HK1071769B - Therapeutic binding molecules - Google Patents

Description

Therapeutic binding molecules

Technical Field

The present invention relates to organic compounds, such as molecules that bind to the CD45 antigen isoform, such as monoclonal antibodies (mabs).

Background

One approach to treating various diseases is to remove or inactivate pathogenic leukocytes and to obtain the potential to induce tolerance to inactivate the pathological immune response.

Organ, cell and tissue transplant rejection and various autoimmune diseases are thought to be caused primarily by T cell-mediated immune responses triggered by helper T cells; helper T cells recognize specific antigens that are captured by antigen presenting cells (APCs, e.g., macrophages and dendritic cells), processed, and presented to the helper T cells in the form of antigen-MHC complexes, i.e., upon recognition of a specific antigen, the helper T cells are stimulated to produce cytokines (e.g., IL-2) and express or up-regulate some cytokine receptors and other activating molecules and expand. Some of these activated helper T cells may act directly or indirectly, i.e. helper effector cells, cytotoxic T cells or B cells, to destroy cells or tissues expressing the selected antigen. After termination of the immune response, some of the mature clonally selected cells remain in the form of memory helper cells and memory cytotoxic T cells, circulating in the body, and recognizing the antigen rapidly when it reappears. If the antigen that triggers this response is a non-toxic environmental antigen, the result is allergy; if the antigen is not a foreign antigen but an autoantigen, the result is an autoimmune disease; if the antigen is an antigen from a transplanted organ, transplant rejection results.

The immune system has evolved to recognize self and heterosis. This property enables organisms to survive in the environment exposed to the daily threat of pathogens. During the development of the T cell pool in the thymus, specificity for isohexia and tolerance to self are developed by the process of positive and negative selection, which also includes the recognition and elimination of autoreactive T cells. Such tolerance is called central tolerance. However, some of these autoreactive cells can escape this selection mechanism and pose a potential risk for the development of autoimmune diseases. To control autoreactive T cells that escape to the periphery, the immune system has a peripheral regulatory mechanism that prevents autoimmunity. These mechanisms are the basis for peripheral tolerance.

Cell surface antigens recognized by a particular monoclonal antibody are generally designated by the CD (cluster of differentiation) number, which is designated by the continuous international leukocyte typing seminar, and the term CD45 as used herein refers to the cell surface leukocyte common antigen CD 45; monoclonal antibodies that recognize this antigen are referred to herein as "anti-CD 45".

Leukocyte Common Antigen (LCA) or CD45 is the major component of anti-lymphocyte globulin (ALG). CD45 belongs to the family of transmembrane tyrosine phosphatases and is a positive and negative regulator of cell activation (depending on the interaction with the receptor). The phosphatase activity of CD45 appears to be required for activation of the Src-kinase family associated with antigen receptors of B and T lymphocytes (Trowbridge IS et al, AnnuRev Immunol.1994; Vol.12: pp.85-116). Thus, CD45 is required for signal 1 in T cell activation, and CD45 deficient cells are severely deficient in TCR-mediated activation events.

The CD45 antigen is a diverse isoform that includes a family of transmembrane glycoproteins. The different CD45 isoforms differ in their extracellular domain structure, which results from the selective splicing of 3 variable exons encoding part of the extracellular region of CD 45. (Streuli MF. et al, J.Exp.Med.1987; Vol. 166: p. 1548-1566). Various CD45 isoforms have different extracellular domains; but have identical transmembrane and cytoplasmic segments, and two homologous, highly conserved phosphatase domains of approximately 300 residues. Different combinations of isoforms are differentially expressed on subsets of T and B lymphocytes (Thomas ML. et al, Immunol. today 1988; Vol. 9: pp. 320-325). Some monoclonal antibodies recognize epitopes common to all of these different isoforms, while others have limited (CD45R) specificity, depending on which of the selectively spliced exons (a, B or C) they recognize. For example, a monoclonal antibody recognizing the exon A product is therefore designated CD45RA, and a monoclonal antibody recognizing various isoforms containing exon B is designated CD45RB (Beverley PCL et al, Immunol. Supp. 1988; Vol.1: pages 3-5). Antibodies such as UCHL1 selectively bind to the 180kDa isoform CD45RO (without either of the variable exons A, B or C), this CD45RO is restricted to appearing on subsets of activated T cells, memory cells and cortical thymocytes and not detected on B cells (Terry LA et al, Immunol.1988; Vol.64: p. 331-336).

Brief Description of Drawings

FIG. 1 shows that the inhibition of primary MLR by "candidate monoclonal antibodies" is dose dependent, ranging from 0.001 to 10. mu.g/ml. The "concentration" is the concentration of the "candidate monoclonal antibody".

FIG. 2 shows a plasmid map of the expression vector HCMV-G1 HuAb-VHQ, expressed at the full expression vector nucleic acid sequence SEQ ID NO: 15 comprises a nucleic acid sequence having the sequence SEQ ID NO: 12 (3921-4274).

FIG. 3 shows a plasmid map of the expression vector HCMV-G1 HuAb-VHE, expressed at the full expression vector nucleic acid sequence SEQ ID NO: 16 comprises a nucleic acid sequence having the nucleic acid sequence SEQ ID NO: 11 (3921-4274).

FIG. 4 shows a plasmid map of expression vector HCMV-K HuAb-humV1, expressed at the full expression vector nucleic acid sequence SEQ ID NO: 17 comprises a nucleic acid sequence having the sequence SEQ ID NO: 14 (3964-.

FIG. 5 shows a plasmid map of the expression vector HCMV-K HuAb-humV2, expressed at the full expression vector nucleic acid sequence SEQ ID NO: 18 comprises a nucleic acid sequence having the nucleic acid sequence SEQ ID NO: 13 (3926) -4246).

Detailed Description

We have now found binding molecules comprising polypeptide sequences that bind CD45RO and CD45RB, hereinafter also referred to as "CD 45RO/RB binding molecules". These binding molecules according to the invention can induce immunosuppression, suppress primary T cell responses and induce T cell tolerance. Furthermore, the binding molecules of the invention inhibit primary Mixed Lymphocyte Reaction (MLR). Cells from CD45RO/RB binding molecule treated cultures also preferably have an impaired proliferative response in secondary MLRs, even in the absence of CD45RO/RB binding molecules in secondary MLRs. These impaired proliferative responses in secondary MLRs indicate the ability of the binding molecules of the invention to induce tolerance. In addition, administration of CD45RO/RB binding molecules in vivo to severe combined immunodeficiency mice (SCID) suffering from xenograft versus host disease (X-GVHD) following injection of human PBMC, compared to control-treated mice, extended survival of mice despite the detection of circulating human T cells in mice treated with CD45RO/RB binding molecules.

"CD 45RO/RB binding molecule" refers to any molecule that, alone or in association with other molecules, specifically binds to the CD45RB and CD45RO isoforms of the CD45 antigen. Binding reactions can be shown by standard methods (qualitative assays) including, for example: any type of binding assay, such as direct or indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay, in combination with fluorescence microscopy or cytofluorimetric analysis (FACS), in which the binding of molecules to cells expressing a particular CD45 isoform can be visualized. In addition, binding of such molecules may result in altered function of cells expressing these isoforms. For example, inhibition of primary or secondary Mixed Lymphocyte Reaction (MLR) can be determined, e.g., in vitro assays or biological assays for determining inhibition of primary or secondary MLR in the presence and absence of CD45RO/RB binding molecules and for determining differences in primary MLR inhibition.

Alternatively, it is also possible to use polyclonal stimulators (e.g.phytohemagglutinin (PHA) or anti-CD 3 and anti-CD 28 antibodies or phorbol esters and Ca), for example after cell activation in MLR, or after stimulation with specific antigens (e.g.tetanus toxin or other antigens), or with polyclonal stimulators²⁺Ionophore) stimulation by measuring PBMC or T cells or CD4⁺Proliferation of T cells, production of cytokines, alteration of cell surface molecule expression to determine in vitro functional regulation. The setup of the culture was similar to that described for MLR, except that generationFor allogeneic cells as stimuli, soluble antigens or polyclonal stimuli (e.g., as mentioned above) are used. Preferably by incorporation in the manner described above³H-thymidine was used to measure T cell proliferation. Cytokine production is preferably measured by a sandwich ELISA, in which cytokine capture antibodies are coated on the surface of 96-well plates, supernatants from cultures are added and incubated at room temperature for 1 hour, detection antibodies specific for a particular cytokine are added, then the corresponding substrate is added after addition of a secondary antibody linked to an enzyme (e.g. horseradish peroxidase) and the absorbance is measured on a plate reader. Preferably, the alteration of a cell surface molecule is measured by direct or indirect immunofluorescence after staining the target cell with an antibody specific for the particular cell surface molecule. The antibody may be directly labeled with a fluorescent dye or a secondary antibody fluorescently labeled specific to the primary antibody may be used and the cells analyzed with a cytofluorometer.

The binding molecules of the invention have binding specificity for both CD45RO and CD45RB ("CD 45RO/RB binding molecules").

Preferably, the binding molecule binds to an isoform of CD45RO with a dissociation constant (Kd) < 20nM, preferably with a Kd < 15nM or < 10nM, more preferably with a Kd < 5 nM. Preferably, the binding molecule binds to an isoform of CD45RB with a Kd < 50nM, preferably with a Kd < 15nM or < 10nM, more preferably with a Kd < 5 nM.

In another preferred embodiment, the binding molecules of the invention bind to the CD45 isoform, which is

(1) Including the a and B epitopes, but not the C epitope, of the CD45 molecule; and/or

(2) Including the B epitope, but not the a and C epitopes, of the CD45 molecule; and/or

(3) Either of the A, B or C epitopes of CD45 molecule were excluded.

In a further preferred embodiment, the binding molecule of the invention does not bind to such CD45 isoforms, which include

(1) All of the A, B and C epitopes of CD45 molecule; and/or

(2) The B and C epitopes, but not the a epitope, of the CD45 molecule.

In other preferred embodiments, the binding molecules of the invention may also be

(1) Recognizing memory T cells and autoreactive T cells in vivo; and/or

(2) Target for binding on human T cells, e.g., PEER cells; wherein preferably said binding has a Kd < 15nM, more preferably Kd < 10nM, most preferably Kd < 5 nM; and/or

(3) Inhibition of autoreactive T cell function in vitro, preferably IC₅₀About 5nM, more preferably IC₅₀About 1nM, most preferably IC₅₀About 0.5nM or even 0.1 nM; and/or

(4) Inducing alloantigen-specific T cells outside the conductor to be tolerant; and/or

(5) Lethal xenograft-versus-host disease (GvHD) induced in SCID mice by injection of effective doses of human PBMC was prevented.

In yet another preferred embodiment, the binding molecules of the invention bind to the same epitope as monoclonal antibody "A6", described in antibody "A6" by Aversa et al, Cellular Immunology, Vol. 158, pp. 314-328 (1994).

Because of the binding properties and biological activities described above, these binding molecules of the invention are particularly useful medically for treatment and/or prevention. Diseases in which the binding molecules of the invention are particularly useful include autoimmune diseases, transplant rejection, psoriasis, inflammatory bowel disease and allergies, as will be further set forth below.

We have found that a polypeptide comprising SEQ ID NO: 1 and polypeptide SEQ ID NO: 2 is a CD45RO/RB binding molecule. We also found a peptide having SEQ ID NO: 1, wherein CDR1 ' has the amino acid sequence Arg-Ala-Ser-Gln-Asn-Ile-Gly-Thr-Ser-Ile-Gln (rasqnigtsiq), CDR2 ' has the amino acid sequence Ser-Glu-Ser-Ile-Ser (sssesis), CDR3 ' has the amino acid sequence gin-Ser-Asn-Thr-Trp-Pro-Phe-Thr (qqsntwpft).

We also found a peptide having SEQ ID NO: 2 wherein CDR1 has the amino acid sequence Asn-Tyr-Ile-His (nyiih), CDR2 has the amino acid sequence Tyr-Phe-Asn-Pro-Tyr-Asn-His-Gly-Thr-Lys-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (yfynhgtkynekfkg), and CDR3 has the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (sgpyfdt).

The CDRs are 3 specific complementarity determining regions, also known as hypervariable regions, which essentially determine the antigen-binding characteristics. These CDRs are, for example, SEQ ID NOs: 1 or SEQ ID NO: 2, wherein the CDRs alternate with Framework Regions (FRs) (e.g., constant regions). In the chimeric antibody according to the present invention, SEQ ID NO: 1 is a light chain such as SEQ ID NO: 3, SEQ ID NO: 2 is a heavy chain such as SEQ ID NO: 4. The CDRs of the heavy chain together with the CDRs of the associated light chain essentially constitute the antigen binding site of the molecules of the invention. It is known that the contribution of the light chain variable region is small compared to the contribution of the relevant heavy chain variable region to the binding thermodynamics; and the isolated heavy chain variable region has antigen binding activity itself. These molecules are commonly referred to as single domain antibodies.

In one aspect, the invention provides molecules comprising at least one antigen binding site, such as CD45RO/RB binding molecules and direct equivalents thereof, wherein said molecules comprise, in order, the hypervariable region 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), said CDR3 having the amino acid sequence Ser-Gly-Pro-Tyr-Ala-Trp-Phe-Asp-Thr (SGPYAWFDT).

In another aspect, the invention provides molecules comprising at least one antigen binding site, such as CD45RO/RB binding molecules and direct equivalents thereof, wherein said molecules comprise

a) A first domain comprising in order 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), 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 ', CDR 2' and CDR3 ', CDR 1' 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 (SSSISS), CDR 3' having the amino acid sequence Gln-Gln-Ser-Asn-Thr-Trp-Pro-Phe-Thr (QQSNTWPFT).

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 of the invention there is provided a polypeptide comprising SEQ ID NO: 1 polypeptide and/or SEQ ID NO: 2, a molecule, such as a CD45RO/RB binding molecule, preferably comprising in one domain the amino acid sequence of seq id NO: 1 and in another domain comprises SEQ ID NO: 2 polypeptides, such as chimeric monoclonal antibodies; in another aspect of the present invention there is provided a polypeptide comprising SEQ ID NO: 3 and/or SEQ id no: 4, such as a CD45RO/RB binding molecule, preferably comprising in one domain the amino acid sequence of SEQ ID NO: 3 and in another domain comprises SEQ ID NO: 4, such as a chimeric monoclonal antibody.

When the antigen binding site comprises the first and second domains, or respectively SEQ ID NO: 1 polypeptide or SEQ ID NO: 3 and SEQ ID NO: 2 or SEQ ID NO: 4, or, preferably, each domain is on a different chain, e.g., the first domain is part of a heavy chain (e.g., an immunoglobulin heavy chain) or fragment thereof, and the second domain is part of a light chain (e.g., an immunoglobulin light chain) or fragment thereof.

We have further found that CD45RO/RB binding molecules according to the invention are CD45RO/RB binding molecules in the in vivo environment of a mammal (e.g., a human). Thus, the CD45RO/RB binding molecule according to the invention can be considered a monoclonal antibody (mAb), wherein the binding activity is mainly determined by the CDR regions as described above, which can be linked, for example, to other molecules of essentially human origin (e.g. frameworks such as constant regions) without binding specificity.

Another aspect of the invention provides CD45RO/RB binding molecules that are not the monoclonal antibody "A6" as described by Aversa et al, Cellular Immunology, Vol.158, p.314-328 (1994) (which is incorporated by reference in view of the paragraph that characterizes A6).

In another aspect, the invention provides a CD45RO/RB binding molecule according to the invention which is a chimeric, humanized or fully human monoclonal antibody.

Examples of CD45RO/RB binding molecules include chimeric or humanized antibodies (e.g., antibodies that can be derived from B cells or hybridomas) and/or any fragment thereof, such as F (ab') 2 and Fab fragments, as well as single chain or single domain antibodies. Single chain antibodies consist of the variable regions of the heavy and light chains of an antibody covalently linked by a peptide linker, which typically consists of 10 to 30 amino acids, preferably 15 to 25 amino acids. Thus, such a structure does not include the constant portions of the heavy and light chains, and it is believed that a small peptide spacer should be less antigenic than the entire constant portion. By chimeric antibody is meant an antibody in which the constant regions of either the heavy or light chain, or both, are of human origin, while the variable regions of both the heavy and light chains are of non-human (e.g., murine) origin. A humanized antibody is one in which the hypervariable regions (CDRs) are of non-human (e.g., murine) origin and all or substantially all other portions (e.g., highly conserved portions of constant and variable regions) are of human origin. However, humanized antibodies may retain several amino acids of the murine sequence in the variable region portion adjacent to the hypervariable region.

The hypervariable regions, i.e.the CDRs according to the invention, may be associated with any type of framework region, for example the constant regions of the heavy and light chains of human origin. Suitable framework regions can be found, for example, in the 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 portion of the human heavy chain may belong to the IgG1 class, including subclasses; preferably, the constant part of the human light chain may belong to the kappa or lambda class, more preferably the kappa class. A preferred constant portion of the heavy chain is the polypeptide SEQ ID NO: 4 (without the CDR1 ', CDR2 ', and CDR3 ' sequence portions described above); a preferred constant portion of the light chain is the polypeptide SEQ ID NO: 3 (without the CDR1, CDR2, and CDR3 sequence portions described above).

We have also invented a humanized antibody comprising: a polypeptide having the amino acids SEQ ID NO: 7 or amino acid SEQ ID NO: 8, a light chain variable region; and a polypeptide having amino acids seq id NO: 9 or amino acid SEQ ID NO: 10, heavy chain variable region.

In another aspect of the invention there is provided a polypeptide comprising SEQ ID NO: 9 or SEQ ID NO: 10 and polypeptide SEQ ID NO: 7 or SEQ ID NO: 8 in a humanized form.

In another aspect of the invention, humanized antibodies are provided which comprise

-polypeptide SEQ ID NO: 9 and the polypeptide SEQ ID NO: 7,

-polypeptide SEQ ID NO: 9 and the polypeptide SEQ ID NO: 8,

-polypeptide SEQ ID NO: 10 and polypeptide SEQ ID NO: 7, or

-polypeptide SEQ ID NO: 10 and polypeptide SEQ ID NO: 8.

a polypeptide according to the invention, e.g. a polypeptide having a sequence as indicated herein, e.g. polypeptide CDR1, CDR2, CDR3, CDR1 ', CDR2 ', CDR3 ', or 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, including direct equivalents of said (poly) peptide (sequence); for example, functional derivatives of the polypeptides are included. The functional derivative may comprise a covalent modification of the specified sequence and/or the functional derivative may comprise an amino acid sequence variant of the specified sequence.

Unless otherwise indicated, "polypeptide" includes any peptide or protein containing amino acids linked to each other by peptide bonds, the amino acid sequence of which starts at the N-terminus and ends at the C-terminus. Preferably, the polypeptide of the invention is a monoclonal antibody, more preferably a chimeric (V-grafted) monoclonal antibody or a humanized (CDR-grafted) monoclonal antibody. The humanized (CDR-grafted) monoclonal antibody may or may not include further mutations introduced into the acceptor antibody Framework (FR) sequences.

Functional derivatives as used herein include molecules having the same qualitative biological activity as the polypeptides of the invention, e.g. having the ability to bind to CD45RO and CD45 RB. Functional derivatives include fragments and peptide analogs of the polypeptides of the invention. Fragments include regions within the sequence of the polypeptide of the invention (e.g., the sequence) and the term "derivative" is used to define amino acid sequence variants and covalent modifications of the polypeptide of the invention (e.g., the sequence). A functional derivative of a polypeptide of the invention (e.g., such a sequence) preferably has at least about 65%, more preferably at least about 75%, even more preferably at least about 85%, and most preferably at least about 95% full sequence homology to the amino acid sequence of a polypeptide of the invention (e.g., such a sequence) and substantially retains the ability to bind to CD45RO and CD45 RB.

The term "covalent modification" includes modification of a polypeptide of the invention (e.g., such sequences) or fragments thereof with organic proteinaceous (proteinaceous) or non-proteinaceous derivatizing agents, fusion with heterologous polypeptide sequences, and post-translational modifications. Covalently modified polypeptides (e.g., such sequences) still have the ability to bind to CD45RO and CD45RB by cross-linking. Covalent modifications may be routinely introduced by reacting the target amino acid residue with an organic derivatizing agent capable of reacting with the selected side chain or terminal residue, or by post-translational modification mechanisms that function within the selected recombinant host cell. Some of the post-translational modifications are the result of the action of the recombinant host cell on the expressed polypeptide. Glutaminyl and asparaginyl residues are often deaminated post-translationally to form the corresponding glutamyl and aspartyl residues. Alternatively, these residues may be deaminated under slightly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of serine, tyrosine or threonine residues, methylation of the alpha-amino group of lysine, arginine and histidine side chains, see, e.g., t.e. creighton, Proteins: structure and Molecular Properties, W.H.Freeman & Co., SanFrancisco, pp.79-86 (1983). For example, covalent modifications include fusion proteins comprising a polypeptide of the invention (e.g., such sequences) and amino acid sequence variants thereof, such as immunoadhesins and N-terminal fusions with heterologous signal sequences.

The term "homologous" in relation to a native polypeptide and functional derivatives thereof is defined herein as: after aligning the sequences and introducing gaps (gaps), if necessary, to achieve the maximum percent homology, the percentage of amino acid residues of a candidate sequence that are identical to the residues of the corresponding native polypeptide, without considering conservative substitutions as part of the sequence identity. N-or C-terminal extension and insertion should not be construed as a factor reducing identity or homology. Methods and computer programs for alignment are known. "amino acid" refers to all natural L-alpha-amino acids and includes D-amino acids. Amino acids are identified by known single or three letter names.

The term "amino acid sequence variant" refers to a molecule that differs somewhat in its amino acid sequence as compared to a polypeptide of the invention (e.g., the sequence). Amino acid sequence variants of a polypeptide according to the invention (e.g. the sequence) still have the ability to bind to CD45RO and CD45 RB. A substitution variant refers to a variant in which at least one amino acid has been removed and a different amino acid has been inserted at the same position in a polypeptide of the invention (e.g., the sequence). These substitutions may be single, in which only one amino acid is substituted within the molecule; or may be multiplexed in which two or more amino acids are substituted within the same molecule.

An insertion variant refers to a variant in which one or more amino acids are inserted immediately adjacent to an amino acid at a particular position in a polypeptide of the invention (e.g., the sequence). By immediately adjacent to an amino acid is meant attached to the alpha-carboxy or alpha-amino functionality of the amino acid. A deletion variant refers to a variant in which one or more amino acids have been removed in a polypeptide of the invention (e.g., the sequence). Generally, deletion variants have one or two amino acid deletions in a particular region of the molecule.

We have also found a polynucleotide sequence which,

GGCCAGTCAGAACATTGGCACAAGCATACAGTG, encoding the amino acid sequence of CDR1,

-TTCTTCTGAGTCTATCTCTGG; an amino acid sequence encoding a CDR2,

ACAAAGTAATACCTGGCCATTCACGTT, encoding the amino acid sequence of CDR3,

TTATATTATCCACTG, encoding the amino acid sequence of CDR 1',

TTTTAATCCTTACAATCATGGTACTAAGTACAATGAGAAGTTCAAAGGCAG, encoding the amino acid sequence of CDR 2',

AGGACCCTATGCCTGGTTTGACACCTG, encoding the amino acid sequence of CDR 3',

-SEQ ID NO: 5, encoding the polypeptide of SEQ ID NO: 1, the light chain variable region of a monoclonal antibody according to the invention;

-SEQ ID NO: 6, encoding the polypeptide of SEQ ID NO: 2, the heavy chain variable region of a monoclonal antibody according to the invention;

-SEQ ID NO: 11, encoding the polypeptide SEQ ID NO: 9, a heavy chain variable region comprising the CDR1, CDR2 and CDR3 of the invention;

-SEQ ID NO: 12, encoding the polypeptide SEQ ID NO: 10, a heavy chain variable region comprising the CDR1, CDR2 and CDR3 of the invention;

-SEQ ID NO: 13, encoding the polypeptide SEQ ID NO: 7, a light chain variable region comprising the CDR1 ', CDR2 ' and CDR3 ' of the present invention; and

-SEQ ID NO: 14, encoding the polypeptide SEQ ID NO: 8, a light chain variable region comprising the CDR1 ', CDR2 ' and CDR3 ' of the present invention.

Another aspect of the present invention provides: an isolated polynucleotide comprising a polynucleotide encoding a CD45RO/RB binding molecule, for example a polynucleotide encoding the amino acid sequence of CDR1, CDR2 and CDR3 of the invention and/or, preferably, a polynucleotide encoding the amino acid sequence of CDR1 ', CDR2 ' and CDR3 ' of the invention;

a polynucleotide comprising the polynucleotide of SEQ ID NO: 5 and/or, preferably, the polynucleotide of SEQ ID NO: 6 of (a); and

a polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8 and polypeptide SEQ ID NO: 9 or SEQ ID NO: 10, e.g. encoding

-polypeptide SEQ ID NO: 7 and the polypeptide SEQ ID NO: 9,

-polypeptide SEQ ID NO: 7 and the polypeptide SEQ ID NO: 10,

-polypeptide SEQ ID NO: 8 and polypeptide SEQ ID NO: 9, or

-polypeptide SEQ ID NO: 8 and polypeptide SEQ ID NO: 10; and

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

-polynucleotide SEQ ID NO: 11 and the polynucleotide SEQ ID NO: 13,

-polynucleotide SEQ ID NO: 11 and the polynucleotide SEQ ID NO: 14,

-polynucleotide SEQ ID NO: 12 and the polynucleotide SEQ ID NO: 13, or

-polynucleotide SEQ ID NO: 12 and the polynucleotide SEQ ID NO: 14.

unless otherwise indicated, "polynucleotide" includes any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA, including but not limited to single and double stranded RNA and RNA as a mixture of single and double stranded regions.

Polynucleotides according to the invention, for example, encode the amino acid sequences of CDR1, CDR2, CDR3, CDR1 ', CDR2 ', CDR3 ', or the amino acid sequences of SEQ ID NOs: 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, such as the polynucleotides of SEQ ID NOs: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ id no: 14, including allelic variants and/or complements thereof; for example, polynucleotides are included which can correspond to the nucleotide sequences 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, hybridizing; e.g. encoding the amino acid sequence corresponding 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, e.g. including functional derivatives of said polypeptides, e.g. said functional derivatives may correspond 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, e.g. the functional derivative may accordingly comprise 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, e.g. the functional derivative may accordingly comprise 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; for example, 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 include sequences which, as a result of the redundancy (degeneracy) of the genetic code, correspondingly also encode the polypeptide 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 or correspondingly encodes a polypeptide having an amino acid sequence corresponding 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 a polypeptide having an amino acid sequence at least 80% identical.

CD45RO/RB binding molecules, such as chimeric or humanized antibodies, can be produced by recombinant DNA techniques. Thus, one or more DNA molecules encoding CD45RO/RB may be constructed, placed under appropriate control sequences, and transformed into an appropriate host (organism) with an appropriate vector for expression.

In another aspect, the invention provides polynucleotides encoding the individual heavy and/or light chains of the CD45RO/RB binding molecules of the invention; also provided is the use of a polynucleotide of the invention for recombinantly producing a CD45RO/RB binding molecule according to the invention.

The CD45RO/RB binding molecules can be obtained according to conventional methods, e.g., similar methods, in combination with the information provided herein, e.g., the amino acid sequences of the hypervariable and variable regions and the polynucleotide sequences encoding these regions. Methods for constructing variable region genes are described, for example, in EP 239400 and can be briefly summarized as follows: genes encoding the variable regions of monoclonal antibodies of any specificity can be cloned. DNA fragments encoding the framework and hypervariable regions were determined and DNA fragments encoding the hypervariable regions were removed. Double-stranded synthetic CDR cassettes are prepared by DNA synthesis according to the CDR and CDR' sequences indicated herein. The cartridges are made sticky-ended so that they can be attached to the desired anthropogenic framework regions at the junctions. Polynucleotides encoding single chain antibodies may also be prepared according to conventional methods, e.g., analogous methods. The polynucleotide of the invention thus prepared can be conveniently transformed into a suitable expression vector.

Suitable cell lines can be found according to conventional methods, such as analogous methods. Expression vectors, for example, containing suitable promoters and genes encoding the heavy and light chain constant regions, are known, e.g., commercially available. Suitable hosts are known or can be found by conventional methods, e.g., analogous methods, including cell cultures or transgenic animals.

In another aspect, the invention provides an expression vector comprising a polynucleotide encoding a CD45RO/RB binding molecule of the invention, e.g., a polynucleotide having the sequence of SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: 18 sequence.

In another aspect of the invention provide

-an expression system comprising a polynucleotide according to the invention, wherein said expression system or part thereof is capable of producing a CD45RO/RB binding molecule of the invention when said expression system or part thereof is present in a compatible host cell; and

-an isolated host cell containing an expression system as defined above.

We have further found that the CD45RO/RB binding molecule according to the invention inhibits primary alloimmune responses in a dose-dependent manner as measured by in vitro MLR. This result indicates that the response of cells that are heteroactivated (alloactivitate) in the presence of the CD45RO/RB binding molecule of the invention to alloantigens (alloantegen) is impaired. This confirms that the CD45RO/RB binding molecules according to the invention are able to act directly on alloreactive effector T-cells and modulate their function. In addition, we further investigated the functional properties of T cells from primary MLRs in secondary MLR restimulation experiments, where specificity of the observed functional effect was assessed with specific cell stimulators or third party stimulators. We found that the ability of cells from primary MLR (in which the CD45RO/RB binding molecule according to the invention is present) to respond to subsequent optimal stimulation with specific cell stimuli is impaired, despite the absence of antibody addition in the second culture. The specificity of the inhibition means was demonstrated by the ability of cells treated with the CD45RO/RB binding molecules of the invention to respond normally to cellular stimuli from unrelated third party donors. Thus, restimulation experiments using T cells from primary MLR cultures showed that cells that were heterologously activated in the presence of the CD45RO/RB binding molecules of the invention had low reactivity, i.e., tolerance, to the original alloantigen. Other biological activities are described in example 7.

We have also found that cell proliferation in cells pretreated with the CD45RO/RB binding molecule of the invention can be rescued by exogenous IL-2. This indicates that treatment of alloreactive T cells with the CD45RO/RB binding molecules of the invention can induce a tolerogenic state. Indeed, the attenuated proliferative response observed in cells treated with the CD45RO/RB binding molecules of the present invention is due to impaired T cell function; these cells were able to respond to exogenous IL-2, indicating that these cells were cell-free immunoreactive, i.e., in a truly unresponsive state. This response was confirmed to be specific due to the ability of cells treated with the CD45RO/RB binding molecules of the invention to respond to normal proliferation of unrelated donor cells to the level of control-treated cells.

Furthermore, experiments have shown that the binding of the CD45RO/RB binding molecule according to the invention to CD45RO and CD45RB can inhibit the memory response of Peripheral Blood Mononuclear Cells (PBMCs) from immunized donors to specific recall antigens. Thus the binding of the CD45RO/RB binding molecule according to the invention to CD45RO and CD45RB may also effectively inhibit the memory response to soluble Ag. Since the CD45RO/RB binding molecule according to the invention was able to inhibit recall responses to tetanus in PBMC (from immunized donors), this suggests that the CD45RO/RB binding molecule according to the invention is able to target and modulate the activation of memory T cells. For example, these data indicate that, in addition to recognizing alloreactively activated T cells, CD45RO/RB binding molecules according to the invention can modulate their function, inducing cell-free immunoreactivity of T cells. This property may be important for the treatment of ongoing immune responses against self-antigens, allergens and possibly alloantigens as may be seen in autoimmune diseases, allergies and chronic rejections as well as diseases such as psoriasis, inflammatory bowel disease, where memory responses play a role in maintaining the state of the disease. This is considered to be an important feature in disease states where memory response to self-antigens may play an important role in disease maintenance, for example in autoimmune diseases.

We have also found that the CD45RO/RB binding molecules according to the invention can modulate T cell proliferative responses in Mixed Lymphocyte Reaction (MLR) in vivo, i.e.experiments have found that the CD45RO/RB binding molecules according to the invention have corresponding inhibitory properties in vivo.

Thus, the CD45RO/RB binding molecules according to the invention have immunosuppressive and tolerogenic properties and can be used to induce tolerance to alloantigens, autoantigens, allergens and antigens of bacterial flora in vivo and ex vivo, e.g. the CD45RO/RB binding molecules according to the invention can be used in the treatment and prevention of diseases, e.g. including autoimmune diseases, such as but not limited to rheumatoid arthritis, autoimmune thyroiditis, graves 'disease, type I and type II diabetes, multiple sclerosis, systemic lupus erythematosus, sjogren's syndrome, scleroderma, autoimmune gastritis, glomerulonephritis; transplant rejection, such as organ and tissue allograft and xenograft rejection, and graft-versus-host disease (GVHD), as well as psoriasis, inflammatory bowel disease, and allergies.

In a further aspect the invention provides the use of a CD45RO/RB binding molecule according to the invention as a medicament, for example in the treatment and/or prevention of autoimmune diseases, transplant rejection, psoriasis, inflammatory bowel disease and allergies.

In a further aspect of the invention there is provided a CD45RO/RB binding molecule according to the invention for use in the manufacture of a medicament for the treatment and prevention of diseases associated with autoimmune diseases, transplant rejection, psoriasis, inflammatory bowel disease and allergies.

In another aspect, the invention provides a pharmaceutical composition comprising a CD45RO/RB binding molecule according to the invention, the composition further comprising at least one pharmaceutically acceptable carrier or diluent.

The pharmaceutical composition may also comprise, for example, an active ingredient such as other immunomodulatory antibodies (such as, but not limited to, anti-ICOS, anti-CD 154, anti-CD 134L) or recombinant proteins (such as, but not limited to, rcla-4 (CD152), rOX40(CD134)) or immunomodulatory compounds (such as, but not limited to, cyclosporin a, FTY720, RAD, rapamycin, FK506, 15-deoxyspergualin, steroids).

In a further 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 invention, for example in the form of a pharmaceutical composition according to the invention.

Autoimmune diseases that can be treated with the binding molecules of the invention also include, but are not limited to, rheumatoid arthritis, autoimmune thyroiditis, Graves 'disease, type I and type II diabetes, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, autoimmune gastritis, glomerulonephritis; transplant rejection, such as organ and tissue allograft and xenograft rejection, and Graft Versus Host Disease (GVHD).

Examples

The present invention may be more fully understood with reference to the following examples. However, it should not be construed as limiting the scope of the invention. In the following examples, all temperatures are expressed in degrees celsius.

A "candidate monoclonal antibody" or "chimeric antibody" is a CD45RO/RB binding molecule according to the invention, which comprises the amino acid sequence of SEQ ID NO: 3 and the light chain of SEQ ID NO: 4, or a heavy chain thereof.

The following abbreviations are used:

ELISA enzyme-linked immunosorbent assay

FACS fluorescence activated cell sorting

FITC fluorescein isothiocyanate

FBS fetal bovine serum

GVHD graft versus host disease

HCMV human cytomegalovirus promoter

IgE immunoglobulin isotype E

IgG immunoglobulin isotype G

PBS phosphate buffered saline solution

PCR polymerase chain reaction

xGVHD xenograft versus host disease

Example 1: primary lymphocyte reaction (MLR)

Cells

Blood samples were from healthy human donors. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from leukocytes from whole peripheral blood, leukopheresis or buffy coats, of known blood type but unknown HLA type, by centrifugation on Ficoll-hypaque (pharmacilkb). In some MLR experiments, PBMCs were used directly as cell stimulators after 40Gy irradiation. In additional experiments, T cells were depleted from PBMCs using CD2 or CD3 Dynabeads (Dynal, Oslo, Norway). Beads and contaminating cells were removed with a magnetic field. After irradiation, T cell depleted PBMCs were used as cell stimulators.

In MLR, PBMC, CD3⁺T or CD4⁺T cells are used as effector cells. Cells from different donors were prepared as cell stimulators. CD3 purified by negative selection using anti-CD 16 monoclonal antibody (Zymed, CA), goat anti-mouse IgG Dynabeads, anti-CD 14 Dynabeads, CD19 Dynabeads⁺T cells. In addition anti-CD 8 Dynabeads were used to purify CD4⁺T cells. The cells obtained were cultured with FACScan or FACSCalibur (Becton Dickinson)&Co, CA) analysis, the purity of the cells obtained was > 75%. Cells were suspended in RPMI1640 medium supplemented with 10% heat-inactivated FBS, penicillin, streptomycin and L-glutamine.

Reagent

A chimeric anti-CD 45RO/RB monoclonal antibody "candidate monoclonal antibody" and an isotype-matched control chimeric antibody were also prepared. Murine (human) control IgG specific for KLH (keyhole limpet hemocyanin)₁Antibodies and recombinant human IL-10 were purchased from BD Pharmingen (San Diego, CA). The anti-human CD154 monoclonal antibody 5c8 is described according to Lederman et al 1992.

Primary Mixed Lymphocyte Reaction (MLR)

Aliquots of 1X 10 were plated in each well of a 96-well plate (Costar, Cambridge, MA) in the presence or absence of the monoclonal antibody⁵PBMC or 5X 10⁴An individual CD3⁺Or CD4⁺Cells and irradiated 1X 10⁵PBMC or 5X 10⁴T-cell depleted and irradiated (50Gy) PBMCs were pooled. In some experiments, 10. mu.g/ml of goat anti-mouse Ig or goat anti-human Ig F (ab')₂Fragment (Jackson ImmunoResearch, West Grove, PA) to ensure optimal in vitro cross-linking with the target CD45 molecule. At 37 ℃ in 5% CO₂Culturing the mixed cells for 4 or 5 days, and culturing for the last 16-20 hr³Cells were pulsed with H-thymidine to determine proliferation. Other experiments were similar to those described above with the following exceptions: 1) the medium used was EX-VIVO (Bio-Whittaker) containing 10% FBS and 1% human plasma; 2) anti-mouse intact IgG (5. mu.g/ml) was used as a second crosslinking step; 3) the cell stimulator was irradiated to 60 Gy.

In the presence of "candidate monoclonal antibodies" or control chimeric IgG₁(10. mu.g/ml) in the presence of a second step reagent, i.e.to the Fc partSpecific sheep anti-human Ig F (ab')₂Fragment (10. mu.g/ml). And control IgG₁The percent inhibition of the "candidate monoclonal antibody" can be calculated as a comparison of cell proliferation in the presence. The results are shown in table 1 below:

TABLE 1

Inhibition of Primary MLR by candidate monoclonal antibodies according to the invention at 10. mu.g/ml

^＊Has significant difference with the control value (P < 0.001)

It can be seen from Table 1 that the candidate monoclonal antibodies according to the invention have an inhibitory effect on primary MLR. In CD4 from four different donors⁺The mean inhibitory effect in T cells was 60.83 ± 6.83%, with statistical significance.

As shown in FIG. 1, the inhibitory effect of the "candidate monoclonal antibody" on primary MLR in the range of 0.001 to 10. mu.g/ml was shown to be dose-dependent. The IC of a "candidate monoclonal antibody" for primary MLR inhibition can be determined from the results of three independent MLR experiments using one donor PBMC as effector cells₅₀. Thus, F (ab') in the presence of "candidate monoclonal antibody" or control chimeric antibody and 10. mu.g/ml sheep anti-human Ig₂Effect CD4 from donors #229 and #219 in the Presence of fragments⁺T cells were mixed with T cell depleted and irradiated PBMCs as a stimulator. The experiment was repeated 3 times and the percentage of proliferation in the presence of "candidate monoclonal antibodies" was calculated compared to the proliferation of T cells in the presence of control antibodies. Origin (V was used.) Determining IC₅₀The value is obtained. Calculated cellular Activity IC₅₀The value was 0.87. + -. 0.35nM (0.13. + -. 0.052. mu.g/ml).

Example 2: secondary MLR

To evaluate whether "candidate monoclonal antibodies" induced CD4⁺T cell unresponsiveness to specific alloantigens, secondary MLR is performed after primary MLC in the absence of any antibodies. CD4 was plated in 96-well plates in the presence of the antibodies⁺T cells were cultured with irradiated allogeneic cell stimulators (T cell depleted PBMCs) for 10 days. Then, cells were harvested, layered on a Ficoll-Hypaque gradient to remove dead cells, washed twice with RPMI, and then restimulated with the same stimulator, third party cell stimulator, or IL-2 (50U/ml). The cells were cultured for 3 days by³The proliferation response was measured by pulsing the cells with H-thymidine for the last 16-20 hours of culture.

Specifically, at 10. mu.g/ml of "candidate monoclonal antibody", control IgG1 chimeric antibody and F (ab') of goat anti-human Ig₂In the presence of the fragment, CD4⁺T cells were cultured with irradiated allogeneic cell stimulators (T cell depleted PBMCs from other donors). Primary MLR proliferation was determined on day 5. For secondary MLRs, effector cells and cell stimulators are cultured in the presence of "candidate monoclonal antibodies" for 10 days; the cells were then harvested, washed twice with RPMI1640 and restimulated with specific stimuli, third party stimuli or IL-2(50U/ml) in the absence of any antibody. Cell proliferation was measured on day 3 and the results are listed in table 2:

TABLE 2

Effect CD4+ T cell Donor #	% inhibition of Secondary MLR
		#211	49.90
#220	59.33
		#227	58.68

^＊Significant differences from control values (p ═ 0.001, determined by t-test, SigmaStat v.2.03). # p ═ 0.046

To examine whether the impaired proliferation was due to unresponsiveness as a result of treatment with a "candidate monoclonal antibody", cells from primary MLRs were cultured in the presence of IL-2 (50U/ml). The addition of IL-2 resulted in the restoration of proliferative responses in primary MLRs to T cells treated with the "candidate monoclonal antibody" to a level similar to that observed in the presence of IgG1 control antibody. These data indicate that the impaired secondary response of "candidate monoclonal antibody" treated T cells is due to altered effector T cell function, which becomes unresponsive to specific cell stimuli.

Percent inhibition was calculated according to the following equation:

statistical analysis was performed using SigmaStat (vers.2.03).

Data were analyzed using a two-way ANOVA followed by Dunnett's method. Probability < 0.05 was considered significant in all tests. The t-test (SigmaStat V.2.03) was used in some experiments.

Example 3: in vivo survival Studies of SCID mice

hu-PBL implanted SCID mice

C.B 17/Gbms Tac-Prkdc to SCID mouse^scid Lyst^bgMice (Taconic, Germantown, NY) were injected intraperitoneally with human Peripheral Blood Mononuclear Cells (PBMCs) at a dose sufficient to induce lethal xenograft-versus-host disease (xGvHD) in > 90% of mice within 4 weeks after cell implantation. This treated SCID mouse is hereinafter referred to as hu-PBL-SCID mouse.

Monoclonal antibody treatment of hu-PBL-SCID mice

On day 0, i.e., immediately after PBMC injection, on days 3, 7 and every other week thereafter, hu-PBL-SCID mice were treated with "candidate monoclonal antibodies" or murine or chimeric isotype-matched monoclonal antibody controls. Monoclonal antibody was administered subcutaneously in 100. mu.l PBS to a final concentration of 5mg/kg body weight. Treatment was terminated when all control mice died.

Evaluation of treatment results

The primary criterion for assessing the efficacy of a "candidate monoclonal antibody" in this study is the survival of hu-PBL-SCID mice. The significance of the results was evaluated by statistical survival analysis methods with the help of the Systat v9.01 software using the log-rank test (Mantel method). The survival assay is a non-parametric test that considers not only whether a particular mouse is still alive, but also whether the cause is not treatment/disease related if it is sacrificed, such as the need for in vitro analysis of its organs/cells. Biopsies of liver, lung, kidney and spleen were obtained from dead mice for further evaluation. In addition, hu-PBL-SCID mice were weighed at the beginning of the experiment (before cell transfer) and during the experiment (every 2 days) for indirect assessment of their health status. Linear regression lines were generated using body weight from each mouse versus day values after PBMC transfer, after which their slopes were compared using a non-parametric Mann-Whitney assay (control versus anti-CD 45 treated mice).

Results

All hu-PBL-SCID mice treated with murine monoclonal antibody control had human leukocytes infiltrated into the lungs, liver and spleen, and the mice died within about 2 to 3 weeks after cell transfer (4/4). Death may be caused by xGvHD. Control monoclonal antibody treated mice also lost weight in a linear fashion, about 10% and more within 3 weeks.

All hu-PBL-SCID mice treated with "candidate monoclonal antibody" survived (4/4) and had no overt signs of any disease for more than four weeks, even if "candidate monoclonal antibody" treatment was stopped after 3 weeks. Mice treated with the "candidate monoclonal antibody" gained weight in a linear fashion, up to about 5% within 4 weeks.

Example 4: expression of the antibodies of the invention

Comprises the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 of human origin Expression of chemoantibodies

Expression vectors were constructed from the plasmid maps (shown in FIGS. 2 to 5) containing the corresponding nucleotides encoding the amino acid sequences of humanized light chain variable region humV1(SEQ ID NO: 7), humanized light chain variable region humV2(SEQ ID NO: 8), humanized heavy chain variable region VHE (SEQ ID NO: 9), or humanized heavy chain variable region VHQ (SEQ ID NO: 10), respectively. These expression vectors have the DNA (nucleotide) sequences of SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 or SEQ ID NO 18, respectively.

Construction of humanized antibody heavy chain and light chain expression vectors

Human kappa light chain expression vectors for VLh and VLm versions

To construct the final expression vector encoding the fully humanized light chain of the human kappa isotype, DNA fragments encoding the complete light chain variable regions (VLh and VLm) were excised from the PCR-Script cloning vector (Stratagene) (VLm region) containing VLh and VLm using HindIII and BglII. Then purifying the gelThe resultant fragment was subcloned into the HindIII and BamHI sites of the C21-HCMV K expression vector which was generated when the humanized anti-IgE antibody TESC-21 was constructed (Kolbinger et al 1993) and originally obtained from M.Bendig (MRC colloidal center, London, UK) (Maeda et al 1991). The ligation product was purified by phenol/chloroform extraction and electroporated into an electroporation-competent EpicurianXL1-Blue strain (Cat. N ° #200228, Stratagene). After plating on LB/amp agar plates, 12 colonies each were picked overnight at 37 ℃ and plasmid DNA was prepared from 3ml of the culture using a BioRobot 9600 (Qiagen). This results in light chain expression vectors for humanized antibody versions VLh and VLm, respectively, as further described in the figure.

Human gamma-1 heavy chain expression vector for VHQ

To construct the VHQ expression vector, a stepwise approach was used. The complete variable region of VHQ was first assembled by PCR according to the method described by Kolbinger et al 1993(Protein Eng.1993 Nov; 6 (8): 971-80) and then subcloned into the C21-HCMV-gamma-1 expression vector in which the C21 insertion had been removed with the same enzyme. The HindIII/BamHI fragment of the PCRscript clone VHQ containing the entire variable region was subcloned into the expression vector C21-HCMV-gamma-1 cut with the same enzyme. This resulted in the final expression vector for the humanized antibody version VHQ.

Human gamma-1 heavy chain expression vector for VHE

The final VHE expression vector encoding the fully humanized heavy chain of the human gamma-1 isotype was constructed as follows: the HindIII and BamHI restriction PCR fragments encoding this variable region were ligated directly into the HindIII and BamHI sites of the C21-HCMV γ -1 expression vector, which was created when the humanized anti-IgE antibody TESC-21 was constructed (Kolbinger et al 1993) and was also originally supplied by M.Bendig (MRC collagen center, London, UK) (Maeda et al 1991).

Transient expression in COS cells

For adherent COS cells in 150ml cell culture dishes, the following transfection protocol was adapted, using SuperFect^TMTransfection reagents (cat. n ° 301305, Qiagen). The four different expression vectors described above were used to transiently transfect cells. To express humanized antibodies, one of two clones containing a heavy chain insert (VHE or VHQ, respectively) was co-transfected into cells with one of two clones encoding a light chain (humV1 or humV2, respectively) such that there were a total of 4 different combinations of heavy and light chain expression vectors (VHE/humV1, VHE/humV2, VHQ/humV1, and VHQ/humV 2). Prior to transfection, the plasmid was linearized with the restriction enzyme PvuI, which cleaves in the region encoding the ampicillin resistance gene. The day before transfection, 4X 10 cells were plated in 150ml cell culture plates⁶One COS cell was inoculated in 30ml of fresh medium. At this cell concentration, a confluence of 80% is usually established 24 hours after inoculation. On the day of transfection, 4 different combinations of linearized heavy and light chain DNA expression vectors (15 μ g each) were diluted in a total volume of 900 μ l fresh medium without serum and antibiotics. Then 180. mu.l of SuperFect transfection reagent was mixed thoroughly with the DNA solution. The DNA mixture was incubated at room temperature for 10 minutes to allow complex formation. After complex formation, growth medium was removed from COS cell cultures and cells were washed once with PBS. Then 9ml of fresh medium (containing 10% FBS and antibiotics) was added to each reaction tube containing the transfection mixture and mixed well. Cell cultures were incubated with the DNA complexes for 3 hours at 37 ℃ and 5% CO 2. After incubation, the medium containing the transfection complex was removed and replaced with 30ml of fresh medium. Culture supernatants were harvested 48 hours after transfection.

Concentration of culture supernatant

For ELISA and FACS analysis, culture supernatants collected from COS cells transfected with heavy and light chain plasmids were concentrated as follows. 10ml of each supernatant was added to a Centriprep YM-50 centrifugal filtration unit (Cat. N ° 4310, Millipore) as described by the manufacturer. Centrifuge Centriprep filter 10 minutes at room temperature 3000 rpm. This centrifugation step was then repeated again with the remaining 20ml of supernatant, with centrifugation for only 5 minutes and monitoring of the progress of the concentration. The intermediate 500 μ l concentrated supernatant was recovered, transferred to a new Microcon centrifugal filtration device (Cat. N ° 42412, Microcon), and further concentrated according to the manufacturer's protocol. The concentrated supernatant was centrifuged 4 times 24 min at 3000rpm, once 10 min at 6000rpm and then 3 times 5 min at room temperature, during which the progress of the concentration was always supervised. The resulting concentrated conditioned medium had a final volume of 100. mu.l, corresponding to a 250 to 300 fold concentration of the original medium, and was stored at 4 ℃ until use. For comparison and control, media from untransfected cells was similarly concentrated using the same centrifugation protocol described above.

Example 5: measurement of recombinant human IgG expression by ELISA

To determine the IgG concentration of recombinant human antibodies expressed in culture supernatants, a sandwich ELISA method was developed and optimized using human IgG as a standard. Flat-bottomed 96-well microtiter plates (Cat. N.degree. 4-39454, Nunc immunollate Maxisorp) were coated with 100. mu.l of goat anti-human IgG (whole molecule, Cat. N.degree. I1011, SIGMA) at a final concentration of 0.5. mu.g/ml in PBS overnight at 4 ℃. The wells were then washed 3 times with rinsing buffer (PBS containing 0.05% Tween 20) and blocked with blocking buffer (0.5% BSA in PBS) for 1.5 hours at 37 ℃. After 3 cycles of rinsing, antibody samples and standard human IgG (cat. No. i4506, SIGMA) were prepared by serial dilution in blocking buffer at 1.5 fold. 100 μ l of the diluted sample or standard was transferred in duplicate to coated plates and incubated for 1 hour at room temperature. After incubation the plates were washed 3 times with rinsing buffer before incubation for 1 hour with 100 μ l goat anti-human IgG kappa-light chain (cat. n ° a-7164, SIGMA) conjugated to horseradish peroxidase diluted in 1/4000 blocking buffer. Control wells were filled with 100. mu.l of blocking buffer or concentrated normal medium. After rinsing, colorimetric quantitative determination of bound peroxidase was performed in samples and standard wells using TMB peroxidase EIA substrate kit (Cat. N.degree.172-1067, Bio-Rad) according to the manufacturer's instructions. Mu.l of this peroxidase mixture was added to each well and incubated for 30 minutes at room temperature in the dark. The color reaction was stopped by adding 100. mu.l of 1M sulfuric acid. The absorbance of each well was read at 450nm using an ELISA plate reader (3350-UV model, BioRad).

The correlation coefficient for the IgG standard curve was 0.998, and the concentrations of the 4 different culture concentrates were determined as follows (about 250-fold 300-fold concentration):

VHE/humV1 supernatant 8.26 μ g/ml

VHE/humV2 supernatant 6.27 μ g/ml

VHQ/humV1 supernatant 5.3 μ g/ml

VHQ/humV2 supernatant 5.56 μ g/ml

Example 6: FACS competition analysis (binding affinity)

The human T cell line PEER was selected as the target cell for FACS analysis because it expresses the CD45 antigen on its cell surface. To analyze the binding affinity of the humanized antibody supernatant, a competition experiment using FITC-labeled chimeric antibody as a reference was performed and compared with the inhibitory effect of purified murine antibody and chimeric antibody. The PEER cell cultures were centrifuged at 3000rpm for 10 seconds and the medium was removed. Cells were resuspended in FACS buffer (PBS containing 1% PBS and 0.1% sodium azide) and plated at a cell density of 1X 10 per well in 96-well round-bottomed microtiter plates⁵One was inoculated. The plate was centrifuged and the supernatant discarded. For blocking studies, 25 μ l of concentrated untransfected medium or isotype-matched control antibody (negative control), unlabeled murine or chimeric antibody (positive control) and concentrated supernatant (sample) containing a combination of different humanized antibodies were first added to each well at the concentrations indicated herein. After 1 hour incubation at 4 ℃, the PEER cells were washed by centrifugation with 200 μ Ι facs buffer. Cells were then incubated with FITC-conjugated chimeric antibody in FACS buffer for 1 hour at 4 ℃ at a final antibody concentration of 20. mu.g/ml. Cells were washed and resuspended in 300. mu.l FACS buffer containing 2. mu.g/ml propidium iodide (to allow gating of live cells). Cell preparations were analyzed on a flow cytometer (FACSCalibur, Becton Dickinson).

FACS analysis showed that blocking of the fluorochrome-labeled chimeric antibody by concentrated humanized antibody culture supernatant was dose dependent. No dose-dependent blocking of the chimeric antibody was seen with the isotype-matched control antibody, indicating that the blocking effect produced by these different humanized antibody combinations is epitope-specific; and this epitope specificity appears to be retained after humanization treatment.

Example 7: biological Activity of CD45RB/RO binding molecules

In this study, we analyzed whether the CD45RB/RO binding chimeric antibody when present in polyclonal activated primary human T cell cultures (1) supports T cell differentiation with a characteristic Treg phenotype; (2) preventing or enhancing apoptosis following T cell activation; and (3) affecting the expression of specific antigen subpopulations and receptors upon restimulation.

CD45RB/RO binding chimeric antibody potentiates cell death in polyclonal activated T cells

Primary T cells (mixture of CD4+ and CD8+ T subsets) were activated with anti-CD 3 plus anti-CD 28 monoclonal antibody in the presence or absence of CD45RB/RO binding chimeric antibody (control). Excess antibody was removed by washing on day 2. 7-amino-actinomycin D (7-AAD), which is taken up by apoptotic and necrotic cells, is used as a DNA dye for determining cell death after activation. The results show that T cell activation in the presence of CD45RB/RO binding chimeric antibody increased the 7-AAD positive cell fraction by 2-fold more than on day 2 after activation. On day 7, the fraction of 7-AAD positive cells in the CD45RB/RO binding chimeric antibody treated cultures and the control cultures was again similar.

CD45RB/RO binding to chimeric antibody treated T cells but not control monoclonal antibody treated T cells showed Phenotype of outgrowth T regulatory cells (Tregs)

The hallmark of Treg cells is increased expression of CD25 and the negative regulatory protein CTLA-4(CD 152). The functional suppression of primary and secondary T cell responses by CD45 RB/RO-binding chimeric antibodies may be due to the induction of Treg cells. To address this problem, T cells were activated with anti-CD 3+ CD28 monoclonal antibody and cultured in the presence of CD45RB/RO binding chimeric antibody or anti-LPS control monoclonal antibody. The time course of CTLA-4 and CD25 expression showed that control and CD45RB/RO binding chimeric antibody treated T cells were significantly different at day 1 and day 3 after the second stimulation, indicating a Treg phenotype.

Intracellular CTLA-4 sustained expression in the presence of CD45RB/RO binding chimeric antibody

It has been reported that there are a considerable amount of CTLA-4 in the cells. Thus, intracellular CTLA-4 expression was analyzed in parallel with surface CTLA-4 staining. Moderate differences between T cell cultures were seen at day 4 post stimulation. However, after prolonged culture, high levels of intracellular CTLA-4 were maintained only in CD45 RB/RO-binding chimeric antibody-treated T cells, whereas control T cells did not.

The CD45RB/RO binding chimeric antibody treated T cells became double positive for CD4 and CD8

Following stimulation, T cells induce and up-regulate the expression of several surface receptors, such as CD25, CD152(CTLA-4), CD154(CD40 ligand), and the like. In contrast, the expression level of CD4 or CD8 was considered relatively stable. We were able to repeatedly observe a strong increase in both surface CD4 and CD8 antigen of CD45 RB/RO-binding chimeric antibody-treated T cells after activation, but not in control antibody-treated T cells. The appearance of a CD4/CD8 double positive T cell population appears to be due to up-regulation of CD4 on the CD8+ subpopulation, and conversely CD8 on the CD4+ subpopulation. In contrast, there was a moderately low percentage of double positive T cells in the control culture.

High expression of IL-2 receptor alpha chain but beta in CD45RB/RO binding chimeric antibody treated T cells The expression of the chain is very low

Treg cells are known to be positive for CD25, IL-2 receptor alpha chain organization. The regulation of other subunits of the trimeric IL-2 receptor on Treg cells is not known. Recently we compared the expression of the beta chain of the IL-2 receptor, such as CD122, on the surface of T cells; the T cells are 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 had about 10-fold lower CD122 expression compared to T cells in control cultures. This difference may indicate that Treg cells require other factors than IL-2 to proliferate.

Example 8: sequences of the invention (CDR sequences of the invention are underlined)

SEQ ID NO：1

Portions of chimeric light chain amino acid sequences

DILLTQSPAILSVSPGERVSFSCRASQNIGTSIQWYQQRTNGSPRLLIRSSSESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNTWPFTFGSGTKLEIK

SEQ ID NO：2

Portions of chimeric heavy chain amino acid sequences

EVQLQQSGPELVKPGASVKMSCKASGYTFTNYIIHWVKQEPGQGLEWIGYFNPYNHGTKYNEKFKGRATLTADKSSNTAYMDLSSLTSEDSAIYYCARSGPYAWFDTWGQGTTVTVSS

SEQ ID NO：3

Chimeric light chain amino acid sequences

DILLTQSPAILSVSPGERVSFSCRASQNIGTSIQWYQQRTNGSPRLLIRSSSESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNTWPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：4

Chimeric heavy chain amino acid sequences

EVQLQQSGPELVKPGASVKMSCKASGYTFTNYIIHWVKQEPGQGLEWIGYFNPYNHGTKYNEKFKGRATLTADKSSNTAYMDLSSLTSEDSAIYYCARSGPYAWFDTWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：5

Encoding the polypeptide of SEQ ID NO: 1 nucleotide sequence

GACATTCTGCTGACCCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTCGGCTCGGGGACCAAGCTTGAAATCAAA

SEQ ID NO：6

Encoding the polypeptide of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof

GAGGTGCAGCTGCAGCAGTCAGGACCTGAACTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCAGGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTAAGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAGACAAATCCTCCAACACAGCCTACATGGACCTCAGCAGCCTGACCTCTGAGGACTCTGCGATCTACTACTGTGCAAGATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO：7

Part of the humanized light chain amino acid sequence designated humV2(humV2 ═ VLm)

DILLTQSPAT LSLSPGERAT FSCRASQNIG TSIQWYQQKT NGAPRLLIRS SSESISGIPS RFSGSGSGTDFTLTISSLEP EDFAVYYCQQ SNTWPFTFGQ GTKLEIK

SEQ ID NO：8

Part of the humanized light chain amino acid sequence designated humV1(humV1 ═ VLh)

DILLTQSPAT LSLSPGERAT LSCRASQNIG TSIQWYQQKP GQAPRLLIRS SSESISGIPS RFSGSGSGTDFTLTISSLEP EDFAVYYCQQ SNTWPFTFGQ GTKLEIK

SEQ ID NO：9

Part of the humanized heavy chain amino acid sequence designated VHE

EVQLVESGAE VKKPGASVKV SCKASGYTFT NYIIHWVKQE PGQGLEWIGY FNPYNHGTKY NEKFKGRATL TANKSISTAY MELSSLRSED TAVYYCARSG PYAWFDTWGQ GTTVTVSS

SEQ ID NO：10

Part of the humanized heavy chain amino acid sequence designated VHQ

QVQLVESGAE VKKPGASVKV SCKASGYTFT NYIIHWVKQE PGQGLEWIGY FNPYNHGTKY NEKFKGRATL TANKSISTAY MELSSLRSED TAVYYCARSG PYAWFDTWGQ GTTVTVSS

SEQ ID NO：11

Encoding the amino acid sequence of SEQ ID NO: 9 nucleotide sequence

GAGGTGCAGCTGGTGGAGTCAGGAGCCGAAGTGAAAAAGCCTGGGGCTTCAGTGAAGGTGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCAGGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTAAGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAAACAAATCCATCAGCACAGCCTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACTGCGGTCTACTACTGTGCAAGATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO：12

Encoding the amino acid sequence of SEQ ID NO: 10 nucleotide sequence

CAGGTGCAGCTGGTGGAGTCAGGAGCCGAAGTGAAAAAGCCTGGGGCTTCAGTGAAGGTGTCCTGCAAGGCCTCTGGATACACATTCACTAATTATATTATCCACTGGGTGAAGCAGGAGCCTGGTCAGGGCCTTGAATGGATTGGATATTTTAATCCTTACAATCATGGTACTAAGTACAATGAGAAGTTCAAAGGCAGGGCCACACTAACTGCAAACAAATCCATCAGCACAGCCTACATGGAGCTCAGCAGCCTGCGCTCTGAGGACACTGCGGTCTACTACTGTGCAAGATCAGGACCCTATGCCTGGTTTGACACCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO：13

Encoding the amino acid sequence of SEQ ID NO: 7 nucleotide sequence

GACATTCTGCTGACCCAGTCTCCAGCCACCCTGTCTCTGAGTCCAGGAGAAAGAGCCACTTTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAAAACAAATGGTGCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTACCATCAGCAGTCTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTCGGCCAGGGGACCAAGCTGGAGATCAAA

SEQ ID NO：14

Encoding the amino acid sequence of SEQ ID NO: 8 nucleotide sequence

GACATTCTGCTGACCCAGTCTCCAGCCACCCTGTCTCTGAGTCCAGGAGAAAGAGCCACTCTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACAGTGGTATCAACAAAAACCAGGTCAGGCTCCAAGGCTTCTCATAAGGTCTTCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTACCATCAGCAGTCTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAACAAAGTAATACCTGGCCATTCACGTTCGGCCAGGGGACCAAGCTGGAGATCAAA

SEQ ID NO：15

Nucleotide sequence of expression vector HCMV-G1 HuAb-VHQ

(in 3921-4274 complete DNA sequence of humanized heavy chain expression vector comprising SEQ ID NO: 12 (VHQ))

1 AGCTTTTTGC AAAAGCCTAG GCCTCCAAAA AAGCCTCCTC ACTACTTCTG

51 GAATAGCTCA GAGGCCGAGG CGGCCTCGGC CTCTGCATAA ATAAAAAAAA

101 TTAGTCAGCC ATGGGGCGGA GAATGGGCGG AACTGGGCGG AGTTAGGGGC

151 GGGATGGGCG GAGTTAGGGG CGGGACTATG GTTGCTGACT AATTGAGATG

201 CATGCTTTGC ATACTTCTGC CTGCTGGGGA GCCTGGTTGC TGACTAATTG

251 AGATGCATGC TTTGCATACT TCTGCCTGCT GGGGAGCCTG GGGACTTTCC

301 ACACCCTAAC TGACACACAT TCCACAGCTG CCTCGCGCGT TTCGGTGATG

351 ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT CACAGCTTGT

401 CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG CGTCAGCGGG

451 TGTTGGCGGG TGTCGGGGCG CAGCCATGAC CCAGTCACGT AGCGATAGCG

501 GAGTGTATAC TGGCTTAACT ATGCGGCATC AGAGCAGATT GTACTGAGAG

551 TGCACCATAT GCGGTGTGAA ATACCGCACA GATGCGTAAG GAGAAAATAC

601 CGCATCAGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT

651 CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT

701 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC

751 CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA

801 TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA

851 GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT TCCCCCTGGA

901 AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT

951 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT

1001 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG

1051 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG

1101 TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA

1151 CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC TACAGAGTTC

1201 TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG TATTTGGTAT

1251 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT

1301 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG

1351 CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT

1401 TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT

1451 TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT TTTAAATTAA

1501 AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA

1551 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT

1601 TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA

1651 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC

1701 ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG

1751 CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT CCAGTCTATT

1801 AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG

1851 CAACGTTGTT GCCATTGCTG CAGGCATCGT GGTGTCACGC TCGTCGTTTG

1901 GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA

1951 TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT

2001 TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC

2051 TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT TTCTGTGACT

2101 GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC GGCGACCGAG

2151 TTGCTCTTGC CCGGCGTCAA CACGGGATAA TACCGCGCCA CATAGCAGAA

2201 CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA

2251 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC

2301 CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA

2351 AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC GACACGGAAA

2401 TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA GCATTTATCA

2451 GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA

2501 AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC

2551 TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC

2601 GAGGCCCTTT CGTCTTCAAG AATTCAGCTT GGCTGCAGTG AATAATAAAA

2651 TGTGTGTTTG TCCGAAATAC GCGTTTTGAG ATTTCTGTCG CCGACTAAAT

2701 TCATGTCGCG CGATAGTGGT GTTTATCGCC GATAGAGATG GCGATATTGG

2751 AAAAATCGAT ATTTGAAAAT ATGGCATATT GAAAATGTCG CCGATGTGAG

2801 TTTCTGTGTA ACTGATATCG CCATTTTTCC AAAAGTGATT TTTGGGCATA

2851 CGCGATATCT GGCGATAGCG CTTATATCGT TTACGGGGGA TGGCGATAGA

2901 CGACTTTGGT GACTTGGGCG ATTCTGTGTG TCGCAAATAT CGCAGTTTCG

2951 ATATAGGTGA CAGACGATAT GAGGCTATAT CGCCGATAGA GGCGACATCA

3001 AGCTGGCACA TGGCCAATGC ATATCGATCT ATACATTGAA TCAATATTGG

3051 CCATTAGCCA TATTATTCAT TGGTTATATA GCATAAATCA ATATTGGCTA

3101 TTGGCCATTG CATACGTTGT ATCCATATCA TAATATGTAC ATTTATATTG

3151 GCTCATGTCC AACATTACCG CCATGTTGAC ATTGATTATT GACTAGTTAT

3201 TAATAGTAAT CAATTACGGG GTCATTAGTT CATAGCCCAT ATATGGAGTT

3251 CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA CCGCCCAACG

3301 ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA

3351 ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC

3401 CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG

3451 ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC

3501 TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT

3551 TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT GGATAGCGGT

3601 TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT

3651 TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC

3701 CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA

3751 TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA

3801 CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA GCCTCCGCAA

3851 GCTTGCCGCC ACCATGGACT GGACCTGGAG GGTGTTCTGC CTGCTGGCCG

3901 TGGCCCCCGG CGCCCACAGC CAGGTGCAGC TGGTGGAGTC AGGAGCCGAA

3951 GTGAAAAAGC CTGGGGCTTC AGTGAAGGTG TCCTGCAAGG CCTCTGGATA

4001 CACATTCACT AATTATATTA TCCACTGGGT GAAGCAGGAG CCTGGTCAGG

4051 GCCTTGAATG GATTGGATAT TTTAATCCTT ACAATCATGG TACTAAGTAC

4101 AATGAGAAGT TCAAAGGCAG GGCCACACTA ACTGCAAACA AATCCATCAG

4151 CACAGCCTAC ATGGAGCTCA GCAGCCTGCG CTCTGAGGAC ACTGCGGTCT

4201 ACTACTGTGC AAGATCAGGA CCCTATGCCT GGTTTGACAC CTGGGGCCAA

4251 GGGACCACGG TCACCGTCTC CTCAGGTGAG TTCTAGAAGG ATCCCAAGCT

4301 AGCTTTCTGG GGCAGGCCAG GCCTGACCTT GGCTTTGGGG CAGGGAGGGG

4351 GCTAAGGTGA GGCAGGTGGC GCCAGCCAGG TGCACACCCA ATGCCCATGA

4401 GCCCAGACAC TGGACGCTGA ACCTCGCGGA CAGTTAAGAA CCCAGGGGCC

4451 TCTGCGCCCT GGGCCCAGCT CTGTCCCACA CCGCGGTCAC ATGGCACCAC

4501 CTCTCTTGCA GCCTCCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT

4551 CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG CCTGGTCAAG

4601 GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC

4651 CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT

4701 CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACCCAGACC

4751 TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG TGGACAAGAA

4801 AGTTGGTGAG AGGCCAGCAC AGGGAGGGAG GGTGTCTGCT GGAAGCCAGG

4851 CTCAGCGCTC CTGCCTGGAC GCATCCCGGC TATGCAGCCC CAGTCCAGGG

4901 CAGCAAGGCA GGCCCCGTCT GCCTCTTCAC CCGGAGGCCT CTGCCCGCCC

4951 CACTCATGCT CAGGGAGAGG GTCTTCTGGC TTTTTCCCCA GGCTCTGGGC

5001 AGGCACAGGC TAGGTGCCCC TAACCCAGGC CCTGCACACA AAGGGGCAGG

5051 TGCTGGGCTC AGACCTGCCA AGAGCCATAT CCGGGAGGAC CCTGCCCCTG

5101 ACCTAAGCCC ACCCCAAAGG CCAAACTCTC CACTCCCTCA GCTCGGACAC

5151 CTTCTCTCCT CCCAGATTCC AGTAACTCCC AATCTTCTCT CTGCAGAGCC

5201 CAAATCTTGT GACAAAACTC ACACATGCCC ACCGTGCCCA GGTAAGCCAG

5251 CCCAGGCCTC GCCCTCCAGC TCAAGGCGGG ACAGGTGCCC TAGAGTAGCC

5301 TGCATCCAGG GACAGGCCCC AGCCGGGTGC TGACACGTCC ACCTCCATCT

5351 CTTCCTCAGC ACCTGAACTC CTGGGGGGAC CGTCAGTCTT CCTCTTCCCC

5401 CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG AGGTCACATG

5451 CGTGGTGGTG GACGTGAGCC ACGAAGACCC TGAGGTCAAG TTCAACTGGT

5501 ACGTGGACGG CGTGGAGGTG CATAATGCCA AGACAAAGCC GCGGGAGGAG

5551 CAGTACAACA GCACGTACCG TGTGGTCAGC GTCCTCACCG TCCTGCACCA

5601 GGACTGGCTG AATGGCAAGG AGTACAAGTG CAAGGTCTCC AACAAAGCCC

5651 TCCCAGCCCC CATCGAGAAA ACCATCTCCA AAGCCAAAGG TGGGACCCGT

5701 GGGGTGCGAG GGCCACATGG ACAGAGGCCG GCTCGGCCCA CCCTCTGCCC

5751 TGAGAGTGAC CGCTGTACCA ACCTCTGTCC CTACAGGGCA GCCCCGAGAA

5801 CCACAGGTGT ACACCCTGCC CCCATCCCGG GATGAGCTGA CCAAGAACCA

5851 GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG

5901 TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT

5951 CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA AGCTCACCGT

6001 GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC

6051 ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG

6101 GGTAAATGAG TGCGACGGCC GGCAAGCCCC CGCTCCCCGG GCTCTCGCGG

6151 TCGCACGAGG ATGCTTGGCA CGTACCCCCT GTACATACTT CCCGGGCGCC

6201 CAGCATGGAA ATAAAGCACC CAGCGCTGCC CTGGGCCCCT GCGAGACTGT

6251 GATGGTTCTT TCCACGGGTC AGGCCGAGTC TGAGGCCTGA GTGGCATGAG

6301 ATCTGATATC ATCGATGAAT TCGAGCTCGG TACCCGGGGA TCGATCCAGA

6351 CATGATAAGA TACATTGATG AGTTTGGACA AACCACAACT AGAATGCAGT

6401 GAAAAAAATG CTTTATTTGT GAAATTTGTG ATGCTATTGC TTTATTTGTA

6451 ACCATTATAA GCTGCAATAA ACAAGTTAAC AACAACAATT GCATTCATTT

6501 TATGTTTCAG GTTCAGGGGG AGGTGTGGGA GGTTTTTTAA AGCAAGTAAA

6551 ACCTCTACAA ATGTGGTATG GCTGATTATG ATCTCTAGTC AAGGCACTAT

6601 ACATCAAATA TTCCTTATTA ACCCCTTTAC AAATTAAAAA GCTAAAGGTA

6651 CACAATTTTT GAGCATAGTT ATTAATAGCA GACACTCTAT GCCTGTGTGG

6701 AGTAAGAAAA AACAGTATGT TATGATTATA ACTGTTATGC CTACTTATAA

6751 AGGTTACAGA ATATTTTTCC ATAATTTTCT TGTATAGCAG TGCAGCTTTT

6801 TCCTTTGTGG TGTAAATAGC AAAGCAAGCA AGAGTTCTAT TACTAAACAC

6851 AGCATGACTC AAAAAACTTA GCAATTCTGA AGGAAAGTCC TTGGGGTCTT

6901 CTACCTTTCT CTTCTTTTTT GGAGGAGTAG AATGTTGAGA GTCAGCAGTA

6951 GCCTCATCAT CACTAGATGG CATTTCTTCT GAGCAAAACA GGTTTTCCTC

7001 ATTAAAGGCA TTCCACCACT GCTCCCATTC ATCAGTTCCA TAGGTTGGAA

7051 TCTAAAATAC ACAAACAATT AGAATCAGTA GTTTAACACA TTATACACTT

7101 AAAAATTTTA TATTTACCTT AGAGCTTTAA ATCTCTGTAG GTAGTTTGTC

7151 CAATTATGTC ACACCACAGA AGTAAGGTTC CTTCACAAAG ATCCGGGACC

7201 AAAGCGGCCA TCGTGCCTCC CCACTCCTGC AGTTCGGGGG CATGGATGCG

7251 CGGATAGCCG CTGCTGGTTT CCTGGATGCC GACGGATTTG CACTGCCGGT

7301 AGAACTCCGC GAGGTCGTCC AGCCTCAGGC AGCAGCTGAA CCAACTCGCG

7351 AGGGGATCGA GCCCGGGGTG GGCGAAGAAC TCCAGCATGA GATCCCCGCG

7401 CTGGAGGATC ATCCAGCCGG CGTCCCGGAA AACGATTCCG AAGCCCAACC

7451 TTTCATAGAA GGCGGCGGTG GAATCGAAAT CTCGTGATGG CAGGTTGGGC

7501 GTCGCTTGGT CGGTCATTTC GAACCCCAGA GTCCCGCTCA GAAGAACTCG

7551 TCAAGAAGGC GATAGAAGGC GATGCGCTGC GAATCGGGAG CGGCGATACC

7601 GTAAAGCACG AGGAAGCGGT CAGCCCATTC GCCGCCAAGC TCTTCAGCAA

7651 TATCACGGGT AGCCAACGCT ATGTCCTGAT AGCGGTCCGC CACACCCAGC

7701 CGGCCACAGT CGATGAATCC AGAAAAGCGG CCATTTTCCA CCATGATATT

7751 CGGCAAGCAG GCATCGCCAT GGGTCACGAC GAGATCCTCG CCGTCGGGCA

7801 TGCGCGCCTT GAGCCTGGCG AACAGTTCGG CTGGCGCGAG CCCCTGATGC

7851 TCTTCGTCCA GATCATCCTG ATCGACAAGA CCGGCTTCCA TCCGAGTACG

7901 TGCTCGCTCG ATGCGATGTT TCGCTTGGTG GTCGAATGGG CAGGTAGCCG

7951 GATCAAGCGT ATGCAGCCGC CGCATTGCAT CAGCCATGAT GGATACTTTC

8001 TCGGCAGGAG CAAGGTGAGA TGACAGGAGA TCCTGCCCCG GCACTTCGCC

8051 CAATAGCAGC CAGTCCCTTC CCGCTTCAGT GACAACGTCG AGCACAGCTG

8101 CGCAAGGAAC GCCCGTCGTG GCCAGCCACG ATAGCCGCGC TGCCTCGTCC

8151 TGCAGTTCAT TCAGGGCACC GGACAGGTCG GTCTTGACAA AAAGAACCGG

8201 GCGCCCCTGC GCTGACAGCC GGAACACGGC GGCATCAGAG CAGCCGATTG

8251 TCTGTTGTGC CCAGTCATAG CCGAATAGCC TCTCCACCCA AGCGGCCGGA

8301 GAACCTGCGT GCAATCCATC TTGTTCAATC ATGCGAAACG ATCCTCATCC

8351 TGTCTCTTGA TCAGATCTTG ATCCCCTGCG CCATCAGATC CTTGGCGGCA

8401 AGAAAGCCAT CCAGTTTACT TTGCAGGGCT TCCCAACCTT ACCAGAGGGC

8451 GCCCCAGCTG GCAATTCCGG TTCGCTTGCT GTCCATAAAA CCGCCCAGTC

8501 TAGCTATCGC CATGTAAGCC CACTGCAAGC TACCTGCTTT CTCTTTGCGC

8551 TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACATT CATCCGGGGT

8601 CAGCACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT TCCTTTAGCA

8651 GCCCTTGCGC CCTGAGTGCT TGCGGCAGCG TGAAGCT

SEQ ID NO：16

Nucleotide sequence of expression vector HCMV-G1 HuAb-VHE

(in 3921-4274 complete DNA sequence of humanized heavy chain expression vector comprising SEQ ID NO: 11 (VHE))

1 AGCTTTTTGC AAAAGCCTAG GCCTCCAAAA AAGCCTCCTC ACTACTTCTG

51 GAATAGCTCA GAGGCCGAGG CGGCCTCGGC CTCTGCATAA ATAAAAAAAA

101 TTAGTCAGCC ATGGGGCGGA GAATGGGCGG AACTGGGCGG AGTTAGGGGC

151 GGGATGGGCG GAGTTAGGGG CGGGACTATG GTTGCTGACT AATTGAGATG

201 CATGCTTTGC ATACTTCTGC CTGCTGGGGA GCCTGGTTGC TGACTAATTG

251 AGATGCATGC TTTGCATACT TCTGCCTGCT GGGGAGCCTG GGGACTTTCC

301 ACACCCTAAC TGACACACAT TCCACAGCTG CCTCGCGCGT TTCGGTGATG

351 ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT CACAGCTTGT

401 CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG CGTCAGCGGG

451 TGTTGGCGGG TGTCGGGGCG CAGCCATGAC CCAGTCACGT AGCGATAGCG

501 GAGTGTATAC TGGCTTAACT ATGCGGCATC AGAGCAGATT GTACTGAGAG

551 TGCACCATAT GCGGTGTGAA ATACCGCACA GATGCGTAAG GAGAAAATAC

601 CGCATCAGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT

651 CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT

701 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC

751 CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA

801 TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA

851 GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT TCCCCCTGGA

901 AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT

951 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT

100 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG

1051 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG

1101 TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA

1151 CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC TACAGAGTTC

1201 TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG TATTTGGTAT

1251 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT

1301 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG

1351 CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT

1401 TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT

1451 TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT TTTAAATTAA

1501 AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA

1551 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT

1601 TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA

1651 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC

1701 ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG

1751 CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT CCAGTCTATT

1801 AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG

1851 CAACGTTGTT GCCATTGCTG CAGGCATCGT GGTGTCACGC TCGTCGTTTG

1901 GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA

1951 TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT

2001 TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC

2051 TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT TTCTGTGACT

2101 GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC GGCGACCGAG

2151 TTGCTCTTGC CCGGCGTCAA CACGGGATAA TACCGCGCCA CATAGCAGAA

2201 CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA

2251 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC

2301 CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA

2351 AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC GACACGGAAA

2401 TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA GCATTTATCA

2451 GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA

2501 AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC

2551 TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC

2601 GAGGCCCTTT CGTCTTCAAG AATTCAGCTT GGCTGCAGTG AATAATAAAA

2651 TGTGTGTTTG TCCGAAATAC GCGTTTTGAG ATTTCTGTCG CCGACTAAAT

2701 TCATGTCGCG CGATAGTGGT GTTTATCGCC GATAGAGATG GCGATATTGG

2751 AAAAATCGAT ATTTGAAAAT ATGGCATATT GAAAATGTCG CCGATGTGAG

2801 TTTCTGTGTA ACTGATATCG CCATTTTTCC AAAAGTGATT TTTGGGCATA

2851 CGCGATATCT GGCGATAGCG CTTATATCGT TTACGGGGGA TGGCGATAGA

2901 CGACTTTGGT GACTTGGGCG ATTCTGTGTG TCGCAAATAT CGCAGTTTCG

2951 ATATAGGTGA CAGACGATAT GAGGCTATAT CGCCGATAGA GGCGACATCA

3001 AGCTGGCACA TGGCCAATGC ATATCGATCT ATACATTGAA TCAATATTGG

3051 CCATTAGCCA TATTATTCAT TGGTTATATA GCATAAATCA ATATTGGCTA

3101 TTGGCCATTG CATACGTTGT ATCCATATCA TAATATGTAC ATTTATATTG

3151 GCTCATGTCC AACATTACCG CCATGTTGAC ATTGATTATT GACTAGTTAT

3201 TAATAGTAAT CAATTACGGG GTCATTAGTT CATAGCCCAT ATATGGAGTT

3251 CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA CCGCCCAACG

3301 ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA

3351 ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC

3401 CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG

3451 ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC

3501 TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT

3551 TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT GGATAGCGGT

3601 TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT

3651 TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC

3701 CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA

3751 TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA

3801 CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA GCCTCCGCAA

3851 GCTTGCCGCC ACCATGGACT GGACCTGGAG GGTGTTCTGC CTGCTGGCCG

3901 TGGCCCCCGG CGCCCACAGC GAGGTGCAGC TGGTGGAGTC AGGAGCCGAA

3951 GTGAAAAAGC CTGGGGCTTC AGTGAAGGTG TCCTGCAAGG CCTCTGGATA

4001 CACATTCACT AATTATATTA TCCACTGGGT GAAGCAGGAG CCTGGTCAGG

4051 GCCTTGAATG GATTGGATAT TTTAATCCTT ACAATCATGG TACTAAGTAC

4101 AATGAGAAGT TCAAAGGCAG GGCCACACTA ACTGCAAACA AATCCATCAG

4151 CACAGCCTAC ATGGAGCTCA GCAGCCTGCG CTCTGAGGAC ACTGCGGTCT

4201 ACTACTGTGC AAGATCAGGA CCCTATGCCT GGTTTGACAC CTGGGGCCAA

4251 GGGACCACGG TCACCGTCTC CTCAGGTGAG TTCTAGAAGG ATCCCAAGCT

4301 AGCTTTCTGG GGCAGGCCAG GCCTGACCTT GGCTTTGGGG CAGGGAGGGG

4351 GCTAAGGTGA GGCAGGTGGC GCCAGCCAGG TGCACACCCA ATGCCCATGA

4401 GCCCAGACAC TGGACGCTGA ACCTCGCGGA CAGTTAAGAA CCCAGGGGCC

4451 TCTGCGCCCT GGGCCCAGCT CTGTCCCACA CCGCGGTCAC ATGGCACCAC

4501 CTCTCTTGCA GCCTCCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT

4551 CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG CCTGGTCAAG

4601 GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC

4651 CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT

4701 CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACCCAGACC

4751 TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG TGGACAAGAA

4801 AGTTGGTGAG AGGCCAGCAC AGGGAGGGAG GGTGTCTGCT GGAAGCCAGG

4851 CTCAGCGCTC CTGCCTGGAC GCATCCCGGC TATGCAGCCC CAGTCCAGGG

4901 CAGCAAGGCA GGCCCCGTCT GCCTCTTCAC CCGGAGGCCT CTGCCCGCCC

4951 CACTCATGCT CAGGGAGAGG GTCTTCTGGC TTTTTCCCCA GGCTCTGGGC

5001 AGGCACAGGC TAGGTGCCCC TAACCCAGGC CCTGCACACA AAGGGGCAGG

5051 TGCTGGGCTC AGACCTGCCA AGAGCCATAT CCGGGAGGAC CCTGCCCCTG

5101 ACCTAAGCCC ACCCCAAAGG CCAAACTCTC CACTCCCTCA GCTCGGACAC

5151 CTTCTCTCCT CCCAGATTCC AGTAACTCCC AATCTTCTCT CTGCAGAGCC

5201 CAAATCTTGT GACAAAACTC ACACATGCCC ACCGTGCCCA GGTAAGCCAG

5251 CCCAGGCCTC GCCCTCCAGC TCAAGGCGGG ACAGGTGCCC TAGAGTAGCC

5301 TGCATCCAGG GACAGGCCCC AGCCGGGTGC TGACACGTCC ACCTCCATCT

5351 CTTCCTCAGC ACCTGAACTC CTGGGGGGAC CGTCAGTCTT CCTCTTCCCC

5401 CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG AGGTCACATG

5451 CGTGGTGGTG GACGTGAGCC ACGAAGACCC TGAGGTCAAG TTCAACTGGT

5501 ACGTGGACGG CGTGGAGGTG CATAATGCCA AGACAAAGCC GCGGGAGGAG

5551 CAGTACAACA GCACGTACCG TGTGGTCAGC GTCCTCACCG TCCTGCACCA

5601 GGACTGGCTG AATGGCAAGG AGTACAAGTG CAAGGTCTCC AACAAAGCCC

5651 TCCCAGCCCC CATCGAGAAA ACCATCTCCA AAGCCAAAGG TGGGACCCGT

5701 GGGGTGCGAG GGCCACATGG ACAGAGGCCG GCTCGGCCCA CCCTCTGCCC

5751 TGAGAGTGAC CGCTGTACCA ACCTCTGTCC CTACAGGGCA GCCCCGAGAA

5801 CCACAGGTGT ACACCCTGCC CCCATCCCGG GATGAGCTGA CCAAGAACCA

5851 GGTCAGCCTG ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG

5901 TGGAGTGGGA GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT

5951 CCCGTGCTGG ACTCCGACGG CTCCTTCTTC CTCTACAGCA AGCTCACCGT

6001 GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC

6051 ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG

6101 GGTAAATGAG TGCGACGGCC GGCAAGCCCC CGCTCCCCGG GCTCTCGCGG

6151 TCGCACGAGG ATGCTTGGCA CGTACCCCCT GTACATACTT CCCGGGCGCC

6201 CAGCATGGAA ATAAAGCACC CAGCGCTGCC CTGGGCCCCT GCGAGACTGT

6251 GATGGTTCTT TCCACGGGTC AGGCCGAGTC TGAGGCCTGA GTGGCATGAG

6301 ATCTGATATC ATCGATGAAT TCGAGCTCGG TACCCGGGGA TCGATCCAGA

6351 CATGATAAGA TACATTGATG AGTTTGGACA AACCACAACT AGAATGCAGT

6401 GAAAAAAATG CTTTATTTGT GAAATTTGTG ATGCTATTGC TTTATTTGTA

6451 ACCATTATAA GCTGCAATAA ACAAGTTAAC AACAACAATT GCATTCATTT

6501 TATGTTTCAG GTTCAGGGGG AGGTGTGGGA GGTTTTTTAA AGCAAGTAAA

6551 ACCTCTACAA ATGTGGTATG GCTGATTATG ATCTCTAGTC AAGGCACTAT

6601 ACATCAAATA TTCCTTATTA ACCCCTTTAC AAATTAAAAA GCTAAAGGTA

6651 CACAATTTTT GAGCATAGTT ATTAATAGCA GACACTCTAT GCCTGTGTGG

6701 AGTAAGAAAA AACAGTATGT TATGATTATA ACTGTTATGC CTACTTATAA

6751 AGGTTACAGA ATATTTTTCC ATAATTTTCT TGTATAGCAG TGCAGCTTTT

6801 TCCTTTGTGG TGTAAATAGC AAAGCAAGCA AGAGTTCTAT TACTAAACAC

6851 AGCATGACTC AAAAAACTTA GCAATTCTGA AGGAAAGTCC TTGGGGTCTT

6901 CTACCTTTCT CTTCTTTTTT GGAGGAGTAG AATGTTGAGA GTCAGCAGTA

6951 GCCTCATCAT CACTAGATGG CATTTCTTCT GAGCAAAACA GGTTTTCCTC

7001 ATTAAAGGCA TTCCACCACT GCTCCCATTC ATCAGTTCCA TAGGTTGGAA

7051 TCTAAAATAC ACAAACAATT AGAATCAGTA GTTTAACACA TTATACACTT

7101 AAAAATTTTA TATTTACCTT AGAGCTTTAA ATCTCTGTAG GTAGTTTGTC

7151 CAATTATGTC ACACCACAGA AGTAAGGTTC CTTCACAAAG ATCCGGGACC

7201 AAAGCGGCCA TCGTGCCTCC CCACTCCTGC AGTTCGGGGG CATGGATGCG

7251 CGGATAGCCG CTGCTGGTTT CCTGGATGCC GACGGATTTG CACTGCCGGT

7301 AGAACTCCGC GAGGTCGTCC AGCCTCAGGC AGCAGCTGAA CCAACTCGCG

7351 AGGGGATCGA GCCCGGGGTG GGCGAAGAAC TCCAGCATGA GATCCCCGCG

7401 CTGGAGGATC ATCCAGCCGG CGTCCCGGAA AACGATTCCG AAGCCCAACC

7451 TTTCATAGAA GGCGGCGGTG GAATCGAAAT CTCGTGATGG CAGGTTGGGC

7501 GTCGCTTGGT CGGTCATTTC GAACCCCAGA GTCCCGCTCA GAAGAACTCG

7551 TCAAGAAGGC GATAGAAGGC GATGCGCTGC GAATCGGGAG CGGCGATACC

7601 GTAAAGCACG AGGAAGCGGT CAGCCCATTC GCCGCCAAGC TCTTCAGCAA

7651 TATCACGGGT AGCCAACGCT ATGTCCTGAT AGCGGTCCGC CACACCCAGC

7701 CGGCCACAGT CGATGAATCC AGAAAAGCGG CCATTTTCCA CCATGATATT

7751 CGGCAAGCAG GCATCGCCAT GGGTCACGAC GAGATCCTCG CCGTCGGGCA

7801 TGCGCGCCTT GAGCCTGGCG AACAGTTCGG CTGGCGCGAG CCCCTGATGC

7851 TCTTCGTCCA GATCATCCTG ATCGACAAGA CCGGCTTCCA TCCGAGTACG

7901 TGCTCGCTCG ATGCGATGTT TCGCTTGGTG GTCGAATGGG CAGGTAGCCG

7951 GATCAAGCGT ATGCAGCCGC CGCATTGCAT CAGCCATGAT GGATACTTTC

8001 TCGGCAGGAG CAAGGTGAGA TGACAGGAGA TCCTGCCCCG GCACTTCGCC

8051 CAATAGCAGC CAGTCCCTTC CCGCTTCAGT GACAACGTCG AGCACAGCTG

8101 CGCAAGGAAC GCCCGTCGTG GCCAGCCACG ATAGCCGCGC TGCCTCGTCC

8151 TGCAGTTCAT TCAGGGCACC GGACAGGTCG GTCTTGACAA AAAGAACCGG

8201 GCGCCCCTGC GCTGACAGCC GGAACACGGC GGCATCAGAG CAGCCGATTG

8251 TCTGTTGTGC CCAGTCATAG CCGAATAGCC TCTCCACCCA AGCGGCCGGA

8301 GAACCTGCGT GCAATCCATC TTGTTCAATC ATGCGAAACG ATCCTCATCC

8351 TGTCTCTTGA TCAGATCTTG ATCCCCTGCG CCATCAGATC CTTGGCGGCA

8401 AGAAAGCCAT CCAGTTTACT TTGCAGGGCT TCCCAACCTT ACCAGAGGGC

8451 GCCCCAGCTG GCAATTCCGG TTCGCTTGCT GTCCATAAAA CCGCCCAGTC

8501 TAGCTATCGC CATGTAAGCC CACTGCAAGC TACCTGCTTT CTCTTTGCGC

8551 TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACATT CATCCGGGGT

8601 CAGCACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT TCCTTTAGCA

8651 GCCCTTGCGC CCTGAGTGCT TGCGGCAGCG TGAAGCT

SEQ ID NO：17

Nucleotide sequence of expression vector HCMV-K HuAb-VL1 hum V1

(in 3964-4284 contains the entire DNA sequence of the humanized light chain expression vector of SEQ ID NO: 14(humV1 ═ VLh))

1 CTAGCTTTTT GCAAAAGCCT AGGCCTCCAA AAAAGCCTCC TCACTACTTC

51 TGGAATAGCT CAGAGGCCGA GGCGGCCTCG GCCTCTGCAT AAATAAAAAA

101 AATTAGTCAG CCATGGGGCG GAGAATGGGC GGAACTGGGC GGAGTTAGGG

151 GCGGGATGGG CGGAGTTAGG GGCGGGACTA TGGTTGCTGA CTAATTGAGA

201 TGCATGCTTT GCATACTTCT GCCTGCTGGG GAGCCTGGTT GCTGACTAAT

251 TGAGATGCAT GCTTTGCATA CTTCTGCCTG CTGGGGAGCC TGGGGACTTT

301 CCACACCCTA ACTGACACAC ATTCCACAGC TGCCTCGCGC GTTTCGGTGA

351 TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG GTCACAGCTT

401 GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG CGCGTCAGCG

451 GGTGTTGGCG GGTGTCGGGG CGCAGCCATG ACCCAGTCAC GTAGCGATAG

501 CGGAGTGTAT ACTGGCTTAA CTATGCGGCA TCAGAGCAGA TTGTACTGAG

551 AGTGCACCAT ATGCGGTGTG AAATACCGCA CAGATGCGTA AGGAGAAAAT

601 ACCGCATCAG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG

651 GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG

701 GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG

751 GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC

801 CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA

851 GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG

901 GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC

951 CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC ATAGCTCACG

1001 CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG

1051 TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT

1101 CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC

1151 CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT

1201 TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT

1251 ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC

1301 TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA

1351 AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC

1401 TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT

1451 TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT

1501 AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT

1551 GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT

1601 ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA

1651 TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC

1701 CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG

1751 GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA

1801 TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG

1851 CGCAACGTTG TTGCCATTGC TGCAGGCATC GTGGTGTCAC GCTCGTCGTT

1901 TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT

1951 GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC

2001 GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC

2051 ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA

2101 CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG

2151 AGTTGCTCTT GCCCGGCGTC AACACGGGAT AATACCGCGC CACATAGCAG

2201 AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT

2251 CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA

2301 CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC

2351 AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA

2401 AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT

2451 CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA

2501 TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG

2551 TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA TAGGCGTATC

2601 ACGAGGCCCT TTCGTCTTCA AGAATTCAGC TTGGCTGCAG TGAATAATAA

2651 AATGTGTGTT TGTCCGAAAT ACGCGTTTTG AGATTTCTGT CGCCGACTAA

2701 ATTCATGTCG CGCGATAGTG GTGTTTATCG CCGATAGAGA TGGCGATATT

2751 GGAAAAATCG ATATTTGAAA ATATGGCATA TTGAAAATGT CGCCGATGTG

2801 AGTTTCTGTG TAACTGATAT CGCCATTTTT CCAAAAGTGA TTTTTGGGCA

2851 TACGCGATAT CTGGCGATAG CGCTTATATC GTTTACGGGG GATGGCGATA

2901 GACGACTTTG GTGACTTGGG CGATTCTGTG TGTCGCAAAT ATCGCAGTTT

2951 CGATATAGGT GACAGACGAT ATGAGGCTAT ATCGCCGATA GAGGCGACAT

3001 CAAGCTGGCA CATGGCCAAT GCATATCGAT CTATACATTG AATCAATATT

3051 GGCCATTAGC CATATTATTC ATTGGTTATA TAGCATAAAT CAATATTGGC

3101 TATTGGCCAT TGCATACGTT GTATCCATAT CATAATATGT ACATTTATAT

3151 TGGCTCATGT CCAACATTAC CGCCATGTTG ACATTGATTA TTGACTAGTT

3201 ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC ATATATGGAG

3251 TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT GACCGCCCAA

3301 CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC

3351 CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT ACGGTAAACT

3401 GCCCACTTGG CAGTACATCA AGTGTATCAT ATGCCAAGTA CGCCCCCTAT

3451 TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC CAGTACATGA

3501 CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT AGTCATCGCT

3551 ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG

3601 GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA

3651 GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC

3701 TCCGCCCCAT TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTCTA

3751 TATAAGCAGA GCTCGTTTAG TGAACCGTCA GATCGCCTGG AGACGCCATC

3801 CACGCTGTTT TGACCTCCAT AGAAGACACC GGGACCGATC CAGCCTCCGC

3851 AAGCTTGATA TCGAATTCCT GCAGCCCGGG GGATCCGCCC GCTTGCCGCC

3901 ACCATGGAGA CCCCCGCCCA GCTGCTGTTC CTGCTGCTGC TGTGGCTGCC

3951 CGACACCACC GGCGACATTC TGCTGACCCA GTCTCCAGCC ACCCTGTCTC

4001 TGAGTCCAGG AGAAAGAGCC ACTCTCTCCT GCAGGGCCAG TCAGAACATT

4051 GGCACAAGCA TACAGTGGTA TCAACAAAAA CCAGGTCAGG CTCCAAGGCT

4101 TCTCATAAGG TCTTCTTCTG AGTCTATCTC TGGGATCCCT TCCAGGTTTA

4151 GTGGCAGTGG ATCAGGGACA GATTTTACTC TTACCATCAG CAGTCTGGAG

4201 CCTGAAGATT TTGCAGTGTA TTACTGTCAA CAAAGTAATA CCTGGCCATT

4251 CACGTTCGGC CAGGGGACCA AGCTGGAGAT CAAACGTGAG TATTCTAGAA

4301 AGATCCTAGA ATTCTAAACT CTGAGGGGGT CGGATGACGT GGCCATTCTT

4351 TGCCTAAAGC ATTGAGTTTA CTGCAAGGTC AGAAAAGCAT GCAAAGCCCT

4401 CAGAATGGCT GCAAAGAGCT CCAACAAAAC AATTTAGAAC TTTATTAAGG

4451 AATAGGGGGA AGCTAGGAAG AAACTCAAAA CATCAAGATT TTAAATACGC

4501 TTCTTGGTCT CCTTGCTATA ATTATCTGGG ATAAGCATGC TGTTTTCTGT

4551 CTGTCCCTAA CATGCCCTGT GATTATCCGC AAACAACACA CCCAAGGGCA

4601 GAACTTTGTT ACTTAAACAC CATCCTGTTT GCTTCTTTCC TCAGGAACTG

4651 TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA GCAGTTGAAA

4701 TCTGGAACTG CCTCTGTTGT GTGCCTGCTG AATAACTTCT ATCCCAGAGA

4751 GGCCAAAGTA CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC

4801 AGGAGAGTGT CACAGAGCAG GACAGCAAGG ACAGCACCTA CAGCCTCAGC

4851 AGCACCCTGA CGCTGAGCAA AGCAGACTAC GAGAAACACA AAGTCTACGC

4901 CTGCGAAGTC ACCCATCAGG GCCTGAGCTC GCCCGTCACA AAGAGCTTCA

4951 ACAGGGGAGA GTGTTAGAGG GAGAAGTGCC CCCACCTGCT CCTCAGTTCC

5001 AGCCTGACCC CCTCCCATCC TTTGGCCTCT GACCCTTTTT CCACAGGGGA

5051 CCTACCCCTA TTGCGGTCCT CCAGCTCATC TTTCACCTCA CCCCCCTCCT

5101 CCTCCTTGGC TTTAATTATG CTAATGTTGG AGGAGAATGA ATAAATAAAG

5151 TGAATCTTTG CACCTGTGGT TTCTCTCTTT CCTCATTTAA TAATTATTAT

5201 CTGTTGTTTA CCAACTACTC AATTTCTCTT ATAAGGGACT AAATATGTAG

5251 TCATCCTAAG GCGCATAACC ATTTATAAAA ATCATCCTTC ATTCTATTTT

5301 ACCCTATCAT CCTCTGCAAG ACAGTCCTCC CTCAAACCCA CAAGCCTTCT

5351 GTCCTCACAG TCCCCTGGGC CATGGTAGGA GAGACTTGCT TCCTTGTTTT

5401 CCCCTCCTCA GCAAGCCCTC ATAGTCCTTT TTAAGGGTGA CAGGTCTTAC

5451 AGTCATATAT CCTTTGATTC AATTCCCTGA GAATCAACCA AAGCAAATTT

5501 TTCAAAAGAA GAAACCTGCT ATAAAGAGAA TCATTCATTG CAACATGATA

5551 TAAAATAACA ACACAATAAA AGCAATTAAA TAAACAAACA ATAGGGAAAT

5601 GTTTAAGTTC ATCATGGTAC TTAGACTTAA TGGAATGTCA TGCCTTATTT

5651 ACATTTTTAA ACAGGTACTG AGGGACTCCT GTCTGCCAAG GGCCGTATTG

5701 AGTACTTTCC ACAACCTAAT TTAATCCACA CTATACTGTG AGATTAAAAA

5751 CATTCATTAA AATGTTGCAA AGGTTCTATA AAGCTGAGAG ACAAATATAT

5801 TCTATAACTC AGCAATCCCA CTTCTAGATG ACTGAGTGTC CCCACCCACC

5851 AAAAAACTAT GCAAGAATGT TCAAAGCAGC TTTATTTACA AAAGCCAAAA

5901 ATTGGAAATA GCCCGATTGT CCAACAATAG AATGAGTTAT TAAACTGTGG

5951 TATGTTTATA CATTAGAATA CCCAATGAGG AGAATTAACA AGCTACAACT

6001 ATACCTACTC ACACAGATGA ATCTCATAAA AATAATGTTA CATAAGAGAA

6051 ACTCAATGCA AAAGATATGT TCTGTATGTT TTCATCCATA TAAAGTTCAA

6101 AACCAGGTAA AAATAAAGTT AGAAATTTGG ATGGAAATTA CTCTTAGCTG

6151 GGGGTGGGCG AGTTAGTGCC TGGGAGAAGA CAAGAAGGGG CTTCTGGGGT

6201 CTTGGTAATG TTCTGTTCCT CGTGTGGGGT TGTGCAGTTA TGATCTGTGC

6251 ACTGTTCTGT ATACACATTA TGCTTCAAAA TAACTTCACA TAAAGAACAT

6301 CTTATACCCA GTTAATAGAT AGAAGAGGAA TAAGTAATAG GTCAAGACCA

6351 CGCAGCTGGT AAGTGGGGGG GCCTGGGATC AAATAGCTAC CTGCCTAATC

6401 CTGCCCTCTT GAGCCCTGAA TGAGTCTGCC TTCCAGGGCT CAAGGTGCTC

6451 AACAAAACAA CAGGCCTGCT ATTTTCCTGG CATCTGTGCC CTGTTTGGCT

6501 AGCTAGGAGC ACACATACAT AGAAATTAAA TGAAACAGAC CTTCAGCAAG

6551 GGGACAGAGG ACAGAATTAA CCTTGCCCAG ACACTGGAAA CCCATGTATG

6601 AACACTCACA TGTTTGGGAA GGGGGAAGGG CACATGTAAA TGAGGACTCT

6651 TCCTCATTCT ATGGGGCACT CTGGCCCTGC CCCTCTCAGC TACTCATCCA

6701 TCCAACACAC CTTTCTAAGT ACCTCTCTCT GCCTACACTC TGAAGGGGTT

6751 CAGGAGTAAC TAACACAGCA TCCCTTCCCT CAAATGACTG ACAATCCCTT

6801 TGTCCTGCTT TGTTTTTCTT TCCAGTCAGT ACTGGGAAAG TGGGGAAGGA

6851 CAGTCATGGA GAAACTACAT AAGGAAGCAC CTTGCCCTTC TGCCTCTTGA

6901 GAATGTTGAT GAGTATCAAA TCTTTCAAAC TTTGGAGGTT TGAGTAGGGG

6951 TGAGACTCAG TAATGTCCCT TCCAATGACA TGAACTTGCT CACTCATCCC

7001 TGGGGGCCAA ATTGAACAAT CAAAGGCAGG CATAATCCAG CTATGAATTC

7051 TAGGATCGAT CCAGACATGA TAAGATACAT TGATGAGTTT GGACAAACCA

7101 CAACTAGAAT GCAGTGAAAA AAATGCTTTA TTTGTGAAAT TTGTGATGCT

7151 ATTGCTTTAT TTGTAACCAT TATAAGCTGC AATAAACAAG TTAACAACAA

7201 CAATTGCATT CATTTTATGT TTCAGGTTCA GGGGGAGGTG TGGGAGGTTT

7251 TTTAAAGCAA GTAAAACCTC TACAAATGTG GTATGGCTGA TTATGATCTC

7301 TAGTCAAGGC ACTATACATC AAATATTCCT TATTAACCCC TTTACAAATT

7351 AAAAAGCTAA AGGTACACAA TTTTTGAGCA TAGTTATTAA TAGCAGACAC

7401 TCTATGCCTG TGTGGAGTAA GAAAAAACAG TATGTTATGA TTATAACTGT

7451 TATGCCTACT TATAAAGGTT ACAGAATATT TTTCCATAAT TTTCTTGTAT

7501 AGCAGTGCAG CTTTTTCCTT TGTGGTGTAA ATAGCAAAGC AAGCAAGAGT

7551 TCTATTACTA AACACAGCAT GACTCAAAAA ACTTAGCAAT TCTGAAGGAA

7601 AGTCCTTGGG GTCTTCTACC TTTCTCTTCT TTTTTGGAGG AGTAGAATGT

7651 TGAGAGTCAG CAGTAGCCTC ATCATCACTA GATGGCATTT CTTCTGAGCA

7701 AAACAGGTTT TCCTCATTAA AGGCATTCCA CCACTGCTCC CATTCATCAG

7751 TTCCATAGGT TGGAATCTAA AATACACAAA CAATTAGAAT CAGTAGTTTA

7801 ACACATTATA CACTTAAAAA TTTTATATTT ACCTTAGAGC TTTAAATCTC

7851 TGTAGGTAGT TTGTCCAATT ATGTCACACC ACAGAAGTAA GGTTCCTTCA

7901 CAAAGATCCG GGACCAAAGC GGCCATCGTG CCTCCCCACT CCTGCAGTTC

7951 GGGGGCATGG ATGCGCGGAT AGCCGCTGCT GGTTTCCTGG ATGCCGACGG

8001 ATTTGCACTG CCGGTAGAAC TCCGCGAGGT CGTCCAGCCT CAGGCAGCAG

8051 CTGAACCAAC TCGCGAGGGG ATCGAGCCCG GGGTGGGCGA AGAACTCCAG

8101 CATGAGATCC CCGCGCTGGA GGATCATCCA GCCGGCGTCC CGGAAAACGA

8151 TTCCGAAGCC CAACCTTTCA TAGAAGGCGG CGGTGGAATC GAAATCTCGT

8201 GATGGCAGGT TGGGCGTCGC TTGGTCGGTC ATTTCGAACC CCAGAGTCCC

8251 GCTCAGAAGA ACTCGTCAAG AAGGCGATAG AAGGCGATGC GCTGCGAATC

8301 GGGAGCGGCG ATACCGTAAA GCACGAGGAA GCGGTCAGCC CATTCGCCGC

8351 CAAGCTCTTC AGCAATATCA CGGGTAGCCA ACGCTATGTC CTGATAGCGG

8401 TCCGCCACAC CCAGCCGGCC ACAGTCGATG AATCCAGAAA AGCGGCCATT

8451 TTCCACCATG ATATTCGGCA AGCAGGCATC GCCATGGGTC ACGACGAGAT

8501 CCTCGCCGTC GGGCATGCGC GCCTTGAGCC TGGCGAACAG TTCGGCTGGC

8551 GCGAGCCCCT GATGCTCTTC GTCCAGATCA TCCTGATCGA CAAGACCGGC

8601 TTCCATCCGA GTACGTGCTC GCTCGATGCG ATGTTTCGCT TGGTGGTCGA

8651 ATGGGCAGGT AGCCGGATCA AGCGTATGCA GCCGCCGCAT TGCATCAGCC

8701 ATGATGGATA CTTTCTCGGC AGGAGCAAGG TGAGATGACA GGAGATCCTG

8751 CCCCGGCACT TCGCCCAATA GCAGCCAGTC CCTTCCCGCT TCAGTGACAA

8801 CGTCGAGCAC AGCTGCGCAA GGAACGCCCG TCGTGGCCAG CCACGATAGC

8851 CGCGCTGCCT CGTCCTGCAG TTCATTCAGG GCACCGGACA GGTCGGTCTT

8901 GACAAAAAGA ACCGGGCGCC CCTGCGCTGA CAGCCGGAAC ACGGCGGCAT

8951 CAGAGCAGCC GATTGTCTGT TGTGCCCAGT CATAGCCGAA TAGCCTCTCC

9001 ACCCAAGCGG CCGGAGAACC TGCGTGCAAT CCATCTTGTT CAATCATGCG

9051 AAACGATCCT CATCCTGTCT CTTGATCAGA TCTTGATCCC CTGCGCCATC

9101 AGATCCTTGG CGGCAAGAAA GCCATCCAGT TTACTTTGCA GGGCTTCCCA

9151 ACCTTACCAG AGGGCGCCCC AGCTGGCAAT TCCGGTTCGC TTGCTGTCCA

9201 TAAAACCGCC CAGTCTAGCT ATCGCCATGT AAGCCCACTG CAAGCTACCT

9251 GCTTTCTCTT TGCGCTTGCG TTTTCCCTTG TCCAGATAGC CCAGTAGCTG

9301 ACATTCATCC GGGGTCAGCA CCGTTTCTGC GGACTGGCTT TCTACGTGTT

9351 CCGCTTCCTT TAGCAGCCCT TGCGCCCTGA GTGCTTGCGG CAGCGTGAAG

SEQ ID NO：18

Nucleotide sequence of expression vector HCMV-K HuAb-VL1 hum V2

(in 3926-4246 contains the complete DNA sequence of the humanized light chain expression vector of SEQ ID NO: 13(humV2 ═ VLm))

1 CTAGCTTTTT GCAAAAGCCT AGGCCTCCAA AAAAGCCTCC TCACTACTTC

51 TGGAATAGCT CAGAGGCCGA GGCGGCCTCG GCCTCTGCAT AAATAAAAAA

101 AATTAGTCAG CCATGGGGCG GAGAATGGGC GGAACTGGGC GGAGTTAGGG

151 GCGGGATGGG CGGAGTTAGG GGCGGGACTA TGGTTGCTGA CTAATTGAGA

201 TGCATGCTTT GCATACTTCT GCCTGCTGGG GAGCCTGGTT GCTGACTAAT

251 TGAGATGCAT GCTTTGCATA CTTCTGCCTG CTGGGGAGCC TGGGGACTTT

301 CCACACCCTA ACTGACACAC ATTCCACAGC TGCCTCGCGC GTTTCGGTGA

351 TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG GTCACAGCTT

401 GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG CGCGTCAGCG

451 GGTGTTGGCG GGTGTCGGGG CGCAGCCATG ACCCAGTCAC GTAGCGATAG

501 CGGAGTGTAT ACTGGCTTAA CTATGCGGCA TCAGAGCAGA TTGTACTGAG

551 AGTGCACCAT ATGCGGTGTG AAATACCGCA CAGATGCGTA AGGAGAAAAT

601 ACCGCATCAG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG

651 GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG

701 GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG

751 GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC

801 CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA

851 GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG

901 GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC

951 CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC ATAGCTCACG

1001 CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG

1051 TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT

1101 CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC

1151 CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT

1201 TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT

1251 ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC

1301 TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA

1351 AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC

1401 TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT

1451 TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT

1501 AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT

1551 GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT

1601 ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA

1651 TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC

1701 CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG

1751 GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA

1801 TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG

1851 CGCAACGTTG TTGCCATTGC TGCAGGCATC GTGGTGTCAC GCTCGTCGTT

1901 TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT

1951 GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC

2001 GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC

2051 ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA

2101 CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG

2151 AGTTGCTCTT GCCCGGCGTC AACACGGGAT AATACCGCGC CACATAGCAG

2201 AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT

2251 CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA

2301 CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC

2351 AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA

2401 AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT

2451 CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA

2501 TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG

2551 TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA TAGGCGTATC

2601 ACGAGGCCCT TTCGTCTTCA AGAATTCAGC TTGGCTGCAG TGAATAATAA

2651 AATGTGTGTT TGTCCGAAAT ACGCGTTTTG AGATTTCTGT CGCCGACTAA

2701 ATTCATGTCG CGCGATAGTG GTGTTTATCG CCGATAGAGA TGGCGATATT

2751 GGAAAAATCG ATATTTGAAA ATATGGCATA TTGAAAATGT CGCCGATGTG

2801 AGTTTCTGTG TAACTGATAT CGCCATTTTT CCAAAAGTGA TTTTTGGGCA

2851 TACGCGATAT CTGGCGATAG CGCTTATATC GTTTACGGGG GATGGCGATA

2901 GACGACTTTG GTGACTTGGG CGATTCTGTG TGTCGCAAAT ATCGCAGTTT

2951 CGATATAGGT GACAGACGAT ATGAGGCTAT ATCGCCGATA GAGGCGACAT

3001 CAAGCTGGCA CATGGCCAAT GCATATCGAT CTATACATTG AATCAATATT

3051 GGCCATTAGC CATATTATTC ATTGGTTATA TAGCATAAAT CAATATTGGC

3101 TATTGGCCAT TGCATACGTT GTATCCATAT CATAATATGT ACATTTATAT

3151 TGGCTCATGT CCAACATTAC CGCCATGTTG ACATTGATTA TTGACTAGTT

3201 ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC ATATATGGAG

3251 TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT GACCGCCCAA

3301 CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC

3351 CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT ACGGTAAACT

3401 GCCCACTTGG CAGTACATCA AGTGTATCAT ATGCCAAGTA CGCCCCCTAT

3451 TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC CAGTACATGA

3501 CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT AGTCATCGCT

3551 ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG

3601 GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA

3651 GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC

3701 TCCGCCCCAT TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTCTA

3751 TATAAGCAGA GCTCGTTTAG TGAACCGTCA GATCGCCTGG AGACGCCATC

3801 CACGCTGTTT TGACCTCCAT AGAAGACACC GGGACCGATC CAGCCTCCGC

3851 AAGCTTGCCG CCACCATGGA GACCCCCGCC CAGCTGCTGT TCCTGCTGCT

3901 GCTGTGGCTG CCCGACACCA CCGGCGACAT TCTGCTGACC CAGTCTCCAG

3951 CCACCCTGTC TCTGAGTCCA GGAGAAAGAG CCACTTTCTC CTGCAGGGCC

4001 AGTCAGAACA TTGGCACAAG CATACAGTGG TATCAACAAA AAACAAATGG

4051 TGCTCCAAGG CTTCTCATAA GGTCTTCTTC TGAGTCTATC TCTGGGATCC

4101 CTTCCAGGTT TAGTGGCAGT GGATCAGGGA CAGATTTTAC TCTTACCATC

4151 AGCAGTCTGG AGCCTGAAGA TTTTGCAGTG TATTACTGTC AACAAAGTAA

4201 TACCTGGCCA TTCACGTTCG GCCAGGGGAC CAAGCTGGAG ATCAAACGTG

4251 AGTATTCTAG AAAGATCCTA GAATTCTAAA CTCTGAGGGG GTCGGATGAC

4301 GTGGCCATTC TTTGCCTAAA GCATTGAGTT TACTGCAAGG TCAGAAAAGC

4351 ATGCAAAGCC CTCAGAATGG CTGCAAAGAG CTCCAACAAA ACAATTTAGA

4401 ACTTTATTAA GGAATAGGGG GAAGCTAGGA AGAAACTCAA AACATCAAGA

4451 TTTTAAATAC GCTTCTTGGT CTCCTTGCTA TAATTATCTG GGATAAGCAT

4501 GCTGTTTTCT GTCTGTCCCT AACATGCCCT GTGATTATCC GCAAACAACA

4551 CACCCAAGGG CAGAACTTTG TTACTTAAAC ACCATCCTGT TTGCTTCTTT

4601 CCTCAGGAAC TGTGGCTGCA CCATCTGTCT TCATCTTCCC GCCATCTGAT

4651 GAGCAGTTGA AATCTGGAAC TGCCTCTGTT GTGTGCCTGC TGAATAACTT

4701 CTATCCCAGA GAGGCCAAAG TACAGTGGAA GGTGGATAAC GCCCTCCAAT

4751 CGGGTAACTC CCAGGAGAGT GTCACAGAGC AGGACAGCAA GGACAGCACC

4801 TACAGCCTCA GCAGCACCCT GACGCTGAGC AAAGCAGACT ACGAGAAACA

4851 CAAAGTCTAC GCCTGCGAAG TCACCCATCA GGGCCTGAGC TCGCCCGTCA

4901 CAAAGAGCTT CAACAGGGGA GAGTGTTAGA GGGAGAAGTG CCCCCACCTG

4951 CTCCTCAGTT CCAGCCTGAC CCCCTCCCAT CCTTTGGCCT CTGACCCTTT

5001 TTCCACAGGG GACCTACCCC TATTGCGGTC CTCCAGCTCA TCTTTCACCT

5051 CACCCCCCTC CTCCTCCTTG GCTTTAATTA TGCTAATGTT GGAGGAGAAT

5101 GAATAAATAA AGTGAATCTT TGCACCTGTG GTTTCTCTCT TTCCTCATTT

5151 AATAATTATT ATCTGTTGTT TACCAACTAC TCAATTTCTC TTATAAGGGA

5201 CTAAATATGT AGTCATCCTA AGGCGCATAA CCATTTATAA AAATCATCCT

5251 TCATTCTATT TTACCCTATC ATCCTCTGCA AGACAGTCCT CCCTCAAACC

5301 CACAAGCCTT CTGTCCTCAC AGTCCCCTGG GCCATGGTAG GAGAGACTTG

5351 CTTCCTTGTT TTCCCCTCCT CAGCAAGCCC TCATAGTCCT TTTTAAGGGT

5401 GACAGGTCTT ACAGTCATAT ATCCTTTGAT TCAATTCCCT GAGAATCAAC

5451 CAAAGCAAAT TTTTCAAAAG AAGAAACCTG CTATAAAGAG AATCATTCAT

5501 TGCAACATGA TATAAAATAA CAACACAATA AAAGCAATTA AATAAACAAA

5551 CAATAGGGAA ATGTTTAAGT TCATCATGGT ACTTAGACTT AATGGAATGT

5601 CATGCCTTAT TTACATTTTT AAACAGGTAC TGAGGGACTC CTGTCTGCCA

5651 AGGGCCGTAT TGAGTACTTT CCACAACCTA ATTTAATCCA CACTATACTG

5701 TGAGATTAAA AACATTCATT AAAATGTTGC AAAGGTTCTA TAAAGCTGAG

5751 AGACAAATAT ATTCTATAAC TCAGCAATCC CACTTCTAGA TGACTGAGTG

5801 TCCCCACCCA CCAAAAAACT ATGCAAGAAT GTTCAAAGCA GCTTTATTTA

5851 CAAAAGCCAA AAATTGGAAA TAGCCCGATT GTCCAACAAT AGAATGAGTT

5901 ATTAAACTGT GGTATGTTTA TACATTAGAA TACCCAATGA GGAGAATTAA

5951 CAAGCTACAA CTATACCTAC TCACACAGAT GAATCTCATA AAAATAATGT

6001 TACATAAGAG AAACTCAATG CAAAAGATAT GTTCTGTATG TTTTCATCCA

6051 TATAAAGTTC AAAACCAGGT AAAAATAAAG TTAGAAATTT GGATGGAAAT

6101 TACTCTTAGC TGGGGGTGGG CGAGTTAGTG CCTGGGAGAA GACAAGAAGG

6151 GGCTTCTGGG GTCTTGGTAA TGTTCTGTTC CTCGTGTGGG GTTGTGCAGT

6201 TATGATCTGT GCACTGTTCT GTATACACAT TATGCTTCAA AATAACTTCA

6251 CATAAAGAAC ATCTTATACC CAGTTAATAG ATAGAAGAGG AATAAGTAAT

6301 AGGTCAAGAC CACGCAGCTG GTAAGTGGGG GGGCCTGGGA TCAAATAGCT

6351 ACCTGCCTAA TCCTGCCCTC TTGAGCCCTG AATGAGTCTG CCTTCCAGGG

6401 CTCAAGGTGC TCAACAAAAC AACAGGCCTG CTATTTTCCT GGCATCTGTG

6451 CCCTGTTTGG CTAGCTAGGA GCACACATAC ATAGAAATTA AATGAAACAG

6501 ACCTTCAGCA AGGGGACAGA GGACAGAATT AACCTTGCCC AGACACTGGA

6551 AACCCATGTA TGAACACTCA CATGTTTGGG AAGGGGGAAG GGCACATGTA

6601 AATGAGGACT CTTCCTCATT CTATGGGGCA CTCTGGCCCT GCCCCTCTCA

6651 GCTACTCATC CATCCAACAC ACCTTTCTAA GTACCTCTCT CTGCCTACAC

6701 TCTGAAGGGG TTCAGGAGTA ACTAACACAG CATCCCTTCC CTCAAATGAC

6751 TGACAATCCC TTTGTCCTGC TTTGTTTTTC TTTCCAGTCA GTACTGGGAA

6801 AGTGGGGAAG GACAGTCATG GAGAAACTAC ATAAGGAAGC ACCTTGCCCT

6851 TCTGCCTCTT GAGAATGTTG ATGAGTATCA AATCTTTCAA ACTTTGGAGG

6901 TTTGAGTAGG GGTGAGACTC AGTAATGTCC CTTCCAATGA CATGAACTTG

6951 CTCACTCATC CCTGGGGGCC AAATTGAACA ATCAAAGGCA GGCATAATCC

7001 AGCTATGAAT TCTAGGATCG ATCCAGACAT GATAAGATAC ATTGATGAGT

7051 TTGGACAAAC CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA

7101 ATTTGTGATG CTATTGCTTT ATTTGTAACC ATTATAAGCT GCAATAAACA

7151 AGTTAACAAC AACAATTGCA TTCATTTTAT GTTTCAGGTT CAGGGGGAGG

7201 TGTGGGAGGT TTTTTAAAGC AAGTAAAACC TCTACAAATG TGGTATGGCT

7251 GATTATGATC TCTAGTCAAG GCACTATACA TCAAATATTC CTTATTAACC

7301 CCTTTACAAA TTAAAAAGCT AAAGGTACAC AATTTTTGAG CATAGTTATT

7351 AATAGCAGAC ACTCTATGCC TGTGTGGAGT AAGAAAAAAC AGTATGTTAT

7401 GATTATAACT GTTATGCCTA CTTATAAAGG TTACAGAATA TTTTTCCATA

7451 ATTTTCTTGT ATAGCAGTGC AGCTTTTTCC TTTGTGGTGT AAATAGCAAA

7501 GCAAGCAAGA GTTCTATTAC TAAACACAGC ATGACTCAAA AAACTTAGCA

7551 ATTCTGAAGG AAAGTCCTTG GGGTCTTCTA CCTTTCTCTT CTTTTTTGGA

7601 GGAGTAGAAT GTTGAGAGTC AGCAGTAGCC TCATCATCAC TAGATGGCAT

7651 TTCTTCTGAG CAAAACAGGT TTTCCTCATT AAAGGCATTC CACCACTGCT

7701 CCCATTCATC AGTTCCATAG GTTGGAATCT AAAATACACA AACAATTAGA

7751 ATCAGTAGTT TAACACATTA TACACTTAAA AATTTTATAT TTACCTTAGA

7801 GCTTTAAATC TCTGTAGGTA GTTTGTCCAA TTATGTCACA CCACAGAAGT

7851 AAGGTTCCTT CACAAAGATC CGGGACCAAA GCGGCCATCG TGCCTCCCCA

7901 CTCCTGCAGT TCGGGGGCAT GGATGCGCGG ATAGCCGCTG CTGGTTTCCT

7951 GGATGCCGAC GGATTTGCAC TGCCGGTAGA ACTCCGCGAG GTCGTCCAGC

8001 CTCAGGCAGC AGCTGAACCA ACTCGCGAGG GGATCGAGCC CGGGGTGGGC

8051 GAAGAACTCC AGCATGAGAT CCCCGCGCTG GAGGATCATC CAGCCGGCGT

8101 CCCGGAAAAC GATTCCGAAG CCCAACCTTT CATAGAAGGC GGCGGTGGAA

8151 TCGAAATCTC GTGATGGCAG GTTGGGCGTC GCTTGGTCGG TCATTTCGAA

8201 CCCCAGAGTC CCGCTCAGAA GAACTCGTCA AGAAGGCGAT AGAAGGCGAT

8251 GCGCTGCGAA TCGGGAGCGG CGATACCGTA AAGCACGAGG AAGCGGTCAG

8301 CCCATTCGCC GCCAAGCTCT TCAGCAATAT CACGGGTAGC CAACGCTATG

8351 TCCTGATAGC GGTCCGCCAC ACCCAGCCGG CCACAGTCGA TGAATCCAGA

8401 AAAGCGGCCA TTTTCCACCA TGATATTCGG CAAGCAGGCA TCGCCATGGG

8451 TCACGACGAG ATCCTCGCCG TCGGGCATGC GCGCCTTGAG CCTGGCGAAC

8501 AGTTCGGCTG GCGCGAGCCC CTGATGCTCT TCGTCCAGAT CATCCTGATC

8551 GACAAGACCG GCTTCCATCC GAGTACGTGC TCGCTCGATG CGATGTTTCG

8601 CTTGGTGGTC GAATGGGCAG GTAGCCGGAT CAAGCGTATG CAGCCGCCGC

8651 ATTGCATCAG CCATGATGGA TACTTTCTCG GCAGGAGCAA GGTGAGATGA

8701 CAGGAGATCC TGCCCCGGCA CTTCGCCCAA TAGCAGCCAG TCCCTTCCCG

8751 CTTCAGTGAC AACGTCGAGC ACAGCTGCGC AAGGAACGCC CGTCGTGGCC

8801 AGCCACGATA GCCGCGCTGC CTCGTCCTGC AGTTCATTCA GGGCACCGGA

8851 CAGGTCGGTC TTGACAAAAA GAACCGGGCG CCCCTGCGCT GACAGCCGGA

8901 ACACGGCGGC ATCAGAGCAG CCGATTGTCT GTTGTGCCCA GTCATAGCCG

8951 AATAGCCTCT CCACCCAAGC GGCCGGAGAA CCTGCGTGCA ATCCATCTTG

9001 TTCAATCATG CGAAACGATC CTCATCCTGT CTCTTGATCA GATCTTGATC

9051 CCCTGCGCCA TCAGATCCTT GGCGGCAAGA AAGCCATCCA GTTTACTTTG

9101 CAGGGCTTCC CAACCTTACC AGAGGGCGCC CCAGCTGGCA ATTCCGGTTC

9151 GCTTGCTGTC CATAAAACCG CCCAGTCTAG CTATCGCCAT GTAAGCCCAC

9201 TGCAAGCTAC CTGCTTTCTC TTTGCGCTTG CGTTTTCCCT TGTCCAGATA

9251 GCCCAGTAGC TGACATTCAT CCGGGGTCAG CACCGTTTCT GCGGACTGGC

9301 TTTCTACGTG TTCCGCTTCC TTTAGCAGCC CTTGCGCCCT GAGTGCTTGC

9351 GGCAGCGTGA AG

Claims (21)

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-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), 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 order 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), 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 ', CDR 2' and CDR3 ', CDR 1' 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 (SSSISS), CDR 3' having the amino acid sequence Gln-Gln-Ser-Asn-Thr-Trp-Pro-Phe-Thr (QQSNTWPFT).

3. A binding molecule according to any of claims 1 or 2, which is a chimeric or humanized monoclonal antibody.

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

5. a binding molecule according to any of claims 1 or 2, comprising the polypeptide SEQ ID NO: 3 and/or the polypeptide SEQ ID NO: 4.

6. a binding molecule according to any one of claims 4 or 5, which is a chimeric monoclonal antibody.

7. A binding molecule which is a humanized antibody comprising

-polypeptide SEQ ID NO: 9 and the polypeptide SEQ ID NO: 7,

-polypeptide SEQ ID NO: 9 and the polypeptide SEQ ID NO: 8,

-polypeptide SEQ ID NO: 10 and polypeptide SEQ ID NO: 7, or

-polypeptide SEQ ID NO: 10 and polypeptide SEQ ID NO: 8.

8. an isolated polynucleotide comprising a polynucleotide encoding a binding molecule according to any one of claims 1 to 7.

9. A polynucleotide according to claim 8 encoding the amino acid sequences of CDR1, CDR2 and CDR3 according to claim 2; and/or amino acid sequences encoding the CDRs 1 ', 2 ' and 3 ' according to claim 2.

10. A polynucleotide comprising the polynucleotide of SEQ ID NO: 5 and/or the polynucleotide of SEQ id no: 6.

11. a polynucleotide comprising a nucleotide sequence encoding (a) a polypeptide of SEQ ID NO: 7 or SEQ ID NO: 8 and (b) the polypeptide of SEQ ID NO: 9 or SEQ ID NO: 10.

12. A polynucleotide comprising (a) the polynucleotide of SEQ ID NO: 11 or SEQ ID NO: 12 and (b) the polynucleotide of SEQ ID NO: 13 or the polynucleotide of SEQ ID NO: 14.

13. an expression vector comprising a polynucleotide according to any one of claims 8 to 12, wherein said expression vector or combination thereof is capable of producing a binding molecule according to any one of claims 1 to 7 when said expression vector or combination thereof is present in a compatible host cell.

14. An isolated host cell comprising the expression vector according to claim 13.

15. A pharmaceutical composition comprising a binding molecule according to any one of claims 1 to 7 and at least one pharmaceutically acceptable carrier or diluent.

16. Use of a binding molecule according to any one of claims 1 to 7 for the preparation of a medicament for the treatment and/or prevention of diseases associated with autoimmune diseases, transplant rejection, psoriasis, inflammatory bowel disease and allergies.

17. Use according to claim 16, wherein the binding molecule binds to an isoform of CD45RO with a dissociation constant (Kd) < 15 nM.

18. Use according to claim 16, wherein the binding molecule binds to an isoform of CD45RB with a dissociation constant (Kd) < 15 nM.

19. Use according to any one of claims 16 to 18, wherein the binding molecule binds to the CD45 isoform, the CD45 isoform

-the a and B epitopes but not the C epitope of the CD45 molecule; and/or

-the B epitope, but not the a and C epitopes, of the CD45 molecule; and/or

Neither of the A, B and C epitopes of the CD45 molecule.

20. Use according to any one of claims 16 to 18, wherein the binding molecule does not bind to the CD45 isoform, the CD45 isoform

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

-the B and C epitopes but not the a epitope of the CD45 molecule.

21. Use according to any one of claims 16 to 18, wherein the binding molecule binds to its target epitope on the PEER cell, wherein the Kd of said binding is < 15 nM.