Activating monoclonal antibodies targeting human CD40

Abstract

This invention belongs to the field of genetic engineering, specifically relating to an activating antibody and its fragment that specifically binds to human CD40; the CD40-specific antibody or its antigen-binding fragment provided by this invention has CD40 agonist function, stimulates dendritic cell maturation, can induce CD40-mediated anti-tumor immune response, and inhibits tumor growth.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to an activating antibody and its fragment that specifically binds to human CD40. Background Technology

[0002] CD40, also known as tumor necrosis factor receptor superfamily member 5 or TNFR5, is a key immune co-stimulatory pathway receptor. It is expressed on the surface of antigen-presenting cells (APCs) in the immune system, such as B cells, macrophages, and dendritic cells, and plays a crucial role in the activation of innate and adaptive immune system mechanisms. Complete activation of somatic cells requires two distinct but synergistic signals. The first signal, delivered via the T cell antigen receptor (TCR) and the antigen-MHC complex on antigen-presenting cells (APCs), is responsible for specific immune responses. The second or co-stimulatory signal is generated through the interaction of CD28 with B7-1 (CD80) / B7-2 (CD86) and CD40 with CD40L (CD154), responsible for acquired T cell responses. The absence of these co-stimulatory signals may lead to T cell unresponsiveness or programmed cell death (apoptosis) after antigen stimulation.

[0003] CD40 binds to CD40L, a major ligand primarily expressed by activated T lymphocytes and platelets, activating antigen-presenting cells and triggering multiple downstream signaling pathways, including immune cell activation and proliferation, as well as the production of cytokines and chemokines, thereby enhancing cellular and immune function (Ara A et al., (2018) Immunotargets Ther 7: 55-61).

[0004] On the other hand, CD40 is also present on non-immune cells and tumors (Costello et al., (1999) Immunol Today 20(11): 488-493; Tong et al., (2003) Cancer Gene Ther 10(1): 1-13; Leeettal., (2014) Curr Cancer Drug Targets 14(7): 610-620; Ara A et al., (2018) ibid.), and has been reported to be associated with a variety of immune diseases (including autoimmune diseases), atherosclerotic thrombosis, cancer, and respiratory diseases. For example, CD40 / CD40L expression is upregulated in atherosclerosis-related cells. CD40 is expressed in almost all B-cell malignancies and up to 70% of solid tumors, and binding to CD40 ligands in some cellular malignancies promotes the increased expression of various factors that protect tumor cells from apoptosis (Lee et al., (1999) ProcNatIAcadSciUSA96:9136-9141).

[0005] Although the role of CD40 in tumorigenesis is very complex, some CD40 antibodies have been developed for potential tumor treatment. CP-870,893, developed fully human IgG2-agonist CD40 antibodies that can activate dendritic cells and have shown clinical efficacy in patients with advanced cancer in various backgrounds (Vonderheide et al., (2007) J Clin Oncol 25(7): 876-883; Gladue et al., (2011) Cancer Immunol Immunother 60(7): 1009-1017; Beatty et al., (2013) Expert Rev Anticancer Ther 17(2): 175-186; Vonderheide et al., (2013) Oncoimmunology 2(1): e23033; Nowak et al., Ann Oncol 26(12): 2483-2490; 2015 U.S. patent no. 7, 338, 660).

[0006] Dacetuzumab, also known as SGN-40, is a humanized IgG1-agonist CD40 antibody developed by Seattle Genetics. It has shown antitumor activity when administered intravenously weekly, particularly in patients with diffuse large B-cell lymphoma. Preclinical data also indicate synergistic effects of Dacetuzumab with other drugs such as the CD20 monoclonal antibody rituximab (Lapalombella et al., (2009) Br J Haematol 144(6): 848-855; Hussein et al., (2010) Haematologica 95(5): 845-848; de Vos et al., (2014) J He-matol Oncol 7: 44). ChiLob 7 / 4, a chimeric IgG1-agonist anti-human CD40 antibody developed by Cancer Research UK, is undergoing initial clinical trials. Of the 21 patients, 11 had stable conditions without any partial or complete remission (Chowdhury et al., (2014) Cancer Immunol Res 2(3): 229-240).

[0007] Apexigen, a clinical-stage biopharmaceutical company dedicated to developing next-generation antibody drugs for the treatment of diseases such as cancer, recently announced that its CD40-targeting monoclonal antibody, APX005M, has received Orphan Drug Designation from the FDA for the treatment of esophageal and gastroesophageal junction cancer (GEJ) and pancreatic cancer. APX005M activates CD40, mimicking the endogenous immune activation process, thereby reversing immunosuppression in cancer patients. Combining it with the immune checkpoint inhibitor IO is expected to activate APCs in the tumor microenvironment, triggering a more effective and durable immune response against the tumor.

[0008] However, there is still a need to develop more effective CD40-targeting antibodies for cancer treatment. The purpose of this invention is to provide two novel activating anti-human CD40 monoclonal antibodies that, by activating immune cells such as dendritic cells (DCs), break immune tolerance, inhibit tumor cell growth, and thus treat cancer. Summary of the Invention

[0009] The purpose of this invention is to provide an isolated monoclonal antibody, such as a chimeric or humanized monoclonal antibody conjugated to CD40, which may be an agonist CD40 antibody that activates the CD40 signaling pathway. This antibody is used to induce growth inhibition and apoptosis in CD40-positive tumor cells and enhance their immunogenicity, thereby achieving the purpose of treating tumors.

[0010] The first aspect of the present invention is to provide a CD40-specific binding antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences.

[0011] In some embodiments, the HCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO: 1, 4, 6 or 9; in some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO: 2, 5, 7 or 10; and in some embodiments, the HCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO: 3, 8 or 11.

[0012] In some embodiments, the heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3.

[0013] In some embodiments, the heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 4, HCDR2 as shown in SEQ ID NO: 5, and HCDR3 as shown in SEQ ID NO: 3.

[0014] In some embodiments, the heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 6, HCDR2 as shown in SEQ ID NO: 7, and HCDR3 as shown in SEQ ID NO: 8.

[0015] In some embodiments, the heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 9, HCDR2 as shown in SEQ ID NO: 10, and HCDR3 as shown in SEQ ID NO: 11.

[0016] In some embodiments, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 sequences. The LCDR1 sequence comprises the amino acid sequence shown in SEQ ID NO: 12, 15, 16, or 19; the LCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO: 13, 17, or 20; and the LCDR3 sequence comprises the amino acid sequence shown in SEQ ID NO: 14, 18, or 21.

[0017] In some embodiments, the light chain variable region includes LCDR1 with the sequence shown in SEQ ID NO: 12, LCDR2 with the sequence shown in SEQ ID NO: 13, and LCDR3 with the sequence shown in SEQ ID NO: 14.

[0018] In some embodiments, the light chain variable region includes LCDR1 with the sequence shown in SEQ ID NO: 15, LCDR2 with the sequence shown in SEQ ID NO: 13, and LCDR3 with the sequence shown in SEQ ID NO: 14.

[0019] In some embodiments, the light chain variable region includes LCDR1 with the sequence shown in SEQ ID NO: 16, LCDR2 with the sequence shown in SEQ ID NO: 17, and LCDR3 with the sequence shown in SEQ ID NO: 18.

[0020] In some embodiments, the light chain variable region includes LCDR1 with the sequence shown in SEQ ID NO: 19, LCDR2 with the sequence shown in SEQ ID NO: 20, and LCDR3 with the sequence shown in SEQ ID NO: 21.

[0021] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 12, LCDR2 as shown in SEQ ID NO: 13, and LCDR3 as shown in SEQ ID NO: 14.

[0022] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 4, HCDR2 as shown in SEQ ID NO: 5, and HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 15, LCDR2 as shown in SEQ ID NO: 13, and LCDR3 as shown in SEQ ID NO: 14.

[0023] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 6, HCDR2 as shown in SEQ ID NO: 7, and HCDR3 as shown in SEQ ID NO: 8; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 16, LCDR2 as shown in SEQ ID NO: 17, and LCDR3 as shown in SEQ ID NO: 18.

[0024] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 9, HCDR2 as shown in SEQ ID NO: 10, and HCDR3 as shown in SEQ ID NO: 11; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 19, LCDR2 as shown in SEQ ID NO: 20, and LCDR3 as shown in SEQ ID NO: 21.

[0025] In some embodiments, a CD40-specific binding antibody or antigen-binding fragment comprises a heavy chain variable region containing an amino acid sequence shown in any one of SEQ ID NO: 22-25, 32, 33; or contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to such sequences.

[0026] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises a light chain variable region containing an amino acid sequence shown in any one of SEQ ID NO: 26-29, 34, 35; or contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to such sequences.

[0027] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain contains the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 22; and the light chain contains the amino acid sequence of the light chain variable region shown in SEQ ID NO: 26. Alternatively, it may contain amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with these sequences.

[0028] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain contains the heavy chain variable region amino acid sequence shown in SEQ ID NO: 23; and the light chain contains the light chain variable region amino acid sequence shown in SEQ ID NO: 27. Alternatively, it may contain amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with these sequences.

[0029] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain contains the heavy chain variable region amino acid sequence shown in SEQ ID NO: 24; and the light chain contains the light chain variable region amino acid sequence shown in SEQ ID NO: 28. Alternatively, it may contain amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with these sequences.

[0030] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain contains the heavy chain variable region amino acid sequence shown in SEQ ID NO: 25; and the light chain contains the light chain variable region amino acid sequence shown in SEQ ID NO: 29. Alternatively, it may contain amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with these sequences.

[0031] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises a light chain, the heavy chain comprising the heavy chain variable region amino acid sequence shown in SEQ ID NO: 32, and the light chain comprising the light chain variable region amino acid sequence shown in SEQ ID NO: 34; or comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with these sequences.

[0032] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises a light chain, the heavy chain comprising the heavy chain variable region amino acid sequence shown in SEQ ID NO: 33, and the light chain comprising the light chain variable region amino acid sequence shown in SEQ ID NO: 35; or comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with these sequences.

[0033] In one embodiment, a CD40-specific binding antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region and a heavy chain constant region, the light chain comprising a light chain variable region and a light chain constant region, wherein the heavy chain variable region and the light chain variable region contain the amino acid sequences described above, the heavy chain constant region contains the amino acid sequence of SEQ ID NO: 30, and the light chain constant region contains the amino acid sequence of SEQ ID NO: 31; or contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with these sequences.

[0034] In some embodiments, a CD40-specific binding antibody or its antigen-binding fragment comprises two heavy chains and two light chains, or is composed of two heavy chains and two light chains, wherein each heavy chain contains the aforementioned heavy chain constant region sequence, heavy chain variable region sequence, or CDR sequence, and each light chain contains the aforementioned light chain constant region sequence, light chain variable region sequence, or CDR sequence. The antibody of the present invention can be a full-length antibody, such as IgG1, IgG2, or IgG4 subtypes, preferably IgG1.

[0035] In some embodiments, the antibody or its antigen-binding portion described in this invention can induce the maturation of dendritic cells, and in some embodiments, the antibody or its antigen-binding portion described in this invention can induce the activation of T lymphocytes.

[0036] Preferably, the antibody is a murine antibody, a human-mouse chimeric antibody, or a humanized antibody.

[0037] In a second aspect, the invention provides a nucleotide molecule that encodes a CD40-specific binding antibody or antigen-binding fragment as described in the first aspect.

[0038] In some embodiments, the nucleotide molecule comprises a light chain variable region nucleotide sequence as shown in SEQ ID NO: 40 and a heavy chain variable region nucleotide sequence as shown in SEQ ID NO: 44.

[0039] In some embodiments, the nucleotide molecule comprises a light chain variable region nucleotide sequence as shown in SEQ ID NO: 41 and a heavy chain variable region nucleotide sequence as shown in SEQ ID NO: 45.

[0040] In some embodiments, the nucleotide molecule comprises a light chain variable region nucleotide sequence as shown in SEQ ID NO: 42 and a heavy chain variable region nucleotide sequence as shown in SEQ ID NO: 46.

[0041] In some embodiments, the nucleotide molecule comprises a light chain variable region nucleotide sequence as shown in SEQ ID NO: 43 and a heavy chain variable region nucleotide sequence as shown in sequence SEQ ID NO: 47.

[0042] In some embodiments, the nucleotide molecule comprises a light chain variable region nucleotide sequence as shown in SEQ ID NO: 48 and a heavy chain variable region nucleotide sequence as shown in sequence SEQ ID NO: 50.

[0043] In some embodiments, the nucleotide molecule comprises a light chain variable region nucleotide sequence as shown in SEQ ID NO: 49 and a heavy chain variable region nucleotide sequence as shown in sequence SEQ ID NO: 51.

[0044] In some embodiments, the nucleotide molecule may comprise a light chain constant region nucleotide sequence as shown in SEQ ID NO: 57 and a heavy chain constant region nucleotide sequence as shown in SEQ ID NO: 56.

[0045] In a third aspect, the present invention provides an expression vector containing the nucleotide molecules described in the second aspect.

[0046] In a fourth aspect, the present invention provides a host cell containing the expression vector described in the third aspect.

[0047] A fifth aspect of the invention provides a method for preparing a CD40-specific antibody or antigen-binding fragment as described in the first aspect. The antibodies of the present invention can be generated using various techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique described in Kohler and Milstein, Nature 256:495 (1975). Somatic cell hybridization is preferred, but other techniques for generating monoclonal antibodies can also be used in principle, such as viral or oncogene transformation of B lymphocytes or phage display using antibody gene libraries. Chimeric or humanized antibodies are also well known in the art, for example, in U.S. patents 4,816,567, 5,225,539, 5,530,101, 5,585,089, 5,693,762, or 6,180,370.

[0048] The preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the mouse system. Hybridoma production in mice is a well-established method. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion methods are also known.

[0049] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are rat and rabbit systems (e.g., described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92: 9348 (1995), see also Rossiet et al., Am. J. Clin. Pathol. 124: 295 (2005)).

[0050] Another strategy for generating monoclonal antibodies is to directly isolate the antibody-encoding gene from the antibody-producing lymphocytes of the defined strategy, for example, see Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of a defined strategy. For details on recombinant antibody engineering, see Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo, Antibody Engineering ISBN 1-58829-092-1.

[0051] In a sixth aspect, the present invention provides two hybridoma cell lines, named Anti-CD40-31A2-2 and Anti-CD40-42D11-8, respectively. The hybridoma cell line Anti-CD40-31A2-2 is deposited with the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 21911, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 22, 2021. It is classified and named a mouse hybridoma cell line, corresponding to the hybridoma cell line named 31A2-2 in the embodiments of the present invention. The hybridoma cell line Anti-CD40-42D11-8 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21912, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 22, 2021. It is classified and named as a mouse hybridoma cell line, which corresponds to the hybridoma cell line named 42D11-8 in the embodiments of this invention.

[0052] A seventh aspect of the invention provides a pharmaceutical composition comprising a CD40-specific binding antibody or an antigen-binding fragment thereof as described in the first aspect, and a pharmaceutically acceptable carrier.

[0053] An eighth aspect of the invention provides a vaccine comprising a CD40-specific antibody or an antigen-binding fragment thereof as described in the first aspect, and optionally an immune adjuvant.

[0054] A ninth aspect of the invention provides the use of a CD40-specific binding antibody or an antigen-binding fragment thereof as described in the first aspect in the preparation of a medicament for the prevention or treatment of CD40-related diseases, such as an oncology medicament.

[0055] In some implementations, the tumors include lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, advanced pancreatic cancer, skin cancer, head or neck cancer, nasopharyngeal carcinoma, melanoma of the skin or eye, uterine cancer, ovarian cancer, cervical cancer, rectal cancer, bladder cancer, gastrointestinal cancer, stomach cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, and hairy cell lung cancer. Leukemia, acute lymphoblastic leukemia, multiple myeloma, esophageal cancer, small intestinal cancer, endocrine system cancer, thyroid cancer, adrenal cancer, soft tissue sarcoma, biliary tract cancer, central nervous system tumors, spinal tumors, brainstem glioma, glioblastoma multiforme, astrocytoma, neuroma, myeloma, meningioma, squamous cell carcinoma, pituitary adenocarcinoma, melanoma, mesothelioma, malignant hematologic malignancies, lymphoma, advanced solid tumors, or their metastases, etc.

[0056] The beneficial effects of the present invention are as follows: The anti-CD40 antibody or antigen-binding part of the present invention can specifically bind to CD40 and has one or more of the following effects: it has CD40 agonist function, stimulates dendritic cell maturation, induces CD40-mediated anti-tumor immune response, and inhibits tumor growth.

[0057] the term

[0058] To facilitate understanding of the invention, some terms used herein are first defined.

[0059] An "antibody" is an immunoglobulin molecule comprising four polypeptide chains: two heavy chains (H) and two light chains (L) linked by disulfide bonds, as well as its polymers (e.g., IgM). Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region contains one domain (CL). VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with conserved regions called framework regions (FRs). In some embodiments, from the N-terminus to the C-terminus, the light and heavy chain variable domains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions.

[0060] An "antigen-binding moiety" of an antibody refers to a portion or segment of the complete antibody molecule responsible for binding an antigen. The antigen-binding domain may contain a heavy chain variable region (VH), a light chain variable region (VL), or both. Antigen-binding fragments of antibodies may be prepared from complete antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering. Non-limiting examples of antigen-binding moieties include: Fab fragments; F(ab')2 fragments; Fd fragments; Fv fragments; single-chain Fv (svFv) molecules; single-domain antibodies; dAb fragments; and minimal recognition units (e.g., isolated CDRs) consisting of amino acid residues mimicking the hypervariable region of an antibody. The term "antigen-binding moiety" also includes other engineered molecules such as biantibodies, triantibodies, tetraantibodies, and microantibodies. For example, the Fd fragment described herein refers to an antibody fragment consisting of a VH and CH1 domain; the Fv fragment consists of a VL and VH domain in one arm of the antibody; and the dAb fragment consists of a VH domain.

[0061] It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. There are two common ways to define the CDR sequence for VH or VL: the Kabat definition and the Chothia definition, for example, see Kabat et al., “Sequences of Proteins of Immunological Interest”, National Institutes of Health, Bethesda, MD. (1991); Al-Lazikan et al., J Mol Biol 273:927-948 (1997); and Martine et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). For a given antibody's variable region sequence, the CDR sequence in the VH and VL sequences can be determined according to the Kabat or Chothia definition. In the embodiments of this application, the Kabat definition of the CDR sequence is used. In this paper, the CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; the CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.

[0062] "Specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope, or the ability of an antibody to bind to a specific antigen with an affinity at least twice that of a non-specific antigen. However, it should be understood that antibodies can specifically bind to two or more sequence-related antigens. For example, the antibodies of the present invention can specifically bind to CD40 in both humans and non-humans (e.g., mice or non-human primates).

[0063] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies comprising the group are identical except for the possibility of naturally occurring mutations in the retina. The monoclonal antibodies described herein specifically include "chimeric" antibodies, wherein a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remaining portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, provided they exhibit the desired biological characteristics.

[0064] "Humanized antibody" refers to any antibody in which all constant domain sequences are human sequences.

[0065] A "chimeric antibody" is an antibody that comprises segments derived from two or more different antibodies. In some embodiments, one or more CDRs are derived from mouse anti-CD40 antibodies. In other embodiments, all CDRs are derived from mouse anti-CD40 antibodies. In some embodiments, the chimeric antibody combines CDRs derived from more than one mouse anti-CD40 antibody. For example, the chimeric antibody may comprise CDR1 from the light chain of a first mouse anti-CD40 antibody, CDR2 from the light chain of a second mouse anti-CD40 antibody, and CDR3 from the light chain of a third mouse anti-CD40 antibody, and CDRs from the heavy chain may be derived from one or more other anti-CD40 antibodies. Furthermore, the framework regions may originate from the same anti-CD40 antibody or from one or more different individuals.

[0066] A "stimulatory" antibody is an antibody that, when added to cells, tissues, or organisms expressing CD40, increases the activity of one or more CD40 molecules by at least about 20%. In some embodiments, antibodies with stimulatory function increase CD40 activity by at least 40%, 50%, or 60%. In some embodiments, dendritic cell assays are used to determine the release of IL-12 to assess the activity of the activated antibody. Throughout this document, the terms "stimulatory antibody," "stimulatory antibody," and "activated antibody" are used interchangeably.

[0067] "Homology" is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and vacancy introduction, reaching the maximum percentage of homology if desired. The methods and computer programs used for alignment are well known in the art. "At least 80% homology" as used herein refers to any value between 80% and 100%, such as 85%, 90%, 95%, 99%, etc.

[0068] "Host cell" includes an individual cell or cell culture that may be or has been a recipient of the vector of the present invention. Host cell includes the offspring of a single host cell. Due to natural, accidental, or intentional mutations, the offspring need not be identical to the original parent cell (morphologically or in terms of genomic or total DNA complementarity). Host cell includes cells transfected in vivo with the vector of the present invention. "Host cell" can refer to a prokaryotic cell, eukaryotic cell, or cell line cultured as a single-cell entity that can be or has been used as a recipient of recombinant vectors or other transfer polynucleotides, and includes the offspring of a transfected original cell. It should be understood that due to natural, accidental, or intentional mutations, the offspring of a single cell need not be identical to the original parent morphologically or in terms of genomic or total DNA complementarity.

[0069] A "vector" is a nucleic acid molecule, preferably self-replicating, that transfers an inserted nucleic acid molecule into a host cell and / or between host cells. This genus includes vectors whose primary function is to insert DNA or RNA into a cell, replication vectors whose primary function is to replicate DNA or RNA, and expression vectors whose function is to transcribe and / or translate DNA or RNA. It also includes vectors that provide more than one of the above functions. An "expression vector" refers to a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. An "expression system" generally refers to a suitable host cell containing an expression vector capable of producing a desired expression product.

[0070] “Treatment” as used herein refers broadly to achieving the desired pharmacological and / or physiological effect. This effect may be preventative in terms of completely or partially preventing the disease or its symptoms, and / or therapeutic in terms of partially or completely stabilizing or curing the disease and / or causing adverse reactions attributable to the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals such as mice, rats, rabbits, pigs, primates, including humans and other apes, particularly humans, and the term includes: (a) preventing the occurrence of a disease or symptoms in subjects who may be susceptible to the disease or symptoms but have not yet been diagnosed; (b) suppressing disease symptoms; (c) halting the development of the disease; (d) alleviating disease symptoms; (e) causing the remission of the disease or symptoms; or any combination thereof.

[0071] The terms “cancer,” “tumor,” and “carcinoma” are used interchangeably in this application to refer to cells that exhibit relatively autonomous growth, resulting in an abnormal growth phenotype characterized by significantly uncontrolled cell proliferation. Typically, target cells used for monitoring or treatment in this application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells.

[0072] "Dendrical cells (DCs)" refers to a group of cells, also known as dendritic leukocytes, that have a strong antigen-presenting function.

[0073] "EC50" refers to the concentration for 50% of the maximal effect, which is the concentration that can cause a 50% maximal effect.

[0074] "KD" refers to the equilibrium dissociation constant of specific antibody-antigen interactions.

[0075] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0076] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated herein, the singular forms "a," "an," and "this" include the plural forms. Within the numerical ranges given in the embodiments, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints may be selected.

[0077] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, this invention can be implemented using any prior art methods, apparatus, and materials similar to or equivalent to those described in the embodiments of this invention, based on the prior art's knowledge and the description of this invention. Attached Figure Description

[0078] Figure 1 : Activation function detection curve of purified mouse monoclonal antibody - 1;

[0079] Figure 2 : Activation function detection curve of purified mouse monoclonal antibody - 2;

[0080] Figure 3 : Activation function detection curve of purified mouse monoclonal antibody - 3;

[0081] Figure 4 : Activation function detection curve of purified mouse monoclonal antibody - 4;

[0082] Figure 5 : Activation function detection curve of purified mouse monoclonal antibody - 5;

[0083] Figure 6 Figure: ELISA results of mouse monoclonal antibody and human CD40;

[0084] Figure 7 FACS assay curve showing the binding of mouse CD40 monoclonal antibody to human CD40 expressed in HEK293T cells;

[0085] Figure 8 : FACS assay curve showing the binding of mouse CD40 monoclonal antibody to monkey CD40 expressed in HEK293T cells;

[0086] Figure 9 : Graph of monoclonal antibody activation function verification experiment;

[0087] Figure 10 : FACS assay curves showing the binding of chimeric antibodies C42 and C31 to 293T-huCD40;

[0088] Figure 11 Figure 1 shows the results of the DC activation assay demonstrating the effect of chimeric antibodies C42 and C31 on the maturation of dendritic cells.

[0089] Figure 12 Figure 2 shows the results of the DC activation assay demonstrating the effect of chimeric antibodies C42 and C31 on the maturation of dendritic cells.

[0090] Figure 13 Figure 3 shows the results of the DC activation assay demonstrating the effect of chimeric antibodies C42 and C31 on the maturation of dendritic cells.

[0091] Figure 14 Figure 4 shows the results of the DC activation assay demonstrating the effect of chimeric antibodies C42 and C31 on the maturation of dendritic cells.

[0092] Figure 15 Figure 5 shows the results of the DC activation assay demonstrating the effect of chimeric antibodies C42 and C31 on the maturation of dendritic cells.

[0093] Figure 16 Figure 6 shows the results of the DC activation assay demonstrating the effect of chimeric antibodies C42 and C31 on the maturation of dendritic cells.

[0094] Figure 17 Figure 7 shows the results of the DC activation assay demonstrating the effect of chimeric antibodies C42 and C31 on the maturation of dendritic cells.

[0095] Figure 18 Figure 8 shows the results of the DC activation assay demonstrating the effect of chimeric antibodies C42 and C31 on the maturation of dendritic cells.

[0096] Figure 19 : FACS assay curves showing the binding of humanized antibodies 42 and 31 to 293T-huCD4O;

[0097] Figure 20 Figure 1 shows the results of the DC activation assay that detected the effects of humanized antibodies 42 and 31 on the maturation of dendritic cells.

[0098] Figure 21 Figure 2 shows the results of the DC activation assay that detected the effects of humanized antibodies 42 and 31 on the maturation of dendritic cells.

[0099] Figure 22 Figure 3 shows the results of the DC activation assay that detected the effects of humanized antibodies 42 and 31 on the maturation of dendritic cells.

[0100] Figure 23 Figure 4 shows the results of the DC activation assay that detected the effects of humanized antibodies 42 and 31 on the maturation of dendritic cells.

[0101] Figure 24 The antitumor effects of humanized CD40 antibodies 31 and 42 in a Raji / CIK / DC co-inoculated subcutaneous xenograft model;

[0102] Figure 25 The therapeutic effects of CD40 humanized antibodies 31 and 42 on a Raji / CIK / DC co-inoculated subcutaneous xenograft model. Detailed Implementation

[0103] The present invention will be further explained and described below with reference to the embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0104] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Unless otherwise stated, the materials and reagents used in the following examples are commercially available.

[0105] The positive control antibodies used in the examples are APX005 and APX005M. APX005 has the human IgG1 / κ constant region and is prepared according to the amino acid sequence in WO2014070934A1. APX005M is prepared according to the amino acid sequence in patent WO2018140831A2.

[0106] Example 1: Preparation of a hybridoma cell line secreting human CD40 antibody

[0107] 1.1 Animal Immunization

[0108] Twenty 6-8 week old female BALB / c mice were cross-injected with recombinant human CD40-his protein. The human CD40(ECD)-his protein was emulsified using equal volumes of complete Freund's adjuvant, incomplete Freund's adjuvant, or PBS via sonication. The initial immunization dose was 100 μg / mouse, supplemented with CFA (complete Freund's adjuvant), followed by a dose of 50 μg / mouse supplemented with IFA (incomplete Freund's adjuvant), with cross-immunization every two weeks. One week after each booster immunization, 50 μl of serum was collected from each mouse, and the titer was determined by ELISA, specifically using recombinant human CD40-his for binding assay. Based on the ELISA and FACS results after the final booster, the seven mice with the highest serum titers were selected for the next step of hybridoma cell line preparation.

[0109] 1.2 Preparation of hybridoma cell lines

[0110] Four days after the final booster, mice were euthanized, and spleens were harvested and prepared into single-cell suspensions in PBS. Spleen cells were washed three times with DMEM. Logarithmic growth phase mouse myeloma cells SP2 / 0 were mixed with the isolated mouse spleen cells at a 1:4 ratio and washed twice with DMEM. Cell fusion was performed using PEG fusion. The fused cells were washed three times with DMEM and resuspended in cell growth medium (RPMI 1640 + 10% FBS + 1×HAT). The cell suspension was plated into 96-well plates at 200 μl per well, incubated at 5×10⁻⁶ cells / well. 4 Cells were cultured in each well at 37°C in a humidified cell culture incubator with 5% CO2 for 7 days. On day 7, the culture medium was replaced with fresh medium (DMEM + 10% FBS + 1×HT). After 2-3 days, the cell culture supernatant was aspirated, and hybridomas were screened by ELISA and FACS.

[0111] 1.2.1 Screening hybridoma cell lines by ELISA

[0112] Hybridoma clones binding to human CD40 were screened using high-throughput ELISA. For ELISA, 100 μl of human CD40-his (0.5 μg / ml) was plated into 96-well ELISA plates and incubated overnight at room temperature. The ELISA plates were washed three times with PBST (PBS + 0.05% Tween 20) solution, blocked for 2 hours at room temperature with 200 μl of blocking buffer (PBS + 1% BSA + 1% goat serum + 0.05% Tween 20), and washed three times with PBST solution. 100 μl of hybridoma cell culture supernatant was added to the sample detection wells. The negative antibody was mouse IgG1, and the positive control antibody was APX005. After incubation at room temperature for 1 hour, the plates were washed three times with PBST. 100 μl of goat anti-mouse Fc-HRP (1:5000) or human Fc-HRP was added to each well, and the plates were incubated at room temperature for 1 hour. After washing three times with PBST, 80 μl of TMB was added for color development. After 5-10 minutes, add 80 μl of 0.16M sulfuric acid to stop the color development. Detect the OD450 value with an ELISA reader. Take the OD value that is 5 times greater than the negative control and screen out 328 candidate hybridoma cell lines.

[0113] 1.2.2 Screening of hybridoma cell lines by FACS detection

[0114] The binding affinity of 328 selected hybridoma cell lines to human and monkey CD40 expressed in HEK293T cells was further tested. First, 1×10⁻⁶ cells were used... 5 HEK293T-human CD40 cells and HEK293T-monkey CD40 cells were added to each well of a 96-well plate. Hybridoma culture supernatant was added to each well (100 μl). Mouse IgG1 was used as a negative control, and APX005 as a positive control antibody. After incubation at 4°C for 1 hour, the cells were washed three times with FACS wash buffer (PBS + 1% BSA + 0.01% Tween 20). The cells were then resuspended in FACS wash buffer and incubated at 4°C for 1 hour with 500-fold diluted APC goat anti-mouse IgG secondary antibody or anti-human IgG secondary antibody. After washing the plate three times with PBS, cell fluorescence was detected using a FACS analyzer. Mean fluorescence intensity (MFI) values ​​were analyzed and calculated using FlowJo (TreeStar) software version 10.0.7. Clones with an MFI greater than or equal to 20 times that of the negative control antibody were considered positive clones, and APX005 was used as a positive reference antibody.

[0115] Based on the above FACS screening, 176 hybridoma clones with high binding affinity to HEK293T / human CD40 cells and HEK293T / monkey CD40 cells were obtained.

[0116] Subcloning was performed on the 176 hybridoma clones mentioned above. During the subcloning process, multiple subclones (n>3) of each clone were selected and their characteristics were confirmed by the ELISA / FACS assays described above. The subclones obtained through this step were identified as monoclonal hybridoma cell lines. Ultimately, 176 subclones exhibiting high binding affinity to human and monkey CD40 were obtained, each subclone derived from a different original maternal clone.

[0117] 1.2.3 Screening hybridoma cell lines using FORTEBIO affinity

[0118] The affinity of 176 hybridoma monoclonal cell culture supernatants for human CD40-his was determined using a Fortebio Octet RED96 instrument, according to the Fortebio instruction manual.

[0119] Weigh 1g of BSA, measure 500μL of Tween 20, and add it to 1000mL of 1×PBS. Mix well. Filter and aliquot for storage. Pipette 0.1mL of 0.1M pH 2.0 glycine solution into 0.9mL of ultrapure water and mix well. Dilute the antibody with KB buffer to 10μg / mL, and dilute the antigen with KB buffer to a series of concentration gradients: 200, 50, 12.5, and 0 nM. Pre-humidify the AMC sensor in the dark for at least 10 minutes before testing the sample plate. After successful testing, proceed according to the preset program. In sample plate 1, 200 μL / well of KB buffer was added to columns 1, 10, and 12; 0.01 M pH 2.0 glycine solution was added to column 11; prepared sample solutions were added to columns 2-8 (one sample per four wells, i.e., two samples per column); and human CD40-his was added to column 9 in descending order of concentration: 200 nM antigen solution to wells 1 and 5; 50 nM antigen solution to wells 2 and 6; 12.5 nM antigen solution to wells 3 and 7; and 0 nM antigen solution to wells 4 and 8. After testing, 25 strains with an affinity KD value ≤ 10 were obtained. -8 The antibodies are shown in Table 1.

[0120] Table 1. Affinity test results of 25 hybridoma supernatants

[0121]

[0122]

[0123] 1.3 Small-scale cell production, cryopreservation and thaw

[0124] 1.3.1 Purification

[0125] First, monoclonal mouse antibodies from 25 selected clones were purified. In short, hybridoma cells from each subclone were grown in T175 cell culture flasks containing 100 ml of fresh serum-free hybridoma medium and 1% HT supplementation. Cells were cultured at 37°C and 5% CO2 for 10 days. Cultures were collected, centrifuged at 3500 rpm for 5 minutes, and filtered through a 0.22 μm filter to remove cell debris. Monoclonal antibodies were enriched and purified using a pre-equilibrated protein-A affinity column. Elution was then performed with elution buffer (20 mM citrate, pH 3.0–3.5). Antibodies were stored in PBS (pH 7.0), and antibody concentrations were monitored using NanoDrop.

[0126] 1.3.2 Cryopreservation

[0127] Each cell line had a viable cell count greater than 90%. After centrifugation at 1000 rpm for 5 min, the supernatant was discarded. The supernatant was then prepared into 0.2–1 × 10⁻⁶ cells / mL solution using cryoprotectant. 7 / ml of cell suspension was dispensed into cryovials, placed in a programmed cooling box, and stored overnight at -80°C. Then, the cells were placed in liquid nitrogen.

[0128] 1.3.3 Cell resuscitation

[0129] Remove the cryovial from the liquid nitrogen and quickly place it in a 37°C water bath. Shake it constantly to accelerate thawing. Add an appropriate amount of culture medium, centrifuge, wash away the cryosol, and transfer it to a bottle for incubation.

[0130] 1.4 Detection of the activation function of purified anti-CD40 mouse antibody

[0131] HT1080 cells expressing CD40 were constructed using conventional techniques in the field, with 10 cells per well. 5 HT1080-CD40 cells were seeded into 96-well plates, with 150 μL of 1640 complete culture medium per well. CD40 antibody was started at 20 μg / mL and diluted 3.16-fold 8-10 times with culture medium, then mixed with HT1080-CD40 cells and incubated for 6 hours. After centrifugation at 1000 rpm for 5 min, 50 μL of the supernatant was collected and stored at 4°C for IL-8 assay. The assay was performed according to the Human IL-8 Immunoassay Kit instructions.

[0132] (1) ELISA plate coating:

[0133] a. Dilute Capture Antibody with PBS to prepare a working concentration. Add 100 μL of Capture Antibody working solution to a 96-well ELISA plate. Seal the plate and incubate at room temperature overnight.

[0134] b. Discard the Capture Antibody working solution and wash the cell plate three times with Wash Buffer. Use 400 μL Wash Buffer per well, thoroughly removing the Wash Buffer with each wash. For the final wash, invert the plate onto clean paper to completely remove the Wash Buffer.

[0135] c. Add 300 μL of Block Buffer to each well. Incubate at room temperature for at least 1 hour.

[0136] d. Repeat the washing process in step b.

[0137] (2) Sample testing:

[0138] a. Add 100 μL of sample (diluted 4-fold with reagent diluent: 25 μL cell culture supernatant + 75 μL Reagent Dilution Buffer) or standard to each well. Seal the plate and incubate at room temperature for 2 hours.

[0139] b. Repeat the washing process in step (1) b.

[0140] c. Add 100 μL of Detection Antibody to each well. Seal the plate and incubate at room temperature for 2 hours.

[0141] d. Repeat the washing process in step b of (1).

[0142] e. Add 100 μL of Streptavidin-HRP working solution to each well. Seal the plate and incubate at room temperature for 20 minutes. Protect from light during this process. f. Repeat the washing process in step (1) b.

[0143] g. Add 100 μL of Substrate Solution to each well. Seal the plate and incubate at room temperature for 20 minutes. Protect from light during this process.

[0144] h. Add 50 μL of Stop Solution to each well. Gently tap the cell plate to ensure thorough mixing.

[0145] i. Measure the OD450 of each well using a multi-plate reader. If wavelength calibration is available, set it to 540 nm or 570 nm. If wavelength calibration is not available, subtract OD540 or OD570 from OD450. This process should be completed within 30 minutes after step h.

[0146] The measurement results are as follows Figure 1-5 As shown, the EC50 values ​​of the antibody activation assay are presented in Table 2. Figure 1-5As shown in Table 2, antibodies 30H7-1 and 35A1-1 are comparable to the positive control APX005, while antibodies 42D11-8 and 31A2-2 have better activation functions than APX005. Therefore, antibodies 30H7-1, 35A1-1, 42D11-8, and 31A2-2 were selected as candidate antibodies for subsequent experiments.

[0147] Table 2. Results of EC50 assay for antibody activation.

[0148]

[0149]

[0150] 1.5 Amplification of variable region sequences of murine antibodies 30H7-1, 35A1-1, 42D11-8 and 31A2-2

[0151] Hybridoma cells of 30H7-1, 35A1-1, 42D11-8, and 31A2-2 were cultured, and RNA was extracted from each cell. cDNA was amplified by RT-PCR. Using primers mentioned in the literature (Juste, Muzard, & Billiald, (2006), Anal Biochem., 1; 349(1): 159-61), the variable region nucleotide sequences of the heavy and light chains of these four antibodies (30H7-1, 35A1-1, 42D11-8, and 31A2-2) were amplified using RACE PCR (GenScript). The PCR products were then subcloned into the pMD18-T vector system (TaKaRa). The insert fragments were verified and sequenced using vector-specific primers. The amino acid / DNA sequences encoding the variable regions of the heavy and light chains of the generated antibodies were obtained. The antibody heavy chain CDR sequence, light chain CDR, and amino acid sequences of the variable regions of the antibody heavy and light chains are shown in SEQ ID NO: 1-29, and the nucleotide sequences are shown in SEQ ID NO: 40-47; and are listed in Tables 3-1 and 3-2. The CDRs are determined according to the Kabat number.

[0152] Table 3-1 Antibody amino acid sequences

[0153]

[0154]

[0155]

[0156] Table 3-2 Antibody Nucleotide Sequences

[0157]

[0158]

[0159]

[0160]

[0161] Example 2: Functional Validation of Candidate Antibodies 30H7-1, 35A1-1, 42D11-8, and 31A2-2

[0162] 2.1 Binding assays of purified mouse CD40 monoclonal antibodies 30H7-1, 35A1-1, 42D11-8, and 31A2-2 with human CD40

[0163] The ELISA test plate was coated overnight at 4°C with 500 ng / mL human CD40-his. Each well was blocked for 2 hours at room temperature with 200 μl of blocking buffer (PBS + 1% BSA + 1% goat serum + 0.05% Tween 20), followed by incubation at room temperature for 1 hour with 100 μl of serially diluted CD40 antibody (maximum concentration 40 μg / mL). The ELISA test plate was washed three times with PBST (PBS + 0.05% Tween 20), then incubated at room temperature for 1 hour with 5000-fold diluted goat anti-mouse IgG-HRP. The plate was then developed with freshly prepared Ultra-TMB at room temperature for 5 minutes. Finally, the plate was analyzed using SpectraMax. R i3X reading at 450 nm. Measurement results are as follows. Figure 6 As shown, from Figure 6 It can be seen that, compared with the positive control antibody APX005, antibodies 30H7-1, 35A1-1, 31A2-2 and 42D11-8 have a stronger binding affinity to human CD40.

[0164] 2.2 Binding assay of mouse CD40 monoclonal antibody to human and monkey CD40 expressed in HEK293T cells

[0165] To further determine whether the CD40 antibody binds to human and monkey CD40 expressed on HEK293T cells, FACS cell binding assays were performed using HEK293T cells stably overexpressing human or monkey CD40, respectively. In short, HEK293T cells expressing human CD40 and monkey CD40 were constructed using conventional techniques in the art, and 10 5 HEK293T-huCD40 or HEK293T-cynoCD40 cells were plated in 96-well plates, and serially diluted CD40 antibody (maximum concentration 40 μg / ml) was added. After incubation at 4°C for 1 hour, the plates were washed three times with PBST. APC-goat anti-mouse IgG, diluted 500-fold, was then added. After incubation at 4°C for 1 hour, the cells were washed three times with PBS, and cell fluorescence was monitored using a FACS analyzer. The results are shown below. Figure 7 , Figure 8 As shown, from Figure 7 , Figure 8 It can be seen that, compared with the control antibody APX005, antibodies 30H7-1, 35A1-1, 42D11-8 and 31A2-2 have higher binding affinity to human and monkey CD40.

[0166] 2.3 Activation function assays of candidate antibodies 30H7-1, 35A1-1, 42D11-8, and 31A2-2

[0167] For specific experimental methods, please refer to the screening section of the activation function experiment in Example 1.4. The results are as follows: Figure 9 ,from Figure 9 It can be seen that monoclonal antibodies 42D11-8 and 31A2-2 have strong CD40 signaling pathway activating activity and are significantly better than the control antibody APX005M; 30H7-1 and 35A1-1 have slightly better CD40 signaling pathway activating activity than the control antibody.

[0168] In summary, the binding and activation functions of antibodies 30H7-1, 35A1-1, 42D11-8, and 31A2-2 were verified. Antibodies 42D11-8 and 31A2-2 were screened for humanization and further verification.

[0169] Example 3: Expression and purification of chimeric anti-CD40 antibody

[0170] The heavy and light chain variable region genes encoding antibodies 42D11-8 and 31A2-2 (as shown in SEQ ID NO: 40 and SEQ ID NO: 44; SEQ ID NO: 41 and SEQ ID NO: 45, respectively) and their respective human IgG1 / κ constant regions (heavy chain constant region sequence as shown in SEQ ID NO: 56, light chain constant region sequence as shown in SEQ ID NO: 57) were inserted into the restriction enzyme site XhoI / BamHI on pCDNA3.4 to construct a mouse-human chimeric expression vector.

[0171] The expression vectors obtained above were transfected into Expi-293TM cells. Specifically, Expi-293TM cells were cultured in Expi-293TM expression medium, and the expression vectors were transfected into the cells using the Expi-293TM transfection kit. The ratio of DNA to Expi-Fectamine was 1:3, and the amount of DNA added was 1.5 μg per milliliter of cell culture medium. The transfected Expi-293TM cells were cultured at 37°C and 5% CO2 at 100 RPM. After 5-7 days, the cell culture supernatant was collected, and the monoclonal antibody was purified according to the procedure in Example 1.3.

[0172] Example 4: Binding assay of CD40 chimeric monoclonal antibody with human CD40 expressed in HEK293T cells.

[0173] The chimeric antibody of antibody 42D11-8 is named antibody C42, and its heavy chain and light chain variable region amino acid sequences are shown in SEQ ID NO: 22 and 26; the mouse-human chimeric antibody of antibody 31A2-2 is named antibody C31, and its heavy chain and light chain variable region amino acid sequences are shown in SEQ ID NO: 23 and 27; the human heavy chain and light chain constant region amino acid sequences are shown in SEQ ID NO: 30 and 31.

[0174] To confirm the accurate VH and VL sequences identified, the binding activity of chimeric antibodies C42 and C31 to human CD40 expressed on 293T cells was further verified using FACS assays on CD40-expressing 293T cells. Specific experimental procedures are detailed in Example 2.2. The results are as follows: Figure 10 As shown, chimeric antibodies C42 and C31 have high affinity for 293T-human CD40 and their binding activity is superior to that of the positive control antibody APX005M.

[0175] Example 5: Application of DC activation assay to detect the effect of chimeric antibodies C42 and C31 on promoting dendritic cell maturation

[0176] Peripheral blood was collected from eight blood donors and placed in anticoagulant tubes. After aseptic transfer and collection, the blood was mixed with an equal volume of sterile PBS. In a 50ml sterile centrifuge tube, 15ml of PBMC separation reagent was slowly added along the tube wall, followed by 30ml of the blood-PBS mixture. The tube was centrifuged at 800g for 20min with an ascent rate of 1 and a deceleration rate of 0. The intermediate layer was collected as the enriched PBMCs. Monocytes were enriched from the PBMCs using a monocyte isolation kit. After centrifugation and counting, the monocytes were resuspended in 1640+10% non-inactivated FBS medium and the cell density was adjusted to 1×10⁻⁶ cells / mL. 6 Add 1% P / S, 100 ng / ml GM-CSF, and 10 ng / ml IL-4 to a final concentration of 1%, and transfer to a suitable cell culture flask. Incubate the flask at 37°C in a 5% CO2 incubator for 3 days, then replace with the fresh culture medium from the previous steps and continue culturing for 4 days.

[0177] DC cells from 8 individuals were treated separately, collected, centrifuged, resuspended in 1640 + 10% inactivated FBS medium, and the cell density was adjusted to 4 × 10⁻⁶ cells / year. 5150 μL of the antibody was seeded into each well of a 96-well plate. The initial concentration of the antibody to be tested was 30 μg / ml, and it was subsequently diluted 4-fold to 12 concentration points (including the 0 concentration point). 50 μL of the antibody was seeded into each well of the plate. The plates were incubated at 37°C in a 5% CO2 incubator for 48 hours. Human IL-12 / IL-23 p40 Valukine was then administered. TM The release of the cytokine IL-12 in the supernatant was detected using an ELISA kit. Data processing and statistical plotting were performed, with the logarithm of concentration on the x-axis and the release amount of IL-12 on the y-axis to create a non-linear fitting curve. Results are as follows: Figures 11-18 As shown, from Figures 11-18 It was found that the activation experiments using peripheral blood-differentiated dendritic cells (DCs) from 8 healthy volunteers all showed that chimeric antibodies C42, C31, and APX005M had a strong ability to activate DCs, namely, stimulating DCs to activate and release IL-12 in a concentration-dependent manner. Data from the 8 healthy volunteers showed that the activation ability of mouse-human chimeric antibodies C42 and C31 on DCs was stronger than that of the positive control antibody APX005M.

[0178] Example 6 Humanization of CD40 antibody

[0179] 6.1 Humanization of CD40 antibody

[0180] The humanization of mouse-derived antibodies was performed using the complementarity-determining region (CDR) transplantation method, as described in US patents 4,816,567, 5,225,539, 5,530,101, 5,585,089, 5,693,762, and 6,180,370. The specific humanization process is as follows.

[0181] The light and heavy chain variable region sequences of 42D11-8 and 31A2-2 were compared with the human immunoglobulin gene database on the NCBI website (http: / / www.ncbi.nlm.nih.gov / igblast / ). Human germline IGVH and IGVK, which have the highest homology and expression levels with 42D11-8 and 31A2-2 and have been used in other existing drugs, were selected as the framework for humanization. The selected light chain germline receptor sequence was human IGKV2D-30*01, and the selected heavy chain germline receptor sequence was human IGHV4*01.

[0182] For the variable domains of 42D11-8 and 31A2-2, the closest antibody crystal structure models (structural resolution higher than 2.5 Å) were retrieved using PDB BLAST. Three-dimensional structural simulations were then performed to identify key framework amino acid residues that may play an important role in maintaining the CDR ring structure, thus enabling the design of reversion mutations for humanized antibodies. In short, the selected structural templates shared the same types of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, and H-CDR3 ring structures as 42D11-8 and 31A2-2, respectively. Using the selected structural templates, humanized structural models of 42D11-8 and 31A2-2 were constructed by replacing the mouse framework with human heavy and light chain framework sequences. Subsequently, three-dimensional structural modeling was performed to identify key framework amino acid residues that may play an important role in maintaining the CDR ring structure or the heavy and light chain linkages. When the mouse antibody framework and the human receptor framework shared the same amino acid residues at a certain site, the human amino acid residues were retained. On the other hand, when the murine and human receptor frames have different amino acid residues at a certain site, structural simulations are used to evaluate the importance of that residue. If it is found that an amino acid residue in the human receptor frame interacts with residues in the CDR region and affects the CDR residues, then that residue will be reverted to a murine residue.

[0183] Antibodies may contain one or more glycosylation sites in the variable regions of the light or heavy chains. These glycosylation sites may cause increased antibody immunogenicity or altered antibody pK values ​​due to altered antigen binding (Marshall et al. (1972) Annu Rev Biochem 41: 673-702; Gala and Morrison (2004) J Immunol 172: 5489-94; Wallick et al. (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al. (1985) Nature 316: 452-7; Mimura et al. (2000) Mol Immunol 37: 697-706). Glycosylation is known to occur in motifs containing NXS / T sequences. In CDR1-NYSVH of 31A2-2-VH, N is a glycosylation site, which is mutated to S.

[0184] 6.2 Expression and purification of humanized antibodies

[0185] Humanized CD40 antibodies 42D11-8 (42D11-8-humanized) and 31A2-2 (31A2-2-humanized) are named 42 and 31, respectively. The nucleotide sequences of the humanized heavy and light chain variable regions of 42 are shown in SEQ NO:48 and 50, and the amino acid sequences are shown in SEQ ID NO:32 and 34. The nucleotide sequences encoding the humanized heavy and light chain variable regions of 31 are shown in SEQ NO:49 and 51, and the amino acid sequences are shown in SEQ ID NO:33 and 35. The nucleotide sequence of the human IgG1 constant region is shown in SEQ NO:56, and the amino acid sequence is shown in SEQ ID NO:30. The nucleotide sequence of the humanized light chain variable region (human K constant region) is shown in SEQ NO:57, and the amino acid sequence is shown in SEQ ID NO:31.

[0186] After homology modeling calculations, the optimal Fab structure was obtained. The sequences encoding the humanized heavy and light chain variable regions of antibodies 42 and 31 were synthesized, and the nucleotide sequences encoding the human IgG1 constant region and the human K constant region encoding the humanized light chain variable region were added. These sequences were then cloned into the pCDNA3.4(+) expression vector using BamHI and XhoI restriction enzyme sites. All expression constructions were confirmed by sequencing: the heavy and light chain amino acid sequences of antibody 42 are shown in SEQ ID NO:36 and 38, and the nucleotide sequences are shown in SEQ ID NO:52 and 54; the heavy and light chain amino acid sequences of antibody 31 are shown in SEQ ID NO:37 and 39, and the nucleotide sequences are shown in SEQ ID NO:53 and 55. The Expi-293F expression system was transfected with the heavy chain and light chain expression vectors, and humanized CD40 antibodies 42 and 31 were transiently expressed, following the steps described in Example 3. The humanized antibodies were purified according to the method described in Example 1.3.

[0187] Example 7: Activity Detection of Humanized CD40 Antibody

[0188] 7.1 FACS assay to detect the binding affinity of humanized antibodies

[0189] The binding affinity of humanized antibodies 42 and 31 to 293T-huCD40 was further verified using FACS experiments. Specific experimental procedures are detailed in Example 2.2. Results are as follows: Figure 19 As shown, from Figure 19 It can be seen that the EC50 values ​​of antibody 31 (0.2967 μg / ml) and antibody 42 (0.2505 μg / ml) are both lower than the EC50 value of the control antibody APX005M (0.4875 μg / ml), indicating that the binding strength of humanized antibodies 42 and 31 to 293T-huCD40 is better than that of the control antibody.

[0190] 7.2 Affinity of humanized CD40 antibody to human CD40

[0191] The affinity of chimeric or humanized CD40 antibodies for human CD40 was determined using the Blitz method according to the Fortebio instrument's instruction manual. An AMC biosensor was used. Anti-CD40 antibody was bound at 10 μg / ml for 120 seconds, followed by dissociation in kinetic buffer for 120 seconds. The mobile phase was antigen huCD40-his, diluted at six gradients: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM. Data analysis was performed using Blitz Pro 1.2 software, obtaining the binding constant (Ka) and dissociation constant (Kd). Finally, the affinity constant KD (Kd / Ka) of the monoclonal antibody was calculated, and the results are shown in Table 4. Table 4 shows that the binding affinity data of the chimeric and humanized antibodies were superior to the positive control APX005M antibody.

[0192] Table 4. Binding affinity of CD40 antibody to human CD40

[0193] APX005M CD40His 3.82E-08 1.40E+05 5.33E-03 31Chimeric CD40His 4.52E-09 8.75E+04 3.95E-04 31Humanized CD40His 1.65E-08 1.35E+05 2.23E-03 42 Chimeric CD40His 1.35E-08 3.89E+05 5.24E-03 42Humanized CD40His 3.32E-08 9.40E+04 3.12E-03

[0194] Example 8: Application of DC activation assay to detect the effects of humanized antibodies 42 and 31 on promoting dendritic cell maturation

[0195] Following the experimental procedures in Example 5, the DC activation assay was used to detect the promoting effect of humanized antibodies 42 and 31 on dendritic cell maturation. The results are as follows: Figure 20-23 As shown, DC cells differentiated from peripheral blood donated by four healthy volunteers were used for activation experiments. The results showed that humanized antibodies 42, 31, and APX005M all had a strong ability to activate DC cells, namely, to stimulate DC cells to activate and release IL-12 in a concentration-dependent manner. The data from the four healthy volunteers showed that the activation ability of humanized antibodies 42 and 31 on DC cells was stronger than that of the positive control antibody APX005M.

[0196] Example 9: In vivo pharmacodynamic study of humanized antibodies

[0197] 9.1 Pharmacodynamic study of humanized antibodies 31 and 42 in NPG mouse xenograft model co-inoculated with human Burkitt's lymphoma Raji cells, CIK cells, and DC cells.

[0198] Female NPG mice aged 7–8 weeks (purchased from Beijing Vitonda Biotechnology Co., Ltd.) were selected. Raji cells in the logarithmic growth phase (purchased from the Cell Bank of the Chinese Academy of Sciences) were collected. Raji cells were mixed with CIK cells and DC cells at a ratio of 20:10:2, and 0.2 ml / mouse was subcutaneously injected into the right flank of NPG mice. The amount of Raji cells injected into each mouse was 5 × 10⁶. 6 On day 3 after tumor inoculation, mice were randomly divided into seven groups (G1-G7) according to body weight: solvent control group, low- and high-dose antibody 31 group, low- and high-dose antibody 42 group, and low- and high-dose APX005M group. Drug administration began after grouping. The solvent control group received an intraperitoneal injection of 10 ml / kg PBS; the low- and high-dose antibody 31 group received intraperitoneal injections of 1 and 10 mpk of antibody 31, respectively; the low- and high-dose antibody 42 group received an intraperitoneal injection of 1 and 10 mpk of antibody 42, respectively; and the low- and high-dose APX005M group received an intraperitoneal injection of 1 and 10 mpk of APX005M, respectively. All groups received drug administration twice a week for three consecutive weeks. The day of drug administration was recorded as day 0. The maximum diameter (D) and minimum diameter (d) of the tumor were measured weekly using electronic calipers, and the tumor volume (mm³) was calculated using the following formula. 3 )=[D×d 2 ] / 2, and calculate the tumor growth inhibition rate (TGI) (%) of each treatment group according to the formula = (1 - volume of treatment group / volume of control group) × 100%.

[0199] The results are as follows Figure 24 As shown, on day 19 after administration, the TGI of antibody 31 (low and high dose groups) was 60.05% and 86.83%, respectively; the TGI of antibody 42 (low and high dose groups) was 66.96% and 87.95%, respectively; and the TGI of APX005M (low and high dose groups) was 58.09% and 85.27%, respectively. Compared with the solvent control group, both the high and low dose groups of antibodies 31 and 42 showed very significant antitumor effects (P<0.01). These results indicate that anti-CD40 humanized antibodies 31 and 42 both exhibit good antitumor effects, with tumor-suppressing effects superior to the control antibody APX005M.

[0200] 9.2 Therapeutic effects of antibodies 31 and 42 on NPG mouse models of subcutaneous xenograft tumors co-inoculated with Raji cells, CIK cells, and DC cells.

[0201] Female NPG mice aged 7–8 weeks (purchased from Beijing Vitonda Biotechnology Co., Ltd.) were selected. Raji cells in the logarithmic growth phase (purchased from the Cell Bank of the Chinese Academy of Sciences) were collected. Raji cells were mixed with CIK cells and DC cells at a ratio of 20:10:2, and 0.2 ml / mouse was subcutaneously injected into the right flank of NPG mice. The amount of Raji cells injected into each mouse was 5 × 10⁶. 6The mice were trained until the average tumor volume reached 100 mm². 3 At approximately 10:00 AM, mice were randomly divided into seven groups according to tumor volume and body weight (solvent control group, low- and high-dose antibody 31 group, low- and high-dose antibody 42 group, and low- and high-dose APX005M group). Drug administration began after grouping. The solvent control group received an intraperitoneal injection of 10 ml / kg PBS; the low- and high-dose antibody 31 groups received intraperitoneal injections of 3 and 10 mpk of antibody 31, respectively; the low- and high-dose antibody 42 groups received intraperitoneal injections of 3 and 10 mpk of antibody 42, respectively; and the low- and high-dose APX005M groups received intraperitoneal injections of 3 and 10 mpk of APX005M, respectively. All groups received administration twice a week for four consecutive weeks. The day of administration was recorded as day 0. The maximum diameter (D) and minimum diameter (d) of the tumor were measured weekly using electronic calipers, and the tumor volume (mm²) was calculated using the following formula. 3 )=[D×d 2 ] / 2, and calculate the tumor growth inhibition rate (TGI) (%) of each treatment group according to the formula = (1 - volume of treatment group / volume of control group) × 100%.

[0202] The results are as follows Figure 25 As shown, on day 31 after administration, the TGI of antibody 31 (low and high doses) was 68.80% and 74.52%, respectively; the TGI of antibody 42 (low and high doses) was 70.05% and 84.95%, respectively; and the TGI of APX005M (low and high doses) was 66.69% and 67.27%, respectively. Compared with the solvent control group, antibodies 31 and 42, as well as the low and high doses of APX005M, all exhibited highly significant antitumor effects (P<0.01). These results indicate that anti-CD40 humanized antibodies 31 and 42 have significant therapeutic effects on the Raji / CIK / DC co-inoculated subcutaneous xenograft model, and their therapeutic effects are superior to those of the control antibody APX005M. sequence list <110> Lunan Pharmaceutical Group Co., Ltd. <120> Activating monoclonal antibodies targeting human CD40 <130> 2020 <160> 57 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <400> 1 Ser Tyr Gly Val His 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <400> 2 Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ser Ala Phe Ile Ser 1 5 10 15 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <400> 3 Val Gly Gly Asp Tyr 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <400> 4 Asn Tyr Ser Val His 1 5 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <400> 5 Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile Ser 1 5 10 15 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <400> 6 Ser Tyr Gly Val Ser 1 5 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <400> 7 Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ser Ala Val Ile Gly 1 5 10 15 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <400> 8 Val Gly Met Asp Tyr 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <400> 9 Ser Tyr Gly Val Tyr 1 5 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <400> 10 Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ser Ala Phe Lys Gly 1 5 10 15 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <400> 11 Val Gly Phe Asp Tyr 1 5 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <400> 12 Arg Ser Ser Gln Ser Leu Val His Ser Arg Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <400> 13 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <400> 14 Phe Gln Thr Thr His Val Pro Trp Thr 1 5 <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <400> 15 Arg Ser Ser Gln Ser Leu Leu His Ser Arg Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 16 <211> 16 <212> PRT <213> Artificial Sequence <400> 16 Arg Ala Ser Gln Ser Leu Val His Ser Arg Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <400> 17 Leu Ala Ser Asn Arg Phe Ser 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <400> 18 Phe Gln Thr Thr His Val Pro Leu Thr 1 5 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <400> 19 Lys Ala Ser Gln Ser Leu Val His Ser Arg Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <400> 20 Trp Thr Ser Asn Arg Phe Ser 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial Sequence <400> twenty one Phe Gln Gln Thr His Val Pro Tyr Thr 1 5 <210> 22 <211> 113 <212> PRT <213> Artificial Sequence <400> 22 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ser Ala Phe Ile 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Phe Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Val Gly Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110 Ser <210> 23 <211> 113 <212> PRT <213> Artificial Sequence <400> 23 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Ser Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Phe Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Val Gly Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110 Ser <210> 24 <211> 113 <212> PRT <213> Artificial Sequence <400> 24 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val Ser Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ser Ala Val Ile 50 55 60 Gly Arg Leu Ser Ile Ser Lys Arg Leu Phe Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Val Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 100 105 110 Ser <210> 25 <211> 113 <212> PRT <213> Artificial Sequence <400> 25 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val Tyr Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ser Ala Phe Lys 50 55 60 Gly Arg Leu Ser Ile Ser Lys Asp Asn Phe Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95 Arg Val Gly Phe Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 100 105 110 Ser <210> 26 <211> 112 <212> PRT <213> Artificial Sequence <400> 26 Asp Ile Val Ile Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Arg Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Leu Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gln Thr 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 27 <211> 112 <212> PRT <213> Artificial Sequence <400> 27 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Arg Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gln Thr 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 28 <211> 112 <212> PRT <213> Artificial Sequence <400> 28 Asp Ile Val Leu Thr Gln Ser Pro Thr Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30 Arg Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Lys Leu Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Leu Glu Ile Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Thr 85 90 95 Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 29 <211> 112 <212> PRT <213> Artificial Sequence <400> 29 Asp Ile Val Leu Thr Gln Ser Pro Thr Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Leu Val His Ser 20 25 30 Arg Gly Asn Thr Tyr Leu His Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Trp Thr Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Glu Thr Leu Arg Ile 65 70 75 80 Ser Arg Leu Glu Ala Leu Asp Leu Gly Val Tyr Phe Cys Phe Gln Gln 85 90 95 Thr His Val Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 30 <211> 330 <212> PRT <213> Artificial Sequence <400> 30 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Glu His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 31 <211> 107 <212> PRT <213> Artificial Sequence <400> 31 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 32 <211> 113 <212> PRT <213> Artificial Sequence <400> 32 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln His Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ser Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Gly Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110 Ser <210> 33 <211> 113 <212> PRT <213> Artificial Sequence <400> 33 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Gly Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110 Ser <210> 34 <211> 112 <212> PRT <213> Artificial Sequence <400> 34 Asp Ile Val Ile Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Arg Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser 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 Phe Cys Phe Gln Thr 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 <210> 35 <211> 112 <212> PRT <213> Artificial Sequence <400> 35 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Arg Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser 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 Phe Cys Phe Gln Thr 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 <210> 36 <211> 443 <212> PRT <213> Artificial Sequence <400> 36 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln His Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ser Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Gly Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Glu His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 340 345 350 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 37 <211> 443 <212> PRT <213> Artificial Sequence <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Gly Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Glu His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 340 345 350 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 38 <211> 219 <212> PRT <213> Artificial Sequence <400> 38 Asp Ile Val Ile Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Arg Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser 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 Phe Cys Phe Gln Thr 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Arg Leu 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 <210> 39 <211> 219 <212> PRT <213> Artificial Sequence <400> 39 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Arg Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser 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 Phe Cys Phe Gln Thr 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Arg Leu 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 <210> 40 <211> 339 <212> DNA <213> Artificial Sequence <400> 40 caggtgcagc tgaagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccatc 60 acctgcacag tctctggttt ctcattaact agctatggtg tacactgggt tcgccagtct 120 ccaggaaagg gtctggagtg gctgggaatg atatggagtg gtggaagcac agactataat 180 tcagctttca tatccagact gagcatcagc aaggacaact tcaagagcca agtcttcttt 240 aaaatgaaca gtctgcaagc tgatgacaca gccatatact attgtgccag agttgggggg 300 gactactggg gccaaggcac cactgtcaca gtctcctca 339 <210> 41 <211> 339 <212> DNA <213> Artificial Sequence <400> 41 caggtgcagc tgaaggagtc aggacctggc ctagtgcagc cctcacagag cctgtccatc 60 acctgcacag tctctggttt ctcattaact aactatagtg tacactgggt tcgccagtct 120 ccaggaaagg gtctggagtg gctgggaatg atatggagtg gtggaagcac agactataat 180 gcagctttca tatccagact gagcatcagc aaggacaact tcaagagcca agtcttcttt 240 aaaatgaaca gtctgcaagc tgatgacaca gccatatact attgtgccag agttgggggg 300 gactactggg gccaaggcac cactgtcaca gtctcctca 339 <210> 42 <211> 339 <212> DNA <213> Artificial Sequence <400> 42 gaggttcagc tgcaacagag tggcgcagag ttggtgcagc cctcacaatc cctgtcaatt 60 acatgcactg tatccggctt ttcattgacc tcttatgggg tctcatgggt cagacagagc 120 cccggcaaag ggttggagtg gcttggaatg atatggtcag gaggatcaac tgactataat 180 tccgcagtca ttggtcgctt gagtattagt aagaggttgt ttaagtctca agtcttcttc 240 aagatgaact cactccaagc agacgacacc gctatgtact attgtgctcg cgtcgggatg 300 gactactggg gtcaggggac tagcgtcact gtgtctagc 339 <210> 43 <211> 339 <212> DNA <213> Artificial Sequence <400> 43 gaagtccaac tccaacagtc aggggcagag cttgttcaac ctagtcagtc cctgagcatt 60 acttgtaccg tctccggctt cagtctcacc tcttatggtg tatactgggt tagacaaagt 120 ccaggcaaag ggttggaatg gttgggtatg atatggagcg gcggtagcac cgactacaat 180 agcgccttca aaggtagatt gagcatatct aaagacaact tcaaaagtca ggtctttttt 240 aagatgaata gtctccaagc tgatgatacc gcagtgtact tctgtgctcg cgttggtttc 300 gattattggg gacaagggac aagtgtcacc gttagttct 339 <210> 44 <211> 336 <212> DNA <213> Artificial Sequence <400> 44 gatattgtga taacccagac tcctctctcc ctgcctgtca gtcttggaga tccagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtcgtg gaaacaccta tttacattgg 120 ttcctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccggttt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaggatc 240 agcagattgg aggctgagga tctgggagtt tatttctgct ttcaaactac acatgttccg 300 tggacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 45 <211> 336 <212> DNA <213> Artificial Sequence <400> 45 gatgttttga tgacccaaac gcctctctcc ctgcctgtca gtcttggaga tcctgcctcc 60 atctcttgca gatctagtca gagccttcta cacagtcgtg gaaacaccta tttacattgg 120 ttcctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaggatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ttcaaactac acatgttccg 300 tggacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 46 <211> 336 <212> DNA <213> Artificial Sequence <400> 46 gacatagtgc tcacccaaag tcctacatcc ctcccagtga gtctgggaga tccagcttcc 60 atttcatgcc gagcatctca aagcctggta catagcaggg gaaatacata tcttcactgg 120 ttccttcaaa agctcggtca aagcccaaag ctccttatat atctggcctc aaaccgcttt 180 agtggggtgc ccgacaggtt tagtggctct ggaagcggta ctgacttcac cctgcgcata 240 tcacgactcg aaattgaaga cttgggagtt tattattgct ttcagacaac tcacgttcct 300 ctcacctttg gagcaggcac caagctcgaa ataaaa 336 <210> 47 <211> 336 <212> DNA <213> Artificial Sequence <400> 47 gatatcgtgc ttactcagag tccaacatct ctccccgtga gtcttggcga tccagcctct 60 atatcttgca aagcaagtca atcattggtg cattccaggg gtaatacata ccttcactgg 120 tttcttcaaa agcctggaca gagccctaaa cttctcatct actggacaag caatagattc 180 tcaggtgtcc ccgacagatt ctccggtagc gggagcggta cagacgaaac acttcggatt 240 tcccggcttg aagccctgga ccttggtgtt tatttttgtt ttcaacaaac acacgtccct 300 tacaccttcg gtgccggtac aaaactcgaa ataaag 336 <210> 48 <211> 339 <212> DNA <213> Artificial Sequence <400> 48 caagtgcagc tgcaagagag cggccctggc ctggtgaagc cttctcagac cctgagcctg 60 acctgcaccg tgagcggctt cagcctgaca agctacggcg tgcactgggt gagacagcac 120 cctggcaagg gcctggagtg gctgggcatg atctggagcg gcggcagcac cgactacaac 180 agcgccttca tcagcagact gacgattagc aaggacaaca gcaagagcca agtgtccctg 240 aagctgagct cggtcaccgc cgccgacacc gccgtgtact actgcgcaag agtaggcggc 300 gattactggg gccaaggcac caccgtgact gtgagcagc 339 <210> 49 <211> 339 <212> DNA <213> Artificial Sequence <400> 49 caggttcagc tgcaagagtc tggccctggc ctggtcaagc cttccgaaac actgtctctg 60 acctgcaccg tgtccggctt ctccctgacc tcttactccg tgcactgggt ccgacagcct 120 ccaggcaaag gattggagtg gctgggcatg atttggagcg gcggctctac cgattacaac 180 gccgccttca tctcccggct gaccatctcc aaggacaact ccaagagcca ggtgtccctg 240 aagctgtcct ctgtgaccgc tgctgatacc gccgtgtact actgtgctag agtcggcggc 300 gattattggg gccagggcac aacagtgacc gtgtcctct 339 <210> 50 <211> 336 <212> DNA <213> Artificial Sequence <400> 50 gacatcgtga tcacacagag ccctctgagc ctgcctgtga ccctgggaca gcctgctagc 60 atcacctgca gaagctctca gagcctggtg cacagcagag gcaacaccta cctgcactgg 120 tttcagcaga gacctggaca gagccctaga ctgctgatct acaaggtgag caataggttc 180 agtggagtgc ccgacagatt cagtggttcc ggtagcggca ccgacttcac cctgaagatc 240 agcagagtgg aggccgagga cgtgggcgtg tacttctgct ttcagaccac ccacgtgcct 300 tggaccttcg gccaaggcac aagactggag atcaag 336 <210> 51 <211> 336 <212> DNA <213> Artificial Sequence <400> 51 gacgtggtca tgacacagag ccctctgagc ctgcctgtga cattgggaca gcctgcctct 60 atcacctgtc ggtcctctca gtccctgctg cactccagag gcaacaccta cctgcactgg 120 ttccagcaga ggcctggcca gtctcctaga ctgctgatct acaaggtgtc caaccggttc 180 tctggcgtgc ccgacagatt ttccggctct ggctctggca ccgacttcac cctgaagatc 240 tccagagtgg aagccgagga cgtgggcgtg tacttctgct tccaaaccac acacgtgccc 300 tggacctttg gccagggcac cagactggaa atcaag 336 <210> 52 <211> 1329 <212> DNA <213> Artificial Sequence <400> 52 caagtgcagc tgcaagagag cggccctggc ctggtgaagc cttctcagac cctgagcctg 60 acctgcaccg tgagcggctt cagcctgaca agctacggcg tgcactgggt gagacagcac 120 cctggcaagg gcctggagtg gctgggcatg atctggagcg gcggcagcac cgactacaac 180 agcgccttca tcagcagact gacgattagc aaggacaaca gcaagagcca agtgtccctg 240 aagctgagct cggtcaccgc cgccgacacc gccgtgtact actgcgcaag agtaggcggc 300 gattactggg gccaaggcac caccgtgact gtgagcagcg ctagcaccaa gggtcctagc 360 gtgttccctt tagcccctag cagcaagagc acaagcggcg gcaccgccgc cctgggctgc 420 ttggtaaagg attacttccc tgagcctgtg accgtatcct ggaacagcgg cgccctgaca 480 agcggcgtgc acaccttccc tgccgtgctg cagagcagcg gcctgtacag cctgagcagc 540 gtagtgaccg tgcctagcag cagcctgggc acacagacct acatctgcaa cgtgaaccac aagcctagca acaccaaggt ggacaagaag gtggagccta agagctgcga caagacccac acctgccctc cttgccctgc ccctgagctg ctgggcggcc ctagcgtgtt tctgtttcct 720 cctaagccta aggacaccct gatgatcagc agaacccctg aggtgacctg cgtggtggtg gacgtggagc acgaggaccc tgaggtgaag ttcaactggt acgtggacgg cgtggaggtg 840 cacaacgcca agaccaagcc rich cagtacaaca gcacctacag agtggtgagc gtgctgaccg tgctgcacca agactggctg aacggcaagg agtacaagtg caaggtgagc 960 aacaaggccc tgcctgcccc tatcgagaag acaataagca aggccaaagg acagcctaga gagcctcaag tgtacaccct gcctcctagc agagacgagc tgaccaaga ccaagtgagc ctgacatgtc ttgtgaaagg gttctaccct agcgacatcg ccgtggagtg ggagagcaac 1140 ggacagcctg agaacaacta caagaccacc cctcctgtgc tggacagcga cggcagcttc 1200 ttcctgtaca gcaagctgac cgtggacaag agcagatggc agcaaggcaa cgtgttcagc 1260 tgctctgtta tgcacgaggc cctgcacaac cactacacac agaagagcct gagcctgagc 1320 cctggcaag 1329 <210> 53 <211> 1329 <212> DNA <213> Artificial Sequence <400> 53 caggttcagc tgcaagagtc tggccctggc ctggtcaagc cttccgaaac actgtctctg 60 acctgcaccg tgtccggctt ctccctgacc tcttactccg tgcactgggt ccgacagcct 120 ccaggcaaag gattggagtg gctgggcatg atttggagcg gcggctctac cgattacaac 180 gccgccttca tctcccggct gaccatctcc aaggacaact ccaagagcca ggtgtccctg 240 aagctgtcct ctgtgaccgc tgctgatacc gccgtgtact actgtgctag agtcggcggc 300 gattattggg gccagggcac aacagtgacc gtgtcctctg cttccaccaa gggaccctct 360 gtgttccctc tggctccttc cagcaagtct acctctggcg gaacagctgc tctgggctgt 420 ctggtcaagg actacttccc tgagcctgtg acagtgtcct ggaactctgg cgctctgaca 480 tccggcgtgc acaccttcc agctgtgctg caatcctccg gcctgtactc tctgtcctcc 540 gtcgtgaccg tgccttctag ctctctgggc acccagacct acatctgcaa tgtgaaccac 600 aagcctagca acaccaaggt ggacaagaag gtggaaccca agtcctgcga caagacccac acctgtcctc catgtcctgc tccagaactg ctcggcggac cttccgtgtt tctgttccct 720 ccaaagccta aggacaccct gatgatctct cggacccctg aagtgacctg cgtggtggtg 780 gatgtggaac acgaggatcc cgaagtgaag ttcaattggt acgtggacgg cgtggaagtg cacaacgcca agaccaagcc tagagagga cagtacaact ccacctacag agtggtgtcc gtgctgaccg tgctgcacca ggattggctg aacggcaaag agtacaagtg caaggtgtcc aacaaggccc tgcctgctcc tatcgaaaag accatcagca aggctaaggg ccagcctcgg gaaccccagg tttacacatt gcctccatct cgggacgagc tgaccaagaa tcaggtttcc ctgacatgcc tcgtgaaggg cttctacccc tccgatatcg ccgtggaatg ggagtccaat 1140 ggccagcctg agaacaacta caagacaacc cctcctgtgc tggactccga cggctcattc 1200 ttcctgtact ccaagctgac agtggacaag tccagatggc agcagggcaa cgtgttctcc 1260 tgctccgtga tgcacgaggc cctgcacaat cactacaccc agaagtccct gtctctgtcc 1320 cctggcaaa 1329 <210> 54 <211> 657 <212> DNA <213> Artificial Sequence <400> 54 gacatcgtga tcacacagag ccctctgagc ctgcctgtga ccctgggaca gcctgctagc 60 atcacctgca gaagctctca gagcctggtg cacagcagag gcaacaccta cctgcactgg 120 tttcagcaga gacctggaca gagccctaga ctgctgatct acaaggtgag caataggttc 180 agtggagtgc ccgacagatt cagtggttcc ggtagcggca ccgacttcac cctgaagatc 240 agcagagtgg aggccgagga cgtgggcgtg tacttctgct ttcagaccac ccacgtgcct 300 tggaccttcg gccaaggcac aagactggag atcaagagaa ccgtggccgc ccctagcgtg 360 ttcatcttcc ctcctagcga cgagcagctg aagagcggca ccgctagcgt ggtgtgcctg 420 ctgaacaact tctaccctag agaggccaag gtgcagtgga aggtggacaa cgccctgcag 480 agcggcaaca gccaagagag cgtgaccgag caagacagca aggacagcac ctacagcctg 540 agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaagtgta cgcctgcgag 600 gtgacccacc aaggcctgag cagccctgtg accaagagct tcaacagagg cgagtgc 657 <210> 55 <211> 657 <212> DNA <213> Artificial Sequence <400> 55 gacgtggtca tgacacagag ccctctgagc ctgcctgtga cattgggaca gcctgcctct 60 atcacctgtc ggtcctctca gtccctgctg cactccagag gcaacaccta cctgcactgg 120 ttccagcaga ggcctggcca gtctcctaga ctgctgatct acaaggtgtc caaccggttc 180 tctggcgtgc ccgacagatt ttccggctct ggctctggca ccgacttcac cctgaagatc 240 tccagagtgg aagccgagga cgtgggcgtg tacttctgct tccaaaccac acacgtgccc 300 tggacctttg gccagggcac cagactggaa atcaagcgga cagtggccgc tccttccgtg 360 ttcatcttcc caccttccga cgagcagctg aagtccggca cagcttctgt cgtgtgcctg 420 ctgaacaact tctaccctcg ggaagccaag gtgcagtgga aggtggacaa tgccctgcag 480 tccggcaact cccaagagtc tgtgaccgag caggactcca aggacagcac ctacagcctg 540 tcctccacac tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgaa 600 gtgacccatc agggcctgtc tagccctgtg accaagtctt tcaaccgggg cgagtgt 657 <210> 56 <211> 990 <212> DNA <213> Artificial Sequence <400> 56 gctagcacca agggtcctag cgtgttccct ttagccccta gcagcaagag cacaagcggc 60 ggcaccgccg ccctgggctg cttggtaaag gattacttcc ctgagcctgt gaccgtatcc 120 tggaacagcg gcgccctgac aagcggcgtg cacaccttcc ctgccgtgct gcagagcagc 180 ggcctgtaca gcctgagcag cgtagtgacc gtgcctagca gcagcctggg cacacagacc 240 tacatctgca acgtgaacca caagcctagc aacaccaagg tggacaagaa ggtggagcct 300 aagagctgcg acaagaccca cacctgccct ccttgccctg cccctgagct gctgggcggc 360 cctagcgtgt ttctgtttcc tcctaagcct aaggacaccc tgatgatcag cagaacccct 420 gaggtgacct gcgtggtggt ggacgtggag cacgaggacc ctgaggtgaa gttcaactgg 480 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ctagagagga gcagtacaac 540 agcacctaca gagtggtgag cgtgctgacc gtgctgcacc aagactggct gaacggcaag 600 gagtacaagt gcaaggtgag caacaaggcc ctgcctgccc ctatcgagaa gacaataagc 660 aaggccaaag gacagcctag agagcctcaa gtgtacaccc tgcctcctag cagagacgag 720 ctgaccaaga accaagtgag cctgacatgt cttgtgaaag ggttctaccc tagcgacatc 780 gccgtggagt gggagagcaa cggacagcct gagaacaact acaagaccac ccctcctgtg 840 ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gagcagatgg 900 cagcaaggca acgtgttcag ctgctctgtt atgcacgagg ccctgcacaa ccactacaca 960 cagaagagcc tgagcctgag ccctggcaag 990 <210> 57 <211> 321 <212> DNA <213> Artificial Sequence <400> 57 agaaccgtgg ccgcccctag cgtgttcatc ttccctccta gcgacgagca gctgaagagc 60 ggcaccgcta gcgtggtgtg cctgctgaac aacttctacc ctagagaggc caaggtgcag 120 tggaaggtgg acaacgccct gcagagcggc aacagccaag agagcgtgac cgagcaagac 180 agcaaggaca gcacctacag cctgagcagc accctgaccc tgagcaaggc cgactacgag 240 aagcacaaag tgtacgcctg cgaggtgacc caccaaggcc tgagcagccc tgtgaccaag 300 agcttcaaca gaggcgagtg c 321

Claims

1. A CD40-specific antibody or its antigen-binding fragment thereof, characterized in that, The antibody or antigen-binding fragment includes LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, selected from any one of the following groups: LCDR1 with sequence as shown in SEQ ID NO: 12, LCDR2 with sequence as shown in SEQ ID NO: 13, LCDR3 with sequence as shown in SEQ ID NO: 14, HCDR1 with sequence as shown in SEQ ID NO: 1, HCDR2 with sequence as shown in SEQ ID NO: 2, and HCDR3 with sequence as shown in SEQ ID NO: 3; Alternatively, the sequence is LCDR1 as shown in SEQ ID NO: 15, LCDR2 as shown in SEQ ID NO: 13, LCDR3 as shown in SEQ ID NO: 14, HCDR1 as shown in SEQ ID NO: 4, HCDR2 as shown in SEQ ID NO: 5, and HCDR3 as shown in SEQ ID NO: 3; Alternatively, LCDR1 with the sequence shown in SEQ ID NO:15, LCDR2 with the sequence shown in SEQ ID NO:13, LCDR3 with the sequence shown in SEQ ID NO:14, HCDR1 with the sequence SYSVH, HCDR2 with the sequence shown in SEQ ID NO:5, and HCDR3 with the sequence shown in SEQ ID NO:

3.

2. The CD40-specific binding antibody or its antigen-binding fragment according to claim 1, characterized in that, It has any of the following sets of heavy chain variable regions and light chain variable regions: The heavy chain variable region shown in SEQ ID NO: 22, and the light chain variable region shown in SEQ ID NO: 26; The heavy chain variable region shown in SEQ ID NO: 23, and the light chain variable region shown in SEQ ID NO: 27; The heavy chain variable region shown in SEQ ID NO: 32, and the light chain variable region shown in SEQ ID NO: 34; The heavy chain variable region shown in SEQ ID NO: 33, and the light chain variable region shown in SEQ ID NO: 35; It contains amino acid sequences that are at least 80% identical to these sequences.

3. The CD40-specific binding antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a murine antibody, a human-mouse chimeric antibody, or a humanized antibody.

4. A polynucleotide encoding a CD40-specific binding antibody or antigen-binding fragment as described in claim 1.

5. A polynucleotide encoding a CD40-specific binding antibody or antigen-binding fragment as described in claim 2.

6. An expression vector comprising the polynucleotide of claim 4.

7. An expression vector containing the polynucleotide of claim 5.

8. A host cell containing the expression vector of claim 6.

9. A host cell containing the expression vector of claim 7.

10. A composition comprising a CD40-specific antibody or an antigen-binding fragment thereof as described in claim 1, and a pharmaceutically acceptable carrier.

11. Use of the CD40-specific antibody of claim 1 or its antigen-binding fragment thereof in the preparation of a medicament for the prevention or treatment of lymphoma, colon cancer, gastric cancer, non-small cell lung cancer, melanoma, rectal cancer, ovarian cancer or pancreatic cancer.

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