Anti-CD40 antibody, its antigen-binding fragment, and pharmaceutical applications

Abstract

The present invention provides an anti-CD40 antibody, an antigen-binding fragment thereof and medical uses thereof, a pharmaceutical composition comprising the anti-CD40 antibody or the antigen-binding fragment thereof, and a method for treating or preventing a disease, in particular a method for treating an autoimmune disease.

Description

[Technical Field]

[0001] This application claims priority to the Chinese patent application filed on 28 June 2021, application number 202110722124.0.

[0002] This disclosure relates to the field of biopharmaceuticals, particularly to the treatment or intervention of diseases related to the CD40 / CD40L signaling pathway. Specifically, this disclosure relates to CD40 antibodies, their antigen-binding fragments and their pharmaceutical compositions, as well as methods for treating autoimmune diseases and related pharmaceutical applications. [Background technology]

[0003] CD40 is a type I transmembrane glycoprotein belonging to the tumor necrosis factor receptor (TNFR) superfamily, located on the cell membrane surface, with a molecular weight of approximately 48 kDa, and plays a crucial role in the immune system. CD40 is expressed in various immune cells, including B cells, dendritic cells, mononuclear cells, and macrophages, as well as platelets, and under certain conditions, can also be expressed in eosinophils and parenchymal cells. The native ligand for CD40 is CD154 or CD40L, a type II transmembrane protein whose expression can be induced in various cell types, including activated CD4+ T cells, NK cells, platelets, and B cells (Pucino V et al., 2020).

[0004] After binding to CD40, CD40L recruits TRAF and mediates downstream signaling via the NF-κB, JNK, and MAPK pathways, resulting in a variety of cell-type-dependent activation outcomes, including activation and proliferation of immune cells and secretion of inflammatory factors and chemokines (Vonderheide RH et al., 2007). For example, signaling via these pathways is required for several important effector functions of the adaptive immune system, including primary T cell-dependent antibody response (TDAR), B cell proliferation, germinal center (GC) formation, immunoglobulin (Ig) isotype conversion, somatic mutation, and differentiation of memory B cells and plasma cells (Foy TM et al., 1993; Foy TM et al., 1994). Beyond its effects on B cells, activation of the CD40 pathway provides important signals for DC maturation and function, as well as for the survival and cytokine secretion of mononuclear cells and macrophages (Caux, C et al., 1994).

[0005] Dysfunction of the CD40 signaling pathway can lead to autoimmune diseases (Karnell JL et al., 2018). The CD40-CD40L signaling pathway has been found to be involved in the function of parenchymal cells in inflammatory tissues: activated epithelial cells, originating from sites such as the kidney, salivary glands, and skin, that can secrete chemokines are capable of responding to CD40. Furthermore, the expression levels of either CD40 or CD40L are elevated in lesion sites in patients with atherosclerosis and preclinical atherosclerosis models. CD40 can stimulate and induce the expression of matrix-degrading enzymes and promote the expression of tissue factor in cell types associated with the pathogenicity of atherosclerosis, such as endothelial cells, smooth muscle cells, and macrophages (Michel NA et al., 2017). The CD40 pathway upregulates the production of inflammatory factors such as IL-1, IL-6, and IL-8, as well as adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule (VCAM). The CD40 / CD40L interaction has also been used to prevent transplant rejection. In a rhesus monkey kidney allograft study, the use of the chimeric anti-CD40 antagonist ch5D12 revealed that the antagonism of CD40 was sufficient to improve disease status and extend the mean survival time by more than 100 days. When ch5D12 was administered only at the start of an allograft study, followed by extension therapy with cyclosporine, a mean survival time of more than 4 years was achieved, demonstrating that this combination can potentially induce immune tolerance (Haanstra et al., 2005).

[0006] Numerous preclinical studies have provided evidence of the crucial role of CD40 / CD40L interaction in promoting T cell-dependent immune responses. Therefore, blocking CD40 signaling is considered an appropriate and necessary therapeutic strategy to inhibit pathogenic autoimmune responses in diseases such as rheumatoid arthritis, systemic lupus erythematosus, and Sjögren's syndrome. Currently, no anti-CD40 antibodies are approved for the treatment of such diseases. Therefore, there remains a strong need in this field for therapeutic agents that can intervene in the CD40-CD40L interaction and block CD40 signaling. This disclosure provides such a therapeutic humanized anti-CD40 antibody, which is specifically capable of binding to CD40 and possesses excellent antigen-binding specificity, affinity, pharmacokinetic and pharmacodynamic properties for intervention or treatment of CD40 signaling pathway-related diseases, particularly autoimmune diseases. Furthermore, it provides the use of a therapeutic humanized anti-CD40 antibody in combination with tacrolimus for the treatment of graft-versus-host disease or mitigation of transplant rejection. [Overview of the Initiative]

[0007] This disclosure provides an anti-CD40 antibody encoding a polynucleotide and its antigen-binding fragment, a vector containing the polynucleotide, host cells, a pharmaceutical composition containing the antibody or its antigen-binding fragment, methods for treating or intervening in autoimmune diseases (including graft-versus-host disease and transplant rejection), and related pharmaceutical applications.

[0008] Anti-CD40 antibody, its antigen-binding fragment According to one aspect, in some embodiments, the present disclosure is provided. Heavy chain HCDR1 containing the sequence shown in sequence number 23, A heavy chain HCDR2 containing the sequence shown in sequence number 24, A heavy chain HCDR3 containing the sequence shown in sequence number 25, The light chain LCDR1 includes the sequence shown in QX1SEDISSNLX2 (sequence number 74), where X1 is selected from A or S and X2 is selected from A or S. A light chain LCDR2 containing the sequence indicated by X3ASNLAS (sequence number 75), where X3 is selected from A or P, QGX4YWX5X6X7SX8FGX9X 10 The sequence includes the sequence shown in (Sequence ID 76), where X4 is selected from A or G, X5 is selected from S or T, X6 is selected from S or G, X7 is selected from T or S, X8 is selected from N or Y, X9 is selected from N, S, T or Q, and X 10 A light chain LCDR3 is selected from V or G, The present invention provides an anti-CD40 antibody and its antigen-binding fragment, which include the above.

[0009] In some specific embodiments, Heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences shown in sequence numbers 23, 24, and 25 respectively, and / or light chains LCDR1, LCDR2, and LCDR3 containing the sequences shown in sequence numbers 26, 27, and 28 respectively, Heavy chains HCDR1, HCDR2, HCDR3, and / or light chain LCDR1, containing the sequence shown in sequence number 29, 24, and 25 respectively, and light chain LCDR2, containing the sequence shown in sequence number 30, QGGYWTSTSNFGX9X 10 The sequence includes the sequence shown in (Sequence No. 73), of which X9 is selected from N, S, T, or Q, and X 10 A light chain LCDR3 selected from V or G, Heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences shown in sequence numbers 23, 24, and 25, respectively, and / or light chains LCDR1, LCDR2, and LCDR3 containing the sequences shown in sequence numbers 29, 27, and 32, respectively. The present invention provides an anti-CD40 antibody or its antigen-binding fragment, which includes the above.

[0010] In some specific embodiments, Heavy chain HCDR1, HCDR2, and HCDR3 each containing the sequences shown by SEQ ID NOs: 23, 24, and 25, light chain LCDR1 containing the sequence shown by SEQ ID NO: 29, light chain LCDR2 containing the sequence shown by SEQ ID NO: 30, and light chain LCDR3 containing any one of the sequences shown by SEQ ID NOs: 69 to 72. Provided is an anti - CD40 antibody or an antigen - binding fragment thereof, which contains .

[0011] In some specific embodiments, the present disclosure provides an anti - CD40 antibody or an antigen - binding fragment thereof, which contains any one of the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, or any combination thereof.

[0012] According to another aspect, in some embodiments, the present disclosure SYGVX 11 (SEQ ID NO: 88), wherein X 11 is a heavy - chain HCDR1 selected from S or T, and X 12 IX 13 SX 14 GX 15 X 16 YYAX 17 WAX 18 S (SEQ ID NO: 89), wherein X 12 is selected from A or G, X 13 is selected from G or A, X 14 is selected from T, S or D, X 15 is selected from T or S, X 16 is selected from T or A, X 17 is selected from S, N, X 18 is a heavy - chain HCDR2 selected from K or R, and GGITX 19 YAX 20 (SEQ ID NO: 90), wherein X 19 is selected from A or V, X 20 is a heavy - chain HCDR3 selected from I or M, and QASX 21 X 22 IX 23 X24 X 25 It includes the sequence indicated by LA (sequence number 91), of which X 21 If Q or E is selected, X 22 If selected from S or D, X 23 is selected from S or T, X 24 is selected from N, Q, S, or T, X 25 A light chain LCDR1 selected from V or G, A light chain LCDR2 containing the sequence shown in sequence number 37, QSYX 26 X 27 SX 28 X 29 TX 30 The sequence includes the sequence shown in (Sequence No. 92), of which X 26 If F or Y is selected, X 27 is selected from S, D, or N, X 28 is selected from S or F, X 29 is selected from S, T, or Y, X 30 A light chain LCDR3 selected from V or I, The present invention provides an anti-CD40 antibody or its antigen-binding fragment, which includes the above.

[0013] In some specific embodiments, Heavy chains HCDR1, HCDR2, HCDR3 containing the sequences shown in sequence numbers 33, 34, and 35, respectively, and / or light chains LCDR1, LCDR2, LCDR3 containing the sequences shown in sequence numbers 36, 37, and 38, respectively. Heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences shown in sequence numbers 39, 40, and 41 respectively, and / or light chains LCDR1, LCDR2, and LCDR3 containing the sequences shown in sequence numbers 42, 37, and 43 respectively, Heavy chains HCDR1, HCDR2, HCDR3 containing the sequences shown in sequence numbers 39, 44, and 35 respectively, and / or light chains LCDR1, LCDR2, LCDR3 containing the sequences shown in sequence numbers 45, 37, and 46 respectively, Heavy chains HCDR1, HCDR2, HCDR3 containing the sequences shown in sequence numbers 39, 47, and 41, respectively, and / or light chains LCDR1, LCDR2, LCDR3 containing the sequences shown in sequence numbers 36, 37, and 48, respectively. Heavy chain HCDR1, HCDR2, HCDR3, and / or QASQSISX, each containing the sequences indicated by sequence numbers 39, 47, and 49, respectively. 24 X 25 It includes the sequence indicated by LA (sequence number 87), of which X 24 is selected from N, Q, S, or T, X 25 A light chain LCDR1 selected from V or G, a light chain LCDR2 containing the sequence shown in SEQ ID NO: 50, a light chain LCDR3 containing the sequence shown in SEQ ID NO: 48, The present invention provides an anti-CD40 antibody or its antigen-binding fragment, which includes the above.

[0014] In some specific embodiments, Heavy chains HCDR1, HCDR2, and HCDR3 each contain the sequences indicated by sequence numbers 39, 47, and 49, respectively; light chain LCDR1 contains the sequence indicated by any one of sequence numbers 83 to 86; and light chains LCDR2 and LCDR3 each contain the sequences indicated by sequence numbers 50 and 48, respectively. The present invention provides an anti-CD40 antibody or its antigen-binding fragment, which includes the above.

[0015] In some specific embodiments, the present disclosure provides an anti-CD40 antibody or an antigen-binding fragment thereof comprising any one of the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, or any combination thereof.

[0016] In another embodiment, in some embodiments, the present disclosure provides an anti-CD40 antibody or antigen-binding fragment thereof comprising heavy chains HCDR1, HCDR2, HCDR3 containing sequences indicated by SEQ ID NOs. 51, 52, and 53, and / or light chains LCDR1, LCDR2, LCDR3 containing sequences indicated by SEQ ID NOs. 54, 55, and 56, respectively.

[0017] In some embodiments, the disclosure provides an anti-CD40 antibody or antigen-binding fragment thereof comprising heavy chains HCDR1, HCDR2, HCDR3 containing sequences indicated by SEQ ID NOs. 57, 58, and 59, respectively, and / or light chains LCDR1, LCDR2, LCDR3 containing sequences indicated by SEQ ID NOs. 60, 61, and 62, respectively.

[0018] In some specific embodiments, the present disclosure provides an anti-CD40 antibody or an antigen-binding fragment thereof comprising any one of the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, or any combination thereof.

[0019] In another embodiment, the disclosure includes a heavy chain variable region (VH) and a light chain variable region (VL), of which, a-1) The above VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by Sequence ID 1, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by Sequence ID 2. a-2) The above VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by Sequence ID 3, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by Sequence ID 4. a-3) The above VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by Sequence ID 5, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by Sequence ID 6. a-4) The above VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by either SEQ ID NO: 67 or 68, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by any one of SEQ ID NOs: 63 to 66. b-1) The above VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by Sequence ID 7, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by Sequence ID 8. b-2) The above VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by sequence number 9, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by sequence number 10. b-3) The above VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by Sequence ID 11, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by Sequence ID 12. b-4) The above VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by Sequence ID 13, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by Sequence ID 14. b-5) The above VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID 15, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID 16. b-6) The above VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID No. 17, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID No. 18. b-7) The above VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by either SEQ ID NO: 81 or 82, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by any one of SEQ ID NOs: 77 to 80. c) The above VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID 19, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID 20, or d) The above VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID 21, and the above VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID 22. Among these, the above-mentioned CDR provides an anti-CD40 antibody or its antigen-binding fragment, as defined by the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the CDR is defined by the Kabat numbering system.

[0020] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment is a recombinant antibody.

[0021] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment is a rabbit antibody, a chimeric antibody, a humanized antibody, a human antibody, or its antigen-binding fragment.

[0022] In some embodiments, when the anti-CD40 antibody or its antigen-binding fragment is a humanized antibody, the heavy chain framework region is derived from IGHV2-26*01, IGHV4-30-4*02, IGHV4-4*08, IGHJ1*01, and / or the light chain framework region is derived from IGkV1-13*02, IGkV1-9*01, IGkV1-6*01, IGKJ4*01. For example, FR1~FR3 of the heavy chain framework region are derived from IGHV2-26*01, IGHV4-30-4*02, IGHV4-4*08, FR4 of the heavy chain framework region is derived from IGHJ1*01, FR1~FR3 of the light chain framework region are derived from IGkV1-13*02, IGkV1-9*01, IGkV1-6*01, and FR4 of the light chain framework region is derived from IGKJ4*01.

[0023] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment comprises VH and VL, of which, A-1) The amino acid sequence of VH is shown in SEQ ID NO: 1 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 2 or has at least 90% identity thereto. A-2) The amino acid sequence of VH is shown in SEQ ID NO: 3 or has at least 90% identity with it, and the amino acid sequence of VL is shown in SEQ ID NO: 4 or has at least 90% identity with it. A-3) The amino acid sequence of VH is shown in SEQ ID NO: 5 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 6 or has at least 90% identity thereto. A-4) The amino acid sequence of VH is shown in SEQ ID NO: 67 or has at least 90% identity with it, and the amino acid sequence of VL is shown in any one of SEQ ID NOs: 63-66 or has at least 90% identity with it. A-5) The amino acid sequence of VH is shown in SEQ ID NO: 68 or has at least 90% identity with it, and the amino acid sequence of VL is shown in any one of SEQ ID NOs: 63-66 or has at least 90% identity with it. B-1) The amino acid sequence of VH is shown in SEQ ID NO: 7 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 8 or has at least 90% identity thereto. B-2) The amino acid sequence of VH is shown in SEQ ID NO: 9 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 10 or has at least 90% identity thereto. B-3) The amino acid sequence of VH is shown in SEQ ID NO: 11 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 12 or has at least 90% identity thereto. B-4) The amino acid sequence of VH is shown in SEQ ID NO: 13 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 14 or has at least 90% identity thereto. B-5) The amino acid sequence of VH is shown in SEQ ID NO: 15 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 16 or has at least 90% identity thereto. B-6) The amino acid sequence of VH is shown in SEQ ID NO: 17 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 18 or has at least 90% identity thereto. B-7) The amino acid sequence of VH is shown in SEQ ID NO: 81 or has at least 90% identity with it, and the amino acid sequence of VL is shown in any one of SEQ ID NOs: 77-80 or has at least 90% identity with it. B-8) The amino acid sequence of VH is shown in SEQ ID NO: 82 or has at least 90% identity with it, and the amino acid sequence of VL is shown in any one of SEQ ID NOs: 77-80 or has at least 90% identity with it. C) The amino acid sequence of VH is shown in SEQ ID NO: 19 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 20 or has at least 90% identity thereto, or D) The amino acid sequence of VH is shown in SEQ ID NO: 21 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 22 or has at least 90% identity thereto.

[0024] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment, for example, an IgG1, IgG2, IgG2, IgG4 antibody or its antigen-binding fragment, for example, an IgG1 antibody having the N297A mutation or its antigen-binding fragment, for example, an IgG1 antibody having one or any combination of L234A, L235A, M252Y, S254T, and T256E or its antigen-binding fragment.

[0025] In some embodiments, the antigen-binding fragment of the anti-CD40 antibody is Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nano antibody, peptide antibody (peptibody), domain antibody, and multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), for example, scFv, Fv, Fab, or Fab' fragment.

[0026] In some embodiments, the full-length heavy chain amino acid sequence of the antigen-binding fragment of the anti-CD40 antibody is represented by SEQ ID NO: 93 or 97 or has at least 90% identity thereto, and the full-length light chain amino acid sequence is represented by SEQ ID NO: 94 or has at least 90% identity thereto, or The full-length heavy chain amino acid sequence is represented by SEQ ID NO: 95 or 98, or has at least 90% identity thereto, and the full-length light chain amino acid sequence is represented by SEQ ID NO: 96, or has at least 90% identity thereto.

[0027] The "at least 90% identity" mentioned above includes, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity.

[0028] In some embodiments, the heavy chain variable region of the anti-CD40 antibody or its antigen-binding fragment has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes, and the light chain variable region has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes. In some specific embodiments, the above amino acid changes are conservative replacements, substitutions or modifications, and / or deletions or additions that do not affect function.

[0029] In some embodiments, the present invention provides an anti-CD40 antibody or antigen-binding fragment that binds to the same epitope as the above-mentioned anti-CD40 antibody or antigen-binding fragment, or that binds to the same epitope competitively.

[0030] In some embodiments, an anti-CD40 antibody or antigen-binding fragment is provided that blocks the binding of the anti-CD40 antibody or its antigen-binding fragment to CD40 (e.g., human CD40).

[0031] In some embodiments, an anti-CD40 antibody or antigen-binding fragment is provided whose binding to CD40 (e.g., human CD40) is blocked by the anti-CD40 antibody or its antigen-binding fragment.

[0032] In some embodiments, the anti-CD40 antibody or antigen-binding fragment is (i) K below 10 nM D And it binds to human CD40, (ii) The absence of clear agonist activity It has at least one of the following clauses.

[0033] In some embodiments, the anti-CD40 antibody or antigen-binding fragment reduces the binding of the CD40 ligand to CD40 by at least 45%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or at least 95%.

[0034] In some embodiments, the anti-CD40 antibody or antigen-binding fragment is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 K below M D It then binds to human CD40.

[0035] In some embodiments, a CD40-binding molecule is provided that contains any of the above-described anti-CD40 antibodies or their antigen-binding fragments.

[0036] In some embodiments, a complex comprising the anti-CD40 antibody or its antigen-binding fragment is provided. For example, the complex is an antibody-drug conjugate.

[0037] Polynucleotides and vectors This disclosure provides isolated polynucleotides encoding the anti-CD40 antibody of this disclosure or an antigen-binding fragment thereof. The isolated polynucleotide may be RNA, DNA, or cDNA. According to some embodiments of this disclosure, the polynucleotide of this disclosure is an isolated polynucleotide.

[0038] This disclosure further provides DNA molecules encoding any of the anti-CD40 antibodies or antigen-binding fragments thereof described in the above disclosure.

[0039] The polynucleotides of this disclosure may be in the form of a vector, present in a vector, and / or part of a vector, such vector being, for example, a plasmid, cosmid, YAC, or viral vector. The vector may be an expression vector, i.e., a vector capable of providing in vitro and / or in vivo expression (i.e., in a suitable host cell, host organism, and / or expression system) of a VEGF-binding molecule or its complex. Such an expression vector typically comprises at least one polynucleotide of this disclosure, which is operably linked to one or more suitable expression regulators (e.g., promoters, enhancers, terminators, etc.). Selecting the above-mentioned elements and their sequences for expression in a particular host is common sense for those skilled in the art. Useful or necessary regulators and other elements for the expression of the anti-CD40 antibody or its antigen-binding fragment of this disclosure include, for example, promoters, enhancers, terminators, embedded factors, selection markers, reader sequences, and reporter genes.

[0040] The polynucleotides of this disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA technology) and / or isolated from suitable natural sources, based on information regarding the amino acid sequences of the polypeptides of this disclosure.

[0041] host cell This disclosure provides recombinant host cells comprising the anti-CD40 antibody or its antigen-binding fragment or complex, or the polynucleotide or vector of this disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0042] Bacterial cells include, for example, Gram-negative strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0043] Fungal cells include, for example, cells of the genera Trichoderma, Neurospora, and Aspergillus, or cells of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0044] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, and COS cells.

[0045] However, the present disclosure may use amphibian cells, insect cells, plant cells and any other cells in the art for expressing heterologous proteins.

[0046] In one embodiment, the host cells used in this disclosure cannot develop into a complete plant or animal organism.

[0047] Preparation method This disclosure provides a method for preparing the anti-CD40 antibody or its antigen-binding fragment, which, - Culture host cells of the present disclosure under conditions that enable the expression of the anti-CD40 antibody or its antigen-binding fragment, - The method includes recovering anti-CD40 antibodies or their antigen-binding fragments expressed by host cells from the culture, and - This includes optionally further purifying and / or modifying the anti-CD40 antibody of the present disclosure or its antigen-binding fragment.

[0048] This disclosure provides a method for preparing a complex, comprising compounding or modifying a drug with the anti-CD40 antibody or its antigen-binding fragment provided herein.

[0049] The anti-CD40 antibody or its antigen-binding fragment of the present disclosure may be produced intracellularly in the above-mentioned cells (e.g., in the cytoplasm, periplasm, or in inclusion bodies), then isolated from the host cell, and optionally further purified; or it may be produced extracellularly (e.g., in the culture medium for the host cell), then isolated from the medium, and optionally further purified. For example, purification may be performed using an A or G Sepharose FF column containing a prepared buffer, nonspecifically bound components may be washed away, the bound antibody may be eluted by a pH gradient, and detected and collected by SDS-PAGE. Selectively, the product may be filtered and concentrated by conventional methods. Soluble mixtures and polymers may be removed by conventional methods such as molecular sieving or ion exchange. The obtained product may be immediately frozen at -70°C or lyophilized.

[0050] Methods and reagents for recombinant polypeptide production, such as specific preferred expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, the protein isolation and purification techniques applicable to the methods for producing the anti-CD40 antibody or its antigen-binding fragment or complex according to this disclosure are well known to those skilled in the art.

[0051] composition This disclosure provides compositions comprising the above-described anti-CD40 antibody or its antigen-binding fragment. For example, it provides a pharmaceutical composition comprising a therapeutic or palliative effective amount of the above-described anti-CD40 antibody or its antigen-binding fragment and at least one medicinal excipient, diluent, or carrier.

[0052] In some specific embodiments, the unit dose of the pharmaceutical composition may contain 0.01 to 99 wt% of anti-CD40 antibody or its antigen-binding fragment, or the content of anti-CD40 antibody or its antigen-binding fragment in the unit dose of the pharmaceutical composition may be 0.1 to 2000 mg, and in some specific embodiments, 1 to 1000 mg.

[0053] In some embodiments, the following are provided: a combination or composition of the anti-CD40 antibody or its antigen-binding fragment with one or more other immunosuppressants. The composition is, for example, a pharmaceutical composition. Selectively, the pharmaceutical composition may further contain pharmaceutically acceptable excipients, diluents, or carriers.

[0054] One embodiment of this disclosure provides a pharmaceutical composition comprising the above-mentioned anti-CD40 antibody or its antigen-binding fragment, tacrolimus, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0055] In some embodiments, a product comprising the anti-CD40 antibody or its antigen-binding fragment is provided. Selectively, the product comprises a container and a label. The container is, for example, a vial, syringe, and test tube. The container contains a composition effective for treating a medical condition. A label on or attached to the container indicates that the composition is used for the treatment of a selected medical condition. The composition contains the anti-CD40 antibody or its antigen-binding fragment. The product may further comprise a second container containing tacrolimus effective for treating a disease.

[0056] In some embodiments, a product comprising the above-mentioned anti-CD40 antibody or its antigen-binding fragment and tacrolimus is provided.

[0057] Treatment methods and pharmaceutical uses This disclosure provides a method for producing the above-mentioned anti-CD40 antibody or its antigen-binding fragment for use in the treatment, intervention, prevention, or diagnosis of a disease or medical condition.

[0058] Specifically, in some embodiments, the Disclosure provides uses of the anti-CD40 antibodies or antigen-binding fragments described herein in the preparation of agents for treating or alleviating autoimmune diseases, graft-versus-host diseases, or mitigating transplant rejection.

[0059] Furthermore, in some embodiments, the Disclosure provides applications for the preparation of agents for treating or alleviating autoimmune diseases, graft-versus-host diseases, or mitigating transplant rejection, using a combination of the anti-CD40 antibody or its antigen-binding fragment described herein with one or more other immunosuppressants.

[0060] In embodiments of this disclosure, the anti-CD40 antibody or its antigen-binding fragment described herein may be administered individually, sequentially, or simultaneously with one or more other immunosuppressants.

[0061] In embodiments of this disclosure, the anti-CD40 antibody or its antigen-binding fragment described herein may be administered before, after, or concurrently with one or more other immunosuppressants.

[0062] Specifically, this disclosure provides applications of the anti-CD40 antibody or its antigen-binding fragment described herein in combination with tacrolimus in the preparation of agents for treating or alleviating autoimmune diseases, graft-versus-host diseases, or mitigating transplant rejection. In some embodiments, methods and related pharmaceutical applications are provided for improving or treating graft-versus-host diseases, organ transplant rejection, etc., which include administering to a subject an improved or therapeutically effective amount of the anti-CD40 antibody or its antigen-binding fragment or the pharmaceutical composition thereof.

[0063] In some embodiments, methods for improving or treating autoimmune diseases, inflammatory diseases, and related pharmaceutical applications are provided, comprising administering to a subject an effective amount of the above-mentioned anti-CD40 antibody or its antigen-binding fragment or a pharmaceutical composition thereof for improvement or treatment.

[0064] In some embodiments, methods for treating CD40-related disorders and related pharmaceutical applications are provided; in some embodiments, methods for inhibiting the growth or differentiation of CD40-related disordered cells and related pharmaceutical applications; in some embodiments, methods for inhibiting the growth and / or differentiation of cells expressing human CD40 antigen and related pharmaceutical applications; in some embodiments, methods for inhibiting antibody production by B cells in a subject and related pharmaceutical applications; and in some embodiments, methods for treating immunodeficiency disorders and related pharmaceutical applications. Each of the above methods comprises administering to a subject or cells a therapeutic or inhibitory amount of the above anti-CD40 antibody or its antigen-binding fragment or a pharmaceutical composition thereof.

[0065] In some embodiments, methods for inducing peripheral B cell depletion and related pharmaceutical applications are provided, which include administering to a subject an effective amount of the anti-CD40 antibody or its antigen-binding fragment or a pharmaceutical composition thereof.

[0066] In some embodiments, methods for treating or alleviating diseases or conditions and related pharmaceutical uses are provided, comprising administering an effective amount of the above anti-CD40 antibody or its antigen-binding fragment to the target subject, wherein the disease or condition may or may not be related to CD40, and includes rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, autoimmune demyelinating diseases (e.g., multiple sclerosis, allergic encephalomyelitis), endocrine eye diseases, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune liver disease, inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), hypersensitivity, anaphylactic reactions, Sjögren's syndrome, type 1 diabetes, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, inflammatory myositis, polyendocrine insufficiency, and Schmidt's syndrome. Dressler's syndrome, autoimmune uveitis, Addison's disease, adrenal thyroiditis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, herpetiform dermatitis, alopecia areata, bullous pemphigoid, scleroderma, systemic progressive sclerosis, CREST syndrome (calcification, Raynaud's phenomenon) (phenomenon), esophageal peristalsis, finger sclerosis and telangiectasia, autoimmune infertility in men and women, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas's disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid syndrome, farmer's lung, erythema multiforme, postoperative syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, avian breeder's lung, toxic epidermal necrolysis, Alport's syndromeSyndrome, alveolitis, allergic alveolitis, fibrotic alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatous granulomatosis, Behcet's disease, Kaplan's syndrome, Kawasaki disease, dengue fever, encephalomyelitis, endocarditis, endocardial fibrosis, endophthalmitis, erythema elevata, psoriasis, erythroblastosis fetus, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome This includes conditions such as filariasis, pilosa, ciliitis, chronic ciliitis, heterochromic ciliitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft-versus-host disease, transplant rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenström macroglobulinemia, Evans syndrome, acute respiratory distress syndrome, pneumonia, osteoporosis, delayed-type hypersensitivity reactions, and autoimmune gonadal dysfunction. Examples include Sjögren's syndrome, multiple sclerosis, and systemic lupus erythematosus.

[0067] In some embodiments, the present invention provides methods and related pharmaceutical applications for treating diseases associated with B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture syndrome, rheumatoid arthritis, and type 1 diabetes), Th1 lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's disease, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft-versus-host disease), or Th2 lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft-versus-host disease), comprising administering an effective amount of the above-mentioned anti-CD40 antibody or its antigen-binding fragment to the target subject as needed.

[0068] In some embodiments, methods for treating tumors or cancer and related pharmaceutical applications are provided, comprising administering an effective amount of the anti-CD40 antibody or its antigen-binding fragment to a target subject of interest, wherein the tumor or cancer may or may not be related to CD40 expression.

[0069] In some embodiments, a method is provided for treating or mitigating graft-versus-host disease or transplant rejection, comprising administering the anti-CD40 antibody or its antigen-binding fragment and tacrolimus to the subject in need.

[0070] In some embodiments, the use of the anti-CD40 antibody or its antigen-binding fragment in the preparation of agents for treating or mitigating graft-versus-host disease or transplant rejection is provided, including in combination with tacrolimus. In some embodiments, the use of tacrolimus in the preparation of agents for treating or mitigating graft-versus-host disease or transplant rejection is provided, including in combination with the anti-CD40 antibody or its antigen-binding fragment.

[0071] In some embodiments, the present invention provides a method for using the above-mentioned anti-CD40 antibody or its antigen-binding fragment in combination with tacrolimus for the treatment or mitigation of graft-versus-host disease or transplant rejection, and uses for the preparation of a drug for the treatment or mitigation of graft-versus-host disease or transplant rejection in combination.

[0072] In some embodiments, the transplant is a solid organ transplant, such as a kidney transplant, liver transplant, heart transplant, lung transplant, pancreas transplant, small intestine transplant, or composite tissue transplant.

[0073] In some embodiments, the above transplantation means transplanting one selected from the group consisting of allogeneic cells, xenogeneic cells, allogeneic tissue, xenogeneic tissue, allogeneic organ, and xenogeneic organ.

[0074] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment inhibits or reverses rejection based on tissue grafts in transplant recipients, or extends or maintains the function of tissue transplanted to transplant recipients, or restores the function of damaged transplant tissue in transplant recipients.

[0075] detection This disclosure provides compositions for detecting CD40, the compositions comprising an anti-CD40 antibody or its antigen-binding fragment according to this disclosure. This disclosure further provides a method, system, or apparatus for detecting CD40 in vivo or in vitro, which comprises treating a sample with the anti-CD40 antibody or its antigen-binding fragment according to this disclosure.

[0076] In some embodiments, the in vitro detection method, system, or apparatus is, for example, (1) Contact the sample with an anti-CD4 antibody or its antigen-binding fragment. (2) To detect a complex formed between a CD40-conjugated antibody or its antigen-conjugated fragment and a sample, and / or (3) Contacting a reference sample (e.g., a control sample) with the antibody, and (4) Determine the degree of complex formation by comparing it with a reference sample. This may include: For example, a change in composite formation in the sample or subject compared to that in the control sample or subject (e.g., a statistically significant change) indicates the presence of CD40 in the sample.

[0077] In some other embodiments, an in vivo detection method, system, or apparatus is: (1) Administer an anti-CD40 antibody or its antigen-binding fragment to the subject, (2) To detect the formation of a complex between an anti-CD40 antibody or its antigen-binding fragment and a target, It may include.

[0078] Detection may include determining the location or time of complex formation. Detection of a substance binding to the CD40 antibody (e.g., CD40) is achieved by labeling the CD40 antibody with a detectable substance and detecting the label. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. The formation of a complex between the CD40-binding antibody or its antigen-binding fragment and CD40 can be detected by measuring or visualizing antibodies that bind to or do not bind to CD40. Conventional detection assays, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), or tissue immunohistochemistry, can be used. For detection, the anti-CD40 antibody or its fragment according to this disclosure may be labeled with a fluorophore chromophore.

[0079] In some embodiments, a reagent kit is further provided, which comprises an anti-CD40 antibody or its antigen-binding fragment, and may further comprise a diagnostic manual. The reagent kit may further comprise at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the antibody can be prepared as a pharmaceutical composition.

[0080] Definition of Terms To make this disclosure more easily understood, several technical and scientific terms are defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings that are ordinarily understood by those skilled in the art.

[0081] Unless otherwise specified in the context, words such as "contain," "possess," and "include" throughout the specification and claims should be understood to have a comprehensive meaning, i.e., "includes, but is not limited to," rather than an exclusive or exhaustive meaning.

[0082] The three-letter and one-letter amino acid codes used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

[0083] "CD40" and "CD40 antigen" refer to a glycoprotein of approximately 48 kD expressed on the surface of normal and neoplastic B cells, which acts as a receptor for signals involved in cell proliferation and differentiation (Ledbetter et al., 1987, J.Immunol. 138:788-785). The cDNA molecule encoding CD40 was isolated from a library prepared using the Burkitt lymphoma cell line Raji (Stamenkovic et al., 1989, EMBO J. 8:1403). Sequence information may be found in Table 2 of this disclosure. Cells endogenously expressing CD40 are any cells characterized by surface expression of CD40, including, but not limited to, normal and neoplastic B cells, digitoid cells, basal epithelial cells, cancer cells, macrophages, endothelial cells, follicular dendritic cells, tonsillar cells, and bone marrow-derived plasma cells.

[0084] The term "antibody" is used in its broadest sense to refer to any antibody that exhibits the desired antigen-binding activity, covering a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions). An antibody may also refer to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to differences in the amino acid composition and sequence order of the heavy chain constant region. Therefore, immunoglobulins can be divided into five types, or immunoglobulin isotypes, called IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain can be divided into a κ chain or a λ chain depending on the constant region. Each of the five types of Ig may have either a κ chain or a λ chain. In the antibody heavy and light chains, the sequence of approximately 110 amino acids near the N-terminus is greatly altered, forming a variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable, forming a constant region (C region). The variable region includes three hypervariable regions (CDRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3, while the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0085] The definitive determination or definition of a CDR can be achieved by elucidating the structure of the antibody and / or the structure of the antibody-ligand complex, thereby achieving a reliable description of the CDR and identification of residues containing the antibody binding site. This can be accomplished by any one of the various techniques known to those skilled in the art, for example, by X-ray crystallography. Various analytical methods can be used for the identification of CDRs, including, but not limited to, the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definition, and conformational definition.

[0086] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural loop regions (see, e.g., Chothia et al., 1986, J.Mol.Biol., 196:901-17, and Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs from Oxford Molecular Group that models antibody structures (see, for example, Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272, “AbMTM, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK, Oxford Molecular, Ltd). The AbM numbering system models the tertiary structure of antibodies from their basic sequences using a combination of a knowledge database and the ab initio method (see “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach” in Samudrala et al., 1999, PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). The contact definition is based on the analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J.Mol.Biol., 5:732-45). In the conformational definition, the position of the CDR can be identified as a residue that makes an enthalpy contribution to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).While the definitions of other CDR boundaries do not necessarily strictly follow one of the methods described above, they still overlap with at least some Kabat CDRs, although they may be shortened or extended, based on predictions or experimental results that certain residues or groups of residues do not significantly affect antigen binding. As used herein, a CDR can refer to a CDR defined by any method (including combinations of methods) known in the art. The correspondences of each numbering system are well known to those skilled in the art and are shown exemplarily in Table 1 below.

[0087] [Table 1]

[0088] The CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment relating to this disclosure conform to known Kabat numbering systems in terms of quantity and position. A "monoclonal antibody" is an antibody obtained from a essentially homogeneous antibody population; that is, apart from small, naturally occurring variations, each antibody in the population is identical. Monoclonal antibodies are highly specific and target a single antigen site. Furthermore, in contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier "monoclonal" indicates, for example, the characteristic of antibodies obtained from an essentially homogeneous antibody population, and does not imply that the antibody needs to be produced by any particular method.

[0089] In this disclosure, the term "rabbit antibody" refers to a monoclonal antibody against human CD40 or its epitope prepared by the knowledge and techniques of the art. During preparation, the CD40 antigen is injected into test rabbits, and then antibodies expressing antibodies with desired sequence or functional properties are isolated. In one specific embodiment of this disclosure, the rabbit anti-human CD40 antibody or its antigen-binding fragment may further comprise the light chain constant region of a rabbit κ, λ chain or a variant thereof, or further comprise the heavy chain constant region of rabbit IgG1, IgG2, IgG3, or IgG4 or a variant thereof.

[0090] The term "fully human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies according to this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis, or in vivo somatic mutation). However, the term "fully human antibody" does not include antibodies (i.e., "humanized antibodies") obtained by transplanting CDR sequences derived from the germline of another mammalian species (e.g., rabbits) into a human skeletal sequence.

[0091] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by transplanting a non-human CDR sequence into a human antibody variable region framework. This allows for overcoming the strong immune response induced by chimeric antibodies due to their large amount of non-human protein components. To avoid decreased activity associated with reduced immunogenicity, activity can be maintained by performing the smallest possible reverse mutations on the fully human antibody variable region.

[0092] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of an antibody of a first species with the constant region of an antibody of a second species, and can reduce the immune response induced by the antibody of the first species. As an example, to create a chimeric antibody, first, rabbits that secrete rabbit-specific monoclonal antibodies are created, the antibodies are isolated, and if necessary, the constant region gene of a fully human antibody is cloned. The rabbit variable region gene and the human constant region gene are then ligated to form a chimeric gene, which is then inserted into a human vector, and finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell line. The constant region of the fully human antibody may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4 or their variants, preferably including the human IgG1 or IgG4 heavy chain constant region, or using IgG1 that does not exhibit ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

[0093] The term "antigen-binding fragment" includes single-chain antibodies (i.e., full-length heavy and light chains), Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH, VL, or VHH), scFv, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and any one of the above epitope-binding fragments (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136, Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for producing and preparing these antibody fragments are known in this field (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv form was first disclosed in WO2009 / 040562, and its disulfide bond-stabilized form, Fab-dsFv, was first disclosed in WO2010 / 035012. The antigen-binding fragments relating to this disclosure further include the Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. The polyvalent antibodies may include multispecific, for example, bispecific, or monospecific (see, for example, WO92 / 22583 and WO05 / 113605), an example of the latter being Tri-Fab (or TFM) described in WO92 / 22583.

[0094] The term "binding to CD40" means being able to interact with CD40 or its epitope, which may be of human origin. The term "antigen-binding site" in this disclosure means a three-dimensional spatial site that is not contiguous in the antigen and is recognized by the antibody or antigen-binding fragment of this disclosure.

[0095] The term “antigen” refers to a molecule used to immunize immunocompetent vertebrates to produce antibodies that recognize the antigen, or to screen expression libraries (e.g., phages, saccharomyces, or ribosome display libraries). In this disclosure, antigens are defined more broadly and include target molecules that are specifically recognized by antibodies, and also include some molecules or mimics used in the immunization process for antibody production or the selection of libraries for antibody selection. With respect to human CD40-conjugated antibodies in this disclosure, monomers and polymers of human CD40 (e.g., dimers, trimers, etc.), and deletion mutants and other mutants of human CD40 are all referred to as antigens.

[0096] The term "epitope" refers to a site in an antigen that binds to an immunoglobulin or antibody. Epitopes may be formed from adjacent amino acids or from non-adjacent amino acids arranged in parallel by tertiary folding of the protein. Epitopes formed from adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. Epitopes typically have a unique spatial conformation and contain at least 3 to 15 amino acids. Methods for determining which epitope is bound by a given antibody are well known in the art and include Western blotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0097] The terms "specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a given antigen. Typically, when human CD40 or its epitope is used as the analyte and an antibody is used as the ligand, and measurement is performed using surface plasmon resonance (SPR) technology in an instrument, the antibody binds to approximately 10 -7 Equilibrium dissociation constant (K) less than or equal to M D) binds to a predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is at least twice as much as its binding affinity to nonspecific antigens other than the predetermined antigen (or its epitope) or closely related antigen (e.g., BSA). The term “antigen-recognizing antibody” may be used interchangeably with the term “specifically binding antibody” in this disclosure.

[0098] In this disclosure, "binding affinity" or "affinity" is used as an indicator of the strength of the non-covalent interaction between two molecules (e.g., an antibody or a part thereof and an antigen). The binding affinity between two molecules is expressed by the dissociation constant (K). D KD can be quantified by determining the rate constants. KD can be determined, for example, by measuring the dynamics of composite formation and dissociation using surface plasmon resonance (SPR) (Biacore). The rate constants corresponding to bonding and dissociation of monovalent composites are called the bonding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D is, K D The dissociation constant is related to ka and kd by the equation =kd / ka. The value of the dissociation constant can be directly determined by well-known methods, and can also be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). D This can be determined by a double-filter nitrocellulose filter binding assay, such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). Other standard assays for evaluating the antibody's binding ability to target antigens are known in the art and include, for example, ELISA, Western blotting, RIA and flow cytometry analysis, and other assays cited elsewhere in this disclosure. Antibody binding kinetics and binding affinity can be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, Biacore. TM The K of each antibody / antigen complex may be evaluated by the system or KinExA. DBy comparing values, we can compare binding affinities related to interactions with different molecules, for example, comparing the binding affinities of different antibodies to a given antigen. Similarly, the specificity of the interaction is the K of the target interaction (e.g., a specific interaction between an antibody and an antigen). D K values ​​and non-target interactions (e.g., known control antibodies that do not bind to CD40) D Evaluation can be performed by determining and comparing values.

[0099] The term "conservative substitution" refers to the substitution of an original amino acid residue with another amino acid residue that has similar properties. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have polar side chains with no charge, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Furthermore, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that when amino acid residues in the group exhibiting the above-mentioned similar properties are substituted, it does not result in any particular change in properties.

[0100] "Inhibition" and "blockage" may be used interchangeably and cover both partial and complete inhibition / blockage. Inhibition / blockage against CD40 preferably reduces or alters the normal level or type of activity that would occur when CD40 binding occurs in the absence of inhibition or blockage. Inhibition and blockage are also intended to include a decrease in any measurable CD40 binding affinity when in contact with an anti-CD40 antibody compared to CD40 not in contact with the anti-CD40 antibody.

[0101] "Inhibition of growth" (for example, with respect to cells) is intended to include any measurable decrease in cell growth.

[0102] "Agonist activity," "agonist behavior," or "agonist properties" refers to the function of an agonist. The binding of an agonist to a cell receptor elicits a reaction or activity similar to or identical to that caused by the receptor's natural ligand. For example, CD40 agonists can induce any or all of the following responses: cell proliferation and / or differentiation, upregulation of cell-cell adhesion by molecules such as ICAM-1, E-selectin, and VCAM; secretion of pro-inflammatory cytokines such as IL-1, IL-6, IL-8, IL-12, and TNF; signaling via pathways such as TRAF (e.g., TRAF2 and / or TRAF3), MAP kinases, e.g., NIK (NF-κB-inducing kinase), 1-κB kinase (IKKα / β), the transcription factor NF-κB, the Ras and MEK / ERK pathway, the PI3K / Akt pathway, and the P38 MAPK pathway; transmission of anti-apoptotic signals by molecules such as XIAP, Mcl-1, and BCLx; production of B and / or T cell memory; production of B cell antibodies; conversion of B cell isotypes; and upregulation of cell surface expression of class II MHC and CD80 / 86. "Anticantal activity," "antagonistic activity," or "antagonism" refers to the function of a substance that can act as an antagonist. For example, a CD40 antagonist can prevent or reduce any response induced by the binding of the CD40 receptor to an agonist ligand, specifically CD40L. An antagonist can reduce one or more responses induced by agonist binding by 5%, 10%, 15%, 20%, 25%, 30%, 35%, preferably 40%, 45%, 50%, 55%, 60%, more preferably 70%, 80%, 85%, and most preferably 90%, 95%, 99%, or 100%. Methods for detecting the binding specificity and antagonistic activity of anti-CD40 antibodies to CD40 ligands are known to those skilled in the art and include, but are not limited to, standard competitive binding assays, assays for monitoring immunoglobulin secretion by B cells, B cell proliferation assays, Banchereau-like B cell proliferation assays, T cell-assisted assays for antibody production, B cell proliferation co-stimulation assays, and assays for upregulating B cell activation markers.

[0103] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art and can be found, for example, in the Antibody Experiment Techniques Manual by Minato Reizei (Chapters 5-8 and 15). For example, mice can be immunized with human CD40 or a fragment thereof, and the resulting antibodies can be restored, purified, and subjected to amino acid sequencing by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments described herein are obtained by genetic engineering by adding one or more human-derived FR regions to a non-human-derived CDR region. Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website.

[0104] Antibodies can be competitively screened for binding to the same epitope by ordinary techniques known to those skilled in the art. For example, competitive and cross-competitive studies can be conducted to obtain antibodies that compete with each other or cross-compete to bind to an antigen. A high-throughput method for obtaining antibodies that bind to the same epitope through such cross-competition is described in International Patent Publication WO03 / 48731. Accordingly, antibodies and their antigen-binding fragments can be obtained by ordinary techniques known to those skilled in the art to compete with the antibody molecules relating to this disclosure for binding to the same epitope in CD40.

[0105] The term “disorder” refers to any medical condition for which treatment with the humanized anti-CD40 antibody of this disclosure may be beneficial. This includes chronic and acute disorders or diseases. Non-limiting examples of disorders to be treated in this disclosure include cancer, hematological malignancies, benign and malignant tumors, leukemia and lymphoid malignancies, as well as inflammatory disorders, angiogenic disorders, autoimmune disorders and immunological disorders.

[0106] The term “CD40-related disorder” or “CD40-related disease” refers to a medical condition characterized by modification or removal of cells that express CD40. These cells include CD40-expressing cells exhibiting abnormal proliferation, or CD40-expressing cells associated with cancerous or malignant proliferation. More specific examples of cancers exhibiting abnormal CD40 antigen expression include B lymphoblastic tumors, Burkitt lymphoma, multiple myeloma, T-cell lymphoma, Kaposi's sarcoma, osteosarcoma, epidermal and endothelial tumors, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer (melanoma), bladder cancer, and kidney cancer. Such disorders include, but are not limited to, leukemia, lymphoma (including B-cell lymphoma and non-Hodgkin lymphoma), multiple myeloma, Waldenström macroglobulinemia, sarcomas such as osteosarcoma and Ewing's sarcoma, malignant melanoma, adenocarcinoma (including ovarian adenocarcinoma), solid tumors including Kaposi's sarcoma / Kaposi's tumor and squamous cell carcinoma. "CD40-related disorders" further include diseases and disorders of the immune system, such as autoimmune disorders and inflammatory disorders. Such conditions include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), pneumonia, asthma, and idiopathic thrombocytopenic purpura (ITP).

[0107] The terms "to inhibit the growth of..." or "growth inhibition" refer to inhibiting the growth or proliferation of cells, particularly neoplastic cell types that express the CD40 antigen. Therefore, growth inhibition, for example, significantly reduces the percentage of S-phase neoplastic cells.

[0108] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biofluids, “give,” “administer,” and “process” mean the contact of exogenous drugs, therapeutic agents, diagnostic agents, or compositions with animals, humans, subjects, cells, tissues, organs, or biofluids. “Give,” “administer,” and “process” may also mean, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. Processing of cells includes contact of reagents with cells and contact of reagents with fluids, where the fluids come into contact with the cells. “Give,” “administer,” and “process” also mean the treatment of, for example, cells in vitro and in vitro with reagents, diagnostics, conjugated compositions, or through other types of cells. “Process” means therapeutic treatment, preventive or precautionary measures, research, and diagnostic applications when applied to humans, veterinary medicine, or research subjects.

[0109] "Treatment" means administering to a subject an internal or external therapeutic agent, such as any one of the antibodies or its antigen-binding fragments or compositions thereof, where the subject has, is at risk of, or is prone to having one or more diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the subject or population being treated is given the therapeutic agent in a dose that effectively alleviates one or more disease symptoms, whether by inducing regression of such symptoms or by inhibiting such symptoms from progressing to any clinically measurable degree. The amount of therapeutic agent that effectively alleviates any specific disease symptom (also called the "therapeutic dose") can vary depending on several factors, including the subject's disease state, age and weight, and the agent's ability to produce the therapeutic effect required by the subject. Whether the disease symptoms have been reduced can be assessed by any clinical detection method commonly used by physicians or other professional healthcare providers to assess the severity and progression of the symptoms. Embodiments of the present disclosure (e.g., a treatment method or product) may be ineffective in alleviating a target disease symptom in a given subject, but have been confirmed to alleviate a statistically significant number of subjects by any statistical test known in the art, such as the Student t-test, chi-squared test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0110] An "effective dose" includes an amount sufficient to improve or prevent the symptoms or condition of a medical disorder. The effective dose also refers to an amount sufficient to allow or promote a diagnosis. The effective dose used for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the method and route of administration and dosage, and the severity of side effects. The effective dose may also be the maximum dose or administration plan that avoids significant side effects or toxic effects.

[0111] "Homologousity" or "identity" refers to the similarity between two polynucleotide sequences or two polypeptides. If the positions in the two sequences being compared are both occupied by the same nucleotide or amino acid monomer subunit—for example, if the positions in two DNA molecules are both occupied by the same nucleotide—then the molecules are homologous at those positions. The percentage of homology between two sequences is a function of dividing the number of matching or homologous positions shared by the two sequences by the number of positions being compared and multiplying by 100%. For example, if the sequences are optimally aligned, and 6 out of 10 positions in the two sequences are matching or homologous, then the two sequences are 60% homologous. Generally, two sequences are compared when they are aligned to obtain the highest possible percentage of homology.

[0112] The terms "cells," "cell lines," and "cell cultures" may be used interchangeably, and any such name includes their offspring. Furthermore, it should be understood that, due to unintentional or unintentional mutations, no offspring can be exactly the same in terms of DNA content. This includes mutant offspring with similar function or biological activity to those screened from the initial transformed cells.

[0113] "Optional" or "optionally" means that the event or situation described thereafter may or may not occur, and this description includes both cases in which the event or situation occurs and cases in which it does not. For example, "optionally containing 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a specific sequence may or may not be present.

[0114] The “CD40-binding molecule” in this disclosure, interpreted to the maximum extent, includes any protein capable of binding to CD40, including the anti-CD40 antibody or its antigen-binding fragment, and falls within the scope of that term. For example, a CD40-binding protein may include, for example, one or more effector molecules in a complex. The “effector molecule” may have therapeutic activity on its own (e.g., antitumor activity or immunoactivating or inhibitory activity) or detection function, and may take any form, such as a physiologically active protein (e.g., an enzyme), another antibody or antibody fragment, synthetic or naturally occurring polymers, polynucleotides and their fragments, for example, DNA, RNA and its fragments, radionuclides (especially radioactive iodides), radioisotopes, chelate metals, nanoparticles and reporter groups (e.g., fluorescent compounds), or compounds detectable by NMR or ESR spectroscopy. The complex configuration of the effector molecule with the anti-CD40 antibody or its antigen-binding fragment can be achieved by conventional methods.

[0115] An "antibody-drug conjugate" refers to a complex formed by linking a drug (e.g., an antitumor agent, a toxin) to an antibody, and the above-mentioned conjugate can be linked by conventional methods, for example, by a cleavable or incleavable linker. [Brief explanation of the drawing]

[0116] [Figure 1A-B] Figures 1A and 1B show the activity results of CD40 antagonist antibodies in a reporter gene system, with results related to 9E6-L4H2 and 2F12-L4H2, respectively. Both Figures 1A and 1B use human IgG1 isotype as a negative control and CFZ533 as a positive control. [Figure 2A-B]Figure 2A shows the inhibitory activity results of CD40 antagonist antibodies in B cell activation experiments. Figure 2B shows the percentage inhibition results of CD19+CD69+ cells in 9E6-L4H2 and 2F12-L4H2 cells, and Figure 2B shows the MFI inhibition results of CD19+CD69+ cells in 9E6-L4H2 and 2F12-L4H2 cells. In both Figures 2A and 2B, human IgG1 isotype was used as a negative control and CFZ533 was used as a positive control. [Figure 3A-D] Figures 3A, 3B, and 3D show the inhibitory activity of CD40 antagonist antibodies in DC cell activation experiments. Figure 3A shows the inhibition of MFI in CD11C+CD80+ cells of 9E6-L4H2 and 2F12-L4H2 cells, Figure 3C shows the inhibition of IL-12 / 23 p40 in 9E6-L4H2 and 2F12-L4H2 cells, and Figure 3D shows the inhibition of TNFα in 9E6-L4H2 and 2F12-L4H2 cells. In all of Figures 3A-3D, human IgG1 isotype was used as a negative control and CFZ533 was used as a positive control. [Figure 4] These are the endogenous agonist activity results of CD40 antagonist antibodies 9E6-L4H2 and 2F12-L4H2 in a B cell activation experimental system, using human IgG1 isotype, CFZ533, and the agonist anti-CD40 antibody 9E5-SELFNS as controls. [Figure 5A-B] Figure 5A shows the activity results of CD40 antagonist antibodies in a mouse T cell-dependent humoral immune response model. Figure 5B shows the detection results on days 7, 14, 21, and 28. [Figure 6A-B] Figure 6A shows the activity results of CD40 antagonist antibodies in a mouse skin graft rejection model, with Figure 6A being a flowchart and Figure 6B showing the skin graft survival rate (%) and skin graft scoring results. [Figure 7]Figure 7A shows the activity results of combining a CD40 antagonist antibody with tacrolimus (FK506) in a mouse skin graft rejection model. Figure 7B shows the skin graft survival rate (%) for 9E6-L4H2 (10 mpk) and its combination with tacrolimus (FK506). Figure 7C shows the skin graft scoring results for 9E6-L4H2 (10 mpk) and its combination with tacrolimus (FK506). Figure 7D shows the skin graft scoring results for 2F12-L4H2 (10 mpk) and its combination with tacrolimus (FK506). [Figure 8] This study shows the results of PK detection in human CD40 transgenic mice using CD40 antagonist antibodies 9E6-L4H2 and 2F12-L4H2, with CFZ533 used as a control. [Figure 9A-B] Figure 9A shows the inhibitory activity results of Fc-mutated CD40 antagonist antibodies in B cell activation experiments. Figure 9B shows the results of MFI inhibition of CD19+CD69+ cells with 9E6-L4H2 and 9E6-L4H2-AAYTE, and Figure 9B shows the results of MFI inhibition of CD19+CD69+ cells with 2F12-L4H2 and 2F12-L4H2-AAYTE. [Figure 10A-D] Figure 10A shows the inhibitory activity of Fc-mutated CD40 antagonists in a DC cell activation system. Figure 10A shows the inhibition of IL-12 / 23 p40 in 9E6-L4H2 and 9E6-L4H2-AAYTE, Figure 10B shows the inhibition of TNFα in 9E6-L4H2 and 9E6-L4H2-AAYTE, Figure 10C shows the inhibition of IL-12 / 23 p40 in 2F12-L4H2 and 2F12-L4H2-AAYTE, and Figure 10D shows the inhibition of TNFα in 2F12-L4H2 and 2F12-L4H2-AAYTE. [Modes for carrying out the invention]

[0117] The present disclosure will be further described below in accordance with the examples, but these examples are not intended to limit the scope of the present disclosure.

[0118] Experimental methods in the examples or test cases of this disclosure that do not specify concrete conditions generally follow normal conditions or conditions recommended by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory, and Modern Molecular Biology Methods, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents whose specific source is not specified are commonly available commercially.

[0119] Example 1. CD40 immunoantigen, sequence for antigen screening, and preparation. The his-tagged human CD40 (h-CD40-his) recombinant protein (product number CD0-H5228), the mouse Fc-tagged human CD40 (h-CD40-mFc) recombinant protein (product number CD0-H525a), the his-tagged and biotin-tagged human CD40 (hCD40-his-avi) recombinant protein (product number CD0-H82E8), and the his-tagged cynomolgus monkey CD40 (cyno-CD40-his) recombinant protein (product number CD0-C52H6) are all purified commercial protein reagents purchased from Acrobiosystems, and the origin of their respective sequences is shown in Table 2. The above protein reagents can be used in the experiments of each of the following examples.

[0120] [Table 2]

[0121] Example 2. Screening of anti-CD40 rabbit monoclonal antibodies and preparation of human-rabbit chimeric antibodies Anti-human CD40 monoclonal antibodies were produced by immunizing two New Zealand white rabbits. The immunoantigen was His-tagged human CD40 recombinant protein (h-CD40-his, prepared to 1 μg / μL in phosphate buffer). Emulsification with Freund's adjuvant: complete Freund's adjuvant (CFA) was used initially, and incomplete Freund's adjuvant (IFA) was used for the remaining additional immunizations. Each immunization was performed using multiple subcutaneous injections of 400 μg of antigen. Immunotherapy times were days 0, 7, 20, and 41. Blood samples were collected on days 27 and 48 for blood detection, and antibody titers in rabbit serum were detected by ELISA and FACS. Rabbits with high and stable antibody titers in their serum were selected: an additional immunization was performed on day 63, in which an antigen solution prepared from phosphate buffer at 400 μg / rabbit was intravenously injected. On day 67, the spleens of these rabbits were collected, and CD40 antigen with a biotin lag was added. Labeled monoclonal B cells were sorted by flow cytometry and placed in 96-well plates. After 14 days of culture, the supernatant was collected and screened by ELISA and FACS for clones capable of binding to human CD40, cynomolgus monkey CD40, and Raji cells (a tumor cell line expressing human CD40), yielding a total of 28 B cell monoclones. RNA was extracted from these monoclonal cells, reverse transcribed, amplified by PCR, and sent to a sequencing company for sequencing. Finally, the sequences of 28 rabbit antibody lines were obtained and screened by affinity and activity identification experiments (see Examples 2-3 for methods) to obtain 11 monoclonal antibody lines. The sequences of the heavy chain and light chain variable regions are shown in Table 3, and the CDR sequence is shown in Table 4.

[0122] [Table 3-1] [Table 3-2] [Table 3-3]

[0123] [Table 4-1] [Table 4-2] [Table 4-3]

[0124] The obtained variable region sequences are ligated to the human antibody IgG1 constant region (with the N297A mutation, using the Eu numbering system) and the human kappa chain constant region to obtain human-rabbit chimeric antibody sequences. By inserting the chimeric antibody sequence into an expression vector using molecular cloning technology and utilizing the HEK293 cell expression system, human-rabbit chimeric antibodies can be obtained.

[0125] Example 3. Human-rabbit chimeric anti-CD40 antibody conjugation experiment with Raji cells. Raji cells are a tumor cell line that overexpresses human CD40. 2E5 Raji cells were inoculated into a 96-well plate. 100 μL of antibody awaiting measurement was added, and the solution was diluted 5-fold to a maximum final concentration of 100 nM, resulting in a total concentration of 8 cells. The cells were incubated at 4°C for 1 hour. After washing once with washing solution, anti-human IgG antibody conjugated to AF647 (Jackson Immunoresearch Laboratories, product number 205-609-088) was added at a dilution of 1:500 and incubated at 4°C for 30 minutes. After washing once with washing solution, fluorescence intensity was read using a flow cytometer. Conjugation EC of anti-CD40 antibody against CD40 50 The values ​​were calculated. The anti-CD40 antagonist antibody CFZ533 (i.e., Iscalimab, Nova) was used as a positive control, and the sequences of the heavy chain and light chain variable region were derived from sequence 5 and sequence 2 of US8277810B, respectively.

[0126] From the results in Table 5, 2F12, 3F6, 4F6, 5A9, 5B8, 9E6, and 10A4 all exhibit stronger binding EC than CFZ533. 50Although it has, other antibodies, such as 8E1 (sequence not shown), have EC250 than CFZ533. 50 It has been shown to be weak.

[0127] [Table 5]

[0128] Example 4: Cell activity experiment of human-rabbit chimeric anti-CD40 antibody in a reporter gene. HEK-Blue CD40L cells were purchased from Invivogen (Cat#hkb-cd40) and were stably transfected with the human CD40 gene and the NF-κB-mediated SEAP genome. By detecting the SEAP secreted in the supernatant using the SEAP substrate QUANTI-Blue, the activation level of the CD40 signaling pathway can be expressed. This experiment detects the activation of HEK-Blue CD40L cells by CD40L, thereby enabling IC (Impulse Control). 50In vitro antagonistic activity of anti-CD40 antibodies was evaluated based on cell size. HEK-Blue CD40L cells were cultured in DMEM medium containing 10% FBS, 100 μg / mL Zeocin, and 30 μg / mL Blasticidin, and passaged 2-3 times per week at a passage ratio of 1:5 or 1:10. During passage, the medium was aspirated and removed, the cell layer was rinsed with 5 mL of 0.25% trypsin, the trypsin was aspirated and removed, the cells were placed in an incubator and digested for 3-5 minutes, and the cells were resuspended with fresh medium. 100 μL of cell suspension was added to a 96-well cell culture plate with a density of 5 × 10^5 cells / mL, and the medium was DMEM containing 10% FBS, 100 μg / mL Zeocin, and 30 μg / mL Blasticidin. Only 100 μL of sterile water was added to the periphery of the 96-well plate. The culture plates were incubated in an incubator for 24 hours (37°C, 5% CO2). After the cells had adhered to the wall, 100 μL of gradient-diluted antibody awaiting measurement was added to each well and incubated at 37°C for 30 minutes. 25 ng / mL of CD40L (R&D, product number 2706-CL) and 2 μg / mL of anti-His antibody (R&D, product number MAB050) were added, and the culture plates were incubated in an incubator for 20-24 hours (37°C, 5% CO2). 20 μL of cell supernatant was taken from each well and placed in a new 96-well flat plate, 180 μL of QUANTI-Blue substrate solution was added, and the culture plates were incubated in the dark for 1-3 hours. The absorbance at 630 nm was measured using a microplate reader (Thermo MultiSkanFc), and IC50 was measured. 50 The values ​​were calculated to evaluate the in vitro cell activity of anti-CD40 antibodies. Referring to Table 6, the results show that 2F12, 3F6, 4D4, 4F6, 5A9, 5B8, 8C12, 8G10, 9E6, 9F10, and 10A4 all correspond to or have slightly stronger reporter gene cell inhibitory activity than CFZ533. 50 Although it has other antibodies, such as 2F1 (sequence not shown), other antibodies have higher IC50 than CFZ533. 50 It has been shown to be weak.

[0129] [Table 6]

[0130] Example 5. Humanization of anti-CD40 antibody The sequences of the heavy and light chain variable regions of rabbit antibodies 2F12, 3F6, 9E6, and 10A4 were compared with the antibody GermLine database to obtain highly homologous human germline templates. See Table 7 for information on the human germline templates used to humanize the final antibodies.

[0131] [Table 7]

[0132] The CDR region of a rabbit antibody was transplanted into a selected human germline template, replacing the human germline variable region and recombining it with the corresponding human IgG constant region (preferably IgG1 with the N297A mutation in the heavy chain and κ in the light chain). Subsequently, based on the three-dimensional structure of the rabbit antibody, embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH were reversed, and potential post-translational modification risk sites were mutated to obtain the final humanized molecule. Exemplary sequences of the humanized light and heavy chain variable regions corresponding to 2F12 and 9E6 are shown (see Tables 8 and 9), where L represents the light chain and H represents the heavy chain, and the numbers following L and H represent different versions of the humanized sequence containing different reverse mutations.

[0133] [Table 8]

[0134] During the humanization process, 9E6 obtained the following LCDR3: >9E6-L1 LCDR3 QGGYWTSTSNFGNV (Sequence ID 69) >9E6-L2 LCDR3 QGGYWTSTSNFGSG (Sequence ID 70) >9E6-L3 LCDR3 QGGYWTSTSNFGTG (Sequence ID 71) >9E6-L4 LCDR3 QGGYWTSTSNFGQG (Sequence ID 72) >9E6-L LCDR3 general type QGGYWTSTSNFGX9X 10 (Sequence number 73), of which X9 is selected from N, S, T or Q, X 10 It is selected from V or G.

[0135] 5A9, 9E6, and 9F10 have the following general formula structure: HCDR1, HCDR2, and HCDR3 are represented by SYDMS (SEQ ID NO: 23), AIGGAGGTYYASWAKS (SEQ ID NO: 24), and GWTRLDL (SEQ ID NO: 25), respectively. LCDR1 is represented by QX1SEDISSNLX2 (sequence number 74), where X1 is selected from A or S, and X2 is selected from A or S. LCDR2 is represented by X3ASNLAS (sequence number 75), where X3 is selected from A or P. LCDR3 is QGX4YWX5X6X7SX8FGX9X 10 (Sequence number 76) shows that X4 is selected from A or G, X5 is selected from S or T, X6 is selected from S or G, X7 is selected from T or S, X8 is selected from N or Y, X9 is selected from N, S, T or Q, X 10 It is selected from V or G.

[0136] [Table 9]

[0137] During the humanization process, 2F12 obtained the following LCDR1: >2F12-L1 LCDR1 QASQSISNVLA (Sequence ID 83) >2F12-L2 LCDR1 QASQSISQGLA (SEQ ID NO: 84) >LCDR1 of 2F12-L3 QASQSISSGLA (SEQ ID NO: 85) >LCDR1 of 2F12-L4 QASQSISTGLA (SEQ ID NO: 86) >General formula of LCDR1 of 2F12-L QASQSISX 24 X 25 LA (SEQ ID NO: 87), wherein X 24 is selected from N, Q, S or T, and X 25 is selected from V or G.

[0138] 4F6, 5B8, 8G10, 8C12, 4D4, 2F12 have the following general formula structure: HCDR1 is SYGVX 11 (SEQ ID NO: 88), wherein X 11 is selected from S or T, HCDR2 is X 12 IX 13 SX 14 GX<000008​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Indicated by LA (sequence number 91), of which X 21 is selected from Q or E, X 22 is selected from S or D, X 23 is selected from S or T, X 24 is selected from N, Q, S, or T, X 25 It is selected from V or G.

[0139] LCDR2 is represented by GASNLAS (SEQ ID NO: 37), LCDR3 is QSYX 26 X 27 SX 28 X 29 TX 30 (Sequence number 92) indicates, of which X 26 is selected from F or Y, X 27 is selected from S, D, or N, X 28 is selected from S or F, X 29 is selected from S, T, or Y, X 30 It is chosen from V or I.

[0140] Two light and two heavy chains from each humanized version of each rabbit antibody were combined, expressed, and purified. The naming convention was a combination of heavy chain and light chain variable region numbers; for example, "2F12-L1H1" refers to an antibody where sequence number 63 was selected as VH and sequence number 67 as VL. The light and heavy chain constant regions were the human IgG1 constant region (with the N297A mutation, using the Eu numbering system) and the human kappa chain constant region, respectively.

[0141] The sequences of exemplary humanized molecules 2F12-L4H2 (using the 2F12-L4 light chain and 2F12-H2 heavy chain) and 9E6-L4H2 (using the 9E6-L4 light chain and 9E6-H2 heavy chain) are as follows, with their light and heavy chain constant regions adopting the human IgG1 constant region (with the N297A mutation, using the Eu numbering system) and the human kappa chain constant region, respectively.

[0142] >9E6-L4H2 HC: QVQVQESGPGLVKPSDTLSLTCTVSGFSLSSYDMSWIRQPPGKGLEWIGAIGGAGGTYYASWAKSRVTISVDTSLNQVSLKLSSVTAADTAVYYCARGWTRLDLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 93 >9E6-L4H2 LC: DIQLTQSPSFLSASVGDRVTITCQASEDISSNLAWYQQKPGKPPKLLIFPASNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQGGYWTSTSNFGQGFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 94 >2F12-L4H2 HC: EVTVKESGPVLVKPTETLTLTCTVSGFSLSSYGVSWIRQPPGKALEWLGGIGSDGSAYYASWAKSRLTISRDTNLKQVVLTMTNMMDPVDTATYYCARGGITVYAMWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 95 >2F12-L4H2 LC: AFQLTQSPSSLSASVGDRVTITCQASQSISTGLAWYQQKPGKPPKLLIVGASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQSYFSSSSTVFGGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 96

[0143] The heavy chain constant region of the above antibody was modified, and the antibody adopted a human IgG1 constant region with five site mutations: L234A, L235A, M252Y, S254T, and T256E (Eu numbering system), while the light chain constant region and light / heavy chain variable regions remained unchanged. The newly produced antibodies are 2F12-L4H2-AAYTE and 9E6-L4H2-AAYTE, and their complete heavy chain sequences are as follows: >9E6-L4H2-AAYTE HC: QVQVQESGPGLVKPSDTLSLTCTVSGFSLSSYDMSWIRQPPGKGLEWIGAIGGAGGTYYASWAKSRVTISVDTSLNQVSLKLSSVTAADTAVYYCARGWTRLDLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 97 >2F12-L4H2-AAYTE HC: EVTVKESGPVLVKPTETLTLTCTVSGFSLSSYGVSWIRQPPGKALEWLGGIGSDGSAYYASWAKSRLTISRDTNLKQVVLTMTNMMDPVDTATYYCARGGITVYAMWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 98 The underlined region above is the heavy chain or light chain steady region.

[0144] Examples of IgG Fc are as follows: > IgG1-Fc(N297A) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 99 > IgG1-Fc(AAYTE) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 100

[0145] Example 6. Experiment on the binding of humanized anti-CD40 antibody to Raji cells. High expression of human CD40 on the surface of Raji cells can be used to detect the binding properties of CD40 antagonist antibodies to human CD40 on the cell surface.

[0146] Raji cells were seeded in plates at 1.5E5 / well, and various concentrations of CD40 antagonist antibodies were added. The cells were incubated at 4°C for 1 hour. After washing twice with FACS buffer (PBS + 2% FBS), a secondary antibody (anti-human IgG(H+L) antibody conjugated to Alexa Flour 488) was added, and the cells were incubated at 4°C for 0.5 hours. After washing twice with FACS buffer, the fluorescence intensity of the cell surface was detected using a flow cytometer (BD FACS Celesta).

[0147] As a result, as shown in Table 10, 2F12-L4H2, 9E6-L4H2, and CFZ533 have similar binding capabilities to human CD40 on the Raji surface.

[0148] [Table 10]

[0149] Example 7. Affinity assay of anti-CD40 antibodies against human CD40 and cynomolgus monkey CD40. After affinity capture of antibodies awaiting measurement onto an anti-human Fc chip, His-tagged human or cynomolgus monkey CD40 antigen was flowed onto the chip surface in a series of concentration gradients. The reaction signal was detected in real time using a Biacore instrument to obtain binding and dissociation curves. The buffer used in the experiment was HBS-EP + 10× buffer solution (Cat. #BR-1006-69, GE) diluted to 1× (pH 7.4) with DIWater. The data obtained from the experiment were fitted with a (1:1) Binding model to obtain affinity values, as shown in Table 11.

[0150] 9E6-L4H2 and 2F12-L4H2 have similar binding constants to human CD40 compared to CFZ533, but 9E6-L4H2 has a relatively strong binding affinity to cynomolgus monkey CD40, while 2F12-L4H2 has a slightly weaker binding affinity to cynomolgus monkey CD40.

[0151] [Table 11]

[0152] Example 8. Cell activity experiment of reporter gene with anti-CD40 antibody. HEK-Blue CD40L cells were purchased from Invivogen (Cat#hkb-cd40) and were stably transfected with the human CD40 gene and the NF-κB-mediated SEAP genome. The activation level of the CD40 signaling pathway can be expressed by detecting the amount of SEAP secreted in the supernatant using the SEAP substrate QUANTI-Blue. This experiment detects the inhibitory effect of a CD40 antagonist antibody on the activation of CD40L-inducible HEK-Blue CD40L cells, thereby enabling IC (Impulse Control). 50 In vitro cell activity of CD40 antagonist antibodies was evaluated based on their size.

[0153] HEK-Blue CD40L cells were cultured in DMEM medium containing 10% FBS, 100 μg / mL Normocin, 100 μg / mL Zeocin, and 30 pg / mL Blasticidin, and passaged 2-3 times per week. HEK-Blue CD40L cells were seeded at 5E4 / well in a 96-well cell culture plate (medium: DMEM, 10% FBS, 100 μg / mL Normocin) and cultured overnight. After the cells had adhered to the wall, 100 μL of gradient-diluted antibody awaiting measurement was added to each well and incubated at 37°C for 1 hour. CD40L-his (R&D, 2706-CL-025) and anti-His antibody (R&D, MAB050-500) were added and cultured overnight. The cells were centrifuged, 20 μL of the cell supernatant was transferred to a new 96-well white plate, 180 μL of QUANTI-Blue substrate solution was added, and the plate was incubated in the dark for 15 minutes. The absorbance at 620 nm was measured using an Envision microplate reader, and IC50 was measured. 50 The values ​​were calculated to evaluate the in vitro cell activity of the CD40 antagonist antibody.

[0154] As a result, as shown in Table 12 and Figures 1A-1B, 9E6-L4H2 and CFZ533 have similar inhibitory activity against the reporter gene system, but 2F12-L4H2 exhibits superior inhibitory activity compared to CFZ533.

[0155] [Table 12]

[0156] Example 9. Inhibitory activity of anti-CD40 antibody in a B cell activation experimental system. CD40 is highly expressed in B cells, and when CD40L binds to CD40, it can induce B cell activation and upregulate the expression of a series of activation markers. CD40 antagonist antibodies disrupt the immune activation process of B cells by blocking the binding of CD40L to CD40.

[0157] Human PBMCs were seeded in a 96-well cell culture plate at 2E5 / well, 50 μL / well (medium: RPMI-1640, 10% FBS, 1% penicillin-streptomycin). 50 μL of gradient-diluted antibody awaiting measurement was added to each well and co-incubated for 0.5 hours at 37°C in 5% CO2. CD40L-his and anti-His antibodies were added to each well and stimulated overnight. The following day, the cells were centrifuged, the supernatant was removed, and the cells were washed twice with FACS buffer. 100 μL of 1:1000 diluted Fixable viability dye EF780 (Invitrogen, 65086514) was added and stained at room temperature for 15 minutes. After washing the cells twice, 100 μL of 1:200 diluted human Fc blocking agent (Fc blocker, BD, 564220) was added and the cells were blocked at room temperature for 10 minutes. After centrifugation, flow antibodies (PerCP / Cyanine 5.5 anti-human CD19 (Biolegend, 302230) and APC anti-human CD69 (Biolegend, 310910)) diluted 1:200 were added, and the cells were incubated at 4°C for 0.5 hours. The supernatant was removed by centrifugation, the cells were washed twice with FACS buffer, and then the cells were resuspended in 200 μL of PBS. The fluorescence intensity of the cell surface was detected using a flow cytometer (BD FACS Celesta).

[0158] As a result, as shown in Table 13 and Figures 2A-2B, 9E6-L4H2 has similar B cell activation inhibitory activity to CFZ533. 2F12-L4H2 exhibits stronger B cell inhibitory activity than CFZ533.

[0159] [Table 13]

[0160] After adding the antibody, the CD19+CD69+ signal fell below the background level, which is likely due to some degree of inhibition of background B cell activation (apart from the inhibition of CD40L-induced signaling).

[0161] Example 10. Inhibitory activity of anti-CD40 antibody in a DC cell activation system. CD40 is highly expressed in dendritic cells (DCs). When CD40L binds to CD40, it induces DC activation, upregulating the expression of multiple activation markers on the surface of DC cells and promoting the secretion of multiple inflammatory factors, thereby further expanding the immune response. CD40 antagonist antibodies block the binding of CD40L to CD40, thereby deactivating the immune activation process in DC cells.

[0162] Mononuclear cells were selected and enriched from fresh primary human peripheral blood PBMCs using the EsaySep™ Human CD14 Selection Reagent Kit (Stemcell, 19359). These cells were differentiated for 6 days in RPMI-1640 medium (10% FBS, 1% penicillin-streptomycin), 50 ng / mL IL-4 (PeproTech, 200-04), and 50 ng / mL GM-CSF (PeproTech, 300-03). On day 7, differentiated DC cells were seeded at 1E5 / well in a 96-well cell culture plate. Gradient-diluted antibodies awaiting measurement were added to each well and co-incubated for 0.5 hours at 37°C and 5% CO2. Final concentrations of CD40L-his and anti-His antibody were then added to each well. After 48 hours of culture, the activation level of DC cells was detected by flow cytometry: Centrifugation removed the supernatant, and the cells were washed twice with FACS buffer (PBS + 2% FBS). 100 μL of 1:1000 diluted Fixable viability dye EF780 (Invitrogen, 65086514) was added and stained at room temperature for 15 minutes. After washing the cells twice, 100 μL of 1:200 diluted human Fc blocking agent (BD, 564220) was added and the cells were blocked at room temperature for 10 minutes. After centrifugation, the cells were stained with 1:200 diluted flow antibodies (Alexa Fluor® 700 anti-human CD11c (Biolegend, 337220), Brilliant Violet 421). 商標Anti-human CD80 (Biolegend, 305221) and APC anti-human CD86 (Biolegend, 305412) were added, and the cells were incubated at 4°C for 0.5 hours. The supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer. The cells were then resuspended in 200 μL of PBS, and the fluorescence intensity of the cell surface was detected using a flow cytometer (BD FACS Celesta).

[0163] Furthermore, after culturing each molecule for 24 hours (TNFα, Cisbio, 62HTNFAPEG) and 48 hours (IL-12 / 23 p40, Novus, VAL121), the secretion levels of cytokines in the supernatant were detected.

[0164] As shown in Table 14 and Figures 3A-3D, the experimental results indicate that 9E6-L4H2 and 2F12-L4H2 have similar DC cell inhibitory activity to the control antibody CFZ533.

[0165] [Table 14]

[0166] Example 11. Agonist activity of humanized anti-CD40 antibody in a B cell activation experimental system. CD40 belongs to the TNF superfamily of receptors, and when bound to ligand CD40L or cross-linked by an antibody, it can mediate specific and nonspecific activation of downstream signaling pathways. Therefore, the background agonist activity of CD40 antagonist antibodies against B cells can be detected under conditions without the addition of CD40L.

[0167] Human PBMCs were seeded in a 96-well cell culture plate at 2E5 / well, 100 μL / well (medium: RPMI-1640, 10% FBS, 1% penicillin-streptomycin). 100 μL of gradient-diluted antibody was added to each well, and the cells were incubated overnight at 37°C in 5% CO2. The following day, the cells were centrifuged, the supernatant was removed, and the cells were washed twice with FACS buffer. 100 μL of 1:1000 diluted Fixable viability dye EF780 was added, and the cells were stained at room temperature for 15 minutes. After washing the cells twice, 100 μL of 1:200 diluted human Fc blocking agent was added, and the cells were blocked at room temperature for 10 minutes. After centrifugation, flow antibodies (PerCP / Cyanine 5.5 anti-human CD19 (Biolegend, 302230) and APC anti-human CD69 (Biolegend, 310910)) diluted 1:200 were added, and the cells were incubated at 4°C for 0.5 hours. The supernatant was removed by centrifugation, the cells were washed twice with FACS buffer, and then the cells were resuspended in 200 μL of PBS. The fluorescence intensity of the cell surface was detected using a flow cytometer (BD FACS Celesta).

[0168] As a result, as shown in Figure 4, the CD40 agonist antibody 9E5-SELFNS (WO2020108611A1) dose-dependently activates B cells, but 9E6-L4H2 and 2F12-L4H2 do not show clear B cell agonist activity even at 2.5 nM.

[0169] Example 12. Mouse T cell-dependent humoral immune response (TDAR) model Female human CD40 transgenic mice, 6 - 7 weeks old, were purchased from Biocytogen JiangSu Co., Ltd. Breeding environment: SPF, production license: SCXK(Su)-2016-0004, certificate number of human CD40 transgenic mice: 320726200100167773. After the animals arrived, they were adaptively bred for 7 days and randomly grouped. On the 0th day of the experiment, one mouse from each group was taken for blood sampling and then administered by intraperitoneal injection. On the 1st day, the mice were immunized by intraperitoneal injection with 50 μg of KLH (KLH: complete Freund's adjuvant CFA = 1:1 emulsified immune complex) / mouse. On the 15th day of the experiment, the mice were secondarily immunized by intraperitoneal injection with 50 μg of KLH (KLH: incomplete Freund's adjuvant IFA = 1:1 emulsified immune complex) / mouse. The drugs in each group were intraperitoneally injected twice a week, and blood was collected from the orbital venous plexus at ~150 μL on the 7th, 14th, 21st, and 28th days respectively. The whole blood was left standing at room temperature for 1 - 4 hours and centrifuged at 7000 rpm at 4°C for 10 minutes to separate the serum, which was stored at -80°C for future use. The specific experimental process is shown in Figure 5.

[0170] As shown in the specific administration plan in Table 15, mouse serum was separated weekly and the specific IgG level against KLH was detected by ELISA.

[0171]

Table 15

[0172] As a result, as shown in Figure 5B and Table 16, the CD40 antagonist antibody at 1 mg / kg significantly inhibited the production of specific IgG against KLH after the second immunization. The low doses of 0.3 mg / kg of 9E6-L4H2 and 2F12-L4H2 had a better inhibitory effect on the production of IgG against KLH after the second immunization compared to the same dose of CFZ533.

[0173]

Table 16

[0174] Example 13. Mouse skin graft rejection model A 6-week-old male balb / c mouse was purchased from the Laboratory Animal Management Department of the Shanghai Institute of Family Planning and Science. Rearing environment: SPF, Certificate number: 20180006023393.

[0175] Female human CD40 transgenic mice were purchased from Biocytogen JiangSu Co., Ltd. at 6-7 weeks of age. Rearing environment: SPF, Certificate of conformity number: 320726200100179778.

[0176] After the animals arrived, they were adaptively reared for 7 days and then randomly divided into groups. Two days before the experiment, mice were intraperitoneally injected with tacrolimus FK506 or CD40 antagonistic antibody. On day 0 of the experiment, donor Balb / C mice and C57BL6 / J mice were anesthetized with 4% chloral hydrate, the tails of the donor mice were removed, and a 1 cm circumference of tail skin was isolated. The dorsal surfaces of the recipient mice were dehaired, an incision was made along the skin layer, an equal area of ​​skin was removed while preserving the fat and connective tissue of the dorsal surface, the donor skin was placed in the incision of the recipient mouse, the marginal skin was sutured with adhesive, and the mice were placed in cages for recovery. The specific experimental process is shown in Figure 6A.

[0177] Mice were administered according to the administration plan in Table 17, and after allowing the mice to recover for 7 days, the viability of the mice's skin was observed daily, and a rejection score was recorded according to the rejection scoring system. The scoring system is as follows: 3: Skin is smooth with no redness; 2: Part of the skin is red, has lost its luster, and is dry; 1: Most of the skin is red, has no stripes, and is shriveled; 0: 80% of the skin has necrotized due to transplant rejection.

[0178] [Table 17-1] [Table 17-2]

[0179] As a result, as shown in FIG. 6B and Tables 18 to 20, the CD40 antagonist antibody can significantly improve the scoring of mouse skin grafts and extend the survival period of skin grafts compared to the model group. In the process of scoring skin grafts, since no differences were found until then, scoring was performed from the 8th day. 9E6-L4H2 and 2F12-L4H2 showed anti-graft rejection activity superior to that of the control antibody CFZ533.

[0180]

Table 18

[0181]

Table 19

[0182]

Table 20

[0183] In addition, as shown in FIG. 7 and Tables 21 to 23, after 9E6-L4H2 and 2F12-L4H2 were combined with tacrolimus, the effect on anti-mouse skin graft rejection was further improved.

[0184]

Table 21

[0185]

Table 22

[0186]

Table 23

[0187] Example 14. PK Detection of Humanized Anti-CD40 Antibody in Human CD40 Transgenic Mice Female human CD40 transgenic mice were purchased from Biocytogen JiangSu Co., Ltd. at 6-7 weeks of age. Rearing environment: SPF, Production license: SCXK(Su)-2016-0004, Human CD40 transgenic mouse conformity certificate number: 320726200100154632. After arrival, the animals were adaptively reared for 7 days and then randomly divided into groups. On day 0 of the experiment, each group of mice was intraperitoneally injected with 10 mg / kg of anti-CD40 antibody. 100-150 μL of blood was collected at 15 minutes, 4 hours, 8 hours, 1 day, 2 days, 4 days, 7 days, 10 days, and 14 days after administration, anticoagulated with 10 μL of EDTA-K2 (0.1 M), and stored on ice. Antibody concentrations in mouse plasma at different time points were detected by ELISA. The specific detection method is as follows: Goat anti-human IgG Fc antibody (Rockland, Cat#609-101-017) was diluted to 2.5 μg / mL in PBS and added to a 96-well plate at 50 μL / well, and incubated overnight at 4°C. After washing three times with washing solution, 50 μL of blocking solution was added to each well and incubated at 37°C for 1 hour. Mouse plasma and the calibration curve of the antibody awaiting detection were added and incubated at 37°C for 2 hours. After washing three times with washing solution, anti-hlgG Fab-HRP (Sigma, Cat#A0293, 1:10000) was added at 50 μL / well and incubated at room temperature for 1 hour. After washing three times with washing solution, 100 μL of TMB was added to each well and reacted in the dark for 5 minutes. 100 μL of 0.16 M sulfuric acid was added to each well. The OD value at 450 nm was read using an Envision microplate reader, and the concentration of the CD40 antagonist antibody was calculated.

[0188] As a result, as shown in Table 24 and Figure 8, 9E6-L4H2, 2F12-L4H2, and the control molecule CFZ533 exhibit similar PK properties in human CD40 transgenic mice.

[0189] [Table 24]

[0190] Example 15. Measurement of the affinity of humanized anti-CD40 antibody to human FcRn. After affinity capture of antibodies awaiting measurement onto an anti-human Fab chip, human FcRn antigen (purchased from AcroBiosystem) was flowed over the chip surface in a series of concentration gradients, and the signal when the reaction reached a steady state was detected in real time using a Biacore instrument. The buffer used in the experiment was HBS-EP + 10× buffer solution (Cat. #BR-1006-69, GE) diluted to 1× (pH 7.4) with DIWater. The data obtained from the experiment were fitted to a steady-state binding model, and affinity values ​​were obtained as shown in Table 25. Compared to the parent antibody, the anti-CD40 antibody with the AAYTE mutation showed higher binding affinity to human FcRn.

[0191] [Table 25]

[0192] Example 16. Inhibitory activity of an anti-CD40 antibody with an AAYTE mutation in Fc in a B cell activation experimental system. Referring to the method in Example 10, the inhibitory activity of anti-CD40 antibodies 2F12-L4H2-AAYTE and 9E6-L4H2-AAYTE, which have the AAYTE mutation in Fc, in a B cell activation experimental system was detected. As shown in Figures 9A to 9B, the Fc-mutated anti-CD40 antibodies have B cell inhibitory activity similar to that of the parental anti-CD40 antibody.

[0193] Example 17. Inhibitory activity of an anti-CD40 antibody with an AAYTE mutation in Fc in a DC cell activation system. Referring to the method in Example 11, the inhibitory activity of anti-CD40 antibodies 2F12-L4H2-AAYTE and 9E6-L4H2-AAYTE, which have the AAYTE mutation in Fc, in a DC cell activation experimental system was detected. As a result, as shown in Figures 10A to 10D, the Fc-mutated anti-CD40 antibodies have DC cell inhibitory activity similar to that of the parental anti-CD40 antibody. This disclosure relates, for example, to the following: [1] An anti-CD40 antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), a-1) The VH includes HCDR1, HCDR2, and HCDR3 in the VH indicated by either SEQ ID NO: 67 or 68, and the VL includes LCDR1, LCDR2, and LCDR3 in the VL indicated by any one of SEQ ID NOs: 63 to 66. a-2) The VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by Sequence ID 1, and the VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by Sequence ID 2. a-3) The VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by Sequence ID No. 3, and the VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by Sequence ID No. 4. a-4) The VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID No. 5, and the VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID No. 6, b-1) The VH includes HCDR1, HCDR2, and HCDR3 in the VH indicated by either SEQ ID NO: 81 or 82, and the VL includes LCDR1, LCDR2, and LCDR3 in the VL indicated by any one of SEQ ID NOs: 77 to 80. b-2) The VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID 7, and the VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID 8. b-3) The VH includes HCDR1, HCDR2, and HCDR3 in VH indicated by Sequence ID 9, and the VL includes LCDR1, LCDR2, and LCDR3 in VL indicated by Sequence ID 10. b-4) The VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID 11, and the VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID 12. b-5) The VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID No. 13, and the VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID No. 14. b-6) The VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID No. 15, and the VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID No. 16. b-7) The VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID No. 17, and the VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID No. 18. c) The VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID 19, and the VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID 20, or d) The VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID No. 21, and the VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID No. 22. Here, the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, preferably defined by the Kabat numbering system. Anti-CD40 antibody or its antigen-binding fragment. [2] 1) Heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 23, 24, and 25 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing the sequences indicated by sequence numbers 74, 75, and 76 respectively, 2) Heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 88, 89, and 90 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing the sequences indicated by sequence numbers 91, 37, and 92 respectively, 3) Heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 51, 52, and 53 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing the sequences indicated by sequence numbers 54, 55, and 56 respectively, or 4) comprising heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 57, 58, and 59 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing the sequences indicated by sequence numbers 60, 61, and 62 respectively, Preferably, the anti-CD40 antibody or its antigen-binding fragment in 1) 1-1) Heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 23, 24, and 25 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing the sequences indicated by sequence numbers 29, 30, and 73 respectively, 1-2) Heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 23, 24, and 25 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing the sequences indicated by sequence numbers 26, 27, and 28 respectively, 1-3) comprising heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 23, 24, and 25 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing the sequences indicated by sequence numbers 29, 27, and 32 respectively, Preferably, the anti-CD40 antibody or its antigen-binding fragment in 2) is 2-1) Heavy chains HCDR1, HCDR2, and HCDR3 containing sequences indicated by sequence numbers 39, 47, and 49 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing sequences indicated by sequence numbers 87, 50, and 48 respectively, 2-2) Heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 33, 34, and 35 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing the sequences indicated by sequence numbers 36, 37, and 38 respectively, 2-3) Heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 39, 40, and 41 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing the sequences indicated by sequence numbers 42, 37, and 43 respectively, 2-4) Heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 39, 44, and 35 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing the sequences indicated by sequence numbers 45, 37, and 46 respectively, 2-5) comprising heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 39, 47, and 41 respectively, and light chains LCDR1, LCDR2, and LCDR3 containing the sequences indicated by sequence numbers 36, 37, and 48 respectively, More preferably, the anti-CD40 antibody or its antigen-binding fragment in 1-1) Each comprises heavy chains HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 23, 24, and 25, respectively; light chain LCDR1 containing the sequence indicated by sequence number 29; light chain LCDR2 containing the sequence indicated by sequence number 30; and light chain LCDR3 containing the sequence indicated by any one of sequence numbers 69 to 72. More preferably, the anti-CD40 antibody or its antigen-binding fragment in 2-1) Each of the following is a heavy chain HCDR1, HCDR2, and HCDR3 containing the sequences indicated by sequence numbers 39, 47, and 49, respectively; a light chain LCDR1 containing the sequence indicated by any one of sequence numbers 83 to 86; a light chain LCDR2 containing the sequence indicated by sequence number 50; and a light chain LCDR3 containing the sequence indicated by sequence number 48. The anti-CD40 antibody or its antigen-binding fragment as described in [1] above. [3] The anti-CD40 antibody or antigen-binding fragment thereof described in [1] or [2] above, which is a recombinant antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. [4] The heavy chain framework region of the humanized antibody or its antigen-binding fragment is derived from IGHV2-26*01, IGHV4-30-4*02, IGHV4-4*08, and IGHJ1*01, and / or the light chain framework region is derived from IGkV1-13*02, IGkV1-9*01, IGkV1-6*01, and IGKJ4*01. Preferably, the heavy chain framework region FR1 to FR3 are derived from IGHV2-26*01, IGHV4-30-4*02, and IGHV4-4*08, the heavy chain framework region FR4 is derived from IGHJ1*01, the light chain framework region FR1 to FR3 are derived from IGkV1-13*02, IGkV1-9*01, and IGkV1-6*01, and the light chain framework region FR4 is derived from IGKJ4*01. The anti-CD40 antibody or its antigen-binding fragment as described in [3] above. [5] A-1) The amino acid sequence of VH is shown in SEQ ID NO: 67 or has at least 90% identity with it, and the amino acid sequence of VL is shown in any one of SEQ ID NOs: 63-66 or has at least 90% identity with it. A-2) The amino acid sequence of VH is shown in SEQ ID NO: 68 or has at least 90% identity with it, and the amino acid sequence of VL is shown in any one of SEQ ID NOs: 63-66 or has at least 90% identity with it. A-3) The amino acid sequence of VH is shown in SEQ ID NO: 1 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 2 or has at least 90% identity thereto. A-4) The amino acid sequence of VH is shown in SEQ ID NO: 3 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 4 or has at least 90% identity thereto. A-5) The amino acid sequence of VH is shown in SEQ ID NO: 5 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 6 or has at least 90% identity thereto. B-1) The amino acid sequence of VH is shown in SEQ ID NO: 81 or has at least 90% identity with it, and the amino acid sequence of VL is shown in any one of SEQ ID NOs: 77-80 or has at least 90% identity with it. B-2) The amino acid sequence of VH is shown in SEQ ID NO: 82 or has at least 90% identity with it, and the amino acid sequence of VL is shown in any one of SEQ ID NOs: 77-80 or has at least 90% identity with it. B-3) The amino acid sequence of VH is shown in SEQ ID NO: 7 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 8 or has at least 90% identity thereto. B-4) The amino acid sequence of VH is shown in SEQ ID NO: 9 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 10 or has at least 90% identity thereto. B-5) The amino acid sequence of VH is shown in SEQ ID NO: 11 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 12 or has at least 90% identity thereto. B-6) The amino acid sequence of VH is shown in SEQ ID NO: 13 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 14 or has at least 90% identity thereto. B-7) The amino acid sequence of VH is shown in SEQ ID NO: 15 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 16 or has at least 90% identity thereto. B-8) The amino acid sequence of VH is shown in SEQ ID NO: 17 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 18 or has at least 90% identity thereto. C) The amino acid sequence of VH is shown in SEQ ID NO: 19 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 20 or has at least 90% identity thereto, or D) The amino acid sequence of VH is shown in SEQ ID NO: 21 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 22 or has at least 90% identity thereto. An anti-CD40 antibody or its antigen-binding fragment as described in any one of the above items [1] to [4]. [6] An anti-CD40 antibody or antigen-binding fragment thereof according to any one of the preceding items, further comprising an Fc region of an IgG antibody, preferably the IgG antibody is an IgG1, IgG2, or IgG4 antibody, and more preferably the Fc region is an Fc region of IgG1 containing the N297A mutation, or an Fc region of IgG1 having one or more mutations of L234A, L235A, M252Y, S254T, or T256E. [7] The anti-CD40 antibody or its antigen-binding fragment according to any one of the preceding items, wherein the antigen-binding fragment is an scFv, Fv, Fab, or Fab' fragment. [8] The full-length heavy chain amino acid sequence is represented by SEQ ID NO: 93 or 97, or has at least 90% identity thereto, and the full-length light chain amino acid sequence is represented by SEQ ID NO: 94, or has at least 90% identity thereto, or The full-length amino acid sequence of the heavy chain is represented by SEQ ID NO: 95 or 98, or has at least 90% identity thereto, and the full-length amino acid sequence of the light chain is represented by SEQ ID NO: 96, or has at least 90% identity thereto. An anti-CD40 antibody or its antigen-binding fragment as described in any one of the preceding paragraphs. [9] i) K ≤ 10 nM D And it binds to human CD40, ii) The absence of clear agonist activity Having at least one of the following, An anti-CD40 antibody or its antigen-binding fragment as described in any one of the above items [1] to [8].

[10] An isolated polynucleotide encoding an anti-CD40 antibody or its antigen-binding fragment as described in any one of the above items [1] to [9].

[11] A vector comprising the polynucleotide described in

[10] above.

[12] A host cell comprising the polynucleotide described in

[10] above or the vector described in

[11] above.

[13] A CD40-binding molecule comprising an anti-CD40 antibody or its antigen-binding fragment as described in any one of the above items [1] to [9].

[14] A pharmaceutical composition comprising an anti-CD40 antibody or its antigen-binding fragment as described in any one of the above items [1] to [9], and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[15] The pharmaceutical composition according to

[14] , further comprising tacrolimus.

[16] A method for treating autoimmune diseases, This includes administering to the target subject a therapeutically effective amount of an anti-CD40 antibody or its antigen-binding fragment described in any one of the above [1] to [9], or the pharmaceutical composition described in

[14] , The aforementioned autoimmune disease is preferably Sjögren's syndrome, multiple sclerosis, or lupus erythematosus, and more preferably systemic lupus erythematosus. method.

[17] Uses of an anti-CD40 antibody or its antigen-binding fragment as described in any one of the above [1] to [9], for use in the preparation of drugs for treating autoimmune diseases, The aforementioned autoimmune disease is preferably Sjögren's syndrome, multiple sclerosis, or lupus erythematosus, and more preferably systemic lupus erythematosus. Use.

[18] A method for treating graft-versus-host disease or mitigating transplant rejection, This includes administering to the target subject a therapeutically effective amount of any one of the anti-CD40 antibody or its antigen-binding fragment described in any one of the above [1] to [9], or a pharmaceutical composition described in any one of the above

[14] to

[15] , The aforementioned transplantation is preferably a solid organ transplant, and more preferably a liver, kidney, heart, or lung transplant. method.

[19] An application in which an anti-CD40 antibody or its antigen-binding fragment described in any one of the above items [1] to [9] is used in combination with tacrolimus to prepare a drug for treating graft-versus-host disease or mitigating transplant rejection, The aforementioned transplantation is preferably a solid organ transplant, and more preferably a liver, kidney, heart, or lung transplant. Use.

Claims

1. An anti-CD40 antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), a-1) The VH includes HCDR1, HCDR2, and HCDR3 in the VH indicated by either SEQ ID NO: 67 or 68, and the VL includes LCDR1, LCDR2, and LCDR3 in the VL indicated by any one of SEQ ID NOs: 63 to 66, or a-2) The VH includes HCDR1, HCDR2, and HCDR3 in VH shown in Sequence ID No. 3, and the VL includes LCDR1, LCDR2, and LCDR3 in VL shown in Sequence ID No. 4, Here, the CDR is defined according to the Kabat numbering system. Anti-CD40 antibody or its antigen-binding fragment.

2. Heavy chain HCDR1, HCDR2, and HCDR3 each containing the sequences shown in SEQ ID NOs. 23, 24, and 25, respectively, light chain LCDR1 shown in SEQ ID NO. 29, light chain LCDR2 shown in SEQ ID NO. 30, and light chain LCDR3 shown in any one of SEQ ID NOs. 69 to 72, including, The anti-CD40 antibody or its antigen-binding fragment according to claim 1.

3. The anti-CD40 antibody or antigen-binding fragment thereof according to claim 1, which is a recombinant antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or an antigen-binding fragment thereof.

4. The heavy chain framework region of the humanized antibody or its antigen-binding fragment is derived from IGHV2-26*01, IGHV4-30-4*02, IGHV4-4*08, and IGHJ1*01, and / or the light chain framework region is derived from IGkV1-13*02, IGkV1-9*01, IGkV1-6*01, and IGKJ4*01. The anti-CD40 antibody or its antigen-binding fragment according to claim 3.

5. A-1) The amino acid sequence of VH is shown in SEQ ID NO: 67 or has at least 90% identity with it, and the amino acid sequence of VL is shown in any one of SEQ ID NOs: 63 to 66 or has at least 90% identity with it. A-2) The amino acid sequence of VH is shown in SEQ ID NO: 68 or has at least 90% identity with it, and the amino acid sequence of VL is shown in any one of SEQ ID NOs: 63 to 66 or has at least 90% identity with it, or A-4) The amino acid sequence of VH is shown in SEQ ID NO: 3 or has at least 90% identity thereto, and the amino acid sequence of VL is shown in SEQ ID NO: 4 or has at least 90% identity thereto. The anti-CD40 antibody or its antigen-binding fragment according to claim 1.

6. The anti-CD40 antibody or antigen-binding fragment thereof according to claim 1, further comprising the Fc region of an IgG antibody.

7. The anti-CD40 antibody or antigen-binding fragment thereof according to claim 6, wherein the Fc region is an Fc region of IgG1 containing the N297A mutation, or an Fc region of IgG1 having one or more mutations of L234A, L235A, M252Y, S254T, T256E, and / or the antigen-binding fragment is an scFv, Fv, Fab, or Fab' fragment.

8. The full-length amino acid sequence of the heavy chain is shown in SEQ ID NO: 93 or 97, or has at least 90% identity thereto, and the full-length amino acid sequence of the light chain is shown in SEQ ID NO: 94, or has at least 90% identity thereto. The anti-CD40 antibody or its antigen-binding fragment according to claim 1.

9. An isolated polynucleotide encoding an anti-CD40 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A vector comprising the polynucleotide described in claim 9.

11. A host cell comprising the polynucleotide described in claim 9 or a vector containing the polynucleotide.

12. A CD40-binding molecule comprising an anti-CD40 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

13. A pharmaceutical composition comprising an anti-CD40 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

14. The pharmaceutical composition according to claim 13, further comprising tacrolimus.

15. A pharmaceutical composition for treating autoimmune diseases, A comprising an anti-CD40 antibody or its antigen-binding fragment according to any one of claims 1 to 8, Pharmaceutical composition.

16. The aforementioned autoimmune diseases are Sjögren's syndrome, multiple sclerosis, or lupus erythematosus. The pharmaceutical composition according to claim 15.

17. The autoimmune disease is systemic lupus erythematosus. The pharmaceutical composition according to claim 15.

18. A pharmaceutical composition for treating graft-versus-host disease or mitigating transplant rejection, A comprising an anti-CD40 antibody or its antigen-binding fragment according to any one of claims 1 to 8, Pharmaceutical composition.

19. The pharmaceutical composition according to claim 18, wherein the transplant is a solid organ transplant.

20. The pharmaceutical composition according to claim 18, wherein the transplant is a transplant of the liver, kidney, heart, or lung.

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