopsies (WHRTB, Gyor) after cell suspension (using a cell dissociation sieve equipped with a size 50 mesh) and standard density gradient procedures. Aliquots of  ̃5×108 Spl are re-suspended in 1.5 ml of RPMI-10% FCS and injected intraperitoneally (i.p.), on experimental day 0, into the SCID-hu Skin mice. These Spl numbers have been found in previous experiments to be sufficient to induce a lethal xeno-GvHD in >90% of the mice within 4-6 weeks after cell transfer.
Antibody Treatment of SCID-hu Skin Mice
SCID-hu Skin mice, reconstituted with human Spl, are treated with CD45RB/RO binding chimeric antibody or with anti-LPS control mAb at day 0, immediately after mononuclear cell injection, at days 3 and 7 and at weekly intervals thereafter. Antibodies are delivered subcutaneously (s.c.) in 100 μl PBS at a final concentration of 1 mg/kg body weight (b.w.).
Evaluation of Anti-CD45 Treatment
The efficacy of CD45RB/RO binding chimeric antibody is assessed by the survival of the transplanted mice and by monitoring the rejection of the skin grafts. The significance of the results is evaluated by the statistical method of survival analysis using the Log-rank test (Mantel method) with the help of Systat v10 software. At the end of the experiment biopsies of human skin grafts and mouse liver, lung, kidney and spleen are obtained from sacrificed mice for histological purposes. All mice are weighed at the beginning (before cell transfer) and throughout the experiment (every two days) as an indirect estimation of their health status. Linear regression lines are generated using the body weight versus days post-PBMC transfer values obtained from each mouse and subsequently, their slopes (control versus anti-CD45 treated mice) are compared using the non parametric Mann-Whitney test.
Results
The human skin grafts are very well tolerated by the SCID mice. Initially, the grafts undergo a period of keratinocyte hyperproliferation resulting in the formation of hyperkeratotic crusts. About 5 weeks after transplantation, the crusts fall off the grafts and reveal a tissue containing all the characteristic structures observed in normal human skin. During this process, the human skin grafts fuse with the adjacent mouse skin and generate a network of freshly grown human vessels that connect the grafts with the underlying mouse tissue. The circulating human Spl transferred into SCID-hu Skin mice (at experimental day 0, approx. 6 weeks after skin transplantation) infiltrate the skin grafts and after recognition of alloantigen molecules expressed on the human skin mount an inflammatory response that in some cases completely destroy the graft.
Treatment of these mice with CD45RB/RO binding chimeric antibody suppresses the inflammatory process and prevents the rejection of the human skin grafts. In contrast, the sample obtained from the control treated mouse shows a massive infiltration with multiple signs of necrosis and a dramatic destruction of the epidermis. This process is easily monitored by eye and documented by simple photography of the mice.
Six out of six SCID-hu Skin mice transferred with allogeneic human Spl and treated with control anti-LPS mAb show a strong inflammatory response clearly visible by eye 23 days after mononuclear cell transfer. All mice show considerable lesions, including erythema, scaling and pronounced pustules. In contrast the skin grafts of all mice treated with CD45RB/RO binding chimeric antibody have a normal appearance. The dramatic differences between the two groups of mice is specifically due to the antibody treatment since the human skin of all mice have an identical look at the beginning of the experiment. This aspect is not changed until the second week after cell transfer, the time at which the control group started to developed skin lesions. The experiment is terminated at day 34 after mononuclear cell transfer. By that time, one of the control mice is already dead (day 30) and four other are sacrificed (days 27, 27, 27 and 30) due to a strong xeno-GvHD. The pathologic reactions observed in the antibody control treated mice also correlates with a loss of body weight in these animals.
In contrast, the CD45RB/RO binding chimeric antibody treated group displays a healthy status during the whole experimentation time.

## Claims

1. An isolated antibody which binds CD45RB/CD45RO comprising the polypeptide of SEQ ID NO:8 and the polypeptide of SEQ ID NO: 31.

2. An isolated antibody which binds CD45RB/CD45RO comprising:
a) a first domain comprising the hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence Asn-Tyr-Ile-Ile-His (NYIIH) (SEQ ID NO:22), said CDR2 having the amino acid sequence Tyr-Phe-Asn-Pro-Tyr-Asn-His-Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (YFNPYNHGTKYNEKFKG) (SEQ ID NO:23) and said CDR3 having the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (SGPYAWFDT) (SEQ ID NO:24); and
b) a second domain comprising the hypervariable regions CDR1′, CDR2′ and CDR3′, said CDR1′ having the amino acid sequence Arg-Ala-Ser-Gln-Asn-Ile-Gly-Thr-Ser-Ile-Gln (RASQNIGTSIQ) (SEQ ID NO:19), CDR2′ having the amino acid sequence Ser-Ser-Ser-Glu-Ser-Ile-Ser (SSSESIS) (SEQ ID NO:20) and CDR3′ having the amino acid sequence Gln-Gln-Ser-Asn-Thr-Trp-Pro-Phe-Thr (QQSNTWPFT) (SEQ ID NO:21).

3. The isolated antibody according to claim 2, which is a chimeric or humanised monoclonal antibody.

4. The isolated antibody according to claim 2, comprising the polypeptide of SEQ ID NO: 31.

5. The isolated antibody according to claim 2, comprising the polypeptide of SEQ ID NO:8.

6. The isolated antibody according to claim 4 which is a chimeric monoclonal antibody.

7. An isolated antibody which binds CD45RB/CD45RO which is a humanised antibody comprising the polypeptide of SEQ ID NO:8 and the polypeptide of SEQ ID NO: 31.

8. A pharmaceutical composition comprising the antibody according to claim 1 in association with at least one pharmaceutically acceptable carrier or diluent.

9. A pharmaceutical composition comprising the antibody according to claim 2 in association with at least one pharmaceutically acceptable carrier or diluent.

10. The isolated antibody according to claim 1, which is a chimeric or humanised monoclonal antibody.

11. An isolated antibody that binds CD45RB/CD45RO comprising the polypeptide of SEQ ID NO: 31.

12. The isolated antibody according to claim 11, which is a chimeric or humanised monoclonal antibody.

13. A pharmaceutical composition comprising an isolated antibody according to claim 11, in association with at least one pharmaceutically acceptable carrier or diluent.