HK1069122B - Antibodies to cd40 - Google Patents

Description

Antibodies to CD40

This application claims the benefit of U.S. provisional application 60/348,980, applied on day 11, 9, 2001.

Prior Art

The CD40 antigen is a 50kDa cell surface glycoprotein belonging to the tumor necrosis factor receptor (TNF-R) family (Stamenkovic et al, EMBO J.8: 1403-10 (1989)). CD40 is expressed in many normal and tumor cell types, including B lymphocytes, dendritic cells, monocytes, macrophages, thymic epidermal cells, endothelial cells, fibroblasts, and smooth muscle cells (Cancer Immunol.20: 23-8 (1985); adv. Exp. Med. biol. 378: 83-83 (1995) to Banchereau J. et al; J.of Exp. Med. 178: 669-74(1993) to Alderson M.R. et al; J.of Exp. Med. 178: 3737-74 (1993) to Ruggero G. et al; J.of Immunol.156: 3737-46(1996) to Hollenbaugh. et al; J.of Exp.182: 33-40 (1995); J.of Yellor. J.312J.et al; J.of Lebiochyte. 58: 1995: 1998: and La9-161. 161: 1998) to Lauru J.7.7.161 (1998). CD40 is expressed in all B-lymphomas and 70% of all solid tumors. Although constitutively expressed, CD40 can upregulate its expression in antigen presenting cells using maturation signals (e.g., LPS, IL-1. beta., IFN-. gamma., and GM-CSF).

CD40 activation plays an important role in regulating humoral and cellular immune responses. Antigen presentation without CD40 activation results in tolerance, and CD40 signaling can reverse such tolerance, enhance antigen presentation by all Antigen Presenting Cells (APCs), aid in secretion of helper cytokines and chemokines, enhance costimulatory molecule expression and signaling, and stimulate cytolytic activity of immune cells.

CD40 plays a key role in B cell proliferation, maturation and classification transformation (FoyT. M. et al Ann. Rev. of Immunol.14: 591-617 (1996)). Disruption of the CD40 signaling pathway results in abnormal distribution of serum immunoglobulin isotypes, lack of CD4+ T cell priming, and second humoral response defects. For example: x-linked hyper-IgM syndrome is a disease associated with mutations in the human CD40L gene and is characterized by the inability of patients to produce antibodies other than the IgM isotype, indicating that a productive interaction between CD40 and CD40L is required for an effective immune response.

The association of CD40 with CD40L results in the binding of the cytoplasmic domain of CD40 to TRAFs (TNF-R related factors) (Proc. Natl. Acad. Sci. USA 96: 1421-6(1999) of Lee H. et al, Biochemistry 37: 11836-45(1998) of Pullen S. et al, J of lmmunol.161: 1183-93(1998) of Grammar A.C. et al, Proc. Natl. Acad. Sci. USA 93: 9437-42(1996) of Ishida T.K. et al, J.of biol. chem.274: 14246-54(1999) of Pullen S. et al). Interaction with TRAFs eventually simultaneously activates NF-. kappa.B and the Jun/AP1 pathway (Tsukamoton et al Proc. Natl.Acad.Sci.USA 96: 1234-9 (1999); Sutherland C.L. et al J.of Immunol.162: 4720-30 (1999)). Depending on the cell type, this signaling enhances cytokine secretion, such as: IL-6 (Jepson J.D. et al J.of Immunol.161: 1738-42 (1998); Uejima Y. et al int. Arch.of Allergy & Immunol.110: 225-32 (1996)), IL-8(GrussH.J. et al Blood 84: 2305-14 (1994); von Leopreading A. et al Cancer Res.59: 1287-94 (1999); Denfeld R.W. et al Europ.J.of Immunol.26: 2329-34(1996)), IL-12(Cella M. et al J.of Exp.Med.184: 747-52 (Europ.J.of Irlin W.G. et al Eup.J.of Europ.28: 1998-31. et al J.of Europ.32: Oc.32. of Chan.32. J.10. 1996; Eur.32. J.of Oc.32. J.32. J.10. of Ocular R.32. 1996; Eur.32. J.32. J.10. of Oc.32. J.10. J.32. of Ocular (1996; Europ.32. J.32. of Ocular mJ.32. of Ocular R.10. J.10, 1996; III. J.32. J.10. of Ocular et al) (Ocular) and J.10. of Ocular R.10. J.10. of Ocular et al (1996; III. of Ocular et al) (Ocular) of Ocular R.10. J.10, III, MIP1 β, RANTES, etc.) (McDyer j.f. et al, j.of immunol.162: 3711-7 (1999); j.of exp.med.188 of schaniel c. et al: 451-63 (1998); j.of immunol.162 to Altenburg a. et al: 4140-7 (1999); j.of the am. society of neuroprology 9, Deckers J.G. et al: 1187-93 (1998)); increasing MHC class I and II expression (CellularImmunol.156: 272-85(1994) by Santos-Argumedol. et al) and increasing adhesion molecule expression (e.g., ICAM) (proc. Natl.Acad.Sci.USA.96: 1421-6(1999), Grousson J. et al, Archives of Dermatoll.Res.290: 325-30 (1998); Europ.J.of lmmunol.26: 192-200(1996) by Kataday. et al; J.of Mayumi M. et al, Allergy & Clin. Immunol.96: 1136-44(1995), Flow-Romo L. et al, Immunol.79: 445-51: 1993) and increasing costimulatory molecule expression (e.g., cell & Clin. Immunol.96: 1136-44: 1995) (Johnol. J.J.J.of Kirym. et al, J.1995; J.35: 1995; J.J.35: 1995; J.J.J.35: 1995) by Mahon J.35. J.J.35: 35: 73: 35: 1996) and J.35. J.J.35 (1996) by Mahon et al). Cytokines induced by the involvement of CD40 potentiate T cell survival and activation.

In addition to enhancing cellular and immune function, the effects of activation of CD40 include: cell recruitment and differentiation of chemokines and cytokines; activating the monocytes; increasing the lytic activity of Cytolytic T Lymphocytes (CTL) and Natural Killer (NK) cells; induction of apoptosis in CD 40-positive tumors; enhancing the immunogenicity of CD 40-positive tumors; and the production of tumor specific antibodies. The role of CD40 activation in cell-mediated immune responses has also been fully established and described in: grewal et al, ann.rev.of immunol.16: 111-35 (1998); j.of Leukocyte biol.63, Mackey et al: 418-28 (1998); and Noelle R.J., Agents & Actions-suppl.49: 17-22, 1998).

Experiments using a cross-priming model system have shown that CD40 activation of APCs can replace helper T cells required during formation of Cytolytic T Lymphocytes (CTL) (Bennett et al, Nature 393: 478-480 (1998)). As can be seen from the lack of evidence from CD40L mice, CD40 signaling is clearly required at the time of helper T cell initialization. (Grewall I.S. et al Science 273: 1864-7 (1996); Grewal I.S. et al Nature 378: 617-20 (1995)). Activation of CD40 converts tolerogenic, antigen-bearing B cells into competent APCs (Immunity 2: 645-53(1995) Buhlmann J.E. et al). CD40 activation induces maturation and differentiation of spinal cord progenitor blood cells into dendritic cells (Flores-Romo L. et al J.of exp. Med.185: 341-9 (1997); Mackey M.F. et al J.of Immunol.161: 2094-8 (1998)). CD40 activation also induces differentiation of monocytes into functional dendritic cells (Brossart P. et al Blood 92: 4238-47 (1998)). In addition, CD40 activation potentiates the cytolytic activity of NK cells by APC-CD 40-induced cytokines (Carbone E. et al J.of exp. Med.185: 2053-60 (1997); Martin-Fontecha A. et al J.of immunol.162: 5910-6 (1999)). These observations suggest that CD40 plays a fundamental role in initiating and enhancing immune responses by inducing APCs maturation, secreting helper cytokines, upregulating co-stimulatory molecules, and enhancing effector function.

The critical role of CD40 signaling in the initiation and maturation of humoral and cytotoxic immune responses makes this system an ideal target for boosting immunity. Such boosting is particularly important for generating an effective immune response against tumor antigens that are normally presented to the immune system by cross-priming of activated APCs. (Huang A.Y. et al, Ciba Foundation Symp.187: 229-44 (1994); ToesR.E.M. et al, Semins in Immunol.10: 443-8 (1998); Albert M.L. et al, Nature 392: 86-9 (1998); Bennett S.R. et al, J.of exp.Med.186: 65-70 (1997)).

Several working groups have demonstrated the effectiveness of CD40 activation in vitro and in vivo for anti-tumor responses (ToesR. E.M. et al, Semins in Immunol.10: 443-8 (1998)). Two of the groups, using lung metastasis models of renal cell carcinoma and subcutaneous tumors of virus-transformed cells, have demonstrated that CD40 activation can reverse tolerance to tumor-specific antigens, resulting in effective anti-tumor initialization of T cells (prime), respectively (Nature Medicine 5: 780-787(1999) by Sotomayore E.M. et al; Nature Medicine 5: 774-9(1999) by Diehll. et al). Treatment of CD40L with anti-CD 40 antibody also demonstrated anti-tumor activity in the absence of immune cells in a human breast cancer cell line model in SCID mice (HiranoA. et al, Blood 93: 2999-3007 (1999)). Recently, it has also been demonstrated in a mouse model that activation of CD40 by anti-CD 40 antibodies can eradicate CD40+ and CD 40-lymphomas. (French R.R. et al Nature Medicine 5: 548-53 (1999)). Furthermore, the previous experiments by Glennie et al led to the conclusion that: the signaling activity produced by anti-CD 40 antibodies is more effective in inducing tumor clearance in vivo than other anti-surface marker antibodies that recruit effectors (Tutt A.L. et al J.of Immunol.161: 3176-85 (1998)). When anti-CD 40 antibodies were tested in vivo for activity against CD40+ tumor cells, it was also observed that most, but not all, of the anti-tumor activity was associated with CD40 signaling, rather than ADCC (Funakoshi S. et al J. of immunotherapy with Emphasis on Tumoral Immunol.19: 93-101 (1996)). In another assay, bone marrow dendritic cells were treated ex vivo with various agents and tested for in vivo anti-tumor activity. Such experiments demonstrated that DCs stimulated with CD40L were the most mature and effective cells that could provide an anti-tumor response.

The basic role of CD40 in anti-tumor immunity has been demonstrated in the results of comparing the response of wild-type and CD 40-/-mice to tumor vaccines. Such experiments show that CD 40-/-mice do not achieve the tumor immunity seen in normal mice (Mackey M.F. et al cancer research 57: 2569-74 (1997)). In another experiment, splenocytes taken from tumor-bearing mice were stimulated ex vivo with tumor cells and treated with an activating anti-CD 40 antibody, which showed enhanced tumor-specific CTL activity (cancer Immunol. Immunother.48: 153-164 (1999) by Donepudii M. et al). Such tests demonstrate that CD40 plays an important role in anti-tumor immunity, whether CD40 positive or negative tumors. Since CD40 is expressed in lymphomas, leukemias, multiple myeloma, most carcinomas of the nasopharynx, bladder, ovary and liver, and some breast and colorectal cancers, activation of CD40 can have a wide range of clinical uses.

anti-CD 40-activating monoclonal antibodies can eradicate tumors through a variety of important mechanisms. Among these are primarily the activation of host dendritic cells to enhance tumor antigen processing and presentation, and the enhancement of antigen presentation or immunogenicity of CD40 positive tumor cells themselves to activate tumor-specific CD4⁺And CD8⁺A lymphocyte. Other anti-tumor activities may be mediated by other immune-potentiating effects of CD40 signaling (production of chemokines and cytokines, recruitment and activation of monocytes, and potentiation of CTL and NK cytolytic activity), and direct elimination of CD40 by inducing apoptosis or stimulating a humoral response to ADCC⁺A tumor. Apoptotic and dead tumor cells can become important sources of tumor-specific antigens that are processed and presented by CD 40-activated APCs.

There is therefore a great need for clinically relevant anti-CD 40 agonist antibodies that are therapeutic.

Brief Description of Drawings

FIGS. 1A-1H show the predicted amino acid sequences of the light and heavy chain variable domains of the isolated anti-CD 40 monoclonal antibody aligned to the germline amino acid sequences of the corresponding light and heavy chain genes. Differences between clone and germline sequences are shaded. Germline CDR1, CDR2, and CDR3 sequences are underlined. In the alignment of the heavy chain sequences, the apparent insertion of the CDR3 region is shown by the dashed line (-) in the germline sequence and the apparent deletion position of the CDR3 region is shown by the dashed line (-) in the cloned sequence.

FIG. 1A: the germline amino acid sequences of the vk ═ A3/a19 and J ═ jk 1 genes, and the predicted kappa-light chain variable region amino acid sequences of mAbs 3.1.1 and 7.1.2;

FIG. 1B: the predicted kappa-light chain variable region amino acid sequence and germline amino acid sequences from clone 15.1.1 (vk ═ A3/a19 and J ═ jk 2);

FIG. 1C: predicted kappa-light chain variable region amino acid sequences and germline amino acid sequences from mabs10.8.3 and 21.4.1 (vk ═ L5(DP5) and J ═ jk 4);

FIG. 1D: the predicted heavy chain variable region amino acid sequence and germline amino acid sequences from mab3.1.1 (VH ═ 3-30+ (DP-49), D ═ D4+ DIR3 and J ═ JH 6);

FIG. 1E: the predicted heavy chain variable region amino acid sequence and germline amino acid sequences from mab7.1.2 (VH ═ 3-30+ (DP-49), D ═ DIR5+ DI-26 and J ═ JH 6);

FIG. 1F: the predicted heavy chain amino acid sequence and germline amino acid sequence from mab10.8.3 (VH ═ 4.35(VIV-4), D ═ DIR3 and J ═ JH 6);

FIG. 1G: the predicted heavy chain variable region amino acid sequence and germline amino acid sequences from mab15.1.1 (VH ═ 4-59(DP-71), D ═ D4-23 and J ═ JH 4); and

FIG. 1H: the predicted heavy chain variable region amino acid sequence from mab21.4.1 and germline amino acid sequences (VH ═ 1-02(DP-75), D ═ DLR1 and J ═ JH 4).

FIGS. 2A-2H show an alignment of the amino acid sequences of the light and heavy chain variable domains in the isolated anti-CD 40 monoclonal antibody with the germline amino acid sequences of the corresponding light and heavy chain genes. Differences between the clone and germline sequences are shown in bold. Germline CDR1, CDR2, and CDR3 sequences are underlined. In the alignment of the heavy chain sequences, the apparent insertion of the CDR3 region is represented by the dashed line (-) in the germline sequence and the apparent deletion position of the CDR3 region is represented by the dashed line (-) in the cloned sequence.

FIG. 2A: predicted kappa-light chain amino acid sequences and germline amino acid sequences from mabs22.1.1, 23.5.1, and 23.29.1 (vk ═ A3/a19 and J ═ jk 1);

FIG. 2B: predicted kappa-light chain amino acid sequences and germline amino acid sequences from mab21.2.1 (V kappa. A3/a19 and J kappa. J3);

FIG. 2C: predicted kappa-light chain amino acid sequences and germline amino acid sequences from mabs23.28.1, 23.28.1L-C92A, and 24.2.1 (vk ═ a27 and J ═ jk 3);

FIG. 2D: predicted heavy and germline amino acid sequences from mAb21.2.1 (V)_H3-30+, D-DIR 3+ D6-19 and J-J_H4)；

FIG. 2E: predicted heavy and germline amino acid sequences from mabs22.1.1, 22.1.1H-C109A (V)_H3-30+, D-D1-1 and J-J_H6)；

FIG. 2F: predicted heavy and germline amino acid sequences from mAb23.5.1 (V)_H3-30+, D-D4-17 and J-J_H6)；

FIG. 2G: predicted heavy and germline amino acid sequences from mAb23.29.1 (V)_H3-30.3, D-D4-17 and J-J_H6) (ii) a And

FIG. 2H: predicted heavy and germline amino acid sequences from mabs23.28.1, 23.28.1H-D16E, and 24.2.1 (V)_H4-59, D-DIR 1+ D4-17 and J-J_H5)。

FIG. 3 is a dose-response curve illustrating the ability of an anti-CD 40 antibody (21.4.1) of the invention to potentiate IL-12p40 production by human dendritic cells.

FIG. 4 is a dose-response curve illustrating the ability of an anti-CD 40 antibody (21.4.1) of the invention to potentiate IL-12p70 production by human dendritic cells.

FIG. 5 illustrates the ability of the anti-CD 40 antibody (21.4.1) of the present invention to enhance the immunogenicity of Jy-stimulated cells and to potentiate CTL activity against Jy target cells.

FIG. 6 is a tumor growth inhibition curve illustrating the reduction in growth of CD40 positive Daudi tumors in SCID-beige mice treated with an anti-CD 40 antibody of the invention (21.4.1).

FIG. 7 is a tumor growth inhibition curve illustrating the reduction in growth of CD40 negative K562 tumors in SCID-beige mice treated with anti-CD 40 antibody of the invention (21.4.1) and human dendritic cells and T cells.

FIG. 8 illustrates the growth inhibition results of CD40 negative K562 tumors in SCID mice treated with different concentrations of the anti-CD 40 agonist mAb 23.29.1.

Figure 9 shows the growth inhibition results of CD40 negative K562 tumors in SCID mice treated with different concentrations of the anti-CD 40 agonist mab3.1.1.

FIG. 10 shows the growth inhibition results of CD40 positive Raji tumors in SCID mice treated with anti-CD 40 agonist mAb in the presence or absence of T cells and dendritic cells.

FIG. 11 shows the growth inhibition results of CD40 positive Raji tumors in SCID mice treated with anti-CD 40 agonist antibody.

FIG. 12 shows the results of growth inhibition of BT474 breast cancer cells in SCID-beige mice treated with anti-CD 40 agonist antibody.

FIG. 13 shows the results of growth inhibition of PC-3 prostate tumor in SCID-beige mice treated with anti-CD 40 agonist antibody.

FIG. 14 is a survival curve for SCID-beige mice that received intravenous Daudi tumor cell injection (iv) and were treated with anti-CD 40 agonist antibody.

Figure 15 is a western blot analysis of anti-CD 40 agonist antibodies against reduced (R) and unreduced (NR) human CD 40.

FIG. 16 is an alignment of the D1-D4 domains of mouse and human CD 40.

Fig. 17 is an alignment of mouse and human CD40 amino acids, showing the fusion position of the chimera.

Fig. 18 is a diagram of a chimeric CD40 construct.

Summary of The Invention

The present invention provides an isolated antibody, or antigen-binding portion thereof, that binds to CD40 and acts as a CD40 agonist.

The present invention provides a composition comprising an anti-CD 40 antibody, or antigen-binding portion thereof, and a pharmaceutically acceptable carrier. The composition may further comprise another ingredient, such as: an antineoplastic agent or an imaging agent. Diagnostic and medical methods are also provided.

The present invention provides an isolated cell line, such as: a hybridoma that produces an anti-CD 40 antibody or antigen-binding portion thereof.

The invention also provides a nucleic acid molecule encoding the heavy and/or light chain of an anti-CD 40 antibody or antigen-binding portion thereof.

The invention provides vectors and host cells comprising the nucleic acid molecules, and methods for recombinantly producing polypeptides encoded by the nucleic acid molecules.

The invention also provides a non-human transgenic animal expressing the heavy and/or light chain of an anti-CD 40 antibody, or antigen-binding portion thereof.

The invention also provides a method of treating an individual in need thereof with an effective amount of a nucleic acid molecule encoding the heavy and/or light chain of the anti-CD 40 antibody or antigen-binding portion thereof.

Detailed description of the invention

Definition and general techniques

Unless defined otherwise, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required herein, singular terms shall include the plural, and plural terms shall include the singular. Generally, the nomenclature used and the techniques for cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry, and hybridization described herein are those known and commonly employed in the art.

The methods and techniques of the present invention are generally performed according to conventional methods known in the art and are described in various general and more specific references that are extracted and discussed in this specification, unless otherwise specified. See, for example: of Sambrook et alMolecular Cloning：A Laboratory Manual2 nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) and Ausubel et alCurrent Protocols in Molecular Biology(Green Publishing Associates (1992)), and of Harlow and LaneAntibodies：A Laboratory Manual(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990)), the contents of which have been incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions and can generally be performed according to methods known in the art or according to the methods described herein. Nomenclature related to analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein, and laboratory procedures and techniques, are those well known and commonly used in the art. Standard techniques are used for chemosynthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and for patient treatment.

Unless otherwise indicated, the following terms have the following definitions:

the term "polypeptide" encompasses natural or artificial proteins, protein fragments, and polypeptide analogs of a protein sequence. The polypeptide may be monomeric or polymeric.

The terms "isolated protein", "isolated polypeptide" or "isolated antibody" refer to a protein, polypeptide or antibody derived from a source that is substantially (1) not associated with a naturally occurring association component with which it is associated in its natural state, (2) free of other proteins from the same species, (3) expressed by cells of a different species, or (4) does not occur naturally. Thus, a polypeptide may be considered to be "isolated" from its natural binding component if it is chemically synthesized or synthesized in a cellular system other than that from which it is naturally derived. Proteins can also be isolated using protein purification techniques known in the art, such that they are substantially free of naturally associated components.

Examples of isolated antibodies include anti-CD 40 antibodies that have been affinity purified using CD40, anti-CD 40 antibodies synthesized in vitro using hybridomas or other cell lines, and human anti-CD 40 antibodies derived from transgenic mice.

A protein or polypeptide is "substantially pure", "substantially homogeneous", or "substantially purified" in the sense that at least about 60 to 75% of the sample is a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. Substantially pure polypeptides or proteins typically constitute about 50%, 60%, 70%, 80%, or 90% W/W protein samples, more usually about 95%, preferably 99% or more pure. Protein purity or homogeneity can be determined by a variety of methods known in the art, such as: single polypeptide bands were examined after polyacrylamide gel electrophoresis from protein samples, by staining the gel with dyes known in the art. For some purposes, it may be possible to provide higher resolution purification using HPLC or other means known in the art.

The term "polypeptide fragment" as used herein refers to a polypeptide in which the amino-terminal and/or carboxy-terminal ends have been deleted, but the remaining amino acid sequence remains identical to the corresponding position in the native sequence. In some embodiments, fragments are at least 5,6, 8, or 10 amino acids in length. In other embodiments, the fragment is at least 14, at least 20, at least 50, at least 70, 80, 90, 100, 150, or 200 amino acids.

The term "polypeptide analogue" as used herein refers to a polypeptide comprising a segment substantially identical to a portion of an amino acid sequence and having the following properties: (1) under suitable binding conditions, binds specifically to CD40, (2) has the ability to activate CD40, (3) has the ability to upregulate the expression of cell surface molecules (e.g., ICAM, MHC-II, B7-1, B7-2, CD71, CD23, and CD83), or (4) has the ability to potentiate the secretion of cytokines, such as: IFN-beta 1, IL-2, IL-8, IL-12, IL-15, IL-18 and IL-23. Typically, polypeptide analogs contain conservative amino acid substitutions (or insertions or deletions) relative to the native sequence. Analogs typically are at least 20 or 25 amino acids in length, preferably at least 50, 60, 70, 80, 90, 100, 150, or 200 amino acids or longer, and often up to the full length of the native polypeptide.

Preferred amino acid substitutions are: (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for formation of protein complexes, and (4) assignment or modification of other physiochemical or functional properties of such analogs. Analogs can include a variety of muteins whose sequences differ from the native peptide sequence. For example: single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the native sequence (preferably in the portion of the polypeptide outside the domain where intermolecular contacts are made). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acid should not break the helix of the parent sequence or disrupt the secondary structure characteristic of the parent sequence). Examples of secondary and tertiary Structures of polypeptides known in the art are described in Proteins, Structures and molecular Principles, edited by Creighton (W.H.Freeman and Company, New York (1984)); edited by C.Branden and J.ToozeIntroduction to Protein Structure(Garland Publishing, New York, n.y. (1991)); and Nature354 by Thornton et al: 105(1991), the contents of which have been incorporated herein by reference.

Non-peptide analogs have properties similar to peptide templates and are therefore commonly used as drugs in the pharmaceutical industry. This type of non-peptide compound is referred to as a "peptidomimetic". Fauchere j.adv.drug res.15: 29 (1986); TINS, p.392(1985) by Veber and Freidinger; and j.med.chem.30 by Evans et al: 1229(1987), the contents of which are incorporated herein by reference. Such compounds are often developed by means of computerized molecular modeling. Peptidomimetics are structurally similar to peptides of therapeutic use and can be used to produce equivalent therapeutic efficacy orPreventive effect. Generally, peptidomimetics are structurally similar to model polypeptides (i.e., polypeptides having desired biochemical properties or pharmaceutical activities), such as: a human antibody, but wherein one or more peptide linkages are optionally replaced by a linkage selected from the group consisting of: -CH₂NH-、-CH₂S-、-CH₂-CH₂-, -CH-CH- (cis and trans) -, -COCH₂-、-CH(OH)CH₂-, and-CH₂SO-. Systematic substitution of one or more amino acids in a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can result in a more stable peptide. In addition, constrained peptides comprising identical sequences or substantially identical sequence variations may be produced according to methods known in the art (Rizo and Gierasch, Ann. Rev. biochem.61: 387(1992), the contents of which are incorporated herein by reference); for example: an internal cysteine residue is added which can form an intramolecular disulfide bond to cyclize the peptide.

An "antibody" refers to a complete antibody or an antigen-binding portion thereof that competes for specific binding with the complete antibody. For a general description thereof, seeFundamental ImmunologyChapter 7 (Paul, w. editions, 2 nd edition, Raven Press, n.y. (1989)) (the contents of which are fully incorporated herein by reference for all purposes). Antigen-binding portions can be produced using recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. The antigen-binding portion comprises: fab, Fab ', F (ab')₂Fd, Fv, dAb, and Complement Determining Region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, double antibodies (diabodies), and polypeptides comprising at least a portion of an antibody sufficient to confer binding of a specific antigen to the polypeptide.

From N-terminus to C-terminus, both the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 regions. The amino acid assignments for each domain are according to KabatSequences of Proteins of Immunological Interest(national institutes of Health, Bethesda, Md. (1987and1991)), or Chothia and Lesk J.mol.biol.196: 901-917 (1987); chokia et al Nature 342: 878-883 (1989).

The antibodies indicated by numbers herein refer to monoclonal antibodies derived from the same numbered hybridomas. For example: monoclonal antibody 3.1.1 was obtained from hybridoma 3.1.1.

As used herein, Fd fragment refers to the antibody fragment consisting of VH and CH1 domains; the Fv fragment consists of the VL and VH domains in a single arm of the antibody; and dAb fragments (Ward et al Nature 341: 544-546(1989)) consist of VH domains.

In some embodiments, the antibody is a single chain antibody (scFv), wherein the VL and VH domains are paired via a synthetic linker that allows the domains to form a single protein chain, to form a monovalent molecule. (Bird et al Science 242: 423-. In some embodiments, the antibody is a diabody (diabodies), i.e., a bivalent antibody in which both the VH and VL domains are expressed on a single polypeptide chain, but in which the linker used is too short to allow pairing between the two domains on the same chain, such that the domains must be paired with complementary domains on the other chain to create two antigen-binding sites. (see, e.g., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993) by Holliger P. et al, and Structure 2: 1121-1123(1994) by Poljak R.J. et al). In some embodiments, one or more CDRs from an antibody of the invention may be introduced into the molecule via covalent or non-covalent means to form an immunoadhesin that specifically binds CD 40. In such embodiments, the cdrs(s) may be introduced as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be introduced non-covalently.

In embodiments having one or more binding sites, such binding sites may be the same or different.

The term "human antibody" as used herein refers to any antibody in which all of the variable and constant domain sequences are human sequences. Such antibodies can be prepared in a variety of ways as described below.

The term "chimeric antibody" as used herein refers to an antibody comprising segments from two or more different antibodies. In one embodiment, one or more CDRs are derived from a human anti-CD 40 antibody. In another embodiment, all of the CDRs are derived from a human anti-CD 40 antibody. In another embodiment, the CDRs from more than one human anti-CD 40 antibody are combined in a chimeric antibody. For example: chimeric antibodies may comprise a CDR1 from the light chain in a first human anti-CD 40 antibody, a CDR2 from the light chain in a second human anti-CD 40 antibody, and CDRs 3 and CDR3 from the light chain in a third human anti-CD 40 antibody, and the CDRs from the heavy chain may be derived from one or more other anti-CD 40 antibodies. Furthermore, the framework regions may be derived from the same anti-CD 40 antibody or from one or more different human bodies.

As used herein, an "activating antibody" (also referred to herein as an "agonist antibody") means that the antibody, when added to a cell, tissue or organism expressing CD40, increases one or more CD40 activities by at least about 20%. In some embodiments, the antibody activates CD40 activity by at least 40%, 50%, 60%, 70%, 80%, 85%. In some embodiments, the activating antibody is added in the presence of CD 40L. In some embodiments, the activity of the activated antibody is measured using a whole blood surface molecular up-regulation assay. See example VII. In another embodiment, dendritic cell assay is used to determine the release of IL-12, to determine the activity of the activated antibody. See example VIII. In another embodiment, the activity of the activated antibody is determined using an in vivo tumor model. See example X.

Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one of ordinary skill in the art following the teachings of the present specification. The preferred amino-and carboxy-termini of fragments or analogs occur at the boundaries of functional domains. Structural and functional domains can be distinguished by aligning nucleotide and/or amino acid sequence data with published or proprietary sequence databases. Preferably, computerized alignment is used to identify sequence motifs or protein conformation domains that are predicted to occur in other proteins of known structure and/or function. Protein sequences that fold into a known three-dimensional structure can be identified using known methods. See Bowie et al Science 253: 164(1991).

As used herein, "surface plasmon resonance" refers to an optical phenomenon whereby real-time biospecific interactions can be analyzed by detecting changes in protein concentration in the biosensor matrix, such as: performed using the BIAcore system (pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further description see Jonsson u. et al, ann.biol.clin.51: 19-26 (1993); biotechniques11 by Jonsson u. et al: 620-627 (1991); j.mol.recognit.8 to Jonsson b.et al: 125-131 (1995); and anal. biochem.198 to Johnsson b. et al: 268-277(1991).

″K_DThe term "refers to the equilibrium dissociation constant for a particular antibody-0 antigen interaction.

The term "epitope" includes any protein determinant that specifically binds to an immunoglobulin or T-cell receptor. Epitopic determinants are typically composed of a population of chemically active surface molecules, such as: amino acid or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is considered to bind specifically to an antigen when the antibody has an equilibrium dissociation constant of 1. mu.M or less, preferably 100nM or less, and most preferably 10nM or less.

The 20 common amino acids and their abbreviations used herein follow their common usage. See alsoImmunology-A Synthesis(second edition, edited by e.s.golub and d.r.gren, Sinauer Associates, Sunderland, Mass. (1991)), the contents of which are incorporated herein by reference.

The term "polynucleotide" as used herein refers to a polymeric nucleotide of at least 10 bases in length, which may be either a ribonucleotide or a deoxynucleotide or a modified version of both types of nucleotides. This term includes single-stranded as well as double-stranded.

As used herein, the term "isolated polynucleotide" refers to a genomic, cDNA, or synthetic polynucleotide, or some combination thereof, that is substantially (1) not associated with all or part of a polynucleotide with which it is naturally found, (2) operably linked to a polynucleotide with which it is not naturally associated, or (3) not occurring as part of a larger sequence in nature.

The term "oligonucleotide" as used herein includes both natural and modified nucleotides that are joined together by a natural and non-natural oligonucleotide linkage. An oligonucleotide is a polynucleotide subsequence, typically 200 bases or less in length. Preferably, the oligonucleotide is 10 to 60 bases in length, most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are typically single stranded, for example: primers and probes, but oligonucleotides can also be double stranded, for example: when used for constructing gene mutants. The oligonucleotide of the invention may be a sense or antisense oligonucleotide.

The term "natural nucleotides" as used herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" as used herein includes nucleotides having modified or substituted sugar groups and the like. "oligonucleotide linkages" herein include such oligonucleotide linkages as: thiophosphate, dithiophosphate, selenophosphate, diselenophosphate, thiopheneaminophosphate, anilinophosphate, phosphoramidate, and the like. See, for example: nucleic acids res.14 of LaPlanche et al: 9081 (1986); stec et al j.am.chem.soc.106: 6077 (1984); nucleic acids res.16 by Stein et al: 3209 (1988); zon et al Anti-Cancer Drug Design 6: 539 (1991); of Zon et alOligonucleotides and Analogues：A Practical ApproachPp.87-108(F. Eckstein eds., Oxford University Press, Oxford England) (1991)); U.S. patent nos.5,151,510; chemical Reviews90 by Uhlmann and Peyman: 543(1990), the contents of which have been incorporated herein by reference. If desired, the oligonucleotide may include a detectable label.

"operably linked" sequences include expression control sequences that are immediately adjacent to the target gene and expression control sequences that have an inverse effect or are at a distance from the target gene. As used herein, "expression control sequence" refers to a polynucleotide sequence to which a coding sequence is ligated that is necessary for expression and processing. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; effective RNA processing signals are as follows: splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences which promote secretion of the protein. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, ribosome binding site, and transcription termination sequence; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequences" shall at least include all components necessary for the expression and processing and may also include other beneficial components such as: the leader sequence and the fusion partner sequence.

The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded DNA, into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector, wherein an additional DNA segment can be ligated into the viral genome. In some embodiments, the vector is capable of autonomous replication in a host cell into which it is introduced (e.g., a bacterial vector having a bacterial origin of replication and an episomal mammalian vector). In other embodiments, the vector (e.g., a non-episomal mammalian vector) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby replicated together with the host genome. In addition, certain vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

The term "recombinant host cell" (or simply "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. It is understood that "recombinant host cell" and "host cell" refer not only to a particular somatic cell, but also to the progeny of such a cell. Since certain modifications may occur in the next generation due to mutation or environmental influences, such progeny are not necessarily identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

"Selective hybridization" refers herein to specific binding that can be detected. Polynucleotides, oligonucleotides and fragments thereof according to the invention can be selectively hybridized to nucleic acid strands under hybridization and washing conditions which minimize detectable binding to non-specific nucleic acids. "high stringency" or "highly stringent" conditions can be used to achieve selective hybridization conditions known in the art and discussed herein. An example of a "high stringency" or "highly stringent" condition is incubation of a polynucleotide with another polynucleotide, wherein one polynucleotide can be immobilized on a solid surface (e.g., a membrane), incubated in hybridization buffer (6 XSSPE or SSC, 50% formamide, 5 XDenhardt's reagent, 0.5% SDS, 100. mu.g/ml denatured salmon sperm DNA fragments) at 42 ℃ hybridization temperature for 12-16 hours, followed by 2 washes with washing buffer (1 XSSC, 0.5% SDS) at 55 ℃. See also Sambrook et al, supra, pp.9.50-9.55.

The term "percent sequence identity" in the description of nucleic acid sequences refers to residues that are the same when the two sequences are aligned for maximum relatedness. The length of the sequence identity comparison is at least about 9 nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. A variety of different algorithms are known in the art for measuring nucleotide sequence identity. For example: the polynucleotide sequences may be aligned using FASTA, Gap or Bestfit, a WisconsinPackageversion 10.0 program of the Genetic Computer Group (GCG) of Madison, Wis. FASTA includes, for example: FASTA2 and FASTA3 programs provide the alignment and percent identity of regions of optimal overlap between query sequences and search sequences (Pearson, Methods enzymol.183: 63-98 (1990); Pearson, Methods mol.biol.132: 185-219 (2000); Pearson, Methods enzymol.266: 227-258 (1996); Pearson, J.mol.biol.276: 71-84 (1998); the contents of which are incorporated herein by reference). Unless otherwise specified, a particular program or algorithm uses default parameters. For example: the percent sequence identity between nucleic acid sequences can be determined using FASTA, with its default parameters (word length 6, using NOPAM factors in the scoring matrix) or using Gap provided in GCG version 6.1, the contents of which are incorporated herein by reference.

Unless otherwise indicated, reference to a nucleotide sequence includes the complementary strand thereof. Thus, it will be appreciated that reference to a nucleic acid having a particular sequence should include its complementary strand having a complementary sequence.

In the field of molecular biology, researchers use the terms "percent sequence identity", "percent sequence similarity", and "percent sequence homology" interchangeably. In the present application, such terms are used only in the context of nucleic acid sequences with the same meaning.

"substantial similarity" or "substantial sequence similarity", when referring to a nucleic acid or fragment thereof, means a nucleotide sequence identity of at least about 85%, preferably at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99%, as determined by any known sequence identity algorithm (e.g., FASTA, BLAST or Gap, supra), when optimally aligned with another nucleic acid (or its complementary strand), after appropriate insertions or deletions.

As used in connection with a polypeptide, the term "substantial identity" refers to the identity of the polypeptide when optimally aligned, such as: the default GAP value is used to determine at least 70, 75 or 80% sequence identity, preferably at least 90 or 95% sequence identity, more preferably at least 97, 98 or 99% sequence identity between two peptide sequences using the programs GAP or BESTFIT. Preferably, the difference in residue positions that are not identical is a conservative amino acid substitution. "conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with another amino acid residue having a side chain R group of similar chemical nature (e.g., charge or hydrophilicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ by conservative substitutions, the percent sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. See, for example: pearson, Methods mol. biol. 243: 307-31(1994). Examples of amino acid groups having side chains of similar chemical nature include: 1) aliphatic side chain: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chain: serine and threonine; 3) amide-containing side chain: asparagine and glutamine; 4) aromatic side chain: phenylalanine, tyrosine, and tryptophan; 5) basic side chain: lysine, arginine, and histidine; 6) acidic side chain: aspartic acid and glutamic acid; and 7) sulfur containing side chains: cysteine and methionine. Preferred conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

Alternatively, conservative substitutions are described in Gonnet et al, Science 256: 1443-45(1992), the contents of which are incorporated herein by reference, gives any change in positive values in the PAM250 log-approximation matrix disclosed. The "moderately conservative" substitution is any change that yields a non-negative value in the PAM250 log-approximation matrix.

Sequence similarity (also referred to as sequence identity) of polypeptides is typically determined using sequence analysis software. Protein analysis software matches similar sequences based on similarity measures assigned by different substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example: GCG contains programs such as: "Gap" and "Bestfit" can use default parameters to determine sequence homology or sequence identity between closely related polypeptides (e.g., homologous peptides from different biological species) or between a wild-type protein and its mutant protein. See, for example: GCG version 6.1. Polypeptide sequences can also be aligned using FASTA (GCG program version 6.1), using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of regions of optimal overlap between the query sequence and the search sequence (Pearson, Methods enzymol.183: 63-98 (1990); Pearson, Methods mol.biol.132: 185-219 (2000)). Another preferred algorithm when aligning sequences of the invention with a database containing a large number of sequences from different organisms is the computer program BLAST, in particular blastp or tblastn, which is determined using default parameters. See, for example: j.mol.biol.215 to Altschul et al: 403-; nucleic Acids Res.25 of Altschul et al: 3389-402 (1997); the contents of which have been incorporated herein by reference.

The length of polypeptide sequences used for alignment homology is generally at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching databases containing sequences from a large number of different organisms, it is desirable to align the amino acid sequences.

The term "label" or "labeled" as used herein refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable label, for example: incorporated into a radiolabeled amino acid or attached to a biotinylated moiety, which can be detected using labeled avidin (e.g., streptavidin containing a fluorescent label or enzyme activity that can be detected using optical or colorimetric methods). In another embodiment, the marker may be medical, for example: drug conjugates or toxins. Various methods for labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include (but are not limited to) the following: radioisotopes or radionuclides (e.g.:³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) fluorescent labels (for example: FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g.: horseradish peroxideChemotherapeutics, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotin groups, predetermined polypeptide epitopes recognized by a second reporter (e.g.: leucine zipper-type pairing sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags), magnetic reagents such as: gadolinium chelates, toxins such as pertussis toxin, paclitaxel, cytochalasin B, brevibacillin D, ethidium bromide, emetine, mitomycin, etoposide, dantroside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroyanthrocin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and analogues or homologues thereof. In some embodiments, the labels are attached using spacer arms of different lengths to reduce potential steric hindrance.

The term patient includes human and animal subjects.

Throughout this specification and the claims, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated entity or group of entities but not the exclusion of any other entity or group of entities.

Human anti-CD 40 antibodies and characterization thereof

Human antibodies can avoid some of the problems associated with antibodies having non-human (e.g., rodent) variable or constant regions. Such problems include rapid clearance of the antibody or an immune response against the antibody. Thus, in one embodiment, the invention provides a humanized anti-CD 40 antibody. In another embodiment, the invention provides human anti-CD 40 antibodies. In some embodiments, the human anti-CD 40 antibody is generated by immunizing a rodent that comprises a human immunoglobulin gene in its genome with a human antibody. Human anti-CD 40 antibodies should minimize the immunogenicity and allergic response to non-human monoclonal antibodies or non-human derived monoclonal antibodies (Mabs), thereby increasing the efficacy and safety of the administered antibodies. Fully human antibodies should be useful in the treatment of chronic and recurrent human diseases to provide substantial advantages, such as: inflammation and cancer may require repeated administration of antibodies.

The present invention provides 11 activated human anti-CD 40 monoclonal antibodies (mAbs) and hybridoma cell lines producing such antibodies. Table A lists the nucleic acids encoding the full length heavy and light chains (including the leader sequences), the deduced corresponding full length amino acid sequences, and the sequence numbers of the nucleotides and deduced amino acid sequences of the heavy and light chain variable regions (SEQ ID NOS:).

TABLE A

The invention further provides human anti-CD40mAb 23.25.1 and hybridoma cell lines producing the antibodies.

The present invention further provides certain of the above-described heavy and/or light chain variants of the human anti-CD 40mAbs, which comprise one or more amino acid substitutions. The present invention provides two heavy chain variants of mab3.1.1. In one, the alanine at residue 78 is replaced with a threonine. In the second variant, the alanine at residue 78 is replaced with threonine and the valine at residues 88 and 97 is replaced with alanine. The invention also provides a light chain variant of mab3.1.1 in which the leucine at residue 4 is replaced with leucine at residue 83 with methionine and valine, respectively. The combination of a heavy chain or light chain variant with a wild-type light chain or heavy chain, respectively, is referred to as a mutant chain. Thus, an antibody comprising a wild-type light chain and a heavy chain in which the alanine at residue 78 is mutated to a threonine is designated 3.1.1H-A78T. However, other embodiments of the invention include antibodies comprising any combination of 3.1.1 heavy and light chain variants.

In addition, the invention provides heavy chain variants of mab22.1.1 in which the cysteine at residue 109 is replaced with alanine. The monoclonal antibody comprising the heavy chain variant and the 22.1.1 light chain was designated mAb 22.1.1H-C109A. The invention also provides two heavy chain variants and one light chain variant of the mab23.28.1. In one of the heavy chain variants, the aspartic acid at residue 16 is replaced by glutamic acid. The mAb containing the heavy chain variant and 23.28.1 light chain was designated 23.28.1H-D16E. The invention also includes 23.28.1 light chain variants in which the cysteine at residue 92 is replaced with alanine. The mAb antibody comprising the 23.28.1 heavy chain and this light chain variant was designated 23.28.1L C92A. The invention also provides mAbs containing a 23.28.1 heavy chain variant and a 23.28.1 light chain variant.

The light chain produced by hybridoma 23.29.1 contained a mutation in the constant region at residue 174. The original lysine at this position was changed to arginine in the light chain produced by this hybridoma. Thus, the invention also provides a 23.29.1 light chain comprising the native lysine at residue 174, and a mAb comprising variants of the 23.29.1 heavy and light chains, designated 23.29.1L-R174K.

In a preferred embodiment, the anti-CD 40 antibodies are 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.28.1L-C92A, 23.29.1L-R174K, and 24.2.1. In some embodiments, the anti-CD 40 antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 8. 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 94, 100 or 102 or a variable region thereof, or encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID No.7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 93, 99 or 101. In some embodiments, the anti-CD 40 antibody comprises a light chain comprising at least one CDR2 from one of the above antibodies, one of the above amino acid sequences (as depicted in FIGS. 1A-1C and 2A-2C), or encoded by one of the above nucleic acid sequences. In another embodiment, the light chain further comprises CDR1 and CDR3 each independently selected from a light chain variable region comprising no more than 10 amino acids from the amino acid sequence encoded by a germline vka 3/a19, L5, or a27 gene, or CDR1 and CDR3 each independently selected from one of CDR1 and CDR3 of: (1) an antibody selected from the group consisting of: 3.1.1, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1L-C92A, 23.29.1L-R174K or 24.2.1; (2) amino acid sequence SEQ ID NO: 4. 12,20, 28, 36, 44, 52, 60, 68, 76, 84, 94, 100, or 102, or (3) encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 3. 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 93, 99, or 101.

In another preferred embodiment, the anti-CD 40 antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 6. 14, 22, 30, 38, 46, 54, 62, 70, 78, or 86 or a variable region thereof, or encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NOS: 5. 13, 21, 29, 37, 45, 53, 61, 69, 77 or 85. In some embodiments, the anti-CD 40 antibody comprises a heavy chain, wherein the heavy chain comprises at least the CDR3 of one of the antibodies listed above, one of the amino acid sequences listed above (as depicted in FIGS. 1A-1C and 2A-2C) or is encoded by one of the nucleic acid sequences listed above. In another embodiment, the heavy chain further comprises CDR1 and CDR2 independently selected from a heavy chain variable region comprising no more than 18 amino acids from the amino acid sequence encoded by a germline VH3-30+, 4-59, 1-02, 4.35, or 3-30.3 gene or CDR1 and CDR2 independently selected from one of CDR1 and CDR2 of seq id no: (1) an antibody selected from the group consisting of: 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.29.1 and 24.2.1; (2) amino acid sequence SEQ ID NO: 2. 10, 18, 26, 34, 42, 50,58, 66, 74, 82, 90, 92, 96 or 98; or (3) encoded by the following nucleic acid sequence: SEQ ID NO: 1. 9,17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 91, 95, or 97. In another embodiment, the anti-CD 40 antibody comprises a heavy chain and a light chain as defined above.

The antibody 3.1.1H-A78T used herein is identical to 3.1.1, but in which the alanine at residue 78 in the heavy chain is changed to threonine. Similarly, in the heavy chain of antibody 3.1.1H-A78T-V88A-V97A, residue 78 was changed to A and the valine at residues 88 and 97 was changed to alanine. Antibody 3.1.1L-L4M-L83V was identical to 3.1.1, but in which the leucine at residue 4 of the light chain was changed to methionine and the leucine at residue 83 was changed to valine. Antibody 22.1.1H-C109A is identical to 22.1.1, but with the cysteine at residue 109 in the heavy chain changed to alanine. Antibodies 23.28.1H-D16E and 23.28.1L-C92A are identical to 23.28.1 except that the aspartic acid at residue 16 in the heavy chain is changed to glutamic acid and the cysteine at residue 92 in the light chain is changed to alanine. Antibody 23.29.1L-R174K is the same as 23.29.1 except that the arginine at residue 174 of the light chain is changed to lysine.

anti-CD 40 antibody types and subtypes

The type and subtype of anti-CD 40 antibody can be determined by any means known in the art. Generally, antibody types and subtypes can be determined using antibodies specific for a particular antibody type and subtype. Such antibodies are commercially available. Types and subtypes can be determined using ELISA, or western blot analysis, among other techniques. Alternatively, type and subtype can be determined by analyzing the sequence of all or a portion of the constant domain of the heavy and/or light chain of an antibody, comparing its amino acid sequence to amino acid sequences from a variety of different known immunoglobulin types and subtypes.

In some embodiments, the anti-CD 40 antibody is a monoclonal antibody. The anti-CD 40 antibody can be an IgG, IgM, IgE, IgA or IgD molecule. In a preferred embodiment, the anti-CD 40 antibody is an IgG and is of the IgG1, IgG2, IgG3, or IgG4 subtype. In another preferred embodiment, the anti-CD 40 antibody is of the IgG2 subtype.

Species and molecular selectivity

In another aspect of the invention, the anti-CD 40 antibody exhibits species and molecular selectivity. In some embodiments, the anti-CD 40 antibody binds to primate and human CD 40. In some embodiments, the anti-CD 40 antibody binds to human, cynomolgus or rhesus CD 40. In other embodiments, the anti-CD 40 antibody does not bind to mouse, rat, dog, or rabbit CD 40. In accordance with the teachings of the present specification, the species selectivity of anti-CD 40 antibodies can be determined using methods known in the art. For example: species selectivity can be determined using western blot analysis, FACS, ELISA or RIA. (see, e.g., example IV).

In some embodiments, the anti-CD 40 antibody is more than 100-fold more selective for CD40 than RANK (receptor activator of nuclear factor- κ B), 4-1BB (CD137), TNFR-1 (tumor necrosis factor receptor-1), and TNFR-2 (tumor necrosis factor receptor-2). In some embodiments, the anti-CD 40 antibody does not have any significant specific binding to any other protein than CD 40. The selectivity of anti-CD 40 antibodies for CD40 can be determined using methods known in the art and in accordance with the teachings of the present specification. For example: selectivity can be determined by Western blot analysis, FACS, ELISA or RIA (see, e.g., example V).

Identification of CD40 epitope recognized by anti-CD 40 antibody

In addition, the present invention provides a human anti-CD 40 monoclonal antibody that binds to CD40 that cross-competes for binding to and/or binds to the same epitope and/or has the same K when bound to CD40 with a human anti-CD 40 antibody selected from the group consisting of_D: antibodies 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.28.IL-C92A, 23.29.1L-R174K or 24.2.1; or a human anti-CD 40 antibody, wherein the heavy chain variable region is comprised of the amino acid sequence SEQ ID NO: 2. 10, 18, 26, 34, 42, 50,58, 66, 74, 82, 90, 92, 96 or 98; or a human anti-CD 40 antibody, wherein the light chain variable region is comprised of the amino acid sequence SEQ ID NO: 4. 12,20, 28, 36, 44, 52, 60, 68, 76, 84, 94, 100 or 102.

Any method known in the art can be used to determine whether an antibody will bind to the same epitope as an anti-CD 40 antibody or cross-compete for binding with an anti-CD 40 antibody. In one embodiment, an anti-CD 40 antibody of the invention can be allowed to bind to CD40 under saturating conditions, and the ability of the test antibody to bind to CD40 is then determined. If the test antibody binds to CD40 at the same time as the anti-CD 40 antibody, the test antibody binds to the same epitope, an overlapping epitope, or an epitope in close proximity to the epitope bound by the human anti-CD 40 antibody. This experiment can be performed by ELISA, FACS or surface plasmon resonance (see, e.g., example VI). In a preferred embodiment, the experiment is performed using surface plasmon resonance. In a more preferred embodiment, BIAcore is used.

Binding affinity of anti-CD 40 antibodies to CD40

In some embodiments of the invention, the anti-CD 40 antibody binds CD40 with very high affinity. In some embodiments, the anti-CD 40 antibody binds to CD40 with a KD of 2X10^-8M or less. In another preferred embodiment, the antibody binds to CD40 with a KD of 2X10^-9、2×10^-10、4.0×10^-11M, or lower. In an even more preferred embodiment, the antibody binds CD40 with a KD of 2.5X 10^-12M, or lower. In some embodiments, the antibody binds to K of CD40_DSubstantially the same as the KD of an antibody selected from the group consisting of: 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.28.1L-C92A, 23.29.1L-R174K or 24.2.1. In another preferred embodiment, the antibody binds CD40 with a KD substantially the same as an antibody comprising a CDR2 from the light chain and/or a CDR3 from the heavy chain of an antibody selected from the group consisting of: 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.28.1L-C92A, 23.29.1L-R174K and 24.2.1. In another preferred embodiment, the antibody binds to CD40_DSubstantially identical to a polypeptide comprising a polypeptide having the amino acid sequence of SEQ ID NO: 2. 10, 18, 26, 34, 42, 50,58, 66, 74, 82, 90, 92, 96 or 98, or a light chain variable region comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 4. 12,20, 28, 36, 44, 52, 60, 68, 76, 84, 94, 100 or 102 has the same KD for the antibody in the light chain variable region. In another preferred embodiment, the KD for binding of an antibody to CD40 is substantially equivalent to a KD comprising a polypeptide having the amino acid sequence of SEQ ID NO: 4. 12,20, 28, 36, 44, 52, 60, 68, 76, 84, 94, 100 or 102 or a light chain variable region comprising CDR2 having the amino acid sequence SEQ ID NO: 2. 10, 18, 26, 34, 42, 50,58, 66, 74, 82, 90, 92, 96 or 98, has the same KD.

In some embodiments, the anti-CD 40 antibody has a low off-rate. In some embodiments, the K of the anti-CD 40 antibody_offIs 2.0X 10^-4Or lower. In some embodiments, K_offIs 2.0X 10^-7Or lower. In some embodiments, K_offSubstantially the same as the antibodies described herein, including antibodies selected from the group consisting of: 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.28.1L-C92A, 23.29.1L-R174K and 24.2.1. In some embodiments, the antibody binds to K of CD40_offSubstantially identical to antibody K comprising CDR3 from the heavy chain or CDR2 from the light chain of the following antibodies_offThe same is that: 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.28.IL-C92A, 23.29.1L-R174K and 24.2.1. In some embodiments, the antibody binds to K of CD40_offSubstantially identical to a polypeptide comprising a polypeptide having the amino acid sequence of SEQ ID NO: 2. 10, 18, 26, 34, 42, 50,58, 66, 74, 82, 90, 92, 96 or 98, or a light chain variable region comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 4. 12,20, 28, 36, 44, 52, 60, 68, 76, 84, 94, 100 or 102, or a pharmaceutically acceptable salt thereof_offThe same is true. In another preferred embodiment, the antibody binds to CD40 with a Koff substantially similar to a binding domain comprising a polypeptide having the amino acid sequence SEQ ID NO: 4. 12,20, 28, 36, 44, 52, 60, 68, 76, 84, 94, 100 or 102 or a light chain variable region comprising CDR2 having the amino acid sequence SEQ ID NO: 6. 1. the following examples of the present invention4. 22, 30, 38, 46, 54, 62, 70, 78, 86, 90, 92, 96 or 98, the Koff of the antibody for CDR3 of the heavy chain variable region is the same.

The binding affinity and off-rate of anti-CD 40 antibodies to CD40 can be determined using methods known in the art. Binding affinity can be performed using competitive ELISAs, RIAs or surface plasmon resonance methods, such as: BIAcore. Dissociation rates can also be determined using surface plasmon resonance. Preferably, the binding affinity and dissociation rate are measured by surface plasmon resonance. More preferably, BIAcore is used^TMThe binding affinity and off-rate were determined. See, for example: example XIV. Light and heavy chain gene utilization

The anti-CD 40 antibodies of the invention may comprise human kappa-or human lambda-light chains or amino acid sequences derived therefrom. Some embodiments comprise a kappa-light chain, the light chain variable domain (VL) being encoded in part by the human A3/A19(DPK-15), L5(DP5) or A27(DPK-22) VK gene.

In some embodiments, the VL of an anti-CD 40 antibody comprises one or more amino acid substitutions relative to the germline amino acid sequence. In some embodiments, the VL of the anti-CD 40 antibody comprises 1, 2, 3,4, 5,6, 7, 8,9, or 10 amino acid substitutions relative to the germline amino acid sequence. In some embodiments, one or more of those germline amino acid substitutions occur in the CDR regions of the light chain. In some embodiments, the amino acid substitution position relative to the germline is the same as the substitution position or positions relative to the germline in any one or more of the VLs of antibodies 3.1.1, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1L-C92A, 23.29.1L-R174K, and 24.2.1. For example: the VL of the anti-CD 40 antibody may comprise one or more amino acid substitutions in antibody 21.4.1 relative to germline, and other amino acid substitutions in antibody 10.8.3 relative to germline, wherein the antibody 10.8.3 uses the same VK gene as antibody 21.4.1. In some embodiments, the amino acid change occurs at one or more of the same positions, but involves a mutation that is different from the reference antibody.

In some embodiments, the position at which an amino acid change occurs relative to the germline is the same as one or more of the changed positions in the VL of any one of antibodies 3.1.1, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1L-C92A, 23.29.1L-R174K, and 24.2.1, but such changes may indicate conservative amino acid substitutions at such positions relative to the amino acid in the reference antibody. For example: if one of the antibodies is altered relative to the germline at a particular position and is glutamate, conservative substitutions with aspartate at that position may be made. Similarly, if an amino acid is substituted for serine relative to the germline, a conservative substitution of serine with threonine may be made at that position. Conservative amino acid substitutions have been discussed above.

In some embodiments, the light chain of the human anti-CD 40 antibody comprises an amino acid sequence that is identical to or has at most a conservative amino acid substitution of the amino acid sequence of antibodies 3.1.1(SEQ ID NO: 4), 3.1.1L-4M-L83V (SEQ ID NO: 94), 7.1.2(SEQ ID NO: 12), 10.8.3(SEQ ID NO: 20), 15.1.1(SEQ ID NO: 28), 21.4.1(SEQ ID NO: 60), 21.2.1(SEQ ID NO: 36), 21.4.1(SEQ ID NO: 44), 22.1.1(SEQ ID NO: 52), 23.5.1(SEQ ID NO: 60), 23.28.1(SEQ ID NO: 68), 23.28.1L-C92A (SEQ ID NO: 100), 23.29.1(SEQ ID NO: 76), 23.29.1L-R174K (SEQ ID NO: 102), 24.24.1L-R174 (SEQ ID NO: 102), or 3.1, 3.8, 3.6, 3.8, 3.1, 10, 3.8, 10, or more conservative amino acid substitutions in total.

In some embodiments, the light chain of an anti-CD 40 antibody comprises at least the light chain CDR2, and may also comprise the CDR1 and CDR3 regions of a germline sequence as described herein. In another embodiment, the light chain may comprise CDRs 1 and 2 independently selected from the group consisting of antibodies: 3.1.1, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1L-C92A, 23.29.1 and 24.2.1, or CDR regions each having less than 8, less than 6, less than 4, or less than 3 conservative amino acid substitutions and/or sharing 3 or less non-conservative amino acid substitutions. In other embodiments, the light chain of the anti-CD 40 antibody comprises at least a light chain CDR2 and may also comprise CDR1 and CDR3 regions, the CDR1 and CDR3 being independently selected from the group consisting of SEQ ID NOS: 4. 12,20, 28, 36, 44, 52, 60, 68, 76, 84, 94 or 100, or a light chain variable region of an antibody consisting of CDR1 and CDR3 of an amino acid sequence selected from SEQ ID NOS: 3. 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 93, or 99.

In the heavy chain aspect, in some embodiments, the variable region of the heavy chain amino acid sequence is partially composed of human V_H3-30+、V_H4-59、V_H1-02、V_H4.35 or V_H3-30.3 genes. In some embodiments, the VH of the anti-CD 40 antibody comprises one or more amino acid substitutions, deletions, or insertions (additions) relative to the germline amino acid sequence. In some embodiments, the variable domain of the heavy chain comprises 1, 2, 3,4, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 mutations relative to the germline amino acid sequence. In some embodiments, the mutation is a non-conservative substitution relative to the germline amino acid sequence. In some embodiments, the mutation is in a CDR region of the heavy chain. In some embodiments, the position of the amino acid change is compared to any one or more V of the following antibodies_HThe position of one or more mutations relative to the germline are identical: 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.29.1 and 24.2.1. In other embodiments, the amino acid change occurs at one or more of the same positions as the reference antibody, but involves a different mutation.

In some embodiments, the heavy chain comprises the amino acid sequence of the variable domain (VH) of an antibody: antibodies 3.1.1(SEQ ID NO: 2), 3.1.1H-A78T (SEQ ID NO: 90), 3.1.1H-A78T-V88A-V97A (SEQ ID NO: 92), 7.1.2(SEQ ID NO: 10), 10.8.3(SEQ ID NO: 18), 15.1.1(SEQ ID NO: 26), 21.2.1(SEQ ID NO: 34), 21.4.1(SEQ ID NO: 42), 22.1.1(SEQ ID NO: 50), 22.1.1H-C109A (SEQ ID NO: 96), 23.5.1(SEQ ID NO: 58), 23.28.1(SEQ ID NO: 66), 23.28.1H-C48G (SEQ ID NO: 98), 23.29.1(SEQ ID NO: 74) and 24.2.1(SEQ ID NO: 82), or with up to 1, 2, 10, 3, 10, or more conservative amino acid substitutions and/or conservative amino acid substitutions in total.

In some embodiments, the heavy chain comprises the heavy chain CDR1, CDR2, and CDR3 regions of: antibodies 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.29.1, and 24.2.1 (as shown in FIG. 1D-1H or 2D-2H), or the above CDR regions each contain less than 8, less than 6, less than 4, or less than 3 conservative amino acid substitutions and/or share 3 or less non-conservative amino acid substitutions.

In some embodiments, the heavy chain comprises CDR3, and may also comprise the CDR1 and CDR2 regions of the germline sequences as described above, or may comprise the CDR1 and CDR2 of an antibody independently selected from the group consisting of antibodies comprising heavy chains selected from the group consisting of: 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.29.1 and 24.2.1. In another embodiment, the heavy chain comprises CDR3, and may also comprise CDR1 and CDR2 regions selected from the group consisting of SEQ ID NOS: 2. 10, 18, 26, 34, 42, 50,58, 66, 74, 82, 90, 92, 96 or 98 (as shown in figures 1D-1H or 2D-2H) or is encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NOS: 1. 9,17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 91, 95, or 97. In another embodiment, the antibody comprises a heavy chain as described above and a light chain as described above.

One amino acid substitution that can be made is to change one or more cysteines (which may be chemically reactive) in the antibody to another residue, such as (but not limited to): alanine or serine. In one embodiment, the cysteine substitution occurs in a framework region or in a constant domain of a variable domain of an antibody. In another embodiment, the cysteine is in a non-canonical region of the antibody. Another amino acid substitution that can be made is to alter any possible proteolytic position in the antibody, particularly those positions that are located in the framework regions of the variable domains, in the constant domains of the antibody, or in atypical regions of the antibody. Substitution and removal of the proteolytic site by cysteine residues reduces the risk of any inhomogeneity in the antibody product and thus improves its homogeneity. Another amino acid substitution that may be made is the elimination of paired asparagine-glycines by altering one or both residues in an asparagine-glycine pair that may form a deamidation site. This process is best performed in the framework regions, constant domains or atypical regions of the antibody.

Activation of CD40 by anti-CD 40 antibodies

Another aspect of the invention relates to an anti-CD 40 antibody that is an activating antibody, i.e., a CD40 agonist. The activating antibody may augment or replace the effect of CD40L on CD 40. In some embodiments, the activatable antibody is substantially a mimetic of CD40L and competes with CD40L for binding to CD 40. In some embodiments, the antibody does not compete with CD40L for binding to CD40, but amplifies the effect of CD40L binding to CD 40. In some embodiments, the anti-CD 40 antibody activates CD40 in the presence or absence of CD 40L.

anti-CD 40 antibodies inhibit tumor growth in vivo

According to some embodiments, the present invention provides an anti-CD 40 antibody that inhibits tumor cell proliferation in vitro or tumor growth in vivo.

In some embodiments, the antibody inhibits tumor growth by at least 50%, 55%, 60%, 65%, 70%, 75%. In some embodiments, the antibody inhibits tumor growth by 75%. In one embodiment, inhibition of tumor growth is detected 14 days after initial antibody treatment. In other embodiments, inhibition of tumor growth is detected 7 days after initial antibody treatment. In some embodiments, the anti-CD 40 antibody is administered to the animal together with another anti-neoplastic agent. In some embodiments, the antineoplastic agent can further inhibit tumor growth. In some embodiments, the antineoplastic agent is doxorubicin or paclitaxel. In some embodiments, co-administration of the anti-tumor agent and the anti-CD 40 antibody inhibits tumor growth by at least 50% relative to untreated animals during a period of 22-24 days after primary treatment.

anti-CD 40 antibody induced apoptosis

In another aspect, the invention provides an anti-CD 40 antibody that induces death of CD40 positive cells. In some embodiments, the antibody can cause apoptosis of CD40 positive cells in vivo or in vitro.

Enhancing expression of cell surface molecules

In some embodiments, the anti-CD 40 antibody potentiates expression of B cell surface molecules, including (but not limited to): ICAM, MHC-II, B7-2, CD71, CD23 and CD 83. In some embodiments, 1. mu.g/ml antibody enhances ICAM expression by at least 2-fold, more preferably by at least 4-fold, in a whole blood B-cell surface molecular up-regulation assay. In some embodiments, 1. mu.g/ml antibody can enhance MHC-II expression by at least 2-fold, or more preferably by at least 3-fold, in a whole blood B-cell surface molecule up-regulation assay. In some embodiments, 1. mu.g/ml antibody enhances CD23 expression by at least 2-fold, or more preferably by at least 5-fold, in a whole blood B-cell surface molecular up-regulation assay. See, for example: example VII, table 25.

In some embodiments, the anti-CD 40 antibody can enhance the expression of dendritic cell surface molecules, including (but not limited to): MHC-II, ICAM, B7-2, CD83 and B7-1. In some embodiments, the range of upregulation is similar to the range of upregulation observed by B cells. See, for example: tables 25 and 26 are provided below. In some embodiments, the methods are performed in relation to, for example: b7-2 and MHC-II, and the antibody preferentially upregulates dendritic cell surface molecule expression of such molecules. See, for example: table 27.

Enhancing cytokine secretion by cells

In some embodiments, the antibody potentiates cytokine secretion by the cell, including (but not limited to): IL-8, IL-12, IL-15, IL-18 and IL-23.

In some embodiments, the antibody enhances cytokine secretion by dendritic cells and adherent monocytes. In some embodiments, cytokine production is further enhanced by co-stimulation with one or more of LPS, IFN- γ, or IL-1 β. In another aspect of the invention, co-stimulation of antibodies with LPS enhances IL-12p70 production, EC, in dendritic cell assays₅₀About 0.48. mu.g/ml. In some embodiments, the antibody enhances IL-12p40 production, EC, in dendritic cells₅₀Is about 0.21 g/ml. (see, e.g., example VIII).

In some embodiments, the antibodies enhance IFN- γ secretion in allogeneic T cell/dendritic cell assays, as illustrated in example VIII. In some embodiments, the antibody enhances IFN- γ secretion in allogeneic T cell/dendritic cell assays, with EC thereof₅₀About 0.3. mu.g/ml. In some embodiments, the antibody enhances IFN- γ secretion in allogeneic T cell/dendritic cell assays, with EC thereof₅₀About 0.2 g/ml. In one embodiment, the antibody enhances IFN- γ secretion in an allogeneic T cell/dendritic cell assay, with EC for the antibody₅₀About 0.03 g/ml.

Method for producing antibodies and cell lines producing antibodies

Immunization method

In some embodiments, the human antibody is generated by inoculating a non-human animal comprising part or all of the human immunoglobulin heavy and light chain loci in its genome with the CD40 antigen. In a preferred embodiment, the non-human animal is XenoMouse^TMAn animal.

XenoMouse^TMMice are genetically engineered mouse species that contain large fragments of the human immunoglobulin heavy and light chain loci and are incapable of producing mouse antibodies. See, for example: green et al Nature Genetics 7: 13-21(1994), and U.S. Pat. Nos.5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364, 6,162,963, and 6,150,584. See also WO91/10741, WO94/02602, WO96/34096, WO96/33735, WO98/16654, WO98/24893, WO98/50433, WO99/45031, WO99/53049, WO00/09560 and WO 00/037504.

In another aspect, the invention provides a method for producing anti-CD 40 antibodies from a non-human, non-mouse animal by vaccinating the non-human transgenic animal comprising a human immunoglobulin locus with CD40 antigen. Such animals can be made using the methods described in the above-mentioned documents. The methods disclosed in such documents may be modified as described in us patent 5,994,619. In a preferred embodiment, the non-human animal is a rat, sheep, pig, goat, cow or horse.

XenoMouseTM mice can produce whole human antibodies similar to adult humans, and produce antigen-specific human antibodies. In some embodiments, XenoMouseTM mice contain about 80% of the human antibody V gene bank via introduction of Yeast Artificial Chromosome (YAC) fragments in a megabase size germline configuration of the human heavy chain locus and the kappa-light chain locus. See Mendez et al, Nature Genetics 15: 146-: 483-495(1998), and WO98/24893, the contents of which are incorporated herein by reference.

In some embodiments, the non-human animal comprising human immunoglobulin genes is an animal containing human immunoglobulin "miniloci (miniloci)". In the mini-locus method, exogenous Ig loci are mimicked by the inclusion of a single gene from an Ig locus. Thus, one or more V are formed in the construct_HGene, one or more D_HGenes, one or more JH genes, a mu-constant domain, and a second constant domain(preferably gamma-constant domains) for insertion into an animal. Such methods are described in U.S. patent nos.5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, and 5,643,763, the contents of which are incorporated herein by reference.

The advantage of the mini-locus method is that constructs containing Ig locus moieties can be rapidly generated and introduced into animals. However, a potential drawback of the mini-locus method is that there may not be sufficient immunoglobulin diversity to support full B-cell development, and thus antibody production may be low.

In another aspect of the invention, the invention provides a method of making a humanized anti-CD 40 antibody. In some embodiments, a non-human animal is vaccinated with the CD40 antigen under conditions that allow for the production of antibodies, according to the methods described below. Antibody-producing cells are isolated from the animal, fused with a myeloma to produce a hybridoma, and nucleic acids encoding the heavy and light chains of the anti-CD 40 antibody of interest are isolated. Such nucleic acids are then treated using techniques known in the art and methods described below and the content of non-human sequences is reduced, i.e., antibodies are humanized, to reduce the immune response in humans.

In some embodiments, the CD40 antigen is isolated and/or purified CD 40. In a preferred embodiment, the CD40 antigen is human CD 40. In some embodiments, the CD40 antigen is a fragment of CD 40. In some embodiments, the fragment of CD40 is the extracellular domain of CD 40. In some embodiments, a fragment of CD40 comprises at least one epitope of CD 40. In other embodiments, the CD40 antigen is a cell that expresses or overexpresses CD40 or an immunogenic fragment thereof on its surface. In some embodiments, the CD40 antigen is a CD40 fusion protein.

Immunization of animals can be carried out by any method known in the art. See, for example: of Harlow and LaneAntibodies：A Laboratory ManualNew York: cold spring Harbor Press, 1990. Non-human animals such as: mouse and ratImmunization of sheep, pigs, goats, cattle and horses are known in the art. See, for example: harlow and Lane, as described above, and U.S. Pat. No.5,994,619. In a preferred embodiment, the CD40 antigen is administered with an adjuvant to stimulate an immune response. Examples of adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulatory complex). Such adjuvants may sequester the polypeptide in a localized site to protect the polypeptide from rapid dispersal, or may include substances that stimulate the secretion by the host of chemotactic factors and other components of the immune system of macrophages. Preferably, where a polypeptide is administered, the immunization regimen will comprise two or more administrations of the polypeptide over a period of weeks.

Example I illustrates the preparation of an anti-CD 40 monoclonal antibody.

Antibodies and methods for producing antibody-producing cell lines

After the animals are inoculated with the CD40 antigen, antibodies and/or antibody-producing cells can be obtained from the animals. In some embodiments, serum containing anti-CD 40 antibodies is obtained using a blood draw or after killing the animal. Serum obtained from the animal may be used, or immunoglobulin fractions may be obtained from the serum, or anti-CD 40 antibodies may be purified from the serum. One of ordinary skill in the art will recognize that the serum or immunoglobulin obtained in this manner will be polyclonal. The disadvantage of using polyclonal antibodies prepared from serum is that the amount of antibody obtained is limited and that polyclonal antibodies exhibit inconsistent properties.

In some embodiments, the antibody-producing immortalized cell line is prepared from cells isolated from an immunized animal. After immunization, the animals are killed and B cells from lymph nodes and/or spleen are immortalized. Methods of cell immortalization include, but are not limited to, the use of cancer gene transfer cells, transfection of cells with oncogenic viruses, culture under conditions that allow for the optional extraction of immortalized cells, treatment with an oncogenic compound and a mutagenic compound, fusion with immortalized cells (e.g., myeloma cells), and removal of tumor suppressor gene activity. See, for example: harlow and Lane are as described above. When myeloma cells are used for fusion, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell lines). Immortalized cells are screened using CD40, a portion thereof, or cells expressing CD 40. In a preferred embodiment, the primary screening is performed using enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay. An example of an ELISA screening method is provided in WO00/37504, the contents of which are incorporated herein by reference.

Selecting cells that produce anti-CD 40 antibodies, for example: hybridomas are cloned and further screened for desirable characteristics, including good growth, high antibody production, and desirable antibody characteristics, as described below. Hybridomas can be found in vivo in syngeneic animals, in animals lacking the immune system, for example: nude mice, or expanded in cell culture in vitro. Methods for screening, cloning and amplifying hybridomas are well known to those of ordinary skill in the art.

In a preferred embodiment, the immunized animal is a non-human animal that expresses human immunoglobulin genes, wherein the splenic B cells are fused to a myeloma cell line from the same species as the non-human animal. In a more preferred embodiment, the immunized animal is a XENOMOUSETM animal and the myeloma cell line is a non-secretory mouse myeloma. In an even more preferred embodiment, the myeloma cell line is P3-X63-AG 8.653. See, for example: example I.

Another aspect of the invention provides hybridomas which produce human anti-CD 40 antibodies. In a preferred embodiment, the hybridoma is a mouse hybridoma as described above. In other embodiments, the hybridoma is produced in a non-human, non-mouse species such as: produced in rats, sheep, pigs, goats, cattle or horses. In another embodiment, the hybridoma is a human hybridoma.

Nucleic acid, vector, host cell and recombinant method for producing antibody

Nucleic acids

The invention also includes nucleic acid molecules encoding anti-CD 40 antibodies. In some embodiments, the nucleic acid molecules encoding the heavy and light chains of the anti-CD 40 immunoglobulin are different. In other embodiments, the nucleic acid molecules encoding the heavy and light chains of the anti-CD 40 immunoglobulin are the same.

In some embodiments, the nucleic acid molecule encoding a variable domain in a light chain comprises the human A3/A19(DPK-15), L5(DP5), or A27(DPK-22) V κ gene sequence or a sequence derived therefrom. In some embodiments, the nucleic acid molecule comprises the A3/A19V kappa gene and J kappa 1, J

The nucleotide sequence of the kappa 2 or jkappa 3 gene or a sequence derived therefrom. In some embodiments, the nucleic acid molecule comprises the nucleotide sequences of the L5V k gene and the jk 4 gene. In some embodiments, the nucleic acid molecule comprises the nucleotide sequences of the a27V k gene and the jk 3 gene.

In some embodiments, the nucleic acid molecule encoding the light chain encodes an amino acid sequence that comprises 1, 2, 3,4, 5,6, 7, 8,9, or 10 mutations relative to the germline amino acid sequence. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a VL amino acid sequence comprising 1, 2, 3,4, 5,6, 7, 8,9, or 10 non-conservative amino acid substitutions and/or 1, 2, or 3 non-conservative substitutions relative to a germline sequence. Substitutions may be made in the CDR regions, framework regions, or constant domains.

In some embodiments, a nucleic acid molecule encoding a variable domain of a light chain (VL) encodes a VL amino acid sequence comprising one or more mutations relative to a germline sequence, such mutations being identical to a mutation in the VL of one of the following antibodies: 3.1.1, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1L-C92A, 23.29.1 and 24.2.1. In some embodiments, the nucleic acid molecule encodes at least 3 amino acid mutations relative to germline sequence that can be found in the VL of one of the following antibodies: 3.1.1, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1L-C92A, 23.29.1 and 24.2.1.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a VL amino acid sequence of a monoclonal antibody, or a portion thereof, as follows: 3.1.1(SEQ ID NO: 4), 3.1.1L-L4M-L83V (SEQ ID NO: 94), 7.1.2(SEQ ID NO: 12), 10.8.3(SEQ ID NO: 20), 15.1.1(SEQ ID NO: 28), 21.2.1(SEQ ID NO: 36), 2.1.4.1(SEQ ID NO: 44), 22.1.1(SEQ ID NO: 52), 23.5.1(SEQ ID NO: 60), 23.28.1(SEQ ID NO: 68), 23.28.1L-C92A (SEQ ID NO: 100), 23.29.1(SEQ ID NO: 76) or 24.2.1(SEQ ID NO: 84). In some embodiments, the portion comprises at least the CDR3 region. In some embodiments, the nucleic acid encodes the amino acid sequence of the light chain CDRs of the antibody. In some embodiments, the portion is a contiguous portion comprising CDRs 1-3.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a nucleic acid sequence set forth in SEQ ID NOS: 4. 12,20, 28, 36, 44, 52, 60, 68, 76, 84, 94, or 100 amino acid sequence, or the sequence lacking a signal sequence. In some preferred embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NOS: 3. 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 93, or 99, or a portion thereof, which optionally lacks a signal sequence.

In some embodiments, the portion encodes the VL region. In some embodiments, the portion encodes at least the CDR2 region. In some embodiments, the nucleic acid encodes the amino acid sequence of the light chain CDRs of the antibody. In some embodiments, the portion encodes a contiguous region of CDRs 1-3.

In some embodiments, the nucleic acid molecule encodes a VL amino acid sequence that hybridizes with the VL amino acid sequence of any one of antibodies 3.1.1, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1L-C92A, 23.29.1, or 24.2.1, or with the VL amino acid sequence of SEQ ID NOS: 4. 12,20, 28, 36, 44, 52, 60, 68, 76, 84, 94, or 100, has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity. The nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions (as described above) to nucleic acids encoding SEQ ID NOS: 4. 12,20, 28, 36, 44, 52, 60, 68, 76, 84, 94 or 100, or with a nucleic acid having the amino acid sequence of SEQ ID NOS: 3. 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 93, or 99.

In another embodiment, the nucleic acid encodes an antibody 3.1.1, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1L-C92A, 23.29.1L-R174K, or 24.2.1, a full length light chain comprising SEQ ID NOS: 8. 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 94, 100, or 102, or a light chain encoding an amino acid sequence comprising a mutation as described herein. In addition, the nucleic acid may comprise the nucleotide sequence SEQ ID NOS: 7. 15, 23, 31, 39, 47, 55, 63, 71, 79 or 87, or a nucleic acid molecule encoding a light chain comprising a mutation as described herein.

In another preferred embodiment, the variable domain of the heavy chain (VH) encoded by the nucleic acid molecule comprises the human 3-30+, 4-59, 1-02, 4.35 or 3-30.3VH gene sequence or a sequence derived therefrom. In various embodiments, the nucleic acid molecule comprises human 3-30+ V_HGene, D4(DIR3) gene and human J_H6 genes; human 3-30+ V_HGene, human D1-26(DIR5) gene and human J_H6 genes; human 4.35V_HGene, human DIR3 gene, and human J_H6 genes; human 4-59V_HGene, human D4-23 gene and human J_H4 gene; human 1-02V_HGene, human DLR1 gene and human J_H4 gene; human 3-30+ V_HGene, human D6-19(DIR3) gene and human J_H4 gene; human 3-30+ V_HGene, human D1-1 gene and human J_H6 genes; human 3-30+ V_HGene, human D4-17 gene and human J_H6 genes; human 3-30.3VH gene, human D4-17 gene and human J_H6 genes; human 4-59V_HGene, human D4-17(DIR1) gene and human J_H5 gene, orSequences derived from such human genes.

In some embodiments, the nucleic acid molecule encodes an amino acid sequence that comprises 1, 2, 3,4, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 mutations relative to the germline amino acid sequence of the human V, D or J gene. In some embodiments, the mutation occurs at V_HIn a zone. In some embodiments, the mutation occurs in a CDR region.

In some embodiments, the nucleic acid molecule encodes one or more amino acid mutations relative to germline sequence, such mutations being associated with V occurring in monoclonal antibodies 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.29.1, or 24.2.1_HThe amino acid mutations in (1) are the same. In some embodiments, the nucleic acid encodes at least 3 amino acid mutations relative to the germline sequence, such mutations being identical to at least 3 amino acid mutations that occur in one of the monoclonal antibodies listed above.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding at least a portion of the amino acid sequence of antibody 3.1.1(SEQ ID NO: 2), 3.1.1H-A78T (SEQ ID NO: 90), 3.1.1H-A78T-V88A-V97A (SEQ ID NO: 92), 7.1.2(SEQ ID NO: 10), 10.8.3(SEQ ID NO: 18), 15.1.1(SEQ ID NO: 26), 21.2.1(SEQ ID NO: 34), 21.4.1(SEQ ID NO: 42), 22.1.1(SEQ ID NO: 50), 22.1.1H-C109A (SEQ ID NO: 96), 23.5.1(SEQ ID NO: 58), 23.28.1(SEQ ID NO: 66), 23.28.1H-D16E (SEQ ID NO: 98), 23.29.1(SEQ ID NO: 74) or 24.2.1(SEQ ID NO: 82), or those sequences having conservative amino acid substitutions and/or a total of 3 or fewer non-conservative amino acid substitutions. In various embodiments, the sequences encode one or more CDR regions, preferably the CDR3 region, all 3 CDR regions, including a contiguous portion of CDRs 1-CDR3, or the entire VH region, with or without a signal sequence.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding SEQ IDNOS: 2. 10, 18, 26, 34, 42, 50,58, 66, 74, 82, 90, 92, 96, or 98, or the sequence lacking a signal sequence. In a preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 1. 9,17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 91, 95, or 97, or at least a portion of such sequence lacking a signal sequence. In some embodiments, the partial code V_HA region (with or without a signal sequence), a CDR3 region, all 3 CDR regions, or a contiguous region comprising CDRs 1-CDR 3.

In some embodiments, the nucleic acid molecule encodes a VH amino acid sequence corresponding to V shown in FIGS. 1A-1C or 2A-2C_HAmino acid sequence or a sequence corresponding to SEQ ID NOS: 2. 10, 18, 26, 34, 42, 50,58, 66, 74, 82, 90, 92, 96 or 98, or a pharmaceutically acceptable salt thereof_HThe amino acid sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical. The nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions (as described above) to nucleic acids encoding SEQ ID NOS: 2. 10, 18, 26, 34, 42, 50,58, 66, 74, 82, 90, 92, 96, or 98, or a nucleic acid sequence that hybridizes to or is complementary to the amino acid sequence of SEQ ID NOS: 1. 9,17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 91, 95, or 97. Nucleic acid molecules of the invention include nucleic acids that hybridize under highly stringent conditions (as described above) to nucleic acid sequences encoding the aforementioned VH.

In another embodiment, the nucleic acid encodes a full length heavy chain of antibodies 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.29.1 and 24.2.1, or a full length heavy chain having the amino acid sequence SEQ ID NOS: 6. 14, 22, 30, 38, 46, 54, 62, 70, 78, or 86, or a heavy chain comprising a mutation as discussed herein. In addition, the nucleic acid may comprise the nucleotide sequence SEQ ID NOS: 5. 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, or 89, or a nucleic acid molecule encoding a heavy chain comprising a mutation as discussed herein.

Encoding an anti-CD 40 antibody or a combination thereofNucleic acid molecules of a portion of the heavy chain or the entire light chain can be isolated from any source that can produce such antibodies. In various embodiments, the nucleic acid molecule is isolated from B cells of an animal that has been immunized with CD40, or from immortalized cells derived from such B cells that express anti-CD 40 antibodies. Methods of isolating mRNA encoding an antibody are known in the art. See, for example: sambrook et al. The mRNA can be used to make cDNA for use in Polymerase Chain Reaction (PCR) or cDNA cloning of antibody genes. In a preferred embodiment, the nucleic acid molecule is isolated from a hybridoma which contains as one of its fusion partners a cell from a non-human transgenic animal which produces human immunoglobulin. In an even more preferred embodiment, the human immunoglobulin-producing cell is from Xenomouse^TMIsolated from animals. In another embodiment, the human immunoglobulin-producing cell is a transgenic animal derived from a non-human, non-mouse, as described above. In another embodiment, the nucleic acid is isolated from a non-human, non-transgenic animal. Nucleic acid molecules isolated from non-human, non-transgenic animals can be used, for example, to humanize antibodies.

In some embodiments, the nucleic acid encoding the heavy chain of an anti-CD 40 antibody of the invention can comprise a nucleotide sequence encoding a VH domain of the invention linked in the sense of a read region to a nucleotide sequence encoding a heavy chain constant domain of any origin. Similarly, a nucleic acid molecule encoding the light chain of an anti-CD 40 antibody of the invention may comprise a nucleotide sequence encoding the VL domain of the invention linked in the sense of a read region to a nucleotide sequence encoding the constant domain of the light chain of any origin.

In another aspect of the invention, the coding heavy chain (V)_H) And light chain (V)_L) The nucleic acid molecule of the variable domain of (a) is "transformed" into a full-length antibody gene. In one embodiment, a nucleic acid molecule encoding a VH or VL domain is converted to a full-length antibody gene by insertion into an expression vector that already encodes a heavy chain constant or light chain constant domain, respectively, such that the VH segment is operably linked to a CH segment within the vector, and the V is_LIn segments and carriersC_LThe segments are operatively connected. In another embodiment, code V_HAnd/or V_LDomain nucleic acid molecules encoding VH and/or VL domains are linked to C encoding C via standard molecular biology techniques_HAnd/or C_LThe nucleic acid molecules of the domains are converted into full-length antibody genes. The nucleic acid sequences of human heavy and light chain immunoglobulin constant domain genes are known in the art. See, for example: sequences of Proteins by Kabat et alof Immunological Interest5 th edition, published by NIH, No.91-3242, 1991. The nucleic acid molecules encoding the full-length heavy and/or light chains may then be expressed by the cells into which they are introduced, and the anti-CD 40 antibody isolated.

The nucleic acid molecules can be used to recombinantly express large quantities of anti-CD 40 antibodies. Nucleic acid molecules can also be used to generate chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, double antibodies, mutant antibodies and antibody derivatives, which are described below. If the nucleic acid molecule is derived from a non-human, non-transgenic animal, the nucleic acid molecule may be used to humanize an antibody, as also described below.

In another embodiment, the nucleic acid molecules of the invention are used as probes or PCR primers for specific antibody sequences. For example: the nucleic acid can be used as a probe for diagnostic methods or as a PCR primer for amplifying a region of DNA that can be used for the isolation of other nucleic acid molecules encoding the variable domain of an anti-CD 40 antibody. In some embodiments, the nucleic acid molecule is an oligonucleotide. In some embodiments, the oligonucleotides are from the highly variable regions of the heavy and light chains of the target antibody. In some embodiments, the oligonucleotide encodes all or a portion of one or more CDRs in antibody 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.28.1L-C92A, 23.29.1, or 24.2.1.

Carrier

The invention provides vectors comprising a nucleic acid molecule encoding the heavy chain or antigen-binding portion thereof of an anti-CD 40 antibody of the invention. The invention also provides vectors comprising nucleic acid molecules encoding the light chains or antigen-binding portions thereof of such antibodies. The invention also provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof.

In some embodiments, expression of the anti-CD 40 antibodies or antigen-binding portions thereof of the invention is achieved by inserting DNAs encoding partial or full-length light and heavy chains (the methods for making such DNAs are described above) into an expression vector such that the gene is operably linked to necessary expression control sequences (e.g., transcriptional and translational control sequences). The expression vector includes: plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as: cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived gene-pairs, and the like. The antibody gene is ligated into a vector such that the transcriptional and translational control sequences within the vector perform their function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences selected should be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors. In a preferred embodiment, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector using standard methods (e.g., ligation at complementary restriction sites on the antibody gene fragment and vector, or blunt-ended ligation if no restriction sites are present).

Convenient vectors may encode functionally complete human CH or CL immunoglobulin sequences which have been manipulated to have appropriate restriction sites so that any VH or VL sequence can be readily inserted and expressed as described above. In such vectors, splicing typically occurs between the splice donor site of the inserted J region and the splice acceptor site preceding the human C domain, and also at the splice region within the human CH exon. The polyadenylation reaction and transcription termination reaction are carried out at natural chromosomal locations downstream of the coding region. Recombinant expression vectors may also encode signal peptides that facilitate secretion of the antibody chain by the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain gene, the recombinant expression vector of the present invention may also carry regulatory sequences that control the expression of the antibody chain gene in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector, including the choice of regulatory sequences, will depend on the host cell selected for transformation, the degree of expression of the desired protein, and the like. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high expression of proteins in mammalian cells, such as promoters and/or enhancers derived from: retroviral LTRs, Cytomegalovirus (CMV) (e.g., CMV promoter/enhancer). Simian virus 40(SV40) (e.g., SV40 promoter and/or enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma virus, and mammalian strong promoters, such as: native immunoglobulin and actin promoters. Further description relating to viral regulatory elements and their sequences can be found, for example, in: U.S. Pat. No.5,168,062, U.S. Pat. No.4,510,245, and U.S. Pat. No.4,968,615. Methods for expressing antibodies in plants, including instructions for promoters and vectors, and transformation of plants are known in the art. See, for example: U.S. patent 6,517,529, the contents of which are incorporated herein by reference. Methods for expressing a polypeptide in a bacterial cell or a fungal cell (e.g., a yeast cell) are also known in the art.

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may comprise other sequences, such as: sequences that regulate the replication of the vector in the host cell (e.g., an origin of replication) and a selectable marker gene. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017). For example: selectable marker genes typically confer drug resistance on host cells into which the vector has been introduced, such as: g418, hygromycin or methotrexate. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for methotrexate selection/amplification in DHFR-host cells), the neo gene (for G418 selection), and the glutamate synthase gene.

Non-hybridoma host cells and methods for recombinantly producing proteins

Nucleic acid molecules encoding anti-CD 40 antibodies and vectors comprising such nucleic acid molecules can be used to transfect suitable mammalian, plant, bacterial, or yeast host cells. The transformation method may be any known method for introducing a polynucleotide into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. In addition, viral vectors can be used to introduce nucleic acid molecules into mammalian cells. Cell transformation methods are known in the art. See, for example: U.S. patent nos.4,399,216, 4,912,040, 4,740,461, and 4,959,455 (the contents of which are incorporated herein by reference). Plant cell transformation methods are known in the art and include, for example: agrobacterium-mediated transformation, biolistic transformation (biolistic transformation), direct injection, electroporation, and viral transformation. Methods for transforming cells with yeast cells are known in the art.

Mammalian cell lines useful as expression hosts are known in the art and include a variety of immortalized cell lines from the American Type Culture Collection (ATCC). Including Chinese Hamster Ovary (CHO) cells, NSO, SP2 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatoma cells (e.g., Hep G2), A549 cells, and various other cell lines. Particularly preferred cell lines are those which have been determined to be highly expressed. Other cell lines that may be used are insect cell lines such as: sf9 cells. When the gene encoding the antibody of the recombinant expression vector is introduced into a mammalian host cell, the host cell is cultured for a sufficient period of time to allow the antibody to be expressed in the host cell to produce the antibody, or more preferably, to secrete the antibody into the medium in which the host cell is grown. The antibody was recovered from the culture medium using standard protein purification methods. Plant host cells include, for example: tobacco (Nicotiana), Arabidopsis (Arabidopsis), duckweed (duckweed), corn, wheat, potato, and the like. Bacterial host cells include E.coli (E.coli) and Streptomyces (Streptomyces species). Yeast host cells include Schizosaccharomyces pombe (Schizosaccharomyces pombe), Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris).

In addition, many known techniques can be used to enhance the expression of the antibodies (or other portions derived therefrom) of the invention by the producer cell line. For example: the glutamine synthetase gene expression system (GS system) is a method commonly used to enhance expression under certain conditions. All or part of the disclosure relating to the GS system is discussed in european patent nos.0216846, 0256055, and 0323997, and european patent application No. 89303964.4.

Antibodies expressed by different cell lines or transgenic animals appear to have glycosylation responses that differ from one another. However, all antibodies encoded by or comprising the amino acid sequences provided herein, regardless of glycosylation reactions, are part of the present invention.

Transgenic animals and plants

The anti-CD 40 antibodies of the invention can also be produced by transgenic methods, i.e., by transgenically transferring the desired immunoglobulin heavy and light chain sequences into a mammal or plant and producing the antibody in recoverable form. When transgenic for production in mammals, anti-CD 40 antibodies can be produced and recovered from the milk of goats, cows, or other mammals. See, for example: U.S. patent nos.5,827,690, 5,756,687, 5,750,172, and 5,741,957. In some embodiments, a non-human transgenic animal comprising a human immunoglobulin locus is immunized with CD40 or an immunogenic portion thereof, as described above. Methods for producing antibodies in plants are described, for example: in US patent 6,046,037 and US5,959,177.

In some embodiments, non-human transgenic animals or plants are prepared by introducing one or more nucleic acid molecules encoding an anti-CD 40 antibody of the invention into an animal or plant using standard transgenic techniques. See Hogan and U.S. patent 6,417,429, supra. The transgenic cell used to make the transgenic animal can be an embryonic stem cell or a somatic cell. Transgenic non-human organisms may be chimeric, non-chimeric, heterozygous and non-chimeric homozygotes. See, for example: of Hogan et alManipulating the Mouse Embryo：A Laboratory Manual2 nd edition, Cold Spring Harbor Press (1999); jackson et alMouse Genetics and Transgenics：A Practical Approach，Oxford University Press (2000); and Pinkert' sTransgenic Animal Technology：A Laboratory Handbook,Academycpress (1999). in some embodiments, transgenic non-human animals are targeted to interruptions (deletions) and substitutions by targeting constructs encoding the desired heavy and/or light chains. In a preferred embodiment, the transgenic animal comprises and expresses nucleic acid molecules encoding heavy and light chains that specifically bind to CD40 (preferably human CD 40). In some embodiments, the transgenic animal comprises a nucleic acid molecule encoding a modified antibody (e.g., a single chain antibody, a chimeric antibody, or a humanized antibody). The anti-CD 40 antibody can be made in any transgenic animal. In a preferred embodiment, the non-human animal is a mouse, rat, sheep, pig, goat, cow or horse. The non-human transgenic animal expresses the encoded polypeptide in blood, milk, urine, saliva, tears, mucus, and other body fluids. Phage display libraries

The present invention provides a method of making an anti-CD 40 antibody or antigen-binding portion thereof, comprising the steps of synthesizing a human antibody library from phage, screening the library using CD40 or a portion thereof, isolating phage that bind to CD40, and obtaining the antibody from the phage. For example: a method of preparing an antibody library for use in phage display technology comprising the steps of immunizing a non-human animal comprising a human immunoglobulin locus with CD40 or an antigenic portion thereof, generating an immune response, extracting antibody-producing cells from the immunized animal, isolating RNA from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector, thereby expressing the antibody on a phage. In this manner, the recombinant anti-CD 40 antibody of the invention was obtained.

Recombinant anti-CD 40 human antibodies of the invention can be isolated by screening libraries of recombinant combinatorial antibodies. The library is preferably a scFv phage display library, which is generated using human VL and VHcDNAs made from mRNA isolated in B cells. Methods of making and screening such libraries are known in the art. Kits for generating Phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, product No. 27-9400-01; and Stratagene SurfZAP^TMPhage display kit, product No. 240612). Other methods and reagents may also be used to generate and screen antibody display libraries (see, e.g., U.S. Pat. No.5,223,409; PCT publication Nos. WO92/18619, WO91/17271, WO92/20791, WO92/15679, WO93/01288, WO92/01047, WO 92/09690; Bio/Technology 9: 1370-1372(1991) to Fuchs et al; Hum. Antibad. Hybridoma 3: 81-85(1992) to Huse et al; Science 246: 1275-1281 (1989); Nature 348: 552-554 to McCafferty et al; EMBO J.12: 725-734(1993) to Griffiths et al; J.Mokini.Biond et al: Achills et al: 1989; Nature 357982: 1985-357982; Nature 3579: 1985-1989; Nature 3579: 1989; Nature 3599: 1985-1985; Nature 3599: Nature et al; Nature 3599: 4152: 1989; Nature 3519: Woo et al; Nature 3579: 35: Gray et al; Nature 3582: 4152: USA: 417980; Nature et al; Nature 3579: 19919: 1989; Nature et al; Nature 3579: 10: Woo: 1989; Nature et al; Nature 3579: Woogra: 3579: 35; Nature et al; Nature).

In one embodiment, to isolate human anti-CD 40 antibodies with desired properties, human anti-CD 40 antibodies described herein are used to select human heavy and light chain sequences having similar binding activity to CD40 using the epitope imprinting method described in PCT publication No. WO93/06213. The antibody library used in this method is preferably according to PCT publication No. WO92/01047, Nature348 of McCafferty et al: 552 (1990); and Griffiths et al EMBO J.12: 725-734 (1993). The scFv antibody library is preferably screened using human CD40 as an antigen.

Once the preliminary human V is selected_LAnd V_HAfter the domains, a "mix and pair" experiment was performed in which the preliminarily selected V was screened for CD40 binding_LAnd V_HDifferent pairing of segments to select preferred V_L/V_HAnd (4) combining. In addition, in order to further improve the quality of the antibody, V is preferable_L/V_HPaired V_LAnd V_HThe segments may be randomly mutated, preferably at V_LAnd/or V_HSimilar to the process of in vivo somatic mutation responsible for affinity maturation of antibodies during innate immune responses. The in vitro affinity maturation can be performed using a peptide which is separately related to V_HCDR3 or V_LPCR primer amplification V with complementary CDR3_HAnd V_L(iii) a domain, some positions of the primer having been "doped" (spiked) with a random mixture of 4 nucleotide bases, such that the resulting PCR product encodes a V_HAnd V_LRandom mutations have been introduced into the segment V_HAnd/or the VLCDR3 region. Such randomly mutated VH and VL segments can be screened again for binding to CD 40.

After screening and isolation of the anti-CD 40 antibodies of the invention from recombinant immunoglobulin display libraries, nucleic acids encoding the selected antibodies can be recovered from the display population (e.g., from a phage genome) and subcloned into other expression vectors using standard recombinant DNA techniques. If desired, the nucleic acid may be further processed to form other antibody forms of the invention as will be described below. When recombinant human antibodies isolated by screening combinatorial libraries are to be expressed, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into mammalian host cells as described above.

Transformation by classification

In another aspect, the invention provides a method of converting a type or subtype of anti-CD 40 antibody to another type or subtype. In some embodiments, code V_LOr V_HBut does not include any coding C_LOr C_HThe nucleic acid molecule of (a) is isolated using methods known in the art. The nucleic acid molecule may then be conjugated to a C-encoding molecule from a desired immunoglobulin class or subtype_LOr C_HThe nucleic acid sequence of (a) is operably linked. This process may be used as described above including C_LOr C_HA vector or nucleic acid molecule for the strand. For example: anti-CD 40 antibodies that were originally IgM may be converted to IgG. In addition, class transformation methods can be used to transform one IgG subtype into another, for example: IgG2 was formed from IgG 1. Another method for producing an antibody of the invention comprising a desired isotype comprises the steps of isolating nucleic acid encoding the heavy chain of an anti-CD 40 antibody and nucleic acid encoding the light chain of an anti-CD 40 antibody, isolating nucleic acid encoding V_HSequence of regions, joining V_HSequences and sequences encoding heavy chain constant domains of the desired isotype, expressing light chain genes and heavy chain constructs in cells, and collecting anti-CD 40 antibodies containing the desired isotype.

Deimmunized (deimmunized) antibodies

Another method of making antibodies with reduced immunogenicity is to remove the immunity of the antibody. In another aspect of the invention, the description may be used, for example, in: PCT publication Nos. WO98/52976 and WO00/34317 (the contents of which are fully incorporated herein by reference) remove the immunity of the antibody.

Mutant antibodies

In another embodiment, the nucleic acid molecules, vectors, and host cells can be used to make mutant anti-CD 40 antibodies. The antibody may be mutated in the variable domain of the heavy and/or light chain, for example: altering the binding of the antibody. For example: mutations may be made in one or more CDR regions to increase or decrease the K of the antibody to CD40_DIncreasing or decreasing K_offOr altering the binding specificity of the antibody. Fixed point mutagenesis methodAre known in the art. See, for example: the above references by Sambrook et al and Ausubel et al. In a preferred embodiment, the mutation is made at an amino acid residue to be changed in the variable domain of the anti-CD 40 antibody relative to the germline. In another embodiment, one or more mutations are made in the CDR or framework regions of the variable domains of monoclonal antibodies 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 22.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.28.1L-C92A, 23.29.1L-R174K and 24.2.1, or in the constant domains, relative to the amino acid residues to be altered. In another embodiment, the polypeptide is produced in a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 4. 12,20, 28, 36, 44, 52, 60, 68, 76, 84, 94, 100, 102, 2, 10, 18, 26, 34, 42, 50,58, 66, 74, 82, 90, 92, 96, 98, 100, or 102, or a nucleic acid sequence thereof of SEQ ID NOS: 3. 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 93, 99, 101, 1,9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 91, 95, 97, 99 or 101, one or more mutations are made at the amino acid residues to be changed relative to the germline in the CDR regions or framework regions of the variable domains.

In one embodiment, the framework regions are mutated such that the framework regions formed have an amino acid sequence corresponding to a germline gene. Mutations may be made in the framework or constant domains to extend the half-life of the anti-CD 40 antibody. See, for example: PCT publication No. WO00/09560, the contents of which are incorporated herein by reference. Mutations may also be made in the framework or constant domains to alter the immunogenicity of the antibody, to provide sites for covalent or non-covalent binding to another molecule, or to alter, for example: complement fixation, FcR binding and ADCC. According to the present invention, a single antibody may be mutated at any one or more of the framework, constant and variable regions.

In some embodiments, the V of the mutated anti-CD 40 antibody is relative to the pre-mutated anti-CD 40 antibody_HOr V_LStructural domainsThere are 1 to 18 (including any number between these two) amino acid mutations. Any of the mutations described above may be present in one or more CDR regions. Furthermore, any mutation may be a conservative amino acid substitution. In some embodiments, the amino acid in the constant domain does not vary by more than 5, 4,3, 2, or 1.

Modified antibodies

In another embodiment, the fusion antibody or immunoadhesin produced may comprise the full length or a portion of an anti-CD 40 antibody of the invention linked to another polypeptide. In a preferred embodiment, only the variable domain of the anti-CD 40 antibody is linked to the polypeptide. In another preferred embodiment, V of the anti-CD 40 antibody_HThe V domain of the anti-CD 40 antibody linked to the first polypeptide_LThe structural domain is connected with a second polypeptide, wherein the second polypeptide and the first polypeptide are combined in a mode that V_HAnd V_LThe domains are allowed to interact to form antibody binding sites. In another preferred embodiment, V_HDomains with V_LThe domains are separated by a linker to allow the interaction of the VH and VL domains (see description of "single chain antibodies" below). Subsequently let V_H-linker-V_LThe antibody is linked to a polypeptide of interest. The fusion antibody is useful for targeting a polypeptide to a CD 40-expressing cell or tissue. The polypeptide may be a therapeutic agent, such as: toxins, growth factors or other regulatory proteins, or may be diagnostic agents such as: enzymes that are easily visually inspected, such as: horseradish peroxidase. Furthermore, a fusion antibody in which two (or more) single-chain antibodies are linked to each other may be formed. It is suitable for the manufacture of bivalent or multivalent antibodies on a single polypeptide chain or for the manufacture of bispecific antibodies.

When producing a single-chain antibody, (scFv) V_H-and V_LThe DNA fragment and the other fragment encoding the elastic linker (for example: the amino acid sequence (Gly)₄-Ser)₃Fragment of (d) an operative linkage, thus V_HAnd V_LThe sequence may be expressed as a continuous single-chain protein, wherein V_LAnd V_HThe structural domain isConnected by the elastic connecting body. See, for example: bird et al Science 242: 423-426 (1988); proc.natl.acad.sci.usa85, Huston et al: 5879-5883 (1988); nature348 by McCafferty et al: 552-554(1990). If a single V is used_HAnd V_LWhen a single chain antibody is used, it may be monovalent, if two V's are used_HAnd V_LWhen a single-chain antibody is bivalent, if more than two V are used_HAnd V_LIn the case of single-chain antibodies, the antibodies are multivalent. Bispecific or multivalent antibodies can be generated that specifically bind CD40 to another molecule.

In other embodiments, other modified antibodies may be made using nucleic acid molecules encoding anti-CD 40 antibodies. For example: "kappa-bodies (kappa bones)" (Protein Eng.10: 949-57(1997) of I11 et al, "Minibodies (Minibodies)" (EMBO J.13: 5303-9(1994) of Martin et al, "double antibodies (diabodies)" (Proc. Natl.Acad.Sci.USA 90: 6444-.

Bispecific antibodies or antigen-binding fragments can be made in a variety of ways, including hybridoma fusion or Fab' fragment ligation. See, for example: songsivilai and Lachmann, clin. exp. immunol.79: 315-: 1547-1553(1992). In addition, bispecific antibodies can form "diabodies" or "Janusins". In some embodiments, the bispecific antibody binds two different epitopes of CD 40. In some embodiments, the bispecific antibody has antibodies derived from monoclonal antibodies 3.1.1, 3.1.1H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.25.1, 23.28.1H-D16E, 23.28.1L-C92A, 23.29.1L-R174K and 24.2.1 first heavy and first light chain, and other antibody heavy and light chains. In some embodiments, the additional light and heavy chains are derived from the monoclonal antibodies described above, but are different from the first heavy and light chains.

In some embodiments, the above-described modified antibodies are prepared using one or more variable domains or CDR regions from a human anti-CD 40 monoclonal antibody provided herein, the amino acid sequence of the monoclonal antibody, or the heavy or light chain encoded by the nucleic acid sequence encoding the monoclonal antibody.

Derivatized and labeled antibodies

The anti-CD 40 antibodies of the invention or antigen-binding portions thereof can be derivatized or linked to another molecule (e.g., another peptide or protein). Typically, the antibody or a portion thereof is derivatized such that CD40 binding is not negatively affected by the derivatization reaction or labeling. Thus, the antibodies and antibody portions of the invention should include both intact and modified forms of the human anti-CD 40 antibodies described herein. For example: antibodies and antibody portions of the invention can be functionally linked (using chemical coupling, gene fusion, non-covalent linking, or other methods) to one or more other molecular entities, such as: another antibody (e.g., a bispecific or dual antibody), a detection agent, a cytotoxic agent, an agent, and/or a protein or peptide that can mediate the binding of an antibody or antibody portion to another molecule (e.g., a streptavidin core region or a polyhistidine tag).

One derivatized antibody is produced by cross-linking two or more antibodies (of the same or different species, e.g., to form a bispecific antibody). Suitable cross-linkers include those heterobifunctional cross-linkers having two independent reactive groups separated by a suitable spacer (e.g.m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional cross-linkers (e.g.disuccinimidyl suberate). Such linkers are available from Pierce chemical company (Rockford, I11.).

Another derivatized antibody is a labeled antibody. Detection agents suitable for derivatizing the antibodies or antigen-binding portions of the invention include fluorescent compounds including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. The antibody may be labelled with an enzyme suitable for detection, such as: horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When a detectable enzyme-labeled antibody is used, detection may be carried out with the addition of other enzymes that may be used to produce reaction products for recognition. For example: when horseradish peroxidase preparations are included, the addition of hydrogen peroxide and diaminobenzidine produces a detectable colored reaction product. Detection can also be accomplished by using a biotin-labeled antibody and indirectly measuring the binding of avidin or streptavidin. The antibody may also be labeled with a predetermined polypeptide epitope and the polypeptide epitope may be recognized using a second reporter (e.g., leucine zipper pair sequence, binding site of a second antibody, metal binding domain, epitope tag). In some embodiments, the labels are attached using spacer arms of various lengths to reduce potential steric hindrance.

The anti-CD 40 antibody may also be labeled with a radiolabeled amino acid. The radioactive markers can be used for diagnostic and medical purposes. For example: the radioactive marker may be used to detect tumors expressing CD40 using X-ray or other diagnostic techniques. In addition, the radioactive marker may be used in medicine as a toxin to cancer cells or tumors. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionuclides:³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I。

anti-CD 40 antibodies can also be derivatized with chemical groups such as: polyethylene glycol (PEG), methyl or ethyl, or carbohydrate groups. Such groups are useful for improving the biological properties of antibodies, for example: extending serum half-life or improving tissue binding.

Pharmaceutical composition and kit

The invention also relates to a composition comprising a human anti-CD 40 agonist antibody for use in treating an individual in need of immune stimulation. Such compositions are useful for treating, preventing, reducing the frequency or severity of infections, including viral and bacterial infections, treating hyperproliferative disorders, including cancer and precancerous conditions, treating genetic immunodeficiency disorders, such as: hyper-IgM syndrome, and the treatment of primary or complex immunodeficiency disorders, including the appearance of neutropenia. Individuals treatable using agonist anti-CD 40 antibody therapy include any individual in need of boosting immunity, including, but not limited to, elderly people and individuals with suppressed immunity (e.g., due to chemotherapy).

Hyperproliferative lesions that can be treated using agonist anti-CD 40 antibodies of the invention can involve any tissue or organ, including, but not limited to, cancers of the brain, lung, squamous cell, bladder, stomach, pancreas, breast, head, neck, liver, kidney, ovary, prostate, colorectal, esophagus, gynecology, throat, or thyroid, melanoma, lymphoma, leukemia, or multiple myeloma. In particular, the human agonist anti-CD 40 antibodies of the invention are useful for the treatment of breast, prostate, colon and lung carcinomas.

The treatment involves administering one or more agonist anti-CD 40 monoclonal antibodies or antigen-binding fragments thereof of the present invention, either alone or in combination with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be desirable to include isotonic agents, for example: saccharides, polyols (e.g., mannitol, sorbitol) or sodium chloride. Other examples of pharmaceutically acceptable substances are humectants or minor amounts of auxiliary substances such as: wetting or emulsifying agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the antibody.

Agonist anti-CD 40 antibodies of the invention and compositions comprising them may be administered in combination with one or more other therapeutic, diagnostic or prophylactic agents. Other therapeutic agents include other antineoplastic, antiangiogenic or chemotherapeutic agents. The other agents may be included in the same composition or may be administered separately. In some embodiments, one or more agonist anti-CD 40 antibodies of the invention can be used as or as an adjuvant to a vaccine.

The compositions of the invention may take a variety of different forms, for example: liquid, semi-solid, and solid dosage forms, such as: liquid solutions (e.g., injections and infusions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the mode of administration and the medical use. Typical preferred compositions are in the form of injections or infusions, such as: compositions similar to those used in passive immunization of humans. Preferably, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Medical compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentrations. The preparation method of the sterile injection comprises the following steps: the desired amount of anti-CD 40 antibody, optionally together with one or more of the foregoing ingredients, is added to an appropriate solvent and then filter sterilized. Generally, the dispersion is prepared by: the active compound is added to the sterile medium which contains the basic dispersion medium and the required other ingredients as described above. The preferred methods for preparing sterile powders for injection are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional desired ingredient from a sterile-filtered solution of the foregoing. Proper fluidity of the solution can be maintained, for example, by: a coating such as lecithin, if a dispersion is used, to maintain the desired particle size, and a surfactant. Absorption delaying agents (e.g., monostearate salts and gelatin) can be included in the compositions to delay absorption of the injectable compositions.

The antibodies of the invention can be administered in a variety of ways known in the art, but for many medical uses, the preferred route/model of administration is still subcutaneous, intramuscular, or intravenous infusion. One skilled in the art will appreciate that the route/model of administration will depend on the desired result.

In certain embodiments, the antibody composition active compound may be prepared using a carrier that prevents rapid release of the antibody, such as: controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as: vinyl acetate, polyanhydrides, polydialkyds, collagen, polyorthoesters, and polylactic acid. Various methods of making such formulations are patented or are well known to those skilled in the art. See, for example:Sustained and Controlled Release Drug Delivery Systems(edited by j.r. robinson, Marcel Dekker, inc., New York, 1978).

In certain embodiments, the anti-CD 40 antibodies of the invention can be administered orally, for example: administration using an inert diluent or an assimilable edible carrier. The compound (and other ingredients if desired) may also be embedded in a hard or soft gelatin capsule, compressed into a tablet, or added directly to the patient's diet. For oral administration, the anti-CD 40 antibody can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Where the compounds of the invention are administered by a route other than parenteral, it may be necessary to coat or co-administer the compounds with a substance that prevents their loss of activity.

Other active compounds may also be added to the composition. In certain embodiments, the anti-CD 40 antibodies of the invention are co-formulated and/or co-administered with one or more other therapeutic agents. Such agents include (but are not limited to): antibodies that bind to other targets (e.g., antibodies that bind to one or more growth factors or cytokines or their cell surface receptors, such as anti-CTL 4-antibodies), anti-tumor agents, chemotherapeutic agents, peptide analogs that activate CD40, soluble CD40L, one or more chemical agents that activate CD40, and/or agents known in the art to potentiate an immune response against tumor cells (e.g., IFN-. beta.1, IL-2, IL-8, IL-12, IL-15, IL-18, IL-23, IFN-. gamma., and GM-CSF). Such combination therapies may require lower doses of anti-CD 40 antibody and co-administered agents, and thus may avoid the toxicity or complications associated with each monotherapy.

Agonist anti-CD 40 antibodies of the invention and compositions comprising them may also be administered in combination with other therapeutic regimens, particularly in combination with radiation therapy.

The compositions of the invention may comprise a "therapeutically effective amount" or a prophylactically effective amount "of an antibody of the invention, or an antigen-binding portion thereof. A "therapeutically effective amount" is an amount that will achieve the desired therapeutic result at the dosages and for the period of time necessary. The therapeutically effective amount of an antibody or antibody portion may vary depending on a number of factors such as: the disease state, the age, sex, and weight of the subject, and the ability of the antibody or antibody portion to elicit a desired response in the subject. A therapeutically effective amount also refers to an amount wherein the beneficial therapeutic effect of the antibody or antibody portion outweighs any toxic or detrimental effect. A "prophylactically effective amount" is an amount that achieves the desired prophylactic result at the dosage employed and for the period of time necessary. Typically, since the subject is administered a prophylactic dose prior to onset or early in the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). For example: a single bolus may be administered, several divided small doses administered over a period of time, or the dose may be proportionally reduced or increased as the medical situation becomes more urgent. It is particularly advantageous to formulate compositions for parenteral administration in unit dosage form for convenient administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages for the individual mammals to be treated; each unit comprising a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specific unit dosage forms of the invention are directly defined as follows: (a) the unique properties of anti-CD 40 antibodies or portions thereof and the particular therapeutic or prophylactic effect to be achieved, and (b) the skill in the art of making such antibodies is itself limited by the sensitivity of the individual to treatment.

Examples of therapeutically or prophylactically effective amounts of an antibody or antibody portion of the invention are from 0.025 to 50mg/kg, more preferably from 0.1-25, 0.1 to 10 or 0.1 to 3 mg/kg. It is noted that this dosage may be varied depending on the type and severity of the condition to be alleviated. It will also be understood that the particular dosage regimen for any particular patient will be adjusted over time according to the individual needs and the professional judgment of the administrator or supervising the administration of the compositions, and that the dosage ranges indicated herein are exemplary only and are not intended to limit the scope or practice of the compositions claimed.

In another aspect, the invention provides a kit comprising an anti-CD 40 antibody or antibody portion of the invention, or a composition comprising such an antibody. In addition to the antibody or composition, the kit may further comprise a diagnostic or therapeutic agent. The kit may also contain instructions for its diagnostic or therapeutic method. In a preferred embodiment, the kit comprises an antibody or a composition comprising the antibody, and a diagnostic agent useful in the methods described below. In another preferred embodiment, the kit comprises an antibody or a composition comprising the antibody, one or more therapeutic agents useful in the methods described below.

The invention also relates to a composition for inhibiting abnormal cell growth in a mammal comprising an amount of an antibody of the invention in combination with an amount of a chemotherapeutic agent, wherein the amount of the compound, salt, solvate, or prodrug, together with the amount of the chemotherapeutic agent, is effective to inhibit abnormal cell growth. A wide variety of chemotherapeutic agents are known in the art. In some embodiments, the chemotherapeutic agent is selected from the group consisting of: mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormonal agents, such as: antiandrogens and antiangiogenic agents.

Anti-angiogenic agents, such as: MMP-2 (matrix metalloproteinase 2) inhibitors, MMP-9 (matrix metalloproteinase 9) inhibitors, and COX-II (cyclooxygenase II) inhibitors may be used in combination with the anti-CD 40 antibodies of the invention. Examples of suitable COX-II inhibitors include CELEBREXTM (celecoxib), valdecoxib, and rofecoxib. Suitable matrix metalloproteinase inhibitors are described in WO96/33172 (published 24/10/1996), WO96/27583 (published 7/3/1996), European patent application No.97304971.1 (published 8/7/1997), European patent application No.99308617.2 (published 29/10/1999), WO98/07697 (published 26/2/1998), WO98/03516 (published 29/1998), WO98/34918 (published 13/8/1998), WO98/34915 (published 13/8/1998), WO 98/3333915 (published 6/8/1998), WO98/30566 (published 16/7/1998), European patent publication 606,046 (published 13/1994), European patent publication 931,788 (published 28/1999), WO 90/0531/1990 (published 5/99/52910 (published 5221/68610/1999), WO 8821/8810/1999 (published 1999/8821/1999), WO99/29667 (published 17.6.1999), PCT International application No. PCT/IB98/01113 (published 21.7.1998), European patent application No.99302232.1 (published 25.3.1999), British patent application No.9912961.1 (published 3.6.1999), U.S. provisional application No.60/148,464 (published 12.8.1999), U.S. patent 5,863,949 (published 26.26.1.1999), U.S. patent 5,861,510 (published 19.1.19.1999), and European patent application 780,386 (published 25.6.1997), the contents of which are incorporated herein by reference in their entirety. Preferred MMP inhibitors are those which have been shown not to cause arthralgia. More preferably those that selectively inhibit MMP-2 and/or MMP-9 relative to other matrix metalloproteinases (i.e., MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Some specific examples of MMP inhibitors suitable for use in the present invention are AG-3340, RO32-3555, RS13-0830 and the following compounds: 3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (1-hydroxycarbamoyl-cyclopentyl) -amino ] -propionic acid; 3-pendant-3- [4- (4-fluoro-phenoxy) -benzenesulfonylamino ] -8-oxa-bicyclo [3.2.1] octane-3-carboxylic acid hydroxyamide; (2R, 3R)1- [4- (2-chloro-4-fluoro-benzyloxy) -benzenesulfonyl ] -3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide; 4- [4- (4-fluoro-phenoxy) -benzenesulfonylamino ] -tetrahydro-pyran-4-carboxylic acid hydroxyamide; 3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (1-hydroxycarbamoyl-cyclobutyl) -amino ] -propionic acid; 4- [4- (4-chloro-phenoxy) -benzenesulfonylamino ] -tetrahydro-pyran-4-carboxylic acid hydroxyamide; (R)3- [4- (4-chloro-phenoxy) -benzenesulfonylamino ] -tetrahydro-pyran-3-carboxylic acid hydroxyamide; (2R, 3R)1- [4- (4-fluoro-2-methyl-benzyloxy) -benzenesulfonyl ] -3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide; 3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (1-hydroxycarbamoyl-1-methyl-ethyl) -amino ] -propionic acid; 3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (4-hydroxycarbamoyl-tetrahydro-pyran-4-yl) -amino ] -propionic acid; 3-pendant-3- [4- (4-chloro-phenoxy) -benzenesulfonylamino ] -8-oxa-bicyclo [3.2.1] octane-3-carboxylic acid hydroxyamide; 3-bridged-3- [4- (4-fluoro-phenoxy) -benzenesulfonylamino ] -8-oxa-bicyclo [3.2.1] octane-3-carboxylic acid hydroxyamide; and (R)3- [4- (4-fluoro-phenoxy) -benzenesulfonylamino ] -tetrahydro-furan-3-carboxylic acid hydroxyamide; and pharmaceutically acceptable salts and solvates of the compounds.

The compounds of the invention may also be used in combination with signal transduction inhibitors, such as: agents which inhibit the EGF-R (epidermal growth factor receptor) response, such as: EGF-R antibodies, EGF antibodies, and molecules that are inhibitors of EGF-R; VEGF (vascular endothelial growth factor) inhibitors, such as: VEGF receptors and molecules that inhibit VEGF; and erbB2 receptor inhibitors, such as: organic molecules or antibodies that bind to the erbB2 receptor, such as: HERCEPTINTM (Genentech, Inc.). EGF-R inhibitors are illustrated, for example: WO95/19970 (published on 7/27 th of 1995), WO

98/14451 (announced at 9.4.1998), WO98/02434 (announced at 22.1.1998), and U.S. Pat. No.5,747,498 (announced at 5.5.1998), and such substances may be used in the present invention as described herein. EGFR-inhibitors include (but are not limited to): monoclonal antibody C225 was combined with anti-EGFR 22Mab (Imclone Systems Incorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200(Merck KgaA), EMD-5590(Merck KgaA), MDX-447/H-477 (Merarex Inc. and Merck KgaA), and the compounds ZD-1834, ZD-1838 and ZD-1839(AstraZeneca), PKI-166(Novartis), PKI-166/CGP-75166(Novartis), PTK787(Novartis), CP701(Cephalon), leflunomide (Pharmax/Sugen), CI-1033 (Warnelambert park Davis), CI-1033/PD183, 805(Warner Lankeevirs CL-Parker, Pharmax-785 (Whrhart-1613), Grner-book-3 (Grne-Gernik-R-3/R), Rorne-33, Gernik-3/W-3, Gernik-Gernik, Gernik-Gerni-R-3, Gernik-Gernik, Gernik-3, Gernik-3, Gernik-3, Gernik-Gernik, Gerni, VRCTC-310(Ventech Research), EGF fusion toxin (Seragen Inc.), DAB-389(Seragen/Lilgand), ZM-252808(Imperial Cancer Fund), RG-50864(INSERM), LFM-A12(Parker Hughes Cancer Center), WHI-P97(Parker Hughes Cancer Center), GW-282974(Glaxo), KT-8391(Kyowa Hakko), and EGF-R vaccine (York Medical/Central de Immunoglogica Molecular (CIM)). These and other EGF-R-inhibitors are useful in the present invention.

VEGF inhibitors, for example: SU-5416 and SU-6668(Sugen Inc.), SH-268(Schering), and NX-1838(NeXStar) may also be used in combination with the compounds of the present invention. VEGF inhibitors have been described, for example, in: WO99/24440 (published 20/5/1999), PCT International application PCT/IB99/00797 (published 3/5/1999), WO95/21613 (published 17/8/1995), WO99/61422 (published 2/12/1999), US patent 5,834,504 (published 10/11/1998), WO98/50356 (published 12/1998), US patent 5,883,113 (published 16/3/1999), US patent 5,886,020 (published 23/1999), US patent 5,792,783 (published 11/1998), WO99/10349 (published 4/1999), WO 97/59648 (published 12/1997), WO97/22596 (published 26/1997), WO 98/54093/1998 (published 12/1998), WO 98/328 (published 631/3222/1999), WO 99/16796 (published 26/1999) and WO 2/856/1998 (published 22/1999), the contents of which have been fully incorporated herein by reference. Some examples of other specific VEGF inhibitors suitable for use in the present invention are IM862 (cytarninc.); anti-VEGF monoclonal antibodies (Genentech Inc.); and angiozyme, a synthetic Ribozyme from Ribozyme and Chiron. These and other VEGF inhibitors may be used in the present invention as described herein. The compounds of the invention may also be combined with ErbB2 receptor inhibitors, such as: GW-282974(Glaxo Wellcome plc), and monoclonal antibodies AR-209(Aronex Pharmaceuticals Inc.) and 2B-1(Chiron) were used in combination, for example: those described in WO98/02434 (published 1998 at 22.1.1999), WO99/35146 (published 1999 at 15.6.15), WO99/35132 (published 1999 at 15.6.1999), WO98/02437 (published 1998 at 22.1.22.1998), WO97/13760 (published 1997 at 17.4.1997), WO95/19970 (published 1995 at 27.6.6), U.S. Pat. No.5,587,458 (published 1996 at 24.12.2), and U.S. Pat. No.5,877,305 (published 1999 at 2.3.1999), the contents of which are fully incorporated herein by reference And those described in the U.S. provisional application, as well as other compounds and substances that inhibit the erbB2 receptor, may be used in combination with the compounds of the present invention according to the present invention.

Anti-residual agents (anti-survival agents) include anti-IGF-IR antibodies and anti-integrin preparations such as: anti-integrin antibodies.

Diagnostic method and use

In another aspect of the invention, a diagnostic method is provided. The anti-CD 40 antibodies can be used to detect CD40 in a biological sample in vitro or in vivo. In one embodiment, the present invention provides a method of diagnosing the presence or location of a tumor expressing CD40 in an individual in need thereof comprising the steps of injecting an antibody into the individual, determining the binding location of the antibody to determine the expression of CD40, and comparing the expression of the CD40 to a normal reference individual or standard to diagnose the presence or location of the tumor.

anti-CD 40 antibodies can be used in conventional immunoassays, including (but not limited to): ELISA, RIA, FACS, tissue immunohistochemistry, Western blotting, or immunoprecipitation. The anti-CD 40 antibodies of the invention can be used to detect CD4 from humans. In another embodiment of the invention, the anti-CD 40 antibody can be used to detect primates from the old world such as: CD40 of rhesus macaque, chimpanzee and ape. The present invention provides a method for detecting CD40 in a biological sample, comprising the steps of contacting the biological sample with an anti-CD 40 antibody of the invention, and detecting the bound antibody. In one embodiment, the detectable label is directly labeled on the anti-CD 40 antibody. In another embodiment, the anti-CD 40 antibody (primary antibody) is unlabeled and the secondary antibody or other molecule that binds to the anti-CD 40 antibody is labeled. It is known to those skilled in the art that the secondary antibody selected should specifically bind to the particular species and type of primary antibody. For example: if the anti-CD 40 antibody is human IgG, the second antibody can be anti-human-IgG. Other molecules that can bind to antibodies include (but are not limited to): protein a and protein G, both of which are commercially available, for example: from Pierce Chemical co.

Suitable labels for the antibody or second antibody have been disclosed in the above documents and include a variety of enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluoroisothiocyanate, rhodamine, dichlorotriazolylaminofluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; and examples of suitable radioactive materials include¹²⁵I、¹³¹I、³⁵S or³H。

In other embodiments, the analysis of CD40 in a biological sample can be determined by a competitive immunoassay using a CD40 standard labeled with a detectable substance and an unlabeled anti-CD 40 antibody. In this assay, a biological sample, a labeled CD40 standard, and an anti-CD 40 antibody are combined, and the amount of binding of the labeled CD40 standard to the unlabeled antibody is determined. The amount of CD40 in the biological sample will be inversely proportional to the amount of labeled CD40 standard bound to the anti-CD 40 antibody.

The immunoassays disclosed above can be used for a variety of purposes. For example: anti-CD 40 antibodies can be used to detect CD40 in cells in cell culture. In a preferred embodiment, anti-CD 40 antibodies are used to determine the amount of CD40 on the surface of cells treated with a variety of different compounds. This method can be used to determine whether a compound is suitable for activating or inhibiting CD 40. According to the invention, one cell sample can be treated with a test compound for a period of time while the other sample remains untreated. To determine the total amount of CD40, the cells were lysed and the total amount of CD40 was determined using one of the immunoassays described above. The total amount of CD40 in the treated group was compared to the untreated group to determine the potency of the test compound.

The preferred immunoassay for determining the total amount of CD40 is ELISA or western blot analysis. To determine the amount of CD40 on the cell surface, the cells were not lysed and the amount of CD40 on the cell surface was determined using one of the immunoassays described above. A preferred immunoassay for measuring the level of cell surface CD40 comprises the steps of labeling a cell surface protein with a detectable label, such as: biotin or¹²⁵I, after immunoprecipitation of CD40 with anti-CD 40 antibody, labeled CD40 was detected. Another preferred immunoassay for determining the location (e.g., cell surface content) of CD40 is by immunohistochemistry. Such as: ELISA, RIA, Western blotting, immunohistochemistry, methods for labeling cell surfaces with integral membrane proteins, and immunoprecipitation are known in the art. See, for example: the above references to Harlow and Lane. In addition, immunoassays can be scaled up for high throughput screening to test whether a large number of compounds can activate or inhibit CD 40.

The anti-CD 40 antibodies of the invention can also be used to determine the level of CD40 in a tissue or cells derived from a tissue. In some embodiments, the tissue is diseased tissue. In some embodiments, the tissue is a tumor or a biopsy sample thereof. In some embodiments of the method, the tissue or biopsy sample thereof is excised from the patient. The tissue or biopsy samples thereof are used in immunoassays using the methods described above for determining, for example: total CD40, cell surface CD40 content, or CD40 location.

The above diagnostic methods can be used to determine whether a tumor expresses significant amounts of CD40, which can be used as an indicator of whether the tumor is the target for treatment with anti-CD 40 antibodies. In addition, the same method can also detect cell death in tumors to follow the therapeutic efficacy of anti-CD 40 antibodies. The diagnostic method may also be used to determine whether a tissue or cell expresses insufficient amounts of CD40 or activated CD40, and thus may determine whether it is a candidate target for treatment with an acceptable activated anti-CD 40 antibody, CD40L, and/or other therapeutic agents for increasing CD40 content or activity.

The antibodies of the invention may also be used in vivo to discriminate between tissues and organs expressing CD 40. In some embodiments, anti-CD 40 antibodies are used to discriminate between tumors expressing CD 40. One advantage of using the human anti-CD 40 antibodies of the invention is that they can be used safely in vivo, without eliciting an immune response to the antibody upon administration, unlike antibodies of non-human origin or humanized antibodies.

The method comprises the steps of administering an anti-CD 40 antibody labeled with a detectable agent or a composition comprising the antibody to a patient in need of such a diagnostic test and subjecting the patient to an imaging assay to determine the location of tissue expressing CD 40. Visualization methods are known in the medical arts and include, but are not limited to, X-ray analysis, Magnetic Resonance Imaging (MRI), or computed tomography (CE). Any reagent suitable for in vivo imaging can be labeled on the antibody, for example: contrast agents (contrast agents), such as: barium, which can be used in X-ray analysis, or magnetic contrast agents, such as: gadolinium chelates, which can be used for MRI or CE. Other labeling agents include, but are not limited to, radioisotopes such as:⁹⁹tc. In another embodiment, the anti-CD 40 antibody is unlabeled and is visualized by administering a secondary antibody or other molecule that is detectable and can bind to the anti-CD 40 antibody. In one embodiment, the biopsyTissue samples were obtained from patients to determine whether the target tissue expressed CD 40.

Medical use

In another aspect, the invention provides medical treatments using the anti-CD 40 antibodies of the invention.

The present inventors' agonist anti-CD 40 antibodies can be administered to human or non-human mammals expressing cross-reactive CD 40. Administration of antibodies to such non-human mammals (i.e., primates, rhesus monkeys, or macaques) is for veterinary use or as an animal model of human disease. Such animal models are useful for assessing the therapeutic efficacy of the antibodies of the invention.

In some embodiments, the anti-CD 40 antibody is administered to a patient having a primary and/or combined immunodeficiency, including CD40 dependent immunodeficiency with excessive IgM syndrome, general multiple immunodeficiency, Bruton's agammaglobulinemia, IgG subtype deficiency, and X-linked SCID (general gamma-chain mutation). In some embodiments, the anti-CD 40 antibody administered is used to treat an immunosuppressed patient, such as: those resulting from chemotherapy, or suffering from immune deficiency, including any acquired immune deficiency, such as: HIV. In some embodiments, anti-CD 40 antibodies are administered to boost immunity in the elderly. In some embodiments, the anti-CD 40 antibody is administered to treat a patient having a bacterial, viral, fungal, or parasitic infection. In some embodiments, the agonist anti-CD 40 antibodies of the present invention may be administered prophylactically to individuals who are susceptible to infection due to age, disease, or general physical condition, to prevent or reduce the number or severity of infections.

In some embodiments, an anti-CD 40 antibody is administered to a patient having a hyperproliferative lesion.

In some embodiments, an anti-CD 40 antibody is administered to treat an individual having a tumor. In some embodiments, the tumor is positive for CD 40. In some embodiments, the tumor is CD40 negative. The tumor may be a solid tumor, or a non-solid tumor, such as: lymphoma. In some embodiments, the anti-CD 40 antibody is administered to a patient having a cancerous tumor. In some embodiments, the antibody can inhibit cancer cell proliferation, inhibit or prevent an increase in tumor weight or volume, and/or reduce tumor weight or volume.

Patients who may be treated with an anti-CD 40 antibody or antibody portion of the invention include, but are not limited to, patients diagnosed with: brain cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colorectal cancer, colon cancer, breast cancer, gynecological tumors (e.g., uterine sarcoma, facial neural tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, or vulvar carcinoma), esophageal cancer, small intestine cancer, cancer of the endocrine system (e.g., thyroid cancer, parathyroid cancer, or adrenal cancer), soft tissue sarcoma, leukemia, myeloma, multiple myeloma, urinary tract cancer, penile cancer, prostate cancer, chronic or acute leukemia, solid tumors of young children, Hodgkin's disease, lymphocytic lymphomas, non-Hodgkin's lymphoma, bladder cancer, liver cancer, kidney or ureter cancer (e.g., renal cell carcinoma, renal pelvis carcinoma), or central nervous system tumors (e.g., primary CNS lymphoma, Spinal tumors, brain stem glioma or pituitary adenomas), glioma or fibrosarcoma.

The antibody may be administered 3 times a day to 1 time every 6 months, and is preferably administered by oral, mucosal, buccal, intranasal, inhalation, intravenous, subcutaneous, intramuscular, parenteral, intratumoral, transdermal or topical routes. The antibody may also be administered continuously using a micro-pump. In the case where the antibody causes the tumor or cancer to stop growing or to reduce weight or volume, the administration time of the antibody is generally maintained until disappearance. The antibody dose range is typically 0.025 to 50mg/kg, more preferably 0.1-20mg/kg, 0.1-10mg/kg, 0.1-5mg/kg or even more preferably 0.1-2 mg/kg. Antibodies can also be administered prophylactically.

In some embodiments, the anti-CD 40 antibody is administered to a patient having a hyperproliferative lesion as part of a treatment regimen that includes another anti-cell proliferative drug or molecule, such as: a cancer or a tumor. Examples of antineoplastic agents include (but are not limited to): mitotic inhibitors, alkylating agents, antimetabolites, intercalating agents, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormonal agents, kinase inhibitors, matrix metalloproteinase inhibitors, genetic therapeutic agents, and anti-androgens. In a more preferred embodiment, the anti-CD 40 antibody is administered in conjunction with an anti-neoplastic agent, such as: doxorubicin or paclitaxel. In some preferred embodiments, the anti-CD 40 therapy is administered in conjunction with radiation therapy, chemotherapy, photodynamic therapy, surgery or other immunotherapy. In some embodiments, the anti-CD 40 antibody is administered with one or more other antibodies. For example: the anti-CD 40 antibody can be administered with antibodies known to inhibit tumor or cancer cell proliferation. Such antibodies include (but are not limited to): antibodies that inhibit CTLA4, erbB2 receptor, EGF-R, IGF-1R, CD20, or VEGF.

In some embodiments, the anti-CD 40 antibody is labeled with a radiolabel, immunotoxin or toxin, or a fusion protein comprising a toxic peptide. The anti-CD 40 antibody or anti-CD 40 antibody fusion protein can direct the radiolabel, immunotoxin, toxin, or toxic peptide to the tumor or cancer cell. In a preferred embodiment, the radiolabel, immunotoxin, toxin, or toxic peptide is internalized by the tumor or cancer cell upon binding of the anti-CD 40 antibody to CD40 on the cell surface.

In another aspect of the invention, anti-CD 40 antibodies may be used therapeutically to induce apoptosis of specific cells in a patient. In many cases, the target cell to be apoptotic is a cancer or tumor cell. Accordingly, the present invention provides a method of inducing apoptosis by administering an anti-CD 40 antibody to a patient in need thereof.

In another aspect, the invention provides a method of administering an activated anti-CD 40 antibody to a patient to increase CD40 activity. The anti-CD 40 antibody is co-administered with one or more other factors that increase CD40 activity. Such factors include CD40L, and/or CD40L analogs that activate CD 40.

In some embodiments, the anti-CD 40 antibody is co-administered with one or more immunopotentiators, including (but not limited to): IFN-beta 1, IL-2, IL-8, IL-12, IL-15, IL-18, IL-23, IFN-gamma, and GM-CSF.

In some embodiments, the present inventors use the anti-CD 40 antibody, a class of agonist, as an adjuvant to boost the efficacy of vaccines. When used in this manner, anti-CD 40 antibodies activate CD40 on antigen presenting cells, including B cells, dendritic cells and monocytes, and potentiate the production of immunoregulatory molecules, such as: cytokines and chemokines. The immunostimulatory effect of the antibodies may enhance the immune response of the immunized individual to the vaccine antigen.

In another aspect, the invention provides a method of producing a dendritic cell vaccine for cancer or dendritic cell immunotherapy. According to the method, dendritic cells from a cancer patient are incubated for 1-5 days with a tumor lysate or homogenate, tumor cells killed by irradiation or other means, or a tumor specific antigen (e.g., an individual idiotypic peptide) and 1-10. mu.g/ml anti-CD 40 antibody. The tumor antigen-pulsed dendritic cells are again injected into the patient to stimulate an anti-tumor immune response, particularly an anti-tumor CTL response. The dendritic cells derived from monocytes used in the present method can be prepared by culturing a peripheral blood sample in IL-4 and GM-CSF. Dendritic cells can also be obtained by magnetic purification or screening of CD34 positive cells from the patient's bone marrow followed by culturing in IL-4 and GM-CSF.

Gene therapy

The nucleic acid molecules of the invention can be administered to a patient in need thereof using gene therapy. The therapy may be performed in vivo or ex vivo. In a preferred embodiment, nucleic acid molecules encoding the heavy and light chains are administered to a patient. In a more preferred embodiment, the nucleic acid molecule administered is stably integrated into the chromosome of the B cell, since such cells have been specialized to produce antibodies. In a preferred embodiment, the precursor B cells are transfected or infected ex vivo and then re-transferred to a patient in need thereof. In another embodiment, precursor B cells or other cells are infected in vivo with a virus known to infect the cell type of interest. Typical vectors for gene therapy include liposomes, plasmids and viral vectors. Examples of viral vectors are retroviruses, adenoviruses and adeno-associated viruses. Following in vivo or ex vivo infection, samples are taken from the treated patients and the extent of antibody expression is followed using any immunoassay known in the art or discussed herein.

In a preferred embodiment, gene therapy comprises the steps of administering an isolated nucleic acid molecule encoding the heavy chain of an anti-CD 40 antibody or an antigen-binding portion thereof and expressing the nucleic acid molecule. In another embodiment, the gene therapy comprises the steps of administering an isolated nucleic acid molecule encoding the light chain of an anti-CD 40 antibody or an antigen-binding portion thereof and expressing the nucleic acid molecule. More preferably, in the methods, gene therapy comprises the steps of administering and expressing an isolated nucleic acid molecule encoding the heavy chain or antigen-binding portion thereof and an isolated nucleic acid molecule encoding the light chain or antigen-binding portion thereof of an anti-CD 40 antibody of the invention. Gene therapy also includes the step of administering another anti-cancer agent, such as: paclitaxel or doxorubicin.

In order to further understand the present invention, the following examples are therefore presented. These examples are merely illustrative and should not be construed as limiting the scope of the invention.

Example I

Method for forming hybridoma producing anti-CD 40 antibody

The antibodies of the invention were prepared, selected and analyzed as follows:

immunization and hybridoma formation

Taking 8-10 weeks old XenoMice^TMMice were inoculated intraperitoneally or on their hind paws with CD40-IgG fusion protein (10. mu.g/dose/mouse) or with 300.19-CD40 cells (10X 10)⁶One cell/agent/cell), which is a transfected cell line expressing human CD40 on the cell membrane. This dose is repeated 5-7 times within 3-8 weeks. 4 days prior to fusion, mice were injected with the final dose of extracellular domain of human CD40 in PBS. Lymphocytes from the spleen and lymph nodes of immunized mice were fused with a non-secretory myeloma P3-X63-Ag8.653 cell line, and the fused cells were HAT selected as described in the literature (Galfre and Methods Enzymol.73: 3-46, 1981, Milstein). Recovering a group of human IgG2 which secretes CD40 specificity uniformly_KHybridomas of antibodies. Of these, 11 hybridomas were selected for further study and designated 3.1.1, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.29.1, and 24.2.1.

The Applicant has deposited 3.1.1, 7.1.2, 10.8.3, 15.1.1 and 21.4.1 under the Budapest treaty at 8/6 in 2001 at the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, VA 20110-2209. Hybridomas 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1, 23.29.1, and 24.2.1 were deposited with the ATCC at 16.7.2002. The hybridoma has the following preservation number:

hybridoma cell Deposit number

3.1.1(LN15848) PTA-3600

7.1.2(LN15849) PTA-3601

10.8.3(LN15850) PTA-3602

15.1.1(LN15851) PTA-3603

21.4.1(LN15853) PTA-3605

21.2.1(LN15874) PTA-4549

22.1.1(LN15875) PTA-4550

23.5.1(LN15855) PTA-4548

23.25.1(LN15876) PTA-4551

23.28.1(LN15877) PTA-4552

23.29.1(LN15878) PTA-4553

24.2.1(LN15879) PTA-4554

Example II

Sequences of anti-CD 40-antibodies prepared according to the invention

To analyze the structure of the antibodies produced according to the present invention, we cloned nucleic acids encoding heavy and light chain fragments from a hybridoma producing an anti-CD 40 monoclonal antibody. Cloning and sequence analysis methods were as follows:

about 2X10 derived from XenoMouseTM mice immunized with human CD40 as described in example I⁵In each hybridoma cell, poly (A) was isolated using Fast-Track kit (Invitrogen)⁺mRNA. Followed by PCT-generating random priming(random primer) cDNA. Using human V_HOr human V_KFamily specific variable region primers (Marks et al "Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design-specific Oligonucleotide primers" Eur. J. Immunol.21: 985. sup. 991(1991)) or the common human VH primer, MG-30, CAGGTGCAGCTGGAGCAGTCIGG (SEQ ID NO: 118), with a primer specific for the human Cj2 constant region, MG-40d, 5 '-GCTGAGGGAGTAGAGTCCTGAGGA-3' (SEQ ID NO: 119) or C_KConstant region (h)_KP2; as in the aforementioned Green et al, 1994). Thus, nucleic acid molecules encoding human heavy and kappa light chain transcripts from anti-CD 40-producing hybridomas were obtained by extracting poly (A) with the above primers⁺) The PCR products generated from the RNA were directly sequenced. PCR products were also cloned into pCRII using a TA cloning kit (Invitrogen) and the sequences of both strands were analyzed using a Prism dye-terminator sequence analysis kit (dye-terminator sequences) and an ABI377 sequencer. All sequences were analyzed using MacVector and Geneworks software programs aligned with a "VBASE sequence directory" (Tomlinson et al, MRC Centre for Protein Engineering, Cambridge, UK.).

In addition, full length DNA cloning and sequencing was performed on monoclonal antibodies 3.1.1, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.28.1, 23.29.1 and 24.2.1. For such sequencing, QIAGEN RNeasy RNA isolation kit (QIAGEN) was used, from about 4X10⁶RNA was isolated from each hybridoma cell. mRNA was reverse transcribed using oligo-dT (18) and Advantage RT/PCR kit (Clonetech). VBase was used to design forward amplification primers, including restriction sites, the most appropriate Kozak sequence, ATG start site, and a portion of the signal sequence of the heavy chain. Table 1 lists the forward amplification primers used to analyze the sequence of the antibody clones.

TABLE 1

Cloning	Forward primer heavy chain
		3.1.1	5′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAGTTTGGGCTGAGCTG-3′(SEQ ID NO：120)
7.1.2	5′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAGTTTGGGCTGAGCTG-3′(SEQ ID NO：121)
		10.8.3	5′-TATCTAAGCTTCTAGACTCGAGCGCCACCATGAAACACCTGTGGTTCTTCC-3′(SEQ ID NO：122)
15.1.1	5′-TATCTAAGCTTCTAGACTCGAGCGCCACCATGAAACATCTGTGGTTCTTCC3′(SEQ ID NO：123)
		21.4.1	5′-TATCTAAGCTTCTAGACTCGAGCGCCACCATGGACTGGACCTGGAGGATCC-3′(SEQ ID NO：124)
21.2.1	5′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAGTTTGGGCTGAGCTG-3′(SEQ ID NO：128)

22.1.1	5′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAGTTTGGGCTGAGCTG-3′(SEQ ID NO：129)
		23.5.1	5′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAGTTTGGGCTGAGCTG-3′(SEQ ID NO：130)
23.28.1	5′-TATCTAAGCTTCTAGACTCGAGCGCCACCATGAAACATCTGTGGTTCTTCC-3′(SEQ ID NO：131)
		23.29.1	5′-TATCTAAGCTTCTAGACTCGACCGCCACCATGGAGTTTGGGCTGAGCTG-3′(SEQ ID NO：132)
24.2.1	5′-TATCTAAGCTTCTAGACTCGAGCGCCACCATGAAACATCTGTGGTTCTTCC-3′(SEQ ID NO：133)

The same procedure was used to design primers including the 3 ' coding sequence, the stop codon (5'-TTCTCTGATCAGAATTCCTATCATTTACCCGGAGACAGGGAGAG-3') for the constant region of IgG 2(SEQ ID NO: 125), and restriction sites.

Primers around the ATG start site of the kappa-chain were also designed using the same method: (5 '-CTTCAAGCTTACCCGGGCCACCATGAGGCTCCCTGCTCAGC-3') (SEQ ID NO: 126). The optimal Kozak sequence (CCGCCACC) was added 5' to the ATG start. This primer was used in PCR to clone the light chains of the following antibody clones: 3.1.1, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1 and 23.29.1. The light chain of clones 23.28.1 and 24.2.1 was cloned using the second forward amplification primer 5'-TCTTCAAGCTTGCCCGGGCCCGCCACCATGGAAACCCCAGCGCAG-3' (SEQ ID NO. 134). The same method was also used to design primers (5'-TTCTTTGATCAGAATTCTCACTAACACTCTCCCCTGTTGAAGC-3') (SEQ ID NO: 127) surrounding the stop codon for the kappa-constant region. cDNAs were amplified using this primer pair using the Advantage High Fidelity PCR kit (Clonetech). The PCR product is cloned into a mammalian expression vector and the cloned sequence is analyzed to confirm somatic mutations, and each clone is subjected to at least 3 reactions to confirm the sequence of both strands thereof, according to standard techniques (e.g., primer walking), using a dye-terminator sequencing kit and an ABI sequencing instrument.

Gene utilization analysis

Table 2 lists the gene utilization confirmed by the antibody hybridoma clones selected according to the invention.

TABLE 2

Heavy and light chain gene utilization

Sequence and mutation analysis

It will be appreciated that gene utilization analysis provides only a limited overview of antibody structure. Due to XenoMouse^TMB-cells in animals randomly produce transcripts of either the V-D-J heavy chain or the V-J kappa light chain, and thus many secondary reactions occur, including (but not limited to) somatic hypermutation, deletion, N-addition, and extension of CDR 3. See, for example: mendez et al Nature Genetics 15: 146-156(1997) and International patent publication WO 98/24893. Therefore, in order to further examine the antibody structure, the amino acid sequence of the antibody was predicted from the cDNAs obtained from the clone. Table a provides the sequence number of each nucleotide and the predicted amino acid sequence of the sequenced antibody.

Tables 3-7 provide the nucleotide and predicted amino acid sequences for the heavy and kappa light chains in antibodies 3.1.1 (table 3), 7.1.2 (table 4), 10.8.3 (table 5), 15.1.1 (table 6) and 21.4.1 (table 7).

Tables 8-13 provide the nucleotide and predicted amino acid sequences of the variable domains of the heavy and kappa-light chains of antibodies 21.2.1 (table 8), 22.1.1 (table 9), 23.5.1 (table 10), 23.28.1 (table 11), 23.29.1 (table 12), and 24.2.1 (table 13).

The DNA sequence of monoclonal antibody 23.28.1 from analyzed full-length sequence is different from V in PCR primary product_HSequencing of the region results in a DNA sequence that is only one base pair (C to G) such that residue 16 of the native heavy chain is changed from D to E.

Tables 14-19 provide the nucleotide and predicted amino acid sequences for the heavy and kappa-light chains of antibodies 21.2.1 (table 14), 22.1.1 (table 15), 23.5.1 (table 16), 23.28.1 (table 17), 23.29.1 (table 18), and 24.2.1 (table 19). In the table, the signal peptide sequence (or its coding base) is underlined.

Applicants generated two mutant antibodies 22.1.1 and 23.28.1. The cysteine residue at position 109 in the heavy chain of antibody 22.1.1 was mutated to an alanine residue. This mutated clone was designated 22.1.1H-C019A. The cysteine residue at position 92 in the light chain of antibody 23.28.1 was also mutated to an alanine residue. This mutated clone was designated 23.28.1L-C92A.

Mutagenesis of specific residues was performed by designing primers and using the QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The mutation was confirmed by automated sequencing and the mutated insert was subcloned into an expression vector.

Table 20 provides the nucleotide and amino acid sequences of the mutant heavy chains in antibody 22.1.1H-C109A. Table 21 provides the nucleotide and amino acid sequences of the mutant light chains in antibody 23.28.1. The mutated DNA codons are in italics. The mutated amino acid residues are indicated in bold font.

TABLE 3: DNA and protein sequences of antibody 3.1.1

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence	ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC TCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATGCGCTGTATCTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGTATTACTGTGTGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein sequence	MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISKDGGNKYHADSVKGRFTISRDNSKNALYLQMNSLRVEDTAVYYCVRRGHQLVLGYYYYNGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain DNA sequence	ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTAATGGATACAACTTTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGATTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
		Light chain protein sequence	MRLPAOLLGLLMLWVSGSSGDIVLTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRLEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Description of the drawings:	sequence (signal sequence underlined):
		mature variable domain of heavy chain DNA sequence	CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATGCGCTGTATCTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGTATTACTGTGTGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
Mature variable domain of heavy chain protein sequence	QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISKDGGNKYHADSVKGRFTISRDNSKNALYLQMNSLRVEDTAVYYCVRRGHQLVLGYYYYNGLDVWGQGTTVTVSS
		Mature variable domain of light chain DNA sequence	GATATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTAATGGATACAACTTTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGATTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
Mature variable domain of light chain protein sequence	DIVLTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRLEAEDVGVYYCMQALQTPRTFGQGTKVEIK
		Heavy chain DNA (variable domain) (3.1.1H-a78T) SEQ ID NO: 89	CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATaCGCTGTATCTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGTATTACTGTGTGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein (variable domain) (3.1.1H-a78T) SEQ ID NO: 90	QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISKDGGNKYHADSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCVRRGHQLVLGYYYYNGLDVWGQGTTVTVSS
Heavy chain DNA (variable domain) (3.1.1H-a78T-V88A-V97A) SEQ ID NO: 91	CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATaCGCTGTATCTGCAAATGAATAGCCTGAGAGcTGAAGACACGGCTGTGTATTACTGTGcGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
		Heavy chain protein (variable domain) (3.1.1H-a78T-V88A-V97A) SEQ ID NO: 92	QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISKDGGNKYHADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGHQLVLGYYYYNGLDVWGQGTTVTVSS
Light chain DNA (variable domain) (3.1.1L-L4M-L83V) SEQ ID NO: 93	GATATTGTGaTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTAATGGATACAACTTTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAgTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
		Light chain protein (variable domain) (3.1.1L-L4M-L83V) SEQ ID NO: 94	DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIK

TABLE 4: DNA and protein sequences of antibody 7.1.2

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence	ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC TCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAATGATGGAGATAATAAATACCATGCAGACTCCGTGTGGGGCCGATTCACCATCTCCAGAGACAATTCCAGGAGCACGCTTTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGAGAAGAGGCATGGGGTCTAGTGGGAGCCGTGGGGATTACTACTACTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein sequence	MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISNDGDNKYHADSVWGRFTISRDNSRSTLYLQMNSLRAEDTAVYYCARRGMGSSGSRGDYYYYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain DNA sequence	ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTAATGGATACAACTTTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
		Light chain protein sequence	MRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Description of the drawings:	sequence (signal sequence underlined):
		mature variable domain of heavy chain DNA sequence	CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAATGATGGAGATAATAAATACCATGCAGACTCCGTGTGGGGCCGATTCACCATCTCCAGAGACAATTCCAGGAGCACGCTTTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGAGAAGAGGCATGGGGTCTAGTGGGAGCCGTGGGGATTACTACTACTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
Mature variable domain of heavy chain protein sequence	QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISNDGDNKYHADSVWGRFTISRDNSRSTLYLQMNSLRAEDTAVYYCARRGMGSSGSRGDYYYYGLDVWGQGTTVTVSS
		Mature variable domain of light chain DNA sequence	GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTAATGGATACAACTTTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
Mature variable domain of light chain protein sequence	DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIK

TABLE 5: DNA and protein sequences of antibody 10.8.3

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence	ATGAAACACCTGTGGTTCTTCCTCCTGCTGGTGGC AGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGATCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAATGGATTGGGCGTGTCTATACCAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGATGGTCTTTACAGGGGGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein sequence	MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWIWIRQPAGKGLEWIGRVYTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDGLYRGYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDLAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain DNA sequence	ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGC TGCTCTGGTTCCCAGGTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGCCTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATTTATTCTGCCTCCGGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGACTGACAGTTTCCCGCTCACTTTCGGCGGCGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
		Light chain protein sequence	MRLPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQPISSWLAWYQQKPGKAPKLLIYSASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTDSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Description of the drawings:	sequence (signal sequence underlined):
		mature variable domain of heavy chain DNA sequence	CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGATCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAATGGATTGGGCGTGTCTATACCAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGATGGTCTTTACAGGGGGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
Mature variable domain of heavy chain protein sequence	QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWIWIRQPAGKGLEWIGRVYTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDGLYRGYGMDVWGQGTTVTVSS
		Mature variable domain of light chain DNA sequence	GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGCCTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATTTATTCTGCCTCCGGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGACTGACAGTTTCCCGCTCACTTTCGGCGGCGGGACCAAGGTGGAGATCAAA
Mature variable domain of light chain protein sequence	DIQMTQSPSSVSASVGDRVTITCRASQPISSWLAWYQQKPGKAPKLLIYSASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTDSFPLTFGGGTKVEIK

TABLE 6: DNA and protein sequences of antibody 15.1.1

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence	ATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGC AGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAAGTTACTACTGGACCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGATATATCTATTACAGTGGGAGCACCAACTACAATCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACATGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTTTATTACTGTGCGAGAAAGGGTGACTACGGTGGTAATTTTAACTACTTTCACCAGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGTCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein sequence	MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSIRSYYWTWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDMSKNQFSLKLSSVTAADTAVYYCARKGDYGGNFNYFHQWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain DNA sequence	ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATACTAATGGATACAACTATTTCGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAACTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
		Light chain protein sequence	MRLPAOLLGLLMLWVSGSSGDIVMTQSPLSLPVTPGEPASISCRSSQSLLHTNGYNYFDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Description of the drawings:	sequence (signal sequence underlined):
		mature variable domain of heavy chain DNA sequence	CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAAGTTACTACTGGACCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGATATATCTATTACAGTGGGAGCACCAACTACAATCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACATGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTTTATTACTGTGCGAGAAAGGGTGACTACGGTGGTAATTTTAACTACTTTCACCAGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
Mature variable domain of heavy chain protein sequence	QVQLQESGPGLVKPSETLSLTCTVSGGSIRSYYWTWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDMSKNQFSLKLSSVTAADTAVYYCARKGDYGGNFNYFHQWGQGTLVTVSS
		Mature variable domain of light chain DNA sequence	GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATACTAATGGATACAACTATTTCGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAACTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTACAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAA
Mature variable domain of light chain protein sequence	DIVMTQSPLSLPVTPGEPASISCRSSQSLLHTNGYNYFDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYSFGQGTKLEIK

TABLE 7: DNA and protein sequences of antibody 21.4.1

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence	ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGG CAGCAGCCACAGGAGCCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein sequence	MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain DNA sequence	ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGC TGCTCTGGTTCCCAGGTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTTACAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATACTGCATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACATTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
		Light chain protein sequence	MRLPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTTTCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Description of the drawings:	sequence (signal sequence underlined):
		mature variable domain of heavy chain DNA sequence	CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
Mature variable domain of heavy chain protein sequence	QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSS
		Mature variable domain of light chain DNA sequence	GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTTACAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATACTGCATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACATTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
Mature variable domain of light chain protein sequence	DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIK

TABLE 8: DNA and protein sequences of the mature variable domain of antibody 21.2.1

Description of the drawings:	sequence of
		Heavy chain DNA sequence	CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAGTAGTAAATACTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATAAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGGGTAAAGCAGTGCCTGGTCCTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAG
Heavy chain protein sequence	QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYVMHWVRQAPGKGLEWVAVMSYDGSSKYYANSVKGRFTISRDNSKNTLYLQINSLRAEDTAVYYCARDGGKAVPGPDYWGQGILVTVSS
		Light chain DNA sequence	GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGTGTTCTGTATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGTTTTACAAACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
Light chain protein sequence	DIVMTQSPLSLPVTPGEPASISCRSSQSVLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQVLQTPFTFGPGTKVDIK

TABLE 9: DNA and protein sequences of the mature variable domain of antibody 22.1.1

Description of the drawings:	sequence of
		Heavy chain DNA sequence	CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATCTGATGGAGGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTACGAGAAGAGGGACTGGAAAGACTTACTACCACTACTGTGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG
Heavy chain protein sequence	QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVISSDGGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRRGTGKTYYHYCGMDVWGQGTTVTVSS
		Light chain DNA sequence	GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATAGTAATGGATATAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACACCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGTTCAGGCACTGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC
Light chain protein sequence	DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPHLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIK

Watch 10: DNA and protein sequences of the mature variable domain of antibody 23.5.1

Description of the drawings:	sequence of
		Heavy chain DNA sequence	CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGTAACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAATTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATGTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGACGCGGTCACTACGGGAGGGATTACTACTCCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG
Heavy chain protein sequence	QVQLVESGGGVVQPGRSLRLSCVASGFTFSNYGMHWVRQAPGKGLEWVAIISYDGSNKYYADSVKGRFTISRDNSKNTLYVQMNSLRAEDTAVYYCARRGHYGRDYYSYYGLDVWGQGTTVTVSS
		Light chain DNA sequence	GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC
Light chain protein sequence	DIVMTQSPLSLPVTPGEPASISCRSSQSLLPGNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIK

TABLE 11: DNA and protein sequences of the mature variable domain of antibody 23.28.1

Description of the drawings:	sequence of
		Heavy chain DNA sequence	CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGACACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTACTACTGGAGCTGGATCCGGCAGCCCCCTGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAACTCTGTGACCGCTGCGGACACGGCCGTGTATTATTGTGCGAGAAAGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
Heavy chain protein sequence	QVQLQESGPGLVKPSDTLSLTCTVSGGSIRGYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARKGGLYGDYGWFAPWGQGTLVTVSS
		Light chain DNA sequence	GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCGACTTAGCCTGGCACCAGCAGAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCACTGTCGTAGCTTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
Light chain protein sequence	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSDLAWHQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHCRSLFTFGPGTKVDIK
		Heavy chain DNA (variable domain) (23.28.1H-D16E) (SEQ ID NO: 97)	CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTACTACTGGAGCTGGATCCGGCAGCCCCCTGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAACTCTGTGACCGCTGCGGACACGGCCGTGTATTATTGTGCGAGAAAGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG

Description of the drawings:	sequence of
		Heavy chain protein (variable domain) (23.28.1H-D16E)(SEQ ID NO：98)	QVQLQESGPGLVKPSETLSLTCTVSGGSIRGYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARKGGLYGDYGWFAPWGQGTLVTVSS

TABLE 12: DNA and protein sequences of the mature variable domain of antibody 23.29.1

Description of the drawings:	sequence of
		Heavy chain DNA sequence	CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTACAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGACGCGGTCACTACGGGAATAATTACTACTCCTATTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG
Heavy chain protein sequence	QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTIYRDNSKNTLYLQMNSLRAEDTAVYYCARRGHYGNNYYSYYGLDVWGQGTTVTVSS
		Light chain DNA sequence	GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGCTCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGATTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC
Light chain protein sequence	DIVMTQSPLSLPVTPGEPASISCRSSQSLLPGNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPRTFGQGTKVEIK

Watch 13: DNA and protein sequences of the mature variable domain of antibody 24.2.1

Description of the drawings:	sequence of
		Heavy chain DNA sequence	CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
Heavy chain protein sequence	QVQLQESGPGLVKPSETLSLTCTVSGGSIRGYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGGLYGDYGWFAPWGQGTLVTVSS
		Light chain DNA sequence	GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCACCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATAGTAGCTTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
Light chain protein sequence	EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSLFTFGPGTKVDIK

TABLE 14: DNA and protein sequences of antibody 21.2.1

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence	ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC~TCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAGTAGTAAATACTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATAAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGGGTAAAGCAGTGCCTGGTCCTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein sequence	MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYVMHWVRQAPGKGLEWVAVMSYDGSSKYYANSVKGRFTISRDNSKNTLYLQINSLRAEDTAVYYCARDGGKAVPGPDYWGQGILVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain DNA sequence	ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGTGTTCTGTATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGTTTTACAAACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
		Light chain protein sequence	MRLPAOLLGLLMLWVSGSSGDIVMTQSPLSLPVTPGEPASISCRSSQSVLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQVLQTPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Watch 15: DNA and protein sequences of antibody 22.1.1

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence	ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC TCTTTTAAGAGGTGTCCAGTGTCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATCTGATGGAGGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTACGAGAAGAGGGACTGGAAAGACTTACTACCACTACTGTGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein sequence	MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVISSDGGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRRGTGKTYYHYCGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain DNA sequence	ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATAGTAATGGATATAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACACCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGTTCAGGCACTGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCYTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
		Light chain protein sequence	MRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPHLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 16: DNA and protein sequences of antibody 23.5.1

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence	ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC TCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGTAACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAATTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATGTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGACGCGGTCACTACGGGAGGGATTACTACTCCTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein sequence	MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCVASGFTFSNYGMHWVRQAPGKGLEWVAIISYDGSNKYYADSVKGRFTISRDNSKNTLYVQMNSLRAEDTAVYYCARRGHYGRDYYSYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain DNA sequence	ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTSTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCYTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA
		Light chain protein sequence	MRLPAOLLGLLMLWVSGSSGDIVMTQSPLSLPVTPGEPASISCRSSQSLLPGNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAXVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 17: DNA and protein sequences of antibody 23.28.1

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence	ATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGC AGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTACTACTGGAGCTGGATCCGGCAGCCCCCTGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAACTCTGTGACCGCTGCGGACACGGCCGTGTATTATTGTGCGAGAAAGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein sequence	MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSIRGYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARKGGLYGDYGWFAPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain DNA sequence	ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCT ACTCTGGCTCCCAGAATCCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCGACTTAGCCTGGCACCAGCAGAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCACTGTCGTAGCTTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
		Light chain protein sequence	METPAOLLFLLLLWLPESTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSDLAWHQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHCRSLFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Watch 18: DNA and protein sequences of antibody 23.29.1

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence	ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGC TCTTTTAAGAGGTGTCCAGTGTCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTACAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGACGCGGTCACTACGGGAATAATTACTACTCCTATTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein sequence	MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTIYRDNSKNTLYLQMNSLRAEDTAVYYCARRGHYGNNYYSYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain DNA sequence	ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGCTCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGATTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTTCAGTGGAGGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
		Light chain protein sequence	MRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTPGEPASISCRSSQSLLPGNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWRVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Description of the drawings:	sequence (signal sequence underlined):
		light chain DNA (23.29.1LR174K) (SEQ ID NO：101)	ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAA TGCTCTGGGTCTCTGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGCTCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGATTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTTCAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
Light chain protein sequence (23.29.1LR174K) (SEQ ID NO: 101)	MRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTPGEPASISCRSSQSLLPGNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGTYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Watch 19: DNA and protein sequences of antibody 24.2.1

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence	ATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGC AGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAAGGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain protein sequence	MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSIRGYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGGLYGDYGWFAPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain DNA sequence	ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCT ACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCACCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATAGTAGCTTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
		Light chain protein sequence	METPAOLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSLFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Watch 20: DNA and protein sequences of the mature variable domain of antibody 22.1.1H-C109A

Description of the drawings:	sequence (signal sequence underlined):
		heavy chain DNA sequence (SEQ ID NO: 95)	CAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATCTGATGGAGGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTACGAGAAGAGGGACTGGAAAGACTTACTACCACTACGCCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG
Heavy chain protein sequence (SEQ ID NO: 96)	QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVISSDGGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRRGTGKTYYHYAGMDVWGQGTTVTVSS

TABLE 21: DNA and protein sequences of the mature variable domain of antibody 23.28.1L-C92A

Description of the drawings:	sequence (signal sequence underlined):
		light chain DNA (SEQ ID NO: 99)	GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCGACTTAGCCTGGCACCAGCAGAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCACGCCCGTAGCTTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
Light chain protein sequence (SEQ ID NO: 100)	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSDLAWHQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHARSLFTFGPGTKVDIK

Example III

Analysis of heavy and light chain amino acid substitutions

FIGS. 1D-1H and 2D-2H provide sequence alignments between the predicted heavy chain variable domain amino acid sequences of monoclonal antibodies 3.1.1, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1H-C109A, 23.5.1, 23.28.1H-D16E, 23.29.1 and 24.2.1 and the germline amino acid sequences of their respective genes. Most of the heavy chain CDR3 regions contain amino acid insertions.

The DLR1 gene used in the VH domain of antibody 21.4.1 encodes two cysteine (Cys) residues. Mass spectrometry and homology models demonstrate that two Cys residues are linked by a disulfide bond, which does not disrupt the antibody structure.

FIGS. 1A-1C and 2A-2C provide sequence alignments between the predicted light chain variable amino acid sequences in the monoclonal antibodies 3.1.1, 7.1.2, 10.8.3, 15.1.1, 21.4.1, 21.2.1, 22.1.1, 23.5.1, 23.28.1L-C92A, 23.29.1 and 24.2.1 clones and the germline amino acid sequences of their respective genes. The light chains of such antibodies are derived from 3 different vk genes. 7 of the 11 antibodies used the A3/A19V kappa gene, of which 6 had two mutations in the CDR1 region. In addition, 5 of the 7 antibodies using the A3/A19V kappa gene also used the J kappa 1 gene; in all such antibodies, the first amino acid derived from the J κ 1 gene was consistently changed from W to R.

It will be appreciated that many of the amino acid substitutions or insertions described above occur in close proximity to or in the CDRs. Such substitutions appear to have some effect on the binding of the antibody to the CD40 molecule, and in addition, such substitutions will have a significant effect on the affinity of the antibody.

Example IV

Species Cross-reactivity of antibodies of the invention

The applicants performed FACS analysis to determine the binding and affinity of the antibodies of the invention to CD40 from different species, particularly certain monkeys of the ancient world. Aliquots of human and monkey whole blood were incubated for 1 hour on ice with increasing concentrations of anti-CD 40 antibody exemplified herein or with anti-Keyhole Limpet Hemocyanin (KLH) antibody as a negative control. The samples were then incubated with anti-human IgG 2-conjugated RPE (phycoerythrin) for 30 minutes on ice. Using a cell flow meterBinding was determined by assaying for CD19/CD20 positive B cells and a histogram of fluorescence intensity (F12-H) versus cell number (Counts) was analyzed using CellQuest software. The binding property (K) of each antibody was estimated from the value of the average fluorescence intensity value with respect to the antibody concentration_D). The consumption of antibody is controlled by measuring the change in binding properties over a range of cell concentrations.

Antibodies 3.1.1, 7.1.2, 10.8.3, 15.1.1 and 21.4.1 were tested for binding to human, rhesus monkey, and cynomolgus monkey B cells. Antibodies 21.2.1, 22.1.1, 23.5.1, 23.25.1, 23.28.1, 23.29.1 and 24.2.1 were also tested for binding to human and rhesus B cells.

Applicants observed that the highest signal binding to monkey cells and half the highest binding concentration were within 2 relative to the factor for the corresponding parameter for human B cells. No binding was observed in similar experiments using blood from mice, rats, rabbits, and dogs.

Example V

Antibody selectivity for CD40

Applicants further performed another in vitro assay to determine the selectivity of the antibodies of the invention for CD 40.

CD40 selective ELISA: materials and methods

The 96-well FluroNUNC plate (Nunc accession 475515) was coated with 4 antigens: CD40/Ig, CD44/Ig, RANK/Ig, 4-1BB/Ig, TNFR-1/Ig and TNFR-2/Ig (antigens produced in the laboratory) were incubated at a concentration of 1. mu.g/ml in 0.1M sodium bicarbonate buffer, pH9.6 (100. mu.l/well) overnight at 4 ℃. The plates were then washed 3 times with PBST (PBS + 0.1% Tween-20) and blocked with PBST + 0.5% BSA (150. mu.l/well). After the plates were kept at room temperature for 1 hour, they were washed 3 times with PBST. Subsequently, the anti-CD 40 antibody produced in example I was diluted to 1. mu.g/ml and the diluted antibody was added to the plate. After the plate was incubated at room temperature for 1 hour, the plate was incubated with PBST was washed 3 times. The wells containing the antibody produced in example I were then treated with anti-human IgG2-HRP (southern Biotech accession number 9070-05) diluted 1: 4000 (100 ml/well). One row of wells was also treated with anti-human IgG diluted to 1: 5000 (Jackson accession No. 209-035-088) with 100. mu.1 per well to normalize plate coatings. One row of wells was also treated with anti-human CD40-HRP (Pharmingen accession 345815/Custom HRP conjugate) diluted to 0.05. mu.g/ml as a positive control. After incubation of the plates at room temperature for 1 hour, the plates were washed 3 times with PBST. Addition of TMB substrate (K)&P Labs) 100. mu.l per well, and incubated for 5 to 10 minutes. Then using Spectra-Max^TMThe plate reader reads the data of the culture plate. The results show that the antibody is at least 100-fold more selective for CD40 than for RANK, 4-1BB, TNFR-1 and TNFR-2, with a CD4 specific signal (minus the background CD40 signal) that is at least 100-fold more selective than the corresponding signal for the other molecules.

Example VI

Epitope classification test

After demonstrating selectivity for the antibodies of the invention to CD40, a competitive binding assay was performed using BIAcore and FACS.

BIAcore competition assay

BIAcore competition assays were performed to determine whether the human anti-CD 40 antibodies of the invention bound to the same or different positions on the CD40 molecule.

Such experiments were performed using a BIAcore2000 instrument, according to the manufacturer's instructions. protein-A was immobilized on the surface of the sensor chip of BIAcore. Saturating concentrations of CD40-Ig containing the extracellular domain of CD40 were allowed to bind to the sensor chip. Then, the first human agonist anti-CD 40 antibody, commercially available anti-CD 40 antibody or CD40L was used to bind to CD40 bound to the sensor chip under saturation conditions. The second human agonist anti-CD 40 antibody of the invention was then assayed for its ability to compete with the primary antibody, a commercially available antibody or CD40L for binding to CD 40. This technique allows the antibodies to be divided into different binding populations. Binding to CD40 indicates that an independent epitope can be recognized. Absence of binding indicates that the recognized epitopes are identical or overlapping.

FACS assay

FACS assays were performed to determine whether the human anti-CD 40 antibodies of the invention bound to the same or different positions on the CD40 molecule and to determine whether their binding positions on the CD40 molecule were the same or different from the binding positions of the commercially available anti-CD 40 antibodies EA5(Alexis accession number ANC-300-050), LOB7/6 (Serotecan MCA/590PE) and 5C3(Pharmingen #555458 (unlabeled) and 555460 (labeled PE for FACS)).

Dendritic cells that had been treated with the anti-CD 40 antibody of the present invention were counterstained with EA5 labeled with PE or LOB7/6 antibody labeled with PE and incubated on ice for 30 minutes. After washing, the staining of the cells was analyzed on a B-D cell counter (caliber cytometer). When the commercial antibody product has reduced binding, it is said that the epitope bound by the commercial antibody product is the same as or overlaps with the test antibody.

Competitive binding analysis of BIAcore with FACS showed that the epitope recognized by the mab21.4.1 antibody overlaps with the epitope recognized by the EA5 antibody, but not with the epitope recognized by the commercial LOB7/6 antibody, nor does it overlap with the binding site of CD 40L. The epitope recognized by the remaining antibodies overlaps with the position where it binds CD 40L.

Table 22 summarizes the results of such epitope classification experiments.

TABLE 22

BIAcore competitive epitope Classification assay results for certain anti-CD 40 antibodies of the invention

EA5

5C3

LOB7/6

3.1.1，21.2.1，22.1.1，23.5.1，23.29.1

21.4.1

23.25.1，23.28.1，24.2.1

CD40L

EA5

X

X

X

X

5C3

X

X

X

X

X

LOB7/6

X

X

X

X

3.1.1，21.2.1，22.1.1，23.5.1，23.29.1

X

X

X

21.4.1

X

X

X

23.25.1，23.28.1，24.2.1

X

X

X

X

CD40L

X

X

X

X

X

X

Example VII

Up-regulation assay of surface molecules with anti-CD 40 antibodies

Applicants performed a whole blood assay to determine whether the human anti-CD 40 antibodies of the invention upregulated the expression of surface molecules on B cells.

Human or primate whole blood was taken and cultured for 24 hours using RPMI medium diluted 1: 1 with various concentrations of CD40 agonist antibody or controls. Cells were stained for 30 minutes (on ice, in the dark) for HLA-DR, ICAM, B7-1, B7-2, CD19/CD20, CD40, CD23, and CD71 using commercially available fluorescent dye-labeled antibody reagents. Cells were then analyzed on a FACS-Caliber (Becton-Dickinson). Screening (gating) on CD19 or CD20 positive cells to discriminate B-cells and determining activation markers for the screening.

The highest fold increase in median fluorescence (. ltoreq.1. mu.g/ml antibody) and mean EC obtained with an anti-CD 40 antibody (21.4.1) patented in the present application₅₀The values are shown in Table 23.

TABLE 23

Results of the upregulation of B-cell surface molecules by the anti-CD 40 antibodies of the invention

	Maximum factor of increase	EC50(ng/ml)
				Mean +/-St.Dev.	Mean +/-St.Dev.
MHCII	4.50+/-0.52	3.85+/-0.35
			CD71	2.30+/-0.77	0.73+/-0.28
ICAM	4.52+/-2.42	15.3+/-7.3
			CD23	69.9+/-25.8	19.0+/-4.4
B7-2	2.74+/-0.14	16.0+/-21.9

Applicants also performed experiments to determine whether the human anti-CD 40 antibodies of the invention upregulated the expression of surface molecules of monocyte-derived dendritic cells.

Method for preparing dendritic cell derived from monocyte

Peripheral blood was collected from normal volunteer humans. Mononuclear cells were isolated using Sigma Accuspin tubes (St. Louis, MO), washed with RPMI medium (Gibco BRL, Rockville, Md.), and plated with complete RPMI medium (containing 100U/ml penicillin/streptomycin, 10mM HEPES buffer, 2mM glutamine, 0.1mM nonessential amino acids; all from GibcoBRL); and 10% fetal bovine serum (Hyclone, Logan, Utah) at a concentration of 5X 10⁶Individual cells/ml. At 37 deg.C (5% CO)₂) After 3 hours of subculture, non-adherent cells were discarded, and a selection column (R) was used&D systems, Minneapolis, MN). The adherent cells were washed with RPMI medium and supplemented with 10ng/ml IL-4 (R)&D systems) with 100ng/ml GM-CSF (R)&D systems) for 7 days in complete RPMI medium. The unattached cells were then isolated and, after washing, used as monocyte-derived dendritic cells (mDCs) for all experiments. The remaining adherent cells were removed using trypsin/EDTA and used in experiments with adherent monocytes.

To determine whether the anti-CD 40 antibodies of the invention upregulated expression of cell surface markers, monocyte-derived dendritic cells were incubated with varying concentrations of agonist antibody for 48-72 hours and then stained for HLA-DR, ICAM, B7-1, B7-2, CD40, and CD83 using commercially available fluorescent dye-labeled antibody reagents (on ice, in the dark for 30 minutes). Cells were then analyzed on a FACS-Caliber (Becton-Dickinson).

The highest fold increase in median fluorescence (. ltoreq.1. mu.g/ml antibody) and mean EC obtained using the anti-CD 40 antibody (21.4.1) patented in the present application₅₀The values are shown in Table 24.

Watch 24

Results of the upregulation of dendritic cell surface molecules by the anti-CD 40 antibodies of the invention

	Maximum factor of increase	EC50(ng/ml)
				Mean +/-St.Dev.	Mean +/-St.Dev.
MHCII	7.7+/-5.6	252+/-353
			CD83	36.3+/-42.2	233+/-262
ICAM	10.4+/-4.8	241+/-140
			B7-2	21.9+/-9.4	71.4+/-44.4

Similar experiments were performed using B cells and mDCs, and the anti-CD 40 antibody of the invention and other markers. The expression of B cell surface molecules (MHC-II, ICAM, B7-1, B7-2 and CD23) was determined as described above, but 1. mu.g/ml of anti-CD 40 antibody was used instead. The results of this experiment are shown in Table 25. After 72 hours, the expression of dendritic cell surface molecules (MHC-II, ICAM, B7-1, B7-2 and CD83) was determined as described above, but 1. mu.g/ml of anti-CD 40 antibody was used instead. The results of this experiment are shown in Table 26. Tables 25-26 show the fold improvement in median intensity +/-standard deviation.

TABLE 25

Results of the upregulation of B-cell surface molecules by the anti-CD 40 antibodies of the invention

Watch 26

Results of the upregulation of dendritic cell surface molecules by the anti-CD 40 antibodies of the invention

Table 27 compares the results of upregulated expression of cell surface molecules in dendritic cells versus B cells as a ratio of the mean fold increase in cell surface molecule expression of dendritic cells to the mean fold increase in expression on B cells.

Watch 27

Comparison of surface molecule up-regulated expression on dendritic cells and B cells

	B7-1(CD80)	B7-2(CD86)	MHCClassII	ICAM(CD54)
					3.1.1	1.08	19.40	1.38	1.15
21.2.1	1.01	7.37	1.49	1.12
					21.4.1	0.77	7.04	1.37	0.74
22.1.1	1.18	16.36	1.61	1.44
					23.5.1	0.83	10.54	1.59	1.06
23.25.1	0.66	2.57	0.85	0.98
					23.28.1	0.71	10.81	2.16	2.57
23.29.1	0.73	9.07	1.66	1.23
					24.2.1	3.48	52.30	2.64	1.35

Example VIII

Potentiation of cytokine secretion

Monocyte-derived dendritic cell assays were performed to determine whether the human anti-CD 40 antibody of the invention potentiated secretion of IL-12p40, IL-12p70, and IL-8.

Monocyte-derived dendritic cells and attached monocytes were prepared as described above. Cells were cultured in the presence of anti-CD 40 antibody of the invention (21.4.1) or using anti-Keyhole Limpet Hemocyanin (KLH) antibody as a negative control. After 24 hours, cytokines were measured in the supernatants by ELISA (R & D systems). In some assays (see Table 28), antibody-treated monocyte-derived dendritic cells were also co-stimulated with 100ng/ml LPS (Sigma), 1000U/ml IFN γ (R & D systems), or 25ng/ml IL-1 β R & D systems.

The anti-CD 40 antibody potentiates IL-12p40, IL-12p70 and IL-8 production in both monocyte-derived dendritic cells and adherent monocytes. The presence of LPS further enhances the production of IL-12p40 and IL-12p 70. Only minimal amounts of cytokines were detected in the supernatant of dendritic cells cultured with isotype control antibody (anti-KLH). Representative results are shown in table 28 and fig. 3 and 4. Table 28 summarizes the major cytokines produced by dendritic cells or adherent monocytes after treatment with 1. mu.g/ml of the anti-CD 40 antibody of the invention (21.4.1) +/-100ng/ml LPS. As shown in FIG. 3, anti-CD 40 antibody potentiated IL-12p40 production by human dendritic cells. FIG. 4 illustrates that human dendritic cells potentiate IL-12p70 production in the presence of antibodies and 100ng/ml LPS.

Watch 28

The anti-CD 40 antibody of the invention has the enhancement effect on secretion of IL-12p40, IL-12p70 and IL-8

ND is not determined

Similar experiments were performed using various anti-CD 40 antibodies of the invention. Monocyte-derived dendritic cells were prepared as described above, cultured in the presence of various concentrations of anti-CD 40 antibody, and co-stimulated with 100ng/ml LPS (Sigma). After 24 hours, by ELISA (R &D systems) assay of IL-12p70 in the supernatant and determination of the respective antibody EC₅₀. The results are shown in Table 29.

Watch 29

Potentiation of IL-12p70 secretion in dendritic cells

Antibody cloning	DCIL-12p70EC 50 Maxμg/ml pg/ml
		21.4.122.1.123.25.123.5.124.2.13.1.123.28.123.29.121.2.1	0.3 1796-70040.1 720-10400.2 540-9600.1 676-11120.2 754-36800.2 668-9600.2 1332-14040.1 852-9000.03 656-872

The anti-CD 40 antibodies of the invention were also tested in allogeneic T-cell/dendritic cell assays for their ability to potentiate IFN- γ secretion by T cells. To perform this assay, T cells and monocytes were isolated from peripheral blood of healthy volunteers. Monocytes were differentiated into dendritic cells according to the above method. 1X 10 from an individual⁵T cells and 1X 10 from different individuals⁵Dendritic cells are cultured in the presence of an anti-CD 40 antibody of the invention or in the presence of a control antibody. After 4 days of culture, the supernatants were analyzed for secretion of IFN-. gamma.by ELISA. The results of this analysis are shown in Table 30.

Watch 30

Potentiation of secreted IFN-gamma by the anti-CD 40 antibodies of the invention

Antibody cloning	AlloDC/TIFNY EC 50 Maxμg/ml pg/ml
		21.4.122.1.123.25.123.5.124.2.13.1.123.28.123.29.121.2.1	0.3 2120.3 110-1800.3 180-2320.2 150-2400.2 111-1940.1 100-1950.2 120-1900.3 134-1500.03 230-256

Example IX

The anti-CD 40 antibody of the present invention induces inflammatory cytokines

As described by Wing et al in therapeutic, immunological, 2: 183-90(1995) Whole blood cytokine release assay antibodies 10.8.3, 15.1.1, 21.4.1 and 3.1.1 were tested to determine whether the antibodies induced inflammatory cytokines at concentrations of 1, 10 and 100. mu.g/ml. Antibodies at the indicated concentrations did not significantly release TNF- α, IL-1 β, IFN- γ or IL-6 as found in the blood of 10 normal donors.

Example X

Potentiation of the immunogenicity of the cell line Jy by the anti-CD 40 antibodies of the invention

CD 40-positive JIYOYE cells (ATCC CCL87) ("Jy cells") were cultured and maintained in RPMI medium. JIYOYE fineCells were cultured with anti-CD 40 antibody of the invention (21.4.1) or isotype-corresponding antibody (anti-KLH) in complete RPMI medium for 24 hours. The cells were then washed and incubated for 60 minutes with 25mg mitomycin C (Sigma)/7ml medium. The cells were then mixed with isolated human T cells at 37 deg.C (5% CO) in a ratio of 1: 100₂) The cells were cultured for 6 days. Then, T cells were collected, washed, and the degree of CTL activity was measured with respect to JIYOYE cells labeled with fresh chromium 51(New England Nuclear, Boston, Mass.). The specific lysis rate ═ percent (lysis jy (cpm) -spontaneous lysis (cpm))/(total lysis (cpm) -spontaneous lysis (cpm)) was calculated for CTL specific activity.

As shown in fig. 5, the anti-CD 40 antibody (21.4.1) of the invention significantly potentiates the immunogenicity of antibody-treated Jy cells.

Example XI

Animal tumor model

To further investigate the anti-tumor activity of the anti-CD 40 antibodies produced by the present invention, applicants designed a SCID-beige mouse model to test the effect of antibodies on tumor growth in vivo.

SCID-beige mice were from Charles River and were acclimated for one week prior to use. Tumor cells (Daudi cell (ATCC CCL213), CD40(-) K562 cell (ATCCCL 243) and CD40(+) Raji cell (ATCC CCL86), BT474 breast cancer cell (ATCC HTB20) or PC-3 prostate cell (ATCC CRL1435)) were injected subcutaneously at a concentration of 1X 10⁷Individual cells/animal. In some instances, T cells (5X 10) from the same human donor are used⁵Seed) and dendritic cells (1X 10)⁵One) was injected with tumor cells. The anti-CD 40 antibody of the invention or an isotype-corresponding control antibody (anti-KLH) was also injected intraperitoneally just prior to tumor injection (only once). Tumor growth was then measured. Specific experiments are described below.

In one experiment, tumors were injected immediately (only injection)Once-shot), an anti-CD 40 antibody of the invention (21.4.1) or an isotype-corresponding control antibody (anti-KLH) was injected intraperitoneally at a dose of 10 mg/kg. Tumor cells (Daudi cells) were injected subcutaneously at a concentration of 1X 10⁷Individual cells/animal. Tumor growth was measured using calipers at 17, 19, 20, 21, 25, 26, 27 and 28 days post-implantation in the presence of human T cells and dendritic cells. As shown in FIG. 6, anti-CD 40 antibody inhibited tumor growth by about [60 ]]％。

In another experiment, an anti-CD 40 antibody of the invention (21.4.1), or an isotype-corresponding control antibody (anti-KLH), was injected intraperitoneally at a dose of 0.1mg/kg, 1mg/kg, or 10mg/kg immediately prior to tumor injection (only one injection). Tumor cells (K562 cells) were injected subcutaneously at a concentration of 1X 10⁷Individual cells/animal. In this experiment, T cells (5X 10) from the same human donor were used⁵Seed) and dendritic cells (1X 10)⁵One) was injected with tumor cells. Tumor growth was measured using calipers at days 17, 19, 20, 21, 25, 26, 27 and 28 post-implantation. As shown in fig. 7, the anti-CD 40 antibody inhibited tumor growth by 60-85%.

In another experiment, an anti-CD 40 antibody of the invention (21.4.1, 23.29.1 or 3.1.1), or an isotype-corresponding control antibody (anti-KLH) was injected intraperitoneally just prior to tumor injection (only one injection). The injected dose of control antibody and antibody 21.4.1 corresponding to the isotype was 1 mg/ml. The injection dose of antibodies 23.29.1 and 3.1.1 was 1, 0.1, 0.01, 0.001 or 0.0001 mg/kg. Tumor cells (K562 cells) were injected subcutaneously at a concentration of 1X 10⁷Individual cells/animal. In this experiment, T cells (5X 10) from the same human donor were used⁵Seed) and dendritic cells (1X 10)⁵One) was injected with tumor cells. Tumor growth was measured 28 days after implantation using calipers. The results of this experiment are shown in FIGS. 8 and 9. Each point in the figure represents a measurement from a single animal.

In another experiment, the anti-CD 40 antibody (21.4.1), or isoform, of the invention was injected intraperitoneally just prior to tumor injection (only once)Corresponding control antibody (anti-KLH). The antibody dose was 1, 0.1, 0.01, 0.001 or 0.0001 mg/kg. Tumor cells (Raji cells) were injected subcutaneously at a concentration of 1X 10⁷Individual cells/animal. In some of these animals, T cells (5X 10) from the same human donor were used⁵Seed) and dendritic cells (1X 10)⁵One) was injected with tumor cells. Tumor growth was measured 28 days after implantation using calipers. The results of this experiment are shown in fig. 10. Each point in the figure represents a measurement from a single animal.

In another experiment, an anti-CD 40 antibody of the invention (21.4.1, 23.28.1, 3.1.1, or 23.5.1), or an isotype-corresponding control antibody (anti-KLH) was injected intraperitoneally just prior to tumor injection (only one injection). The injection dose of the antibody was 1 or 0.1 mg/kg. Tumor cells (Raji cells) were injected subcutaneously at a concentration of 1X 10⁷Individual cells/animal. Tumor growth was measured 28 days after implantation using calipers. The results of this experiment are shown in fig. 11. Each point in the figure represents a measurement from a single animal.

In another experiment, an anti-CD 40 antibody of the invention (21.4.1, 23.29.1 or 3.1.1), or an isotype-corresponding control antibody (anti-KLH) was injected intraperitoneally just prior to tumor injection (only one injection). The injection dose of the antibody was 1 mg/kg. Tumor cells (BT474 breast cancer cells) were injected subcutaneously at a concentration of 1X 10⁷Individual cells/animal. T cells (5X 10) from the same human donor⁵Seed) and dendritic cells (1X 10)⁵One) was injected with tumor cells. Tumor growth was measured 39 days after implantation using calipers. As shown in fig. 12, all antibodies inhibited breast cancer tumor growth. Each point in the figure represents a measurement from a single animal.

In another experiment, the anti-CD 40 antibody of the invention (3.1.1), or an isotype-corresponding control antibody (anti-KLH), was injected intraperitoneally just prior to tumor injection (only one injection). The injection dose of the antibody was 1 mg/kg. Tumor cells (PC-3 prostate cells) were injected subcutaneously at a concentration of 1X 10⁷Individual cells/animal. Using calipersTumor growth was measured 41 days after implantation. As shown in fig. 13, the anti-CD 40 antibody inhibited prostate tumor growth by about 60%. Each point in the figure represents a measurement from a single animal.

Example XII

SCID-beige mice treated with injected Daudi tumor cells and anti-CD 40 antibodies of the invention Viability of

In another experiment, the anti-CD 40 antibody of the invention, or an isotype-corresponding control antibody, was injected intraperitoneally (only once) just prior to tumor injection. The injection dose of the antibody was 1 or 0.1 mg/ml. Tumor cells (Daudi cells) were injected intravenously at a dose of 5X 10⁶Individual cells/animal. The survival of the animals was then followed. As shown in figure 14, all of the anti-CD 40 antibodies tested extended survival of mice receiving tumor injections for at least 6 days.

Table 31 lists the ED of anti-CD 40 antibodies in different solid tumor models described in example XI₅₀. Table 31 summarizes the in vivo anti-tumor activity of some of the anti-CD 40 antibodies of the invention in SCID mice. In addition, the ED of the anti-CD 40 antibody in the Daudi systemic tumor model described in example XII is shown₅₀。

Watch 31

ED of the anti-CD 40 antibodies of the invention in SCID mice Using different in vivo tumor models ₅₀

Antibodies	CD40(-) CD40(+)RajiK562 &T/DC&T/DC subcutaneous(mg/kg)(mg/kg)	CD40(+) RajiCD40(+) Daudi subcutaneous intravenous (mg/kg)
			21.4.122.1.123.25.123.5.124.2.13.1.123.28.123.29.121.2.1	0.005 0.00080.01 ND≥1.0 ND>1.0 ND>1.0 ND0.02 ND>1.0 ND0.009 ND≤1.0 ND	0.016 0.1>1.0 0.1>1.0 ND≥1.0 ND>1.0 ND≥0.1 ≤0.1≥1.0 0.1>1.0 ≤0.1ND ND

Not made as ND

Example XIII

Determination of the affinity constant (K) of fully human anti-CD 40 antibody by BIAcore _D )

The affinity of the purified antibody was determined by surface plasmon resonance using a BIAcore3000 instrument, according to the manufacturer's instructions.

Biosensor biosensing biospecific interaction assay instruments (BIAcore) use surface plasmon resonance to determine molecular interactions on CM5 sensor chips. The change in refractive index between the two media (glass and carboxymethylated dextran) caused by the interaction of the molecule with the dextran side of the sensor chip was determined as the change in the free Reflection Unit (RU), which was specified in the manufacturer's instructions.

The carboxymethylated dextran surface of the fluidic chamber on the sensor chip was activated by derivatization with 0.05 MN-hydroxysuccinimide in the medium of 0.2 MN-ethyl-N' - (dimethylaminopropyl) carbodiimide for 7 minutes. The CD40-Ig fusion protein (described in example I) was contained in 10mM sodium acetate, pH3.5, at a concentration of 5g/ml, injected manually into the fluid chamber at a rate of 5. mu.l/min, and covalently immobilized on the surface of the fluid chamber containing the desired amount of RU. Unreacted N-hydroxysuccinimide ester was deactivated using 1M ethanolamine hydrochloride, pH 8.5. After fixation, 5 μ l each of 50mM NaOH was injected 5 times until a stable baseline was reached to clear the fluidic chamber of any unreacted or poorly bound material. Fluid chamber 2 is a high density surface, about 300RU measured after the surface treatment, and fluid chamber 3 is a low density surface, about 150RU measured. The fluid chamber 1 is an activated blank surface and 35. mu.l of 10mM sodium acetate buffer is injected instead of antigen during the immobilization. The fluid compartment 4 contains approximately 450RU of immobilized CTLA4-Ig, an irrelevant antigen control.

A series of dilutions of each antibody were prepared with a half-log (halflog) concentration ranging from 100. mu.g/ml to 0.1. mu.g/ml. The flow rate was set at 5. mu.l/min, 25. mu.l of each concentration of sample was injected onto the sensor chip, and 5. mu.l of 50mM NaOH was injected between antibody injections at each concentration. Data were analyzed using BIA analysis software 3.0.

In reverse kinetic experiments, antibody 21.4.1 was immobilized on the surface of the sensor chip as described above. anti-KHL was used as a control antibody surface. The injection concentration of the antigen CD40-Ig fusion protein ranged from 100. mu.g/ml to 0.1. mu.g/ml.

Table 32 lists affinity measurements for representative anti-CD 40 antibodies of the invention:

watch 32

Affinity measurement of anti-CD 40 antibody of the invention

Antibodies	Kon(1/Ms)	Koff(1/s)	KD(M)
				3.1.1	1.12×106	3.31×10-5	3.95×10-11
10.8.3	2.22×105	4.48×10-7	2.23×10-12
				15.1.1	8.30×104	2.83×10-7	4.05×10-12
21.4.1	8.26×104	2.23×10-5	3.48×10-10
				22.1.1	9.55×105	1.55×10-4	2.79×10-10
23.25.1	3.83×105	1.65×10-7	7.78×10-12
				23.28.1	7.30×105	8.11×10-5	1.61×10-10
23.29.1	3.54×105	3.90×10-5	7.04×10-11

Example XIV

Epitope mapping of anti-CD 40 antibodies

Binding assays were performed using protein A purified CD 40-human IgG1Fc fusion antigen. Human CD40-IgG1Fc fusion protein was cloned by Pfizer. The human CD40IgG1 fusion protein was expressed in mammalian cell lines and purified on a protein A column. The purity of the fusion antigen was analyzed by SDS/PAGE.

The structure of CD40 is a typical type I transmembrane protein. The mature molecule consists of 277 amino acids. The extracellular domain of CD40 consists of 4 cysteine-rich domains of the TNFR-like. See, for example: TIBS23 by Neismith and Sprang: 74-79 (1998); j.leukocyte biol.67, by vankoaten and Banchereau: 2-17 (2000); EMBO J.8 by Stamenkovic et al: 1403-1410(1989).

Binding of anti-CD 40 antibodies to reduced or unreduced human CD40

Since the extracellular domain of CD40 is composed of 4 cysteine-rich domains, the intramolecular linkage, when disrupted by a reducing agent, alters antibody reactivity. To determine whether intramolecular binding disrupted by the reducing agent would alter the reactivity of the anti-CD 40 antibody selected for use in the present invention, purified CD40-hIgG was added to SDS/PAGE (4-20% gel) under either unreduced (NR) or reduced (R) conditions. SDS/PAGE was performed using a mini-gel system according to Laemmli. The separated proteins were transferred to nitrocellulose membranes. After blocking the membrane with 5% (w/v) skim milk powder in PBS for at least 1 hour, color was developed and probed with each antibody for 1 hour. anti-CD 40 antibody was detected using HRP-conjugated goat anti-human immunoglobulin (dilution 1: 8,000; Sigma, accession number A-8667). Use of enhanced Chemiluminesence (Amersham bioscience), the membrane was developed according to the manufacturer's instructions.

The 4 anti-CD 40 antibodies of the invention were then used: western blotting was performed using 21.4.1, 23.25.1, 23.29.1 and 24.2.1 (1. mu.g/ml) as probes, followed by HRP-conjugated goat anti-human IgG (diluted 1: 8000). The results of this experiment are shown in FIG. 15. This result shows that antibodies 21.4.1, 23.25.1, 23.29.1 and 24.2.1 bind to unreduced CD40 but not to reduced CD 40. Thus, the antibody recognizes a spatial epitope.

Binding of anti-CD 40 antibody to human CD40 domain deleted protein:

the extracellular region of CD40 includes 4 TNFR-like repeat domains (designated D1-D4). See, for example: TIBS23 by Neismith and Sprang: 74-79 (1998); j.leukoytebil.67, vankoaten and Banchereau: 2-17 (2000); EMBOJ.8 by Stamenkovic et al: 1403-1410(1989). FIG. 16 shows the amino acid sequence of domains D1-D4 of mouse and human CD 40. To explore the contribution of different regions of the CD40 molecule to epitope presentation, several domain-deletion mutants were constructed.

To make the human CD40 deletion construct, PCR was performed using sequence-specific primers to amplify the entire extracellular domain (amino acids 1-193) of human CD40 from human B cell (CD19+) cDNA (multiple tissue cDNA set, accession number K1428-1, Clontech) and add a 6 XHis-tag at the C-terminus. The full-length and truncated CD40 molecules were cloned using human CD405 'primers 5' -GCAAGCTTCACCAATGGTTCGTCTGCCTCTGCAGTG-3 '(SEQ ID NO: 135) in combination with different 3' primers. The 3' primers used to clone the full-length extracellular domain of human CD40 were: 5 '-TCAGTGATGGTGATGGTGATGTCTCAGCCGATCCTGGGGACCA-3' (SEQ ID NO: 136). The 3' primers used to clone the D1-D3 domain of human CD40 were: 5 '-TCAGTGATGGTGATGGTGATGTGGGCAGGGCTCGCGATGGTAT-3' (SEQ ID NO: 137). The 3' primers used to clone the D1-D2 domain of human CD40 were: 5 '-TCAGTGATGGTGATGGTGATGACAGGTGCAGATGGTGTCTGTT-3' (SEQ ID NO: 138). After the constructs of all such truncated CD40 cdnas were formed, expression was performed in 293F cell line using the pcr3.1 vector (Invitrogen). The CD40-6XHis fusion protein was purified by elution on a nickel column.

The amino acid sequences of these 4 deletion mutants are shown in Table 33.

Watch 33

CD40 His-tag fusion protein

Deletion mutants	Amino acid sequence (leader sequence underlined)
		Human CD40-6XHis (full-Length extracellular Domain)	MVRLPLOCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRHHHHHH(SEQ ID NO：139)
Human CD40(D1-D3) -6XHis	MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPHHHHHH(SEQ IDNO：140)
		Deletion mutants	Amino acid sequence (leader sequence underlined)
Human CD40(D1-D2) -6Xhis	MVRLPLOCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCHHHHHH(SEQ ID NO：141)

To express such human CD40 deletion constructs, the constructs were cloned into the pcr3.1 vector (Invitrogen) and their expression was analyzed in a variety of different stably and transiently transfected 293F cell lines. Supernatants from transiently transfected 293F cell lines were analyzed for binding to antibodies 21.4.1, 23.25.1, 23.29.1 and 24.2.1 by ELISA and Western blotting.

Supernatants from 293F cells transfected with different CD40 constructs were taken for ELISA analysis. Either a goat anti-human CD40 polyclonal antibody (R & D, accession AF632) or a goat anti-mouse CD40 polyclonal antibody (R & D accession AF440) diluted to 1. mu.g/ml in ELISA assay plate coating buffer was coated on the ELISA assay plate. Detection was performed using biotinylated goat anti-human CD40(R & D accession number BAF632), goat anti-mouse CD40(R & D accession number BAF440), or HRP-conjugated anti-His (C-terminal) antibody (Invitrogen, accession numbers 46-0707) to confirm expression of the CD40 construct in 293F cells. Binding of anti-CD 40 human antibodies was detected using HRP conjugated goat anti-human IgG (FC-specific Caltag H10507) diluted to 1: 2000. The results are shown in table 34, which shows that most, if not all, of the epitopes recognized by mabs21.4.1, 23.28.1 and 23.29.1 are located in the D1-D2 region of CD40, while the epitope of mabs 24.2.1 is located at least in part in the domains D3-D4. Human CD 40-rabbit Fc fusion protein was used as a control to confirm the specificity of antibody binding.

Watch 34

ELISA: binding of antibodies to CD40 deletion mutants

	Human CD40(D1-D2) -6Xhis	Human CD40(D1-D3) -6XHis	Human CD40-6XHis
				21.4.1	+	+	+
23.25.1	+	+	+
				23.29.1	+	+	+
24.2.1		+	+
				anti-His	+	+	+
anti-RbIg	ND	ND	ND

The CD40 deletion constructs were also analyzed using western blot analysis. The results are shown in Table 35. The ELISA results showed that the binding positions of antibodies 21.4.1, 23.25.1, 23.29.1 and 24.2.1 involved domains D1-D3. The results also show that the binding position of antibodies 21.4.1, 23.25.1 and 23.29.1 involves domain D1-D2 and the binding position of antibody 24.2.1 involves domain D3.

Watch 35

Western blot analysis: binding of antibodies to CD40 deletion mutants

	Human CD40(D1-D3) -6Xhis	Human CD40-6Xhis
			21.4.1	+	+
23.25.1	+	+
			23.29.1	+	+
24.2.1	+	+
			-His	+	+
-RbIg	ND	ND

Binding of anti-CD 40 antibody to mouse CD40：

The ability of antibodies 21.4.1, 23.25.1, 23.29.1 and 24.2.1 to bind mouse CD40 was determined.

In this experiment, mouse CD40 was amplified from mouse B cell cDNA. The mouse CD40(D1-D3) -6XHis fusion protein was cloned into pCR3.1, which uses the CMV promoter to drive transcription. The 5' primers used to clone the extracellular domain of mouse CD40 were: 5'-TGCAAGCTTCACCATGGTGTCTTTGCCTCGGCTGTG-3' are provided. The 3' primers used to clone the D1-D3 domain of mouse CD40 were: 5'-GTCCTCGAGTCAGTGATGGTGATGGTGATGTGGGCAGGGATGACAGAC-3' are provided. Transient transfection of mouse and human cDNA constructs into 293F cells. The expression of recombinant CD40 was detected by ELISA using polyclonal antibodies against mouse and human CD40, anti-His antibodies, and anti-CD 40 antibodies 21.4.1, 23.25.1, 23.29.1, and 24.2.1. The results of such experiments are shown in table 36. This experiment shows that all antibodies are specific for human CD40 and do not cross-react with mouse CD 40.

Watch 36

Cross-reactivity of mouse and human CD40

	Mouse CD40(D1-D3) -6Xhis	Human CD40(D1-D3) -6XHis
			21.4.1	No	Yes
23.25.1	No	Yes
			23.29.1	No	Yes
24.2.1	No	Yes
			Goat anti-human CD40CD40	No	Yes
Goat anti-mouse CD40CD40	Yes	No
			anti-His	Yes	Yes

Binding of anti-CD 40 bodies to human/mouse chimeric CD40

Since antibodies 21.4.1, 23.25.1, 23.29.1 and 24.2.1 do not bind mouse CD40, human/mouse chimeric CD40 proteins were constructed to more definitively map the epitopes of such antibodies.

To construct an in-frame fusion of the human and murine CD40 chimeric proteins, unique restriction sites at the boundary of the CD40 domain at the same position on both human and mouse CD40 cDNAs were used. Different cDNA constructs were generated using the terminal EcoRI restriction site at domain 1 (nucleotide 244, amino acid 64) and the terminal BanI restriction site at domain 2 (nucleotide 330, amino acid 94) (fig. 17).

The different CD40 domains were amplified using PCR and ligated. This approach can exchange various different domains of mouse CD40 for homologous domains from human CD 40. The resulting construct is shown in FIG. 18.

Whether antibodies 21.4.1, 23.25.1, 23.29.1 and 24.2.1 can bind to the mouse/human chimeric CD40 protein was then determined by ELISA. The results are shown in Table 37. As can be seen from table 37, the epitopes recognizable by mabs21.4.1 and 23.25.1 are partially located at D1 and partially located at D2; the epitope recognized by mab23.29.1 is mostly (if not entirely) located at D2; and the epitope recognized by mab24.2.1 is located at D2 and D3.

Watch 37

Antibodies that bind to chimeric CD40 proteins

Antibodies

HuDl

HuD2

HuD3

HuDl，D2

HuD2，D3

HuDl，D3

21.4.1

No

No

No

Yes

No

No

23.25.1

No

No

No

Yes

No

No

23.29.1

No

Yes

No

Yes

Yes

No

24.2.1

No

No

No

No

Yes

No

The contents of all publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if the contents of each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the invention.

Claims (19)

1. A human monoclonal antibody or antigen-binding portion thereof that specifically binds to and activates CD40, wherein the antibody or antigen-binding portion thereof comprises a heavy chain comprising the heavy chain CDR1, CDR2, and CDR3 amino acid sequences of antibody 21.4.1(ATCC accession No. PTA-3605), and a light chain comprising the light chain CDR1, CDR2, and CDR3 amino acid sequences of antibody 21.4.1.

2. The monoclonal antibody, or antigen-binding portion thereof, of claim 1, wherein the heavy chain comprises the heavy chain variable region amino acid sequence of antibody 21.4.1(ATCC accession No. PTA-3605) and the light chain comprises the light chain variable region amino acid sequence of antibody 21.4.1.

3. A monoclonal antibody, wherein the antibody comprises:

(a) a heavy chain comprising the heavy chain amino acid sequence of antibody 21.4.1(ATCC accession number PTA-3605); and

(b) a light chain comprising the amino acid sequence of the light chain of antibody 21.4.1.

4. A human monoclonal antibody or antigen-binding portion thereof that specifically binds to and activates CD40, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, and wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 42, and CDR1, CDR2, and CDR3, the light chain comprising the amino acid sequence of SEQ ID NO: 44, CDR1, CDR2, and CDR 3.

5. The monoclonal antibody or antigen binding portion according to claim 4, wherein the antibody comprises:

(a) comprises the amino acid sequence SEQ ID NO: 42, a heavy chain variable region; and

(b) comprises the amino acid sequence SEQ ID NO: 44, light chain variable region.

6. The monoclonal antibody or antigen binding portion of claim 5, wherein the antibody or portion comprises:

(a) SEQ ID NO: 46 without a signal sequence, and

(b) SEQ ID NO: 48, and an amino acid sequence not having a signal sequence.

7. A monoclonal antibody, wherein the antibody comprises:

(a) consisting of the amino acid sequence SEQ ID NO: 46; and

(b) consisting of the amino acid sequence SEQ ID NO: 48, or a light chain.

8. The monoclonal antibody or antigen binding portion of claim 1, wherein the antibody or portion has at least one property selected from the group consisting of:

a) does not bind to mouse, rat, dog and/or rabbit B cells;

b) will bind to human, cynomolgus and/or rhesus B cells;

c) its selectivity for CD40 is at least 100-fold higher than its selectivity for receptor activators of the nuclear receptors kappa-B (RANK), 4-1BB (CD137), tumor necrosis factor receptor-1 (TNFR-1) and tumor necrosis factor receptor-2 (TNFR-2);

d) k binding to CD40_DIs 4x10^-10M or below;

e) it K_offIs 2x10^-4(ii) s or less;

f) inhibit tumor growth in vivo in the presence of human T cells and/or human dendritic cells;

g) inhibits CD 40-positive tumor growth in the absence of human immune cells;

h) increasing expression of ICAM, MHC-II, B7-2, CD71, CD23 and/or CD71 on the surface of human B-cells;

i) increasing the secretion of IL-12p40, IL-12p70 and/or IL-8 by human dendritic cells;

j) increasing expression of ICAM, MHC-II, B7-2, and/or CD83 on the surface of human dendritic cells;

k) increasing interferon-gamma expression in human T cells during allogeneic gene stimulation;

1) binds to human CD40 in the presence of human CD 40L; and

m) will bind to an epitope of human CD40 contained in domain 1, domain 2 or domain 3 of the extracellular domain of CD 40.

9. The monoclonal antibody or antigen-binding portion according to any one of claims 1, 2, 4,5 and 8 which is a Fab fragment, F (ab')₂Fragment, F_vFragments or single chain antibodies.

10. A pharmaceutical composition comprising a monoclonal antibody or antigen-binding portion according to any one of claims 1-9, and a pharmaceutically acceptable carrier.

11. Use of a monoclonal antibody or antigen-binding portion according to any one of claims 1-9 in the manufacture of a medicament for treating a hyperproliferative disorder in a human in need thereof.

12. Use of an antibody or antigen-binding portion according to any one of claims 1-9 in the manufacture of a medicament for treating cancer or enhancing an immune response in a human in need thereof.

13. Use of a monoclonal antibody or antigen-binding portion according to any one of claims 1-9 in the manufacture of a medicament for the treatment of a human CD 40-negative tumor.

14. An isolated cell line that produces an antibody or antigen-binding portion according to any one of claims 1-9.

15. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain of a monoclonal antibody, or antigen-binding portion thereof, according to any one of claims 1-9, and a nucleotide sequence encoding the light chain of a monoclonal antibody, or antigen-binding portion thereof, according to any one of claims 1-9, or antigen-binding portion thereof.

16. The isolated nucleic acid molecule according to claim 15, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding the amino acid sequence of the heavy chain or antigen-binding portion thereof of an antibody comprising the amino acid sequence of heavy chain CDR1, CDR2, and CDR3 of antibody 21.4.1(ATCC accession No. PTA-3605), and a nucleotide sequence encoding the amino acid sequence of the light chain or antigen-binding portion thereof of an antibody comprising the amino acid sequence of light chain CDR1, CDR2, and CDR3 of antibody 21.4.1;

(b) encoding a nucleotide sequence comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of antibody 21.4.1;

(c) nucleotide sequences encoding the heavy and light chain amino acid sequences of antibody 21.4.1;

(d) encoding the amino acid sequence of SEQ ID NO: 42 or 46 and the heavy chain amino acid sequence of SEQ ID NO: 44 or 48; alternatively, the nucleic acid sequence encoding SEQ ID NO: 42 or 46 and the heavy chain amino acid sequence of SEQ ID NO: 44 or 48, if said amino acid sequence has a signal sequence, a nucleotide sequence encoding said amino acid sequence lacking a signal sequence; or

(e) SEQ ID NO: 41 or 45 and the heavy chain nucleotide sequence of SEQ ID NO: 43 or 47; alternatively, SEQ ID NO: 41 or 45 and the heavy chain nucleotide sequence of SEQ ID NO: 43 or 47, which is absent a signal sequence-encoding nucleotide sequence if said nucleotide sequence has a signal sequence-encoding nucleotide sequence.

17. A vector comprising a nucleotide sequence encoding a heavy chain or antigen-binding portion thereof of a monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-9, and a nucleotide sequence encoding a light chain or antigen-binding portion thereof of a monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-9.

18. A host cell comprising a nucleotide sequence encoding a heavy chain or antigen-binding portion thereof of a monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-9, and a nucleotide sequence encoding a light chain or antigen-binding portion thereof of a monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-9; or comprising a vector according to claim 17.

19. A method of making an antibody or antigen-binding portion thereof that specifically binds to and activates CD40, comprising culturing a host cell according to claim 18 or a cell line according to claim 14 under suitable conditions and recovering the antibody or antigen-binding portion.