US20050069538A1

US20050069538A1 - Therapeutic binding molecules

Info

Publication number: US20050069538A1
Application number: US10/666,332
Authority: US
Inventors: Gregorio Aversa; Frank Kolbinger; Jose Carballido Herrera; Andras Aszodi; Jose Saldanha; Bruce Hall
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-09-18
Filing date: 2003-09-18
Publication date: 2005-03-31
Also published as: US20110076270A1; CN100422213C; US7825222B2; ZA200601418B; CN1849339A; US20060088525A1

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-Gin (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.

Description

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-deficicient 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 P C L 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 CD45RO/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 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 IC₅₀of about less than 100 nM, preferably less than 50 nM or 30 nM, more preferably with an IC₅₀of about 10 or 5 nM, most preferably with an IC₅₀of 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 admistiered 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 naive 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-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. 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 GIn-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 specifity, 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 polpypeptide 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 deamidated 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

- GGCCAGTCAGAACATTGGCACMGCATACAGTG, encoding the amino acid sequence of CDR1,
- TTCTTCTGAGTCTATCTCTGG; encoding the amino acid sequence of CDR 2,
- ACAMGTMTACCTGGCCATTCACGTT encoding the amino acid sequence of CDR 3,
- TTATATTATCCACTG, encoding the amino acid sequence of CDR1′,
- TTTTMTCCTTACAATCATGGTACTMGTACMTGAGMGTTCAAAGGCAG 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 binding molecule, e.g. encoding the amino acid sequence of CDR1, CDR2 and CDR3 according to the present invention and/or, preferably and, polynucletides 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:1 1 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 framework 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 promotor(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 impairement 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 rCTLA-4 (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.
Autoimune 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
IgG 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
T_regT 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 FicoII-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., CA) 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-CD45RO/RB mAb “candidate mAb” and an isotype matched control chimeric antibody is also generated. Mouse (Human) control IgG₁antibody 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 1×10⁵PBMC or 5×10⁴of CD3⁺ or CD4⁺ cells are mixed with 1×10⁵irradiated PBMC or 5×10⁴T 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′)₂fragment 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% CO₂and 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 IgG, (10 μg/ml) both with a second step reagent, F(ab′)₂fragment 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 IC₅₀for 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′)₂fragment 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. IC₅₀value is determined using Origin (V. 6.0®). The cellular activity IC₅₀value 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 FicoII-Hypaque gradient to remove dead cells, washed twice with RPMI, and restimulated with the same stimulator, 3^rdparty 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′)₂fragment 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 2^ryMLR

#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 unresponsivess 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 IgG, control Ab. These data indicate that the impaired secondary response in T cells treated with a “candidate mAb” is due to 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: $\frac{c . p . m . with control Ab - c . p . m . {with}^{″} candidate mAb}{c . p . m . with control Ab} \times 100$
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-Prkdc^scidLyst^bgmice (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 BgIII. 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. N° #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 SuperFect™ Transfection Reagent (Cat. N°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, 4×10⁶COS 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. N° 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. N° 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 8×10⁶live 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. N° 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. 10⁵cells 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. N° 109-005-098, Jackson Labs) and a peroxidase-coupled antibody against human kappa light chain (Cat. N° 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. N° 4-39454, Nunc Immunoplate Maxisorp) are coated overnight at 4° C. with 100 μl of goat anti-human IgG (whole molecule, Cat. N° I101 1, SIGMA) at the final concentration of 0.5 μg/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. I4506, SIGMA) are prepared by serial 1.5-fold dilution in blocking buffer. 100 μl of diluted samples or standard are transfered 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. N° A-7164, SIGMA) diluted at {fraction (1/4000)} in blocking buffer. Control wells received 100 μl of blocking buffer or concentrated normal culture medium. After washing, the calorimetric quantification of bound peroxidase in the sample and standard wells is performed, using a TMB Peroxidase EIA Substrate Kit (Cat. N° 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 calorimetric 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 microtitter plate at a cell density of 1×10⁵cells 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 1.5×10⁵PEER 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 V HQ/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-AAD) 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-AAD positive cells than two-fold on day 2 after activation. On day 7, the portion of 7-AAD 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)



Part of the amino acid sequence of chimeric light chain

DILLTQSPAILSVSPGERVSFSCRASQNIGTSIQWYQQRTNGSPRLLIRSSSESISGIPSRFSG	SEQ ID NO:1

SGSGTDFTLSINSVESEDIADYYCQQSNTWPFTFGSGTKLEIK

Part of the amino acid sequence of chimeric heavy chain

EVQLQQSGPELVKPGASVKMSCKASGYTFTNYIIHWVKQEPGQGLEWIGYFNPYNHGTKY	SEQ ID NO:2

NEKFKGRATLTADKSSNTAYMDLSSLTSEDSAIYYCARSGPYAWFDTWGQGTTVTVSS

Amino acid sequence of chimeric light chain

DILLTQSPAILSVSPGERVSFSCRASQNIGTSIQWYQQRTNGSPRLLIRSSSESISGIPSRFSG	SEQ ID NO:3

SGSGTDFTLSINSVESEDIADYYCQQSNTWPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKS

GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE

KHKVYACEVTHQGLSSPVTKSFNRGEC

Amino acid sequence of chimeric heavy chain

EVQLQQSGPELVKPGASVKMSCKASGYTFTNYIIHWVKQEPGQGLEWIGYFNPYNHGTKY	SEQ ID NO:4

NEKFKGRATLTADKSSNTAYMDLSSLTSEDSAIYYCARSGPYAWFDTWGQGTTVTVSSAS

TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV

SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGK

Nucleotide sequence encoding a polypeptide of SEQ ID NO:1

GACATTCTGCTGACCCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCA	SEQ ID NO:5

GTTTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAGA

ACAAATGGTTCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCCT

TCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGA

GTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTCGG

CTCGGGGACCAAGCTTGAAATCAAA

Nucleotide sequence encoding a polypeptide of SEQ ID NO:2

GAGGTGCAGCTGCAGCAGTCAGGACCTGAACTGGTAAAGCCTGGGGCTTCAGTGAAG	SEQ ID NO:6

ATGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCA

GGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTA

AGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAGACAAATCCTCCAACACA

GCCTACATGGACCTCAGCAGCCTGACCTCTGAGGACTCTGCGATCTACTACTGTGCAA

GATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTC

CTCA

Part of amino acid sequence of humanised light chain designated humV2 (humV2 =

VLm)

DILLTQSPAT LSLSPGERAT FSCRASQNIG TSIQWYQQKT NGAPRLLIRS SSESISGIPS	SEQ ID NO:7

RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SNTWPFTFGQ GTKLEIK

Part of amino acid sequence of humanised light chain designated humV1 (humV1 =

VLh)

DILLTQSPAT LSLSPGERAT LSCRASQNIG TSIQWYQQKP GQAPRLLIRS SSESISGIPS	SEQ ID NO:8

RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SNTWPFTFGQ GTKLEIK

Part of amino acid sequence of humanised heavy chain designated VHE

EVQLVESGAE VKKPGASVKV SCKASGYTFT NYIIHWVKQE PGQGLEWIGY	SEQ ID NO:9

FNPYNHGTKY NEKFKGRATL TANKSISTAY MELSSLRSED TAVYYCARSG

PYAWFDTWGQ GTTVTVSS

Part of amino acid sequence of humanised heavy chain designated VHQ

QVQLVESGAE VKKPGASVKV SCKASGYTFT NYIIHWVKQE PGQGLEWIGY	SEQ ID NO:10

FNPYNHGTKY NEKFKGRATL TANKSISTAY MELSSLRSED TAVYYCARSG

PYAWFDTWGQ GTTVTVSS

Nucleotide sequence encoding amino acid sequence SEQ ID NO:9

GAGGTGCAGCTGGTGGAGTCAGGAGCCGAAGTGAAAAAGCCTGGGGCTTCAGTGAAG	SEQ ID NO:11

GTGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCA

GGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTA

AGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAAACAAATCCATCAGCACA

GCCTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACTGCGGTCTACTACTGTGCAA

GATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTC

CTCA

Nucleotide sequence encoding amino acid sequence SEQ ID NO:10

CAGGTGCAGCTGGTGGAGTCAGGAGCCGAAGTGAAAAAGCCTGGGGCTTCAGTGAAG	SEQ ID NO:12

GTGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCA

GGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTA

AGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAAACAAATCCATCAGCACA

GCCTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACTGCGGTCTACTACTGTGCAA

GATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTC

CTCA

Nucleotide sequence encoding amino acid sequence SEQ ID NO:7

GACATTCTGCTGACCCAGTCTCCAGCCACCCTGTCTCTGAGTCCAGGAGAAAGAGCCA	SEQ ID NO:13

CTTTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAAA

ACAAATGGTGCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCC

TTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTACCATCAGCAGTCTGG

AGCCTGAAGATTTTGCAGTGTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTC

GGCCAGGGGACCAAGCTGGAGATCAAA

Nucleotide sequence encoding amino acid sequence SEQ ID NO:8

GACATTCTGCTGACCCAGTCTCCAGCCACCCTGTCTCTGAGTCCAGGAGAAAGAGCCA	SEQ ID NO:14

CTCTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAAA

CCAGGTCAGGCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCC

TTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTACCATCAGCAGTCTGG

AGCCTGAAGATTTTGCAGTGTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTC

GGCCAGGGGACCAAGCTGGAGATCAAA

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	SEQ ID NO:15

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	AAAAPACTTA	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

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	SEQ ID NO:16

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	TCCGCTCGGT

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	GAPAAAAATG	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

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	SEQ ID NO:17

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

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	SEQ ID NO:18

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

902	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., CA) 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 pg/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 ED₅₀value of 148±71 nM, followed by VHQ/humV1 with 377±219 nM. CD45R0/RB binding chimeric antibody is less effective with an ED₅₀value of 2440±1205 nM.
In the presence of a cross-linking antiserum, the ED₅₀values 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×10⁵PBMC or 5×10⁴of CD4⁺ cells are mixed with 1×10⁵or 5×10⁴T 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 IC₅₀values (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 (IC₅₀of 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-Prkdc^scidLyst^bgmice (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 (SpI) 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 −5×10⁸SpI 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 SpI 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 SpI, 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 SpI 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 SpI 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. A binding 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).

2. A binding molecule according to claim 1 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′, said 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).

3. A binding molecule according to claim 1, which is a chimeric or humanised monoclonal antibody.

4. A binding molecule according to claim 1, comprising a polypeptide of SEQ ID NO:1 and/or a polypeptide of SEQ ID NO:2.

5. A binding molecule according to claim 1, comprising a polypeptide of SEQ ID NO:3 and/or a polypeptide of SEQ ID NO:4.

6. A binding molecule according to claim 4 which is a chimeric monoclonal antibody.

7. A binding molecule which is 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.

8. A binding molecule which is 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.

9. Isolated polynucleotides comprising polynucleotides encoding a binding molecule according to claim 1.

10. Polynucleotides according to claim 9 encoding the amino acid sequence of CDR1, CDR2 and CDR3, according to 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).

11. Polynucleotides comprising a polynucleotide of SEQ ID NO: 5 and/or a polynucleotide of SEQ ID NO: 6.

12. Polynucletides 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.

13. Polynucleotides comprising a polynucleotide of SEQ ID NO:1 1 or of SEQ ID NO:12 and a polynucleotide of SEQ ID NO:13 or a polynucleotide of SEQ ID NO:14.

14. An expression vector comprising polynucleotides according to claim 9.

15. An expression system comprising a polynucleotide according to claim 9, wherein said expression system or part thereof is capable of producing a binding 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), when said expression system or part thereof is present in a compatible host cell.

16. An isolated host cell which comprises an expression system according to claim 15.

17. Use of a molecule or of a humanised antibody according to claim 1 as a pharmaceutical.

18. Use according to claim 17 in the treatment and/or prophylaxis of autoimmune diseases, transplant rejection, psoriasis, inflammatory bowel disease and allergies.

19. A pharmaceutical composition comprising a molecule or a humanised antibody according to claim 1 in association with at least one pharmaceutically acceptable carrier or diluent.

20. 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 molecule or a humanised antibody according to claim 1.

21. Use of a binding molecule having a binding specificity for both CD45RO and CD45RB in medicine.

22. The use according to claim 21, wherein the binding molecule is a chimeric, a humanised or a fully human monoclonal antibody.

23. The use according to claim 21, wherein the binding molecule binds to a CD45RO isoform with a dissociation constant (Kd) <15 nM.

24. The use according to claim 21, wherein the binding molecule binds to a CD45RB isoform with a dissociation constant (Kd) <15 nM.

25. The use according to claim 21, wherein the binding molecule binds CD45 isoforms which

include the A and B epitopes, but not the C epitope of the CD45 molecule; and/or

include the B epitope, but not the A and not the C epitope of the CD45 molecule; and/or

isoforms which do not include any one of the A, B or C epitopes of the CD45 molecule.

26. The use according to claim 21, wherein the binding molecule does not bind CD45 isoforms which

include all of the A, B and C epitopes of the CD45 molecule; and/or

include both the B and C epitopes, but not the A epitope of the CD45 molecule.

27. The use according to claim 21, wherein the binding molecule binds to its target epitope on PEER cells, and wherein said binding is with a Kd<15 nM.

28. Polynucleotides encoding the amino acid sequence of CDR1′, CDR2′ and CDR3′, according to said 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-GIn-Ser-Asn-Thr-Trp-Pro-Phe-Thr (QQSNTWPFT).

29. An expression system comprising a polynucleotide according to claim 10, wherein said expression system or part thereof is capable of producing a binding 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), when said expression system or part thereof is present in a compatible host cell.

30. An expression system comprising a polynucleotide according to claim 11, wherein said expression system or part thereof is capable of producing a binding 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), when said expression system or part thereof is present in a compatible host cell.

31. An expression system comprising a polynucleotide according to claim 12, wherein said expression system or part thereof is capable of producing a binding 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), when said expression system or part thereof is present in a compatible host cell.

32. An expression system comprising a polynucleotide according to claim 13, wherein said expression system or part thereof is capable of producing a binding 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), when said expression system or part thereof is present in a compatible host cell.

33. An expression system comprising a polynucleotide according to claim 28, wherein said expression system or part thereof is capable of producing a binding molecule of 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), when said expression system or part thereof is present in a compatible host cell.