RU2779128C2 - Antibody to cd40, its antigene-binding fragment and its medical use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and is an antibody to CD40 or its antigene-binding fragment containing a variable region of the light chain of the antibody and a variable region of the heavy chain of the antibody, where the variable region of the light chain of the antibody contains LCDR1, LCDR2 and LCDR3 having certain sequences. The invention also relates to a pharmaceutical composition for the treatment and prevention of a disease or condition mediated by CD40 or CD40L containing the said antibody against CD40 or its antigene-binding fragment and a pharmaceutically acceptable auxiliary substance, diluent or carrier.

EFFECT: invention makes it possible to effectively treat diseases or conditions mediated by CD40 or CD40L.

23 cl, 11 tbl, 11 ex, 4 dwg

Description

The present application claims priority under Chinese Patent Application No. CN201710402559.0 filed June 1, 2017. The entire content of the above application is hereby incorporated by reference.

Field of invention

The present invention relates to an anti-CD40 antibody, an antigen-binding fragment thereof, a chimeric antibody or a humanized antibody containing the CDR regions of an anti-CD40 antibody, and a pharmaceutical composition containing an anti-human CD40 antibody and an antigen-binding fragment thereof, and its use as an anticancer drug.

Background of the invention

As one of the major threats to health, cancer is a major problem that humanity will face in the long term. Therapies such as conventional surgery, chemotherapy, and radiation therapy often have limited effect in the treatment of disseminated solid tumors. Tumor immunotherapy, especially T-cell immunotherapy, is a subject of increased interest in the field of tumor therapy. Tumor immunotherapy ensures the destruction of tumors through the complete activation of killer T cells in tumor patients, which may be the most effective and safe way to treat tumors. Tumor immunotherapy has now shown promise in the treatment of several different types of cancer, including disseminated metastatic tumors.

The activation of T cells in the human body involves a system that includes two signaling pathways. In addition to providing a first signal to T cells by presenting MHC antigen peptides via an antigen presenting cell (APC), a number of co-stimulatory molecules are required to provide a second signal, thereby activating normal T cell immune responses. This dual signaling pathway system plays a critical role in the balance of the in vivo immune system, which tightly regulates the body to activate different immune responses to self and foreign antigens. The absence of a second signal provided by the co-stimulatory molecule will lead to disruption in T cell responses or loss of sustained specific immune responses, resulting in immune tolerance. Therefore, the second signaling pathway plays a very important role in the regulation of the entire immune response process.

CD40 is one of the glycoproteins expressed on the cell surface. It is a 48 kDa type I membrane internal glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) superfamily and plays a critical role in the immune system. It is expressed in various immune cells such as B cells, dendritic cells, monocytes and macrophages. When signaling is mediated by CD40, specialized antigen-presenting cells will be activated. The natural ligand for CD40 is designated CD154 or CD40L and is known to be predominantly expressed in mature T lymphocytes. CD40L-mediated signaling can initiate a number of cellular biological events, including activation and proliferation of immune cells, as well as the production of cytokines and chemokines. Signaling involving CD40 is extremely important for T-cell dependent immune responses, especially in the context of the tumor environment. CD40-stimulated dendritic cells are able to activate tumor-specific effector T cells, which have the potential to kill tumor cells.

CD40 expression has been found in a variety of normal cells and tumor cells, including B-lymphocytes. For example, melanoma is a CD40-expressing tumor, while 30% to 70% of solid tumors also express CD40. It is now known that CD40 activation can effectively trigger antitumor responses (Tong et al., Cancer Gene Therapy, 2003, 10:1-13), including immune activation of tumor-specific T-cell responses, direct apoptosis of CD40-positive tumors, and humoral stimulation-induced ADCC response. In addition, the observed tumor eradication is closely associated with the appearance of cytotoxic T-lymphocytes with tumor specificity. Meanwhile, one should also not ignore the fact that systemic administration of anti-CD40 antibodies is usually associated with various side effects, such as shock syndrome and cytokine release syndrome (van Mierlo et al., Proc. Nat1. Acad. Sci. USA, 2002, 99: 5561-5566).

Currently, a number of international pharmaceutical companies are developing anti-CD40 monoclonal antibodies that specifically stimulate immune activation, maximizing the response of patients' own immune system against tumors, thereby achieving the goal of killing tumor cells. Связанные патенты включают CN1198647, CN1369015, CN1582165, CN100430419, CN101014386, CN101237882, CN101289510, CN101490086, CN103842382, CN104918957, WO2002028904, WO2011123489, WO2012149356, WO2013034904, WO2015091853, WO2016196314, WO2017040932 и WO2017004006 и т.д. To date, anti-CD40 antibodies from Pfizer (related products have been licensed by Roche), Alligator and other companies that have been found to have good antitumor effects in preclinical animal models have entered Phase I clinical trials.

The present invention is directed to providing an anti-CD40 antibody with high affinity, high selectivity and high biological activity, as well as a drug/composition for cancer therapy and a method based thereon for activating immune responses by stimulating CD40 and its pathway.

Brief description of the invention

The present invention provides an anti-CD40 antibody, or antigen-binding fragment thereof, comprising an antibody light chain variable region comprising at least one LCDR selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO : 52, SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 60; and/or

an antibody heavy chain variable region comprising at least one HCDR selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 55, or SEQ ID NO: 56, or SEQ ID NO: 57.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises an LCDR1 having the sequence of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 42, SEQ ID NO: 50 or SEQ ID NO: 58.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises an LCDR2 having the sequence of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 43, SEQ ID NO: 51 or SEQ ID NO: 59.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises an LCDR3 having the sequence of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 44, SEQ ID NO: 52 or SEQ ID NO: 60.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises HCDR1 having the sequence of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 39, SEQ ID NO: 47 or SEQ ID NO: 55.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises HCDR2 having the sequence of SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 40, SEQ ID NO: 48 or SEQ ID NO: 56.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the heavy chain variable region of the antibody comprises HCDR3 having the sequence of SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 41, SEQ ID NO: 49 or SEQ ID NO: 57.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 having the sequence of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 having the sequence of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 having the sequence of SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44 respectively.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 having the sequence of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52 respectively.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 having the sequence of SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60 respectively.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 having the sequence of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 respectively.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 having the sequence of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 respectively.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 having the sequence of SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41 respectively.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 having the sequence of SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49 respectively.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 having the sequence of SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 respectively.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 having the sequence of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively, or LCDR1, LCDR2 and LCDR3 having the sequence under SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively, or LCDR1, LCDR2 and LCDR3 having the sequence under SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44 respectively, or LCDR1, LCDR2 and LCDR3 having the sequence under SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, respectively, or LCDR1, LCDR2 and LCDR3 having the sequence under SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, respectively; and

the variable region of the heavy chain of the antibody contains HCDR1, HCDR2 and HCDR3 having the sequence under SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, or HCDR1, HCDR2 and HCDR3 having the sequence under SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively, or HCDR1, HCDR2, and HCDR3 having the sequence under SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, respectively, or HCDR1, HCDR2, and HCDR3 having the sequence under SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49 respectively, or HCDR1, HCDR2 and HCDR3 having the sequence under SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, respectively.

A particularly preferred anti-CD40 antibody, or antigen-binding fragment thereof, may be selected from the group consisting of the following:

(1) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 having the sequence of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, and the antibody heavy chain variable region contains HCDR1, HCDR2, and HCDR3, having the sequence under SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively;

(2) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 having the sequence of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, and the antibody heavy chain variable region contains HCDR1, HCDR2, and HCDR3, having the sequence under SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively;

(3) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 having the sequence of SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively, and the antibody heavy chain variable region contains HCDR1, HCDR2, and HCDR3, having the sequence under SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively;

(4) the antibody light chain variable region contains LCDR1, LCDR2 and LCDR3 having the sequence of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, respectively, and the antibody heavy chain variable region contains HCDR1, HCDR2 and HCDR3, having the sequence under SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, respectively;

(5) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 having the sequence of SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, respectively, and the antibody heavy chain variable region contains HCDR1, HCDR2, and HCDR3, having the sequence under SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, respectively.

In a preferred embodiment of the present invention, the light chain variable region of an antibody has the sequence of SEQ ID NO: 2 or SEQ ID NO: 10; the heavy chain variable region of an antibody has the sequence of SEQ ID NO: 1 or SEQ ID NO: 9.

The aforementioned anti-CD40 antibody or antigen-binding fragment thereof may be a mouse antibody or a chimeric antibody.

Preferably, the amino acid sequence of the heavy chain variable region of the mouse antibody or chimeric antibody is shown under SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown under SEQ ID NO: 2; or

the amino acid sequence of the heavy chain variable region is shown under SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown under SEQ ID NO: 10; or

the amino acid sequence of the light chain variable region (LCVR) is shown under SEQ ID NO: 38, and the amino acid sequence of the heavy chain variable region (HCVR) is shown under SEQ ID NO: 37; or

the amino acid sequence of the light chain variable region (LCVR) is shown under SEQ ID NO: 46, and the amino acid sequence of the heavy chain variable region (HCVR) is shown under SEQ ID NO: 45; or

the amino acid sequence of the light chain variable region (LCVR) is shown under SEQ ID NO: 54, and the amino acid sequence of the heavy chain variable region (HCVR) is shown under SEQ ID NO: 53.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen-binding fragment thereof as described above, wherein the antibody or antigen-binding fragment thereof is a mouse antibody or fragment thereof.

In a preferred embodiment, the present invention provides a mouse antibody or fragment thereof as described above, wherein the light chain variable region of the antibody further comprises a light chain FR region from the mouse κ, λ chain, or variants thereof.

In a preferred embodiment of the present invention, a mouse antibody or fragment thereof as described above is provided, further comprising a light chain constant region from the mouse κ, λ chain, or variants thereof.

In a preferred embodiment, the present invention provides a mouse antibody or fragment thereof as described above, wherein the heavy chain variable region of the antibody further comprises a heavy chain FR region from mouse IgG1, IgG2, IgG3, IgG4, or variants thereof.

In a preferred embodiment of the present invention, a mouse antibody or fragment thereof as described above is provided, further comprising a heavy chain constant region from mouse IgG1, IgG2, IgG3, IgG4, or variants thereof.

In a preferred embodiment of the present invention, an anti-CD40 antibody or antigen-binding fragment thereof as described above is provided, which may be a chimeric antibody or fragment thereof.

In a preferred embodiment of the present invention, a chimeric anti-CD40 antibody or fragment thereof is provided, as described above, wherein the light chain variable region sequence is shown under SEQ ID NO: 2 or SEQ ID NO: 10.

In a preferred embodiment of the present invention, a chimeric anti-CD40 antibody or fragment thereof is provided, as described above, wherein the heavy chain variable region sequence is shown under SEQ ID NO: 1 or SEQ ID NO: 9.

In a preferred embodiment, the present invention provides a chimeric anti-CD40 antibody or fragment thereof as described above, further comprising a light chain constant region from the human κ, λ chain, or variants thereof.

In a preferred embodiment, the present invention provides a chimeric anti-CD40 antibody or fragment thereof as described above, further comprising a heavy chain constant region from human IgG1, IgG2, IgG3, IgG4, or variants thereof.

In a preferred embodiment of the present invention, an anti-CD40 antibody or antigen-binding fragment thereof as described above is provided, which is a human antibody or fragment thereof.

In a preferred embodiment of the present invention, an anti-CD40 antibody or antigen-binding fragment thereof as described above is provided, which is a humanized antibody or fragment thereof.

In a preferred embodiment, the present invention provides an anti-human CD40 antibody or antigen-binding fragment thereof as described above, wherein the human antibody has the sequence of SEQ ID NO: 18 or SEQ ID NO: 20, or variants thereof; preferably the variants are characterized by 0-10 amino acid mutations in the light chain, more preferably the mutations are at positions 2 and 3 and both mutations result in amino acids, preferably I, V or L.

In a preferred embodiment, the present invention provides an anti-human CD40 antibody or antigen-binding fragment thereof as described above, wherein the human antibody has the sequence of SEQ ID NO: 17 or SEQ ID NO: 19, or variants thereof; preferably the variants are characterized by 0-10 amino acid mutations in the light chain, more preferably the mutations are at positions 6 and 8 and both mutations result in amino acids, preferably I, A or L.

In a preferred embodiment of the present invention, a human anti-CD40 antibody or antigen-binding fragment thereof, as described above, further comprising an IgG1, IgG2, IgG3, IgG4 constant region or variants thereof is provided.

In a preferred embodiment, the present invention provides a humanized anti-CD40 antibody or antigen-binding fragment thereof as described above, further comprising a light chain FR region from the human κ, λ chain, or variants thereof.

In a preferred embodiment, the present invention provides a humanized anti-CD40 antibody or fragment thereof as described above, wherein the sequence of the light chain FR region in the light chain variable region of the humanized antibody is derived from the human germline IGkV1-33 light chain sequence shown under SEQ ID NO: 22, or derived from the human IGkV2-28 germline light chain sequence shown under SEQ ID NO: 24.

In a preferred embodiment, the present invention provides a humanized anti-CD40 antibody or fragment thereof as described above, wherein the light chain sequence of the humanized antibody is the sequence shown under SEQ ID NO: 33 or SEQ ID NO: 34, or variants thereof.

In a preferred embodiment, the present invention provides a humanized anti-CD40 antibody or fragment thereof as described above, wherein the light chain sequence of the humanized antibody is the sequence shown under SEQ ID NO: 18 or SEQ ID NO: 20, or variants thereof.

In a preferred embodiment, the present invention provides a humanized anti-CD40 antibody or fragment thereof as described above, wherein the light chain variable region variants of the humanized antibody preferably have 0-10 amino acid mutations in the light chain, more preferably the mutations are at positions 2 and 3, and as a result both mutations are replaced by amino acids, preferably I, V or L.

In a preferred embodiment, the present invention provides a humanized anti-CD40 antibody, or fragment thereof, as described above, further comprising a human κ-, λ-chain light chain constant region, or variants thereof.

In a preferred embodiment, the present invention provides a humanized anti-CD40 antibody or fragment thereof as described above, further comprising a heavy chain FR region from human IgG1, IgG2, IgG3, IgG4, or variants thereof.

In a preferred embodiment, the present invention provides a humanized anti-CD40 antibody or fragment thereof as described above, wherein the heavy chain variable region FR region from the heavy chain is derived from the human germline heavy chain sequence IGHV1-69 set forth under SEQ ID NO: 21, or derived from the human IGHV1-2 germline heavy chain sequence shown under SEQ ID NO: 23.

In a preferred embodiment of the present invention, a humanized anti-CD40 antibody or fragment thereof is provided, as described above, wherein the heavy chain sequence of the humanized antibody is shown under SEQ ID NO: 26 or SEQ ID NO: 30, or variants thereof.

In a preferred embodiment, the present invention provides a humanized anti-CD40 antibody or fragment thereof as described above, wherein the heavy chain sequence of the humanized antibody is shown under SEQ ID NO: 17 or SEQ ID NO: 19, or variants thereof, preferably the variants are characterized by 0-10 amino acid mutations in the variable region of the heavy chain, more preferably, the mutations are at positions 6 and 8, and the mutation results in a change to amino acids, preferably I, A or L.

In a preferred embodiment, the present invention provides a humanized anti-CD40 antibody or fragment thereof as described above, further comprising a human IgG1, IgG2, IgG3, IgG4 heavy chain constant region or variants thereof, preferably an IgG1, IgG2 or IgG4 heavy chain FR region, more preferably FR region of the heavy chain of IgG1 or IgG2.

In a preferred embodiment, the present invention provides an anti-CD40 antibody or antigen binding fragment as described above, wherein the antigen binding fragment is a Fab, Fv, sFv, F(ab') ₂ , a linear antibody, a single chain antibody, a nanobody, a domain antibody, or a polyspecific antibody.

The present invention further provides a DNA sequence encoding an anti-CD40 antibody or antigen-binding fragment, polyspecific antibody or single chain antibody as described above.

The present invention further provides an expression vector containing the DNA sequence described above.

The present invention further provides host cells transformed with the expression vector described above.

In a preferred embodiment of the present invention, host cells are provided as described above, wherein the host cell is a bacterium, preferably Escherichia coli.

In a preferred embodiment of the present invention, the host cells described above are yeast, preferably Pichia pastoris.

In a preferred embodiment of the present invention, the host cells described above are mammalian cells, preferably Chinese Hamster Ovary (CHO) cells or Human Embryonic Kidney (HEK) 293 cells.

The present invention further provides a single chain antibody comprising an anti-CD40 antibody, or antigen-binding fragment thereof, as described above.

The present invention further provides a polyspecific antibody comprising an anti-CD40 antibody or antigen-binding fragment thereof as described above.

The present invention further provides an antibody-drug conjugate comprising a light chain variable region and/or a heavy chain variable region of an anti-CD40 antibody described above. Antibody-drug conjugates are well known in the art and are formed by antibody-linker-drug (toxin) coupling, and known linkers include cleavable linkers, sub-cleavable linkers such as linkers including, but not limited to, SMCC, SPDP, etc. P. Toxins are also well known in the art, such as DM1, DM4, MMAE, MMAF, and the like.

The present invention further provides a pharmaceutical composition comprising an anti-CD40 antibody or antigen-binding fragment thereof, a polyspecific antibody or a single chain antibody, and a pharmaceutically acceptable excipient, diluent or carrier as described above.

The present invention further provides for the use of an anti-CD40 antibody or antigen-binding fragment thereof, a polyspecific antibody, a single chain antibody, an antibody drug conjugate, or a pharmaceutical composition as described above in the manufacture of a medicament for the treatment or prevention of a CD40 or CD40L mediated disease or condition; where the disease is preferably cancer; wherein the cancer is most preferably lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer , multiple myeloma, leukemia, gallbladder cancer, glioblastoma and melanoma.

The present invention further provides a method for treating and preventing a disease or condition mediated by CD40 or CD40L, the method comprising administering to a subject a therapeutically effective dose of an anti-CD40 antibody or antigen-binding fragment thereof, a polyspecific antibody, a single chain antibody, an antibody-drug conjugate, or a pharmaceutical composition as described above, where the disease is preferably cancer; the cancer is most preferably lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma and melanoma.

The present invention further provides for the use of an anti-CD40 antibody or antigen-binding fragment thereof, a polyspecific antibody, a single chain antibody, an antibody-drug conjugate or a pharmaceutical composition as described above in the manufacture of a medicament for improving symptoms in a patient with an autoimmune disease.

The present invention further provides for the use of an anti-CD40 antibody or antigen-binding fragment thereof, a polyspecific antibody, a single chain antibody, an antibody-drug conjugate, or a pharmaceutical composition as described above in the manufacture of a medicament for improving symptoms in a patient with an inflammatory disease.

Brief description of graphic materials

In FIG. 1 shows activation of a mouse anti-human CD40 antibody on DC cells based on CD80 activating molecules.

In FIG. 2 shows activation of a mouse anti-human CD40 antibody on DC cells based on CD86 activating molecules.

Fig. 3 is a graph showing tumor growth curves of transplanted Raji lymphoma co-grafted with human PBMC and DC cells.

Fig. 4 is a graph showing body weight changes in NOG mice transplanted with Raji-grafted lymphoma co-grafted with human PBMC and DC cells.

Detailed Description of the Preferred Embodiment

1. Definitions

To make it easier for those skilled in the art to understand the present invention, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise elsewhere in this document, all other technical and scientific terms used in this document have the meaning that is generally known to a person skilled in the art to which the present invention pertains.

The amino acid three-letter code and one-letter code used in the present invention are described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody" as used in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure formed by linking two identical heavy chains and two identical light chains through interchain disulfide bonds. Since the constant region of the heavy chain of an immunoglobulin has a different composition and order of amino acids, the antigenicity of different immunoglobulins is different. Accordingly, immunoglobulins can be classified into five classes or can be referred to as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and whose respective heavy chains are μ-chain, δ-chain, γ-chain, α-chain and ε chain respectively. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region, as well as in the number and position of heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3 and IgG4. Light chains can be classified as κ-chain or λ-chain according to the difference in the constant region. Each of the five Ig classes can have a κ chain or a λ chain.

In the present invention, the light chain of an antibody of the present invention may further comprise a light chain constant region that provides for a human or mouse κ, λ chain, or variants thereof.

In the present invention, the heavy chain of an antibody of the present invention may further comprise a heavy chain constant region that provides for human or mouse IgG1, IgG2, IgG3, IgG4, or variants thereof.

The variable region (V-region), consisting of about 110 amino acids close to the N-terminus of the heavy and light chains of an antibody, varies greatly in its amino acid sequence. The constant region (C-region), consisting of the remaining amino acid sequence close to the C-terminus of the antibody, is relatively stable. The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions that determine the specificity of an antibody are also referred to as the complementarity determining region (CDR). Each of the light chain variable region (VL) and the heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three light chain CDR regions are LCDR1, LCDR2 and LCDR3; the three heavy chain CDR regions are HCDR1, HCDR2 and HCDR3. The number and position of the amino acid residues of the CDRs of the VL and VH regions of the antibodies or antigen-binding fragments of the present invention correspond to known IMGT numbering criteria.

The term "antigen presenting cell" or "APC" is a cell that displays on its surface a foreign antigen that forms a complex with the MHC. T cells recognize this complex through the T cell receptor (TCR). Examples of APCs include, without limitation, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes, B lymphoblasts, and monocyte-derived dendritic cells (DCs). The term "antigen presentation" refers to the process by which APCs capture antigens and allow them to be recognized by T cells, eg as a component of MHC-I/MHC-II conjugates.

The term "CD40" refers to a cell surface receptor that is a member of the TNF receptor superfamily, also known as TNFRSF5. CD40 is ubiquitously expressed on the surface of dendritic cells, B cells, and macrophages and is a molecule essential for the development and maintenance of T cell immunity. The term "CD40" includes any CD40 variant or isoform that is naturally expressed by a cell. The antibodies of the present invention may be cross-reactive with non-human CD40. Alternatively, the antibody may be specific for human CD40 and may not be cross-reactive with other species. CD40, or any variant or isoform thereof, may be isolated from cells or tissues in which it is naturally expressed, or produced by recombinant techniques using techniques known in the art and described herein. Preferably, the anti-CD40 antibody targets human CD40 with a normal glycosylation profile.

The term "recombinant human antibody" includes a human antibody that is produced, expressed, engineered, or isolated using recombinant techniques, and the related techniques and methods are well known in the art, for example, (1) an antibody isolated from transgenic transchromosomal animals (e.g., mice ) containing the immunoglobulin gene or hybridomas derived from it; (2) an antibody isolated from host cells transformed to express antibodies, such as a transfectoma; (3) an antibody isolated from a recombinant human combinatorial antibody library; and (4) an antibody produced, expressed, constructed, or isolated by splicing a human immunoglobulin gene sequence with other DNA sequences, and the like. Such a recombinant human antibody contains variable regions and constant regions, in which not only the specific human immunoglobulin germline sequences encoded by the germline genes, but also subsequent rearrangements and mutations, such as those occurring during maturation of the antibody, are contemplated.

The term "mouse antibody" in the present invention is a monoclonal antibody to human CD40, obtained in accordance with the knowledge and skill in the art. The test subject is injected with the CD40 antigen at the time of preparation, and then a hybridoma expressing an antibody having the desired sequence or functional properties is isolated. In a preferred embodiment of the present invention, the mouse anti-CD40 antibody or antigen-binding fragment thereof may further comprise a light chain constant region from mouse κ-, λ-chain or variants thereof, or further comprise a heavy chain constant region from mouse IgG1, IgG2, IgG3 or IgG4 or its options.

The term "human antibody" includes antibodies having variable and constant regions of human immunoglobulin germline sequences. The human antibody of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation). However, the term "human antibody" does not include an antibody in which a CDR sequence derived from a germ type of another mammalian species, such as a mouse, has been grafted onto human framework sequences (i.e., "humanized antibody").

The term "humanized antibody", also known as a CDR-grafted antibody, refers to an antibody obtained by grafting a mouse CDR sequence into the framework regions of a human antibody variable region. It can overcome the intense immune response caused by a chimeric antibody carrying a large number of mouse protein components. To avoid a decrease in activity caused by a decrease in immunogenicity, the variable region of a human antibody can be subjected to minimal backmutation in order to maintain activity.

The term "chimeric antibody" is an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can provide attenuation of the immune response induced by the mouse antibody. To construct a chimeric antibody, a hybridoma that secretes a mouse specific monoclonal antibody is first constructed and selected, then the variable region gene is cloned from the mouse hybridoma cell. The human antibody constant region gene is then cloned as needed. The mouse variable region gene and the human constant region gene are ligated into a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of a human antibody can be selected from a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, and preferably comprises a heavy chain constant region of human IgG1 or IgG2.

The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analog, which typically includes at least a portion of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody. An antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of its original binding activity when specified in moles. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , Fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and polyspecific antibodies. Designed antibody variants are discussed in Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

The "Fab fragment" consists of the light chain and CH1 and the variable region of the heavy chain. The heavy chain of a Fab molecule cannot form disulfide(s) bond(s) with another heavy chain molecule.

The "Fc" region contains two heavy chain fragments containing the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interaction of the CH3 domains.

The "Fab'fragment" contains a light chain and a heavy chain portion containing a VH domain, a CH1 domain, and a region between the CH1 and CH2 domains. Thus, interchain disulfide bonds can form between two heavy chains to form an F(ab') ₂ molecule.

The "F(ab') ₂ fragment" contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains, thereby forming disulfide bonds between the two heavy chains. Thus, the F(ab') ₂ fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

The "Fv region" contains both heavy and light chain variable regions, but no constant region.

The term "multispecific antibody" is used in its broadest sense to encompass antibodies having specificity for multiple epitopes. These multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit is specific for multiple epitopes; antibodies having two or more VL and VH regions, with each VH-VL unit binding to different targets or different epitopes on the same target; antibodies having two or more single variable regions, each single variable region binding to different targets or different epitopes of the same target; full length antibodies, antibody fragments, diabodies, bispecific diabodies and tribodies, antibody fragments that are covalently or non-covalently linked to each other, and the like.

The term "antibody-drug conjugate" (ADC) refers to an antibody or antibody fragment conjugated to one or more heterologous chemically synthesized molecules, including, without limitation, an antibody or antibody fragments conjugated to cytotoxic agents.

The term "single chain antibody" is a single chain recombinant protein formed by joining the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody through a linker peptide, which is the smallest fragment of an antibody with complete antigen-binding regions.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region chain. In some instances, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment may target the same or different antigens.

As used herein, the term "binding to CD40" refers to the ability to interact with human CD40. As used in the present invention, the term "antigen-binding site" refers to a three-dimensional spatial area that has discreteness on the antigen and is recognized by the antibody or antigen-binding fragment of the present invention.

The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. An epitope can be formed by contiguous amino acids, non-contiguous amino acids located side by side due to tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually preserved after exposure to denaturing solvents, while epitopes formed by tertiary folding are usually destroyed after treatment with denaturing solvents. Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitopes are associated with a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, and the like. Methods for determining the spatial conformation of epitopes include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

As used herein, the terms "specifically bind" or "selectively bind" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, when recombinant human CD40 is used as an analyte and an antibody is used as a ligand, the antibody binds to a predetermined antigen with an equilibrium dissociation constant (K _D ) of less than about 10 ^-7 M or even less when measured with using Surface Plasmon Resonance (SPR) techniques in the instrument, and its binding affinity for a predetermined antigen is at least twice that of its binding affinity for a non-specific antigen (such as BSA, etc.) other than the predetermined antigen or a closely related antigen. The term "antibody(s) that recognize(s) an antigen" may be used herein interchangeably with the term "antibody(s) that specifically bind(s)".

The term "cross-reactivity" refers to the ability of an antibody of the present invention to bind to CD40 from different species. For example, an antibody of the present invention that binds to human CD40 can also bind to CD40 from another species. Cross-reactivity is measured by determining the specific reactivity of antibodies with purified antigens in binding assays (eg SPR and ELISA) or by antibody binding or functional interactions with cells that physiologically express CD40. Methods for determining cross-reactivity include the standard binding assays described herein, such as surface plasmon resonance (SPR) analysis or flow cytometry.

The terms "suppress", "suppression", "blockade" or "blockage" are used interchangeably and cover both partial and complete suppression/blockade. Suppression/blockade of the ligand preferably reduces or alters the normal level or pattern of activity, which occurs when ligand binding occurs without suppression or blockade. Inhibition and blockade are also expected to include any measurable reduction in the binding affinity of a ligand upon contact with an anti-CD40 antibody compared to a ligand that is not in contact with an anti-CD40 antibody.

The term "growth inhibition" (eg, involving cells) is intended to include any measurable reduction in cell growth.

The terms "inducing an immune response" and "enhancing an immune response" may be used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a particular antigen. The term "inducing", especially in the case of inducing CDC or ADCC, refers to the stimulation of a specific mechanism of direct cell killing.

"ADCC" as described in the present invention, that is, antibody-dependent cell-mediated cytotoxicity, means that cells expressing Fc receptors directly kill antibody-coated target cells by recognizing the Fc segment of an antibody. The effector function of an ADCC antibody can be reduced or eliminated by modifying the Fc segment to IgG. Modification refers to mutations in the heavy chain constant region of the antibody, such as mutations selected from the group consisting of N297A, L234A, L235A in the case of IgG1; chimeras IgG2/4, F235E in case of IgG4 and mutation L234A/E235A.

Methods for preparing and purifying antibodies and antigen binding fragments are well known and can be found in the art, for example, in Using Antibodies: A Laboratory Manual, Chapters 5-8 and 15, Cold Spring Harbor. For example, a mouse can be immunized with human CD40 or a fragment thereof, and the resulting antibody can be annealed, purified, and subjected to amino acid sequencing in a conventional manner. The antigen binding fragment can also be obtained in a conventional manner. The antibodies or antigen-binding fragments of the present invention are genetically engineered to introduce one or more human FR regions into a non-human CDR region. Human germline FR sequences are available from the ImMunoGeneTic (IMGT) website http://imgt.cines.fr or from The Immunoglobulin FactsBook, 2001 ISBN 014441351.

The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. CHO cells can be stably transfected with a recombinant immunoglobulin expression vector. As a more preferred method, well known in the art, mammalian expression systems will provide antibody glycosylation, in particular at the highly conserved N-terminus of the FC region. Stable clones are obtained by carrying out the expression of antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in a bioreactor to obtain antibodies. The culture medium into which the antibody is secreted can be purified and harvested by conventional techniques. The antibody can be concentrated by conventional filtration. Soluble mixtures and multimers can also be removed by traditional methods such as molecular sieves, ion exchange. The resulting product must be immediately frozen, for example at -70°C, or lyophilized.

The antibody of the present invention relates to a monoclonal antibody. The monoclonal antibody (mAb) of the present invention refers to an antibody derived from a single clonal cell line, and the cell line is not limited to a eukaryotic, prokaryotic or phage clonal cell line. Monoclonal antibodies or antigen-binding fragments can be generated recombinantly using, for example, hybridoma technology, recombinant techniques, phage display technology, synthetic techniques (eg, CDR grafting), or other prior art techniques.

When applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, the terms "administration" and "treatment" refer to bringing an exogenous drug, therapeutic agent, diagnostic agent, or composition into contact with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes bringing the reagents into contact with the cells, as well as bringing the reagents into contact with a liquid, where the liquids are in contact with the cells. "Introduction" and "treatment" also means the treatment of, for example, cells in vitro and ex vivo with reagents, diagnostics, binding compositions or another cell. "Treatment", when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

"Therapy" means the administration of an internal or external therapeutic agent, such as a composition containing any of the binder compounds of the present invention, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount that provides effective relief of the symptoms of one or more diseases in the patient or population being treated, whether by inducing degeneration of such symptoms or suppressing the progression of such symptoms to some degree not clinically measurable. The amount of a therapeutic agent (also referred to as a "therapeutically effective amount") effective to relieve the symptoms of a particular disease may vary depending on a variety of factors such as the condition of the disease, the age and weight of the patient, and the ability of the drug to produce the desired effect in patient. How much the symptoms of a disease have been reduced can be assessed using any clinical trial method commonly used by a physician or other healthcare professionals to assess the severity or progression of a disease. Although embodiments of the present invention (e.g., therapeutic methods or formulations) may not be effective in alleviating the symptoms of the target disease common to each patient, it has been found that the symptoms of the target disease should be relieved in a statistically significant number of patients in accordance with any methods. statistical tests known in the art, such as Student's t-test, chi-square test, Mann-Whitney U-test, Kruskal-Wallis (H-test), Jonkheer-Terpstra test, and Wilcoxon test.

"Conservative modification" or "conservative substitution or substitution" refers to the replacement of amino acids in proteins with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that changes could often be made without altering the biological activity of the protein. It is known to those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not significantly alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co. ., Page 224 (4th ^edition )). In addition, substitutions of structurally or functionally similar amino acids are unlikely to impair biological activity. Common conservative amino acid substitutions are as follows.

The term "consisting essentially of" or variations thereof, as used throughout the specification and claims, includes all such elements or groups of elements, and does not necessarily include other elements that are similar or different in nature from the said elements, provided that the other elements do not substantially change basic or novel features of a given dosing regimen, method or composition. As a non-limiting example, a binding compound consisting essentially of the specified amino acid sequence may also include one or more amino acids that do not significantly affect the properties of the binding compound.

The term "naturally occurring" as applied to an object in accordance with the present invention refers to the fact that the object can be found in nature. For example, a polypeptide sequence or polynucleotide sequence present in an organism (including viruses) that can be isolated from a natural source and that has not been deliberately artificially modified in a laboratory is naturally occurring.

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or sign of a disease state. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on such factors as the condition being treated, the general health of the patient, the route and dosage of the route of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage schedule to avoid significant side effects or toxic effects.

"Exogenous" refers to a substance that is produced outside of a living organism, cell, or human body according to its environment. "Endogenous" refers to a substance produced in a cell, organism, or human body according to its environment.

"Homology" refers to the sequence similarity between two polynucleotide or polypeptide sequences. If positions in both compared sequences are occupied by the same monomeric subunit, which is a base or amino acid, for example, if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, when sequences are optimally aligned, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. In general, comparisons are made when the maximum percentage of homology has been obtained by aligning two sequences.

As used herein, the expressions "cell", "cell line" and "cell culture" may be used interchangeably, and all such names include their progeny. Thus, the words "transformant" and "transformed cells" include the primary test cells and cultures derived from them, regardless of the number of transfers. It should also be understood that all progeny may not be completely identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as those screened in the originally transformed cell are included. When different names are meant, they are clearly distinguishable from the context.

"Optional" or "optional" means that the event or environment described hereinafter may, but need not, occur, including if the event or environment does or does not occur. For example, "optionally containing 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a particular sequence may be present, but need not be.

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein, or their physiologically/pharmaceutically acceptable salt or prodrug and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to provide an administration to the body that promotes absorption of the active ingredient and thereby exerts biological activity.

Detailed Description of the Preferred Embodiment

The following examples further illustrate the present invention and should not be construed as limiting the present invention. Experimental methods without specifying specific conditions in the embodiments of the present invention are usually carried out in accordance with conventional conditions, such as in Using Antibodies: A Laboratory Manual, or Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; or in accordance with the conditions recommended by the manufacturer of the raw material or product. Reagents without source indication are common commercially available reagents.

Example 1 Immune Antigen, Screened Antigen Sequence and Production

His-tagged recombinant human CD40 protein (h-CD40-his), Fc-tagged recombinant human CD40 protein (h-CD40-Fc), His-tagged recombinant mouse CD40 protein (m-CD40-his), and His-tagged macaque CD40 -rhesus (rhesus-CD40-his), recombinant protein (#CD0-C52H7) was a purified industrial protein purchased from Acrobiosystems and the sources of their respective sequences are shown in Table 1. These proteins can be used in the experiments of the following examples.

Пример 2. Получение гибридомы антител

Example 2 Obtaining an antibody hybridoma

Anti-human CD40 monoclonal antibodies were obtained by immunizing mice, which were C57BL/6 experimental mice, female, 6-8 weeks old [Zhao Yan (Suzhou) New Drug Research Center Co., Ltd., animal production license number: 201503259 ]. Feeding environment: SPF level. Mice were purchased and fed in a laboratory environment for 1 week with a 12/12 hour light/dark cycle at 20-25°C and 40-60% humidity. Mice that adapted to the environment were divided into 2 cages with 5 mice in each cage.

The immune antigen was an Fc-labeled modified recombinant human CD40 protein (h-CD40-Fc formulated in phosphate buffer at a concentration of 1 µg/µl). Freund's adjuvant (Sigma, lot #: F5881/F5506) was used for emulsification, where complete Freund's adjuvant (CFA) was used for primary immunization and nucleic acid (CpG, Sangon Biotech) and aluminum adjuvant for injection (Imject Alum), Thermo, Lot No: PH203866) for the remaining booster. Immunization was performed on day 0, day 14, day 28, day 42, day 56, and day 70. Blood was collected on days 21, 35, 49, 63, and 77 for blood analysis, and serum mouse blood was detected using ELISA to determine the antibody titer in mouse serum.

After the fourth immunization, spleen cells were fused from mice in which the antibody titer was high and tended to be stable in serum. Booster immunization was performed 3 days prior to confluence and 10 μg of phosphate buffered antigen solution was administered intraperitoneally (IP) to each mouse. Spleen lymphocytes were fused with Sp2/0 cell myeloma cells (ATCC® CRL-8287TM) using an optimized PEG-mediated fusion step to obtain hybridoma cells and five monoclonal hybridoma cell lines with good in vitro activity were selected.

Example 3 ELISA Binding Assay

The binding properties of antibodies to CD40 were detected using ELISA analysis. The his-tagged recombinant CD40 protein was used directly for coating, after adding the antibody, the binding activity of the antibody to the antigen was detected by adding a secondary antibody (HRP-conjugated anti-Fc antibody) and TMB substrate for HRP.

A 96-well microtiter plate was coated with 100 µl/well of human or rhesus monkey CD40-his protein at 0.5 µg/ml and incubated overnight at 4°C. The plate was washed three times with wash buffer, 250 µl per well. With each wash, the plate was shaken for 10 seconds to ensure sufficient cleaning. 200 μl blocking solution was added to each well and incubated for 2 hours at room temperature. The plate was then washed three times with 250 µl per well. The plate was shaken for 10 seconds with each rinse to ensure sufficient cleaning. 100 µl of test anti-CD40 antibody, diluted according to the dilutions, was added to each well and incubated for 1 hour at room temperature, then the plate was washed three times with 250 µl of wash buffer per well. 100 µl of HRP-labeled goat anti-human IgG secondary antibody diluted 1:20,000 with diluent was added to each well and incubated for 1 hour at room temperature. The plate was then washed three times with 250 µl per well. 100 μl TMB was added to each well and the reaction was allowed to proceed for 15 minutes in the dark. 50 µl of 0.16 M sulfuric acid was added per well. The OD value was measured at 450 nm using a Thermo MμltiSkanFc plate reader and the anti-CD40 antibody EC50 binding to CD40 was calculated.

Table 2 ELISA Binding of Mouse Hybridoma Antibodies to Different CD40 Germ Types

Вариант осуществления 4. Антитело к CD40 блокирует связывание CD40 и CD40L

Embodiment 4 Anti-CD40 Antibody Blocks CD40 and CD40L Binding

In this experiment, a screened anti-human CD40 antibody was found to block binding of human CD40 and human CD40L using an in vitro blocking assay. Specifically, Fc-labeled recombinant CD40 protein (h-CD40-Fc) was coated on a 96-well microtiter plate. After the anti-CD40 antibody was added to fully bind and capture the epitope, his-tagged CD40L was added. By detecting his-tag, the amount of bound CD40 to CD40L was calculated, and then the IC50 value of the CD40 antibody blocking the CD40 active site was calculated.

A 96-well microtiter plate was coated with 100 µl/well of human or rhesus monkey CD40-Fc protein at 1 µg/ml and incubated overnight at 4°C. The plate was washed three times with wash buffer, 250 μl per well. With each wash, the plate was shaken for 10 seconds to ensure sufficient cleaning. 200 μl blocking solution was added to each well and incubated for 2 hours at room temperature. The plate was then washed three times with 250 µl per well. The plate was shaken for 10 seconds with each rinse to ensure sufficient cleaning. 100 µl of test anti-CD40 antibody, diluted according to the dilutions, was added to each well and incubated for 1 hour at room temperature, then the plate was washed three times with 250 µl of wash buffer per well. 100 µl of HRP-labeled goat anti-human IgG secondary antibody diluted 1:2000 with diluent was added to each well and incubated for 1 hour at room temperature. The plate was then washed three times with 250 µl per well. 100 μl TMB was added to each well and the reaction was allowed to proceed for 15 minutes in the dark. 50 µl of 0.16 M sulfuric acid was added per well. The OD value was measured at 450 nm using a Thermo MμltiSkanFc plate reader and the IC50 value of the CD40 antibody blocking the binding of CD40 to CD40L was calculated.

Table 3 Blocking ELISA results with human hCD40/hCD40L

Embodiment 5 Affinity Determination with Biacore

The captured human antibody was covalently bound to the CM5 biosensor chip of the Biacore instrument (Biacore X100, GE) according to the method described in the description of the Human Antibody Capture Kit (Cat. No. BR-1008-39, GE), thus capturing a certain amount chimeric or humanized antibodies to be tested based on affinity. A concentration gradient series of CD40 antigens (purchased from Acrobiosystems) was then run across the surface of the chip and Biacore X100 (GE) was used to detect reaction progress signals in real time to generate binding and dissociation curves. After completion of each dissociation cycle, the biochip was rinsed and regenerated with the regeneration solution provided in the human antibody capture kit. Amine coupling kits (p/n BR-1000-50, GE) were purchased from GE, and HBS-EP buffer + buffer 1× (pH 7.4) was prepared from HBS-EP + buffer 10× (p/n BR-1006-69, GE) by dilution with water D.I. Experimental data were fitted with BiacoreX100 Evaluation Software 2.0 (GE software) with a binding model (1:1) to obtain affinity values as shown in Tables 10 and eleven.

Embodiment 6. Assay for cellular activity of an anti-CD40 antibody based on a reporter gene

HEK-Blue CD40L cells were purchased from Invitrogen (Cat. No. hkb-cd40), which are stably transfected with the human CD40 gene and the NF-κB-mediated SEAP genome. Thus, the level of activation of the signaling pathway involving CD40 can be characterized by detecting secreted SEAP in the supernatant using QUANTI-Blue (SEAP substrate). In this experiment, in vitro CD40 antibody cell viability was assessed according to EC50 value by detecting CD40L activation of HEK-Blue cells. HEK-Blue CD40L cells were cultured in DMEM containing 10% FBS, 100 μg/ml Zeocin and 30 μg/ml blasticidin and passaged 2-3 times a week, and the passage ratio was 1:5 or 1:10. When subculturing the cells, the medium was removed by pipetting and the cell layer was rinsed with 5 ml of 0.25% trypsin. The trypsin was then removed by pipetting, the cells were digested in an incubator for 3-5 minutes, followed by suspension of the cells with fresh medium. 100 μl of the cell suspension was added to a 96-well cell culture plate at a density of 5 x 10 ⁵ cells/ml and the culture medium was DMEM containing 10% FBS, 100 μg/ml zeocin and 30 μg/ml blasticidin. Meanwhile, only 100 µl of sterile water was added to the periphery of the 96-well plate. The plates were incubated in an incubator for 24 hours (37°C, 5% CO ₂ ). After the cells adhered to the wall, 100 μl of the gradient-diluted antibody to be tested was added to each well. The plates were incubated in an incubator for 20-24 hours (37°C, 5% CO ₂ ). Then 40 µl of cell supernatant was taken from each well into a new 96-well flat bottom plate, 160 µl of QUANTI-Blue substrate solution was added and the plate was incubated in an incubator for 1-3 hours in the dark. Absorbance at 620 nm was measured with a microplate reader (Thermo M μlti SkanFc) and an EC ₅₀ value was calculated to assess in vitro cell viability for anti-CD40 antibody.

Table 4. Indicators of cellular activity of the anti-CD40 antibody based on the reporter gene

Embodiment 7. DC Cell Activation Assay with Anti-cd40 Antibody

PBMCs were isolated from normal human peripheral blood and then monocytes were sorted using CD14 MACS beads. RPMI 1640 medium containing 10 ng/ml IL4 and 100 ng/ml GM-CSF was added to the cells and cultured for 6 days, thereby inducing MoDC (monocyte-derived dendritic cells) cell culture. After 6 days, cells were harvested, 1×10 ⁵ cells were taken and stained with CD209-PE, CD1a-PerCP/Cy5.5 and CD14-PE/Cy7 and FACS was used to analyze whether MoDC was successfully induced (all of the above operations are traditional operations in the art). Successfully induced DCs were pooled and a range of test antibodies and reference antibodies were added, respectively, with appropriate concentration gradients (see FIG. 1 for antibody concentration gradient). After 48 hours of culture, cells were harvested and then stained with CD80, CD86 and HLA-DR and data was collected by FACS analysis. Based on primary DC cell activation assay data, all five mouse antibodies showed significant activity and activated CD80 and CD86 activation molecules on the DC surface in a dose-dependent manner. The overall effect of the five antibodies is comparable to or slightly better than the two reference antibodies (CP-870, 893 from Pfizer and ADC-1013 from Alligator Bioscience). See fig. 1 and FIG. 2.

Embodiment 8: Anti-CD40 Antibody Cloning and Sequencing

Hybridoma subclones of the five antibodies screened and identified above were selected and hybridoma cells that were in logarithmic growth phase were harvested. RNA was extracted using Trizol (Invitrogen, 15596-018) according to kit instructions and reverse transcribed (PrimeScript™ reverse transcriptase, Takara, Cat # 2680A). The resulting cDNA was then amplified by PCR using a mouse Ig-Primer kit (Novagen, TB326 Rev. B 0503) and sent to a sequencing company for sequencing. Finally, the sequences of 5 mouse antibodies were obtained.

The sequences of the heavy and light chain variable regions of the mouse mAb 2H6 are as follows:

HCVR 2H6

QVQLQQSGAELVRPGTSVKVSCKASGYAFSDYLIEWAKQRPGQGLEWIGVINPGSGGSNYNEK

IKDRATLTADKSSSTAYMQLSSLTSEDSAVYFCARGGGGFTYWGQGTLVTVSA

SEQ ID NO: 1

LCVR 2H6

EIQLTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTIKLLLNFASRLHSGVPSRFS GSGSGTDFFLTISNLEQDDIATYFCQQGSTLPWTFGGGTKLEIK

SEQ ID NO: 2

The CDR sequences of mAb 2H6 are shown in Table 5 below.

Table 5

The sequences of the mouse 9E5 heavy and light chain variable regions are as follows.

HCVR 9E5

QVQLQQPGADLVKPGASVKMSCKASGYILTTYWITWVKQRPGQGLEWIGDIHPGSGSTKYNEK FKSKATLTVDTSSSTAYMQLTRLSSEDSAVYYCARRDYWGQGTTLTVSS

SEQ ID NO: 9

LCVR9E5

DVLMTQSPLSLPVSLGDQASISCRSSQNIVNSQGNTYLEWYLQKPGESPKLLIYKVTNRFSGV PDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASLVPWTFGGGTKLEIK

SEQID NO：10

The 9E5 CDR sequences are shown in Table 6 below.

Table 6

Последовательности вариабельных областей тяжелой и легкой цепей 1D9 являются следующими.

The sequences of the variable regions of the 1D9 heavy and light chains are as follows.

HCVR 1D9

QVRLQQSGAELVRPGTSMRVSCKASGYAFTNYLINWVKQRPGQGLEWIGILNPGSGGTNYNEN FKDKATLTADKSSNTAYMQLSSLTSEDSAVYFCIRGSPGFAYWGQGTLVTVSA

SEQ ID NO: 37

LCVR 1D9

DIQMTQTTSSLSASLGDRVTISCRASQDINIYLNWYQQKPDGTVKLLIYSTSGLHSGVPSRFN GSGSGTDYSLTISNLEQEDIATYFCQQGYTLPYTFGGGTKLEIK

SEQ ID NO: 38

The 1D9 CDR sequences are shown in Table 7 below.

Table 7

The sequences of the variable regions of the 14C10 heavy and light chains are as follows.

HCVR 14C10

FKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARGGGGFTYWGQGTLVTVSA

SEQ ID NO：45

LCVR 14C10

HIQMTQTTSSLSASLGDRVTISCRASQDISSHLNWYQQKPDGTVKLLISYTSRLHSGVPSRFS GSGSGADYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK

SEQ ID NO：46

The 14C10 CDR sequences are shown in Table 8 below.

Table 8

The sequences of the variable regions of the 38B4 heavy and light chains are as follows.

HCVR38B4

QVRLKQSGAELVRPGASVKVSCKASGYTFTDYYINWVKQRPGQGLEWIAGIYPGTGNTYYNEK FKGKATLTAERSSSTAYMQLTSLTSEDSAVYFCTRRGLPSLCFDYWGQGTTLTVSS

SEQ ID NO：53

LCVR38B4

DFQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFS GSGSGTDYSLTISNLEPEDIATYYCQQYSKLPPTFGGGTKLEIK

SEQ ID NO：54

The 38B4 CDR sequences are shown in Table 9 below.

Table 9

Among them, two optimal antibodies, 2H6 and 9E5, were subjected to further development. The obtained variable region sequences were ligated with the human IgG1 antibody constant region sequence, respectively, to obtain a chimeric antibody sequence with human and mouse sequences, and the chimeric antibody sequence was inserted into the expression vector pCP (purchased from MabSpace biology Co., Ltd) using a molecular cloning technique. These sequences were sequenced and identified after amplification by PCR (molecular biological working methods such as molecular cloning are performed according to traditional working conditions and can be referred specifically to "Molecular Cloning: A Laboratory Manual", and a cell-based expression system HEK293 can be used to generate chimeric antibodies with human and mouse sequences, 2H6-C and 9E5-C Various in vitro activity assays were performed to characterize chimeric antibodies purified by MabSelect SuRe affinity chromatography (GE Lifesciences) Data are shown in Table 10.

Table 10. In vitro activity of chimeric antibodies

Embodiment 9. Mouse Antibody Humanization Experiment

Based on the typical VH/VLCDR structure of the resulting murine 2H6 and 9E5 antibodies, the heavy and light chain variable region sequences were compared with a germ-type antibody database to obtain a human germ-type template with high homology. While the human light chain framework region of the germline type is derived from the human κ light chain gene, the light chain framework region of the antibody of the present invention is preferably a human Vk1-33/JK4 (2H6) or Vk2-28/JK4 (9E5) light chain template. embryonic type. The human heavy chain framework region of the germline type is derived from a human heavy chain, and the heavy chain framework region of the antibody of the present invention is preferably a human VH1-69/JH6 (2H6) or VH1-2/JH6 (9E5) germline heavy chain template as shown below.

The 2H6 heavy chain framework region is preferably the human IGHV1-69 germline heavy chain template (SEQ ID NO: 21):

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQK FQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR.

The 2H6 light chain framework region is preferably a human germline type IGkV1-33 light chain template (SEQ ID NO: 22):

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFS GSGSGTDFTFTISSLQPEDIATYYCQQYDNLP.

The 9E5 heavy chain framework is preferably a human germline heavy chain IGHV1-2 template (SEQ ID NO: 23):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQK FQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR.

The 9E5 light chain framework region is preferably a human germline type IGkV2-28 light chain template (SEQ ID NO: 24):

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGV PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP.

The mouse antibody CDR regions were grafted onto the selected humanized template with the replacement of the humanized variable region and then recombined with the corresponding human IgG constant region (preferably the heavy chain is IgG1 and the light chain is κ). Then, based on the three-dimensional structure of the mouse antibody, inserted residues, residues that have direct interaction with CDRs, and residues that have an important influence on VL and VH conformation were backmutated, and chemically unstable amino acid residues of the CDR regions were optimized to obtain the final humanized molecule. Their heavy chain variable region sequences are shown under SEQ ID NOs: 25-30 and their light chain variable region sequences are shown under SEQ ID NOs: 31-36.

hu2H6-H1a（SEQ ID NO: 25）：

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYLIEWVRQAPGQGLEWMGVINPGSGGSNYNEK IKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGGGFTYWGQGTLVTVSS

hu2H6-H1b（SEQ ID NO: 26）：

QVQLVQSGAEVKKPGSSVKVSCKASGYAFSDYLIEWVRQAPGQGLEWMGVINPGSGGSNYNEK IKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARGGGGFTYWGQGTLVTVSS

hu2H6-H1c（SEQ ID NO: 27）：

QVQLVQSGAEVKKPGSSVKVSCKASGYAFSDYLIEWVRQAPGQGLEWIGVINPGSGGSNYNEK IKDRATLTADKSTSTAYMELSSLRSEDTAVYYCARGGGGFTYWGQGTLVTVSSFGQGTKLEIK

hu9E5-H1a（SEQ ID NO: 28）：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWITWVRQAPGQGLEWMGDIHPGSGSTKYNEK FKSRVTMTVDTSISTAYMELSRLRSEDTAVYYCARRDYWGQGTTVTVSS

hu9E5-H1b (SEQ ID NO: 29):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWITWVRQAPGQGLEWMGDIHPGSGSTKYNEK FKSRVTLTVDTSISTAYMELSRLRSEDTAVYYCARRDYWGQGTTVTVSS

hu9E5-H1c (SEQ ID NO: 30):

QVQLVQSGAEVKKPGASVKVSCKASGYILTTYWITWVRQAPGQGLEWMGDIHPGSGSTKYNEK FKSRVTLTVDTSISTAYMELSRLRSEDTAVYYCARRDYWGQGTTVTVSS

hu2H6-L1a (SEQ ID NO: 31):

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLLNFASRLHSGVPSRFS GSGSGTDFTFTISSLQPEDIATYYCQQGSTLPWTFGGGTKVEIK

hu2H6-L1b (SEQ ID NO: 32):

DIQLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLLNFASRLHSGVPSRFS GSGSGTDFTFTISSLQPEDIATYYCQQGSTLPWTFGGGTKVEIK

hu2H6-L1c (SEQ ID NO: 33):

DIQLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKTIKLLLNFASRLHSGVPSRFS GSGSGTDFTFTISSLQPEDIATYYCQQGSTLPWTFGGGTKVEIK

hu9E5-L1a (SEQ ID NO: 34):

DIVMTQSPLSLPVTPGEPASISCRSSQNIVNSQGNTYLEWYLQKPGQSPQLLIYKVTNRFSGV PDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQASLVPWTFGGGTKVEIK

hu9E5-L1b (SEQ ID NO: 35):

DVVMTQSPLSLPVTPGEPASISCRSSQNIVNSQGNTYLEWYLQKPGQSPQLLIYKVTNRFSGV PDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQASLVPWTFGGGTKVEIK

hu9E5-L1c (SEQ ID NO: 36):

DVLMTQSPLSLPVTPGEPASISCRSSQNIVNSQGNTYLEWYLQKPGQSPQLLIYKVTNRFSGV PDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQASLVPWTFGGGTKVEIK

The final humanized antibody molecules hu2H6 (containing H1b heavy chain and L1c light chain) and hu9E5 (containing H1c heavy chain and L1a light chain) were selected using the above expression studies and comparison of the number of backmutations of combinations of light and heavy chains, and the complete sequences of light and heavy the chains of which are given under SEQ ID NO: 17-20, respectively.

HC hu2H6

QVQLVQSGAEVKKPGSSVKVSCKASGYAFSDYLIEWVRQAPGQGLEWMGVINPGSGGSNYNEKIKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARGGGGFTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 17

LChu2H6

DIQLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKTIKLLLNFASRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGSTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQECKDSTYSLSSTLSKADVEKHKVYASSEQ ID1

HC hu9E5

QVQLVQSGAEVKKPGASVKVSCKASGYILTTYWITWVRQAPGQGLEWMGDIHPGSGSTKYNEKFKSRVTLTVDTSISTAYMELSRLRSEDTAVYYCARRDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 19

LChu9E5

DIVMTQSPLSLPVTPGEPASISCRSSQNIVNSQGNTYLEWYLQKPGQSPQLLIYKVTNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQASLVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYKVY2

Embodiment 10 Humanized Antibody Assay Data

Binding activity, blocking activity, and the like. humanized hu2H6 and hu9E5 antibodies of the present invention against human CD40, rhesus monkeys are shown in Table 11. The results showed that the binding and blocking activity in ELISA of the humanized anti-human CD40 antibody of the present invention was comparable to that of the Pfizer/Alligator positive control antibody. In particular, the affinity of hu9E5 for human CD40 measured by Biacore was 9-fold higher than that of the Alligator positive control reference antibody and 3-fold higher than that of the Pfizer reference.

Table 11 In vitro activity of humanized hu2H6 and hu9E5 antibodies

Embodiment 11 Anti-CD40 Antibody Suppression of Tumor Growth in Mice

Normal human peripheral blood was taken and healthy human PBMCs were isolated by density gradient centrifugation. Monocytes were sorted using the CD14 ⁺ microbead kit and CD14 ⁺ monocytes were isolated according to the protocol provided with the kit, ie. 20 μl of microcarriers to CD14 was added for every 10 ⁷ cells and incubated at 4°C for 15 minutes. The cells were then added to a magnetic column and rinsed three times. Cells in a magnetic column, ie CD14 ⁺ monocytes, were collected. RPMI 1640 medium containing 10 ng/ml IL4 and 100 ng/ml GM-CSF was added to CD14 ⁺ monocytes, thus culturing the monocytes for 6 days (the culture method is a conventional method in the art) to effect cell culture induction MoDC. RPMI 1640 containing IL-2 was added to the remaining cells, and the suspended cells were harvested after culture (the culture method and the cell harvest method are conventional in the art). T cells were sorted using a set of CD3 ⁺ microbeads. Six days later, MoDC cells and CD3 ⁺ T cells were harvested and mixed with Raji cells (Shanghai Institute of Biotechnology cell bank cultured in RPMI1640 medium containing 10% fetal bovine serum) at a ratio of 1:5:20, respectively, and inoculated into each mouse NOG (Nanjing Galaxy Biopharma, adaptive nutrition for 5 days) subcutaneously. Experimental animals were kept in a separate ventilated box with constant temperature and humidity. The temperature in the breeding room was 18.0-26.0°C, the humidity was 40-70%, ventilation was performed 10-20 times/h, and day and night switched at 12h/12h.

The experimental groups included a control group of human IgG1 antibody, hu2H6, hu9E5 and reference antibody G12, and the dose for each group was 3 mg/kg. Five mice in each group were injected with cells once a week for six weeks using three consecutive injections. During the whole experiment, the long axis diameter and the broad axis diameter of the tumor were measured twice a week using a vernier caliper and the tumor volume (mm ³ ) = 0.5 × (tumor long axis diameter × tumor wide axis diameter ² ) was calculated. Relative degree of tumor suppression TGI (%): TGI% = (1-T/C) × 100%. T/C% is the relative tumor growth rate, that is, the percentage of the relative tumor volume or tumor mass in the treatment group and the control group at a certain point in time. T and C are tumor volume (TV) or tumor weight (TW) in the treatment group and IgG1 control group at a specific time point, respectively.

The results showed that the humanized anti-CD40 hu2H6 and hu9E5 antibodies had a very significant anti-tumor effect compared to the IgG1 control, and the tumor was almost completely eliminated 21 days after administration; the antitumor effect of hu2H6 and hu9E5 was generally equivalent to or slightly better than the G12 reference antibody, as shown in FIG. 3 and FIG. four.

Although specific embodiments of the present invention have been described above, those skilled in the art should understand that they are merely illustrative and various changes or modifications may be made to the present invention without departing from the principles and spirit of the present invention, and these equivalent forms are also included. to the extent defined by the appended claims of the present application.

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SEQUENCE LIST

<110> JIANGSU HENGRUI MEDICINE CO., LTD.;

SHANGHAI HENGUI PHARMACEUTICAL CO.,LTD

<120> ANTIBODY TO CD40, ITS ANTIGEN-BINDING FRAGMENT AND ITS

MEDICAL APPLICATION

<130> P19413048EN

<150> CN201710402559.0

<151> 01-06-2017

<160> 60

<170> PatentIn version 3.5

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Gly Val Ile Asn Pro Gly Ser Gly Gly Ser Asn Tyr Asn Glu Lys Ile

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Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

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Ala Arg Gly Gly Gly Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val

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Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Ile Lys Leu Leu Leu

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Asn Phe Ala Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Asp Phe Phe Leu Thr Ile Ser Asn Leu Glu Gln

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Asp Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Trp

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Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Asp Ile His Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe

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Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Thr Arg Leu Ser Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Asn Ser

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Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Glu Ser

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Pro Lys Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro

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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asp Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Ser Asn Tyr Asn Glu Lys Ile

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Gly Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

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Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Ile Lys Leu Leu Leu

35 40 45

Asn Phe Ala Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Leu Thr Thr Tyr

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Trp Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asp Ile His Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

115 120 125

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

130 135 140

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

145 150 155 160

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

165 170 175

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

180 185 190

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

195 200 205

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

340 345 350

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

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Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

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Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Asn Ser

20 25 30

Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala

85 90 95

Ser Leu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

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Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210> 22

<211> 95

<212> PROTEIN

<213> Human germ type

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro

85 90 95

<210> 23

<211> 98

<212> PROTEIN

<213> Human germ type

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210> 24

<211> 100

<212> PROTEIN

<213> Human germ type

<400> 24

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro

100

<210> 25

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody heavy chain variable region

<400> 25

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asp Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Ser Asn Tyr Asn Glu Lys Ile

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Gly Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 26

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody heavy chain variable region

<400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asp Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Ser Asn Tyr Asn Glu Lys Ile

50 55 60

Lys Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Gly Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 27

<211> 126

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody heavy chain variable region

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asp Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Ser Gly Gly Ser Asn Tyr Asn Glu Lys Ile

50 55 60

Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Gly Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210> 28

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody heavy chain variable region

<400> 28

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asp Ile His Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 29

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody heavy chain variable region

<400> 29

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asp Ile His Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 30

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody heavy chain variable region

<400> 30

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Leu Thr Thr Tyr

20 25 30

Trp Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asp Ile His Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

<210> 31

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody light chain variable region

<400> 31

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu

35 40 45

Asn Phe Ala Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 32

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody light chain variable region

<400> 32

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu

35 40 45

Asn Phe Ala Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 33

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody light chain variable region

<400> 33

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Ile Lys Leu Leu Leu

35 40 45

Asn Phe Ala Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 34

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody light chain variable region

<400> 34

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Asn Ser

20 25 30

Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala

85 90 95

Ser Leu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 35

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody light chain variable region

<400> 35

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Asn Ser

20 25 30

Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala

85 90 95

Ser Leu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 36

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Humanized antibody light chain variable region

<400> 36

Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Asn Ser

20 25 30

Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala

85 90 95

Ser Leu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 37

<211> 116

<212> PROTEIN

<213> Mus musculus

<400> 37

Gln Val Arg Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Met Arg Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Glu Trp Ile

35 40 45

Gly Ile Leu Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Asn Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ile Arg Gly Ser Pro Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala

115

<210> 38

<211> 107

<212> PROTEIN

<213> Mus musculus

<400> 38

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Ile Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Ser Thr Ser Gly Leu His Ser Gly Val Pro Ser Arg Phe Asn Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 39

<211> 10

<212> PROTEIN

<213> Mus musculus

<400> 39

Gly Tyr Ala Phe Thr Asn Tyr Leu Ile Asn

1 5 10

<210> 40

<211> 17

<212> PROTEIN

<213> Mus musculus

<400> 40

Ile Leu Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Asn Phe Lys

1 5 10 15

asp

<210> 41

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 41

Gly Ser Pro Gly Phe Ala Tyr

fifteen

<210> 42

<211> 11

<212> PROTEIN

<213> Mus musculus

<400> 42

Arg Ala Ser Gln Asp Ile Asn Ile Tyr Leu Asn

1 5 10

<210> 43

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 43

Ser Thr Ser Gly Leu His Ser

fifteen

<210> 44

<211> 9

<212> PROTEIN

<213> Mus musculus

<400> 44

Gln Gln Gly Tyr Thr Leu Pro Tyr Thr

fifteen

<210> 45

<211> 116

<212> PROTEIN

<213> Mus musculus

<400> 45

Gln Val Gln Val Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Glu Phe Gly Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Gly Gly Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala

115

<210> 46

<211> 107

<212> PROTEIN

<213> Mus musculus

<400> 46

His Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Ser His

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Ser Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 47

<211> 10

<212> PROTEIN

<213> Mus musculus

<400> 47

Gly Tyr Ala Phe Thr Asn Tyr Leu Ile Glu

1 5 10

<210> 48

<211> 17

<212> PROTEIN

<213> Mus musculus

<400> 48

Val Ile Asn Pro Glu Phe Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

gly

<210> 49

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 49

Gly Gly Gly Gly Phe Thr Tyr

fifteen

<210> 50

<211> 11

<212> PROTEIN

<213> Mus musculus

<400> 50

Arg Ala Ser Gln Asp Ile Ser Ser His Leu Asn

1 5 10

<210> 51

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 51

Tyr Thr Ser Arg Leu His Ser

fifteen

<210> 52

<211> 9

<212> PROTEIN

<213> Mus musculus

<400> 52

Gln Gln Gly Asn Thr Leu Pro Trp Thr

fifteen

<210> 53

<211> 119

<212> PROTEIN

<213> Mus musculus

<400> 53

Gln Val Arg Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Ala Gly Ile Tyr Pro Gly Thr Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Glu Arg Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Thr Arg Arg Gly Leu Pro Ser Leu Cys Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 54

<211> 107

<212> PROTEIN

<213> Mus musculus

<400> 54

Asp Phe Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 55

<211> 10

<212> PROTEIN

<213> Mus musculus

<400> 55

Gly Tyr Thr Phe Thr Asp Tyr Tyr Ile Asn

1 5 10

<210> 56

<211> 17

<212> PROTEIN

<213> Mus musculus

<400> 56

Gly Ile Tyr Pro Gly Thr Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Lys

1 5 10 15

gly

<210> 57

<211> 10

<212> PROTEIN

<213> Mus musculus

<400> 57

Arg Gly Leu Pro Ser Leu Cys Phe Asp Tyr

1 5 10

<210> 58

<211> 11

<212> PROTEIN

<213> Mus musculus

<400> 58

Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 59

<211> 7

<212> PROTEIN

<213> Mus musculus

<400> 59

Tyr Thr Ser Ser Leu His Ser

fifteen

<210> 60

<211> 9

<212> PROTEIN

<213> Mus musculus

<400> 60

Gln Gln Tyr Ser Lys Leu Pro Pro Thr

fifteen

<---

Claims (33)

1. An anti-CD40 antibody or antigen-binding fragment thereof comprising an antibody light chain variable region and an antibody heavy chain variable region, wherein the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the sequence of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; and the variable region of the heavy chain of the antibody contains HCDR1, HCDR2 and HCDR3 having the sequence under SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; or the antibody light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the sequence of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; and the heavy chain variable region of the antibody comprises HCDR1, HCDR2, and HCDR3 having the sequence of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; or the antibody light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the sequence of SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively; and the variable region of the heavy chain of the antibody contains HCDR1, HCDR2 and HCDR3 having the sequence under SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively; or the antibody light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the sequence of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively; and the variable region of the heavy chain of the antibody contains HCDR1, HCDR2 and HCDR3 having the sequence under SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, respectively; or the antibody light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the sequence of SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, respectively; and the heavy chain variable region of the antibody comprises HCDR1, HCDR2, and HCDR3 having the sequence of SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively. 2. An anti-CD40 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a mouse antibody or a chimeric antibody. 3. An anti-CD40 antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of the heavy chain variable region of the mouse antibody or chimeric antibody is shown under SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown under SEQ ID NO: 2; or the amino acid sequence of the heavy chain variable region is shown under SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown under SEQ ID NO: 10; or the amino acid sequence of the heavy chain variable region is shown under SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown under SEQ ID NO: 38; or the amino acid sequence of the heavy chain variable region is shown under SEQ ID NO: 45, and the amino acid sequence of the light chain variable region is shown under SEQ ID NO: 46; or the amino acid sequence of the heavy chain variable region is shown under SEQ ID NO: 53, and the amino acid sequence of the light chain variable region is shown under SEQ ID NO: 54. 4. An anti-CD40 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or a human antibody. 5. The anti-CD40 antibody or antigen-binding fragment thereof according to claim 4, wherein the sequence of the light chain FR region in the light chain variable region of the humanized antibody is derived from the human germ-type light chain IGkV1-33 sequence shown under SEQ ID NO: 22, or the sequence IGkV2-28 germline light chain shown under SEQ ID NO: 24. 6. An anti-CD40 antibody or antigen-binding fragment thereof according to claim 4, wherein the light chain sequence of the antibody from the humanized antibody is the sequence shown under SEQ ID NO: 18 or SEQ ID NO: 20, or variants thereof; wherein the variants are preferably characterized by 0-10 amino acid mutations in the variable region of the light chain, more preferably the mutations are introduced at positions 2 and 3 and the mutation results in amino acid substitution, preferably I, V or L. 7. The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 4 to 6, wherein the heavy chain variable region of the humanized antibody further comprises a human IgG1, IgG2, IgG3, or IgG4 heavy chain FR region or variants thereof, preferably comprises a heavy chain FR region. human IgG1, IgG2, IgG3 or IgG4 chain, more preferably contains a human IgG1 or IgG2 heavy chain FR region. 8. The anti-CD40 antibody or antigen-binding fragment thereof according to claim 7, wherein the sequence of the heavy chain FR region in the heavy chain variable region of the humanized antibody is derived from the human IGHV1-69 germline heavy chain sequence shown under SEQ ID NO: 21 or human the IGHV1-2 germline heavy chain sequence shown under SEQ ID NO: 23. 9. An anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 4 to 8, wherein the humanized antibody heavy chain sequence is the sequence shown under SEQ ID NO: 17 or SEQ ID NO: 19, or variants thereof, preferably the variants are characterized by 0 -10 amino acid mutations in the heavy chain variable region, more preferably the mutations are introduced at positions 6 and 8, and the mutation results in a change to amino acids, preferably I, A or L. 10. The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 4 to 9, wherein the humanized antibody is a humanized hu9E5 or hu2H6 antibody, wherein the sequence of the variable region of the humanized antibody is as follows: for the humanized hu2H6 antibody, the heavy chain variable region sequence is shown under SEQ ID NO: 26 and the light chain variable region sequence is shown under SEQ ID NO: 33; for the humanized hu9E5 antibody, the heavy chain variable region sequence is shown under SEQ ID NO: 30, and the light chain variable region sequence is shown under SEQ ID NO: 34. 11. An anti-CD40 antibody or antigen-binding fragment thereof according to claim 10, wherein the humanized hu9E5 antibody contains an antibody heavy chain sequence under SEQ ID NO: 19 and an antibody light chain sequence under SEQ ID NO: 20; wherein the humanized hu2H6 antibody contains the antibody heavy chain sequence of SEQ ID NO: 17 and the antibody light chain sequence of SEQ ID NO: 18. 12. A single chain antibody for the treatment and prevention of a disease or condition mediated by CD40 or CD40L, comprising a light chain variable region or a heavy chain variable region of an anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 1-11. 13. An antibody-drug conjugate for the treatment and prevention of a disease or condition mediated by CD40 or CD40L, wherein the antibody comprises a light chain variable region and/or a heavy chain variable region of an anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 1-11. 14. DNA encoding an anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 1-11. 15. DNA encoding the single chain antibody of claim 12. 16. An expression vector containing DNA according to claim 14 or 15. 17. A host cell for treating and preventing a disease or condition mediated by CD40 or CD40L, wherein the host cell is transformed with the expression vector of claim 16. 18. The host cell according to claim 17, where the host cell is a bacterium, preferably Escherichia coli ; or is a yeast, preferably Pichia pastoris ; or is a mammalian cell, preferably a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) 293 cell. 19. A pharmaceutical composition for the treatment and prevention of a disease or condition mediated by CD40 or CD40L, comprising an anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, a single chain antibody according to claim 12, or an antibody-drug conjugate according to claim 13 and a pharmaceutically acceptable excipient, diluent or carrier. 20. The use of an anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, a single chain antibody according to claim 12, an antibody-drug conjugate according to claim 13, or a pharmaceutical composition according to claim 19 in the manufacture of a medicament for treatment or prevention a disease or condition mediated by CD40 or CD40L; where the disease is preferably cancer; wherein the cancer is most preferably lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer , multiple myeloma, leukemia, gallbladder cancer, glioblastoma and melanoma. 21. A method of treating and preventing a disease or condition mediated by CD40 or CD40L, the method comprising administering to a subject a therapeutically effective dose of an anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, a single chain antibody according to claim 12, an antibody conjugate a drug according to claim 13 or a pharmaceutical composition according to claim 19, wherein the disease is preferably cancer; wherein the cancer is most preferably lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer , multiple myeloma, leukemia, gallbladder cancer, glioblastoma and melanoma. 22. The use of an anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, a single chain antibody according to claim 12, an antibody-drug conjugate according to claim 13, or a pharmaceutical composition according to claim 19 in the manufacture of a medicinal product intended to improve regarding symptoms in a patient with an autoimmune disease. 23. The use of an anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, a single chain antibody according to claim 12, an antibody-drug conjugate according to claim 13 or a pharmaceutical composition according to claim 19 in the manufacture of a medicinal product intended to improve in relation to symptoms in a patient with an inflammatory disease.

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