Compositions and Pharmaceutical Uses of CD40 Binding Molecules

Abstract

Compositions and pharmaceutical uses of CD40 binding molecules. Specifically, the compositions include an anti-CD40 antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, and a buffer.

Description

[Technical Field]

[0001] This application claims priority to a Chinese patent application filed on December 28, 2022, with application number 202211698820.3.

[0002] The present disclosure relates to the field of drug formulations, and in particular to pharmaceutical compositions comprising CD40 binding molecules and their pharmaceutical uses. [Background technology]

[0003] CD40, a type I transmembrane glycoprotein of the tumor necrosis factor receptor (TNFR) superfamily, is located on the cell membrane surface and has a molecular weight of approximately 48 kDa. It plays an important role in the immune system. CD40 is expressed on various immune cells, including B cells, dendritic cells, monocytes, and macrophages, as well as on platelets. Under certain conditions, it can also be expressed on eosinophils and parenchymal cells. The natural ligand of 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 TRAFs and mediates downstream signaling via the NF-kB, JNK, and MAPK pathways, resulting in various cell type-dependent activation outcomes, including immune cell activation and proliferation, and secretion of inflammatory factors and chemokines (Vonderheide RH et al., 2007). For example, signaling through these pathways is required for several important effector functions of the adaptive immune system, including primary T cell-dependent antibody responses (TDARs), B cell proliferation, germinal center (GC) formation, immunoglobulin (Ig) isotype switching, somatic mutation, and differentiation of memory B cells and plasma cells (Foy TM et al., 1993; Foy TM et al., 1994). In addition to affecting B cells, activation of the CD40 pathway also provides important signals for DC maturation and function, and for the survival and cytokine secretion of monocytes and macrophages (Caux, C et al., 1994).

[0005] Dysregulation 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 inflamed tissues, and activated epithelial cells capable of secreting chemokines from sites such as the kidney, salivary gland, and skin can respond to CD40. Furthermore, CD40 and CD40L are both expressed at elevated levels in lesions of atherosclerosis patients and preclinical atherosclerosis models. CD40 can stimulate and induce the expression of matrix metalloproteinases and promote the expression of tissue factor in cell types involved in the pathogenesis 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 (ICA M-1), E-selectin, and vascular cell adhesion molecule (VCA M). CD40 / CD40L interactions have also been used to prevent transplant rejection, and the use of the chimeric anti-CD40 antagonist ch5D12 in rhesus monkey kidney allograft studies has shown that CD40 antagonism is sufficient to ameliorate disease and extend median survival to over 100 days. When ch5D12 was combined with an anti-CD86 antibody and administered only at the beginning of the allograft study, followed by extended treatment with cyclosporine, a median survival of over 4 years was achieved, indicating that this combination can potentially induce immune tolerance (Haanstra et al., 2005).

[0006] Numerous preclinical studies have provided evidence of the important role of CD40 / CD40L interactions in promoting T cell-dependent immune responses. Therefore, blocking CD40 signaling is considered an appropriate and necessary therapeutic strategy for inhibiting pathogenic autoimmune responses in diseases such as rheumatoid arthritis, systemic lupus erythematosus, and Sjögren's syndrome. Currently, no anti-CD40 antibodies have been approved for the treatment of such diseases. Therefore, there remains a strong need in the field for therapeutic agents that can be used to intervene in the CD40-CD40L interaction and block CD40 signaling.

[0007] The applicant's patent application WO2023274201 provides anti-CD40 antibodies with novel structures that can specifically bind to CD40 and are used to intervene in or treat diseases associated with the CD40 signaling pathway.

[0008] Antibody drugs are an important class of biological agents due to their large molecular weight, complex structure, and susceptibility to physical and chemical influences during preparation, storage, and use, which can lead to degradation and polymerization, resulting in reduced or ineffective activity. Therefore, developing excellent antibody formulations is of great importance. Summary of the Invention

[0009] The present disclosure provides pharmaceutical compositions comprising CD40 binding molecules that have excellent stability.

[0010] The present disclosure provides pharmaceutical compositions comprising a CD40 binding molecule and a buffer, wherein the buffer is selected from trishydroxymethylaminomethane (Tris) buffer, acetate buffer, succinate buffer, phosphate buffer, histidine salt buffer, acetate buffer, citrate buffer, tartrate buffer, fumarate buffer, glycylglycine buffer, and citrate buffer. In some embodiments, the buffer is selected from acetate buffer, citrate buffer, histidine salt buffer, and phosphate buffer. In some embodiments, the buffer is a histidine salt buffer. In some embodiments, the histidine salt buffer is selected from a histidine-hydrochloride buffer or a histidine-acetate buffer. In some embodiments, the buffer is a histidine-hydrochloride buffer. In some embodiments, the buffer is a histidine-histidine hydrochloride buffer.

[0011] Anti-CD40 antibody or its antigen-binding fragment In one embodiment, a CD40 binding molecule according to the present disclosure is an anti-CD40 antibody or an antigen-binding fragment thereof.

[0012] In one aspect, the present disclosure provides a pharmaceutical composition comprising an anti-CD40 antibody or antigen-binding fragment thereof, which has excellent stability.

[0013] The present disclosure provides a pharmaceutical composition comprising an anti-CD40 antibody or antigen-binding fragment thereof and a buffer, wherein the buffer is selected from trishydroxymethylaminomethane (Tris) buffer, acetate buffer, succinate buffer, phosphate buffer, histidine salt buffer, acetate buffer, citrate buffer, tartrate buffer, fumarate buffer, glycylglycine buffer, and citrate buffer. In some embodiments, the buffer is selected from acetate buffer, citrate buffer, histidine salt buffer, and phosphate buffer. In some embodiments, the buffer is a histidine salt buffer. In some embodiments, the histidine salt buffer is selected from a histidine-hydrochloride buffer or a histidine-acetate buffer. In some embodiments, the buffer is a histidine-hydrochloride buffer. In some embodiments, the buffer is a histidine-histidine hydrochloride buffer. In some embodiments, the buffer is a histidine-histidine hydrochloride buffer.

[0014] In the pharmaceutical compositions described herein, the anti-CD40 antibodies and antigen-binding fragments thereof are Heavy chain HCDR1, HCDR2, HCDR3 containing the sequences shown in SEQ ID NOs: 3, 4, and 5, respectively, and / or light chain LCDR1 containing the sequence shown in SEQ ID NO: 6, light chain LCDR2 containing the sequence shown in SEQ ID NO: 7, QGGYWTSTSNFGX9X 10 (SEQ ID NO: 19), wherein X is selected from N, S, T, or Q; 10 is selected from V or G.

[0015] In one embodiment, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof is It comprises heavy chain HCDR1, HCDR2, and HCDR3 having the sequences shown in SEQ ID NOs: 3, 4, and 5, respectively; light chain LCDR1 having the sequence shown in SEQ ID NO: 6; light chain LCDR2 having the sequence shown in SEQ ID NO: 7; and light chain LCDR3 having the sequence shown in any one of SEQ ID NOs: 15 to 18.

[0016] In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises HCDR1, HCDR2, and HCDR3 in the VH shown in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2, and LCDR3 in the VL shown in SEQ ID NO: 2, wherein the CDRs are defined according to the Kabat, I MGT, Chothia, Ab M, or Contact numbering systems. In one embodiment, the CDRs are defined according to the Kabat numbering system.

[0017] In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof is a recombinant antibody.

[0018] In certain embodiments, the anti-CD40 antibody or antigen-binding fragment thereof is a rabbit antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antigen-binding fragment thereof.

[0019] In certain embodiments, when the anti-CD40 antibody or antigen-binding fragment thereof is a humanized antibody, the heavy chain framework regions are derived from IGHV2-26*01, IGHV4-30-4*02, IGHV4-4*08, or IGHJ1*01, and / or the light chain framework regions are derived from IGkV1-13*02, IGkV1-9*01, IGkV1-6*01, or IGKJ4*01. For example, FR1 to FR3 of the heavy chain framework region are derived from IGHV2-26*01, IGHV4-30-4*02, and IGHV4-4*08, FR4 of the heavy chain framework region is derived from IGHJ1*01, FR1 to FR3 of the light chain framework region are derived from IGkV1-13*02, IGkV1-9*01, and IGkV1-6*01, and FR4 of the light chain framework region is derived from IGKJ4*01.

[0020] In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein: A-1) The amino acid sequence of VH is set forth in SEQ ID NO: 1 or has at least 90% identity thereto, and the amino acid sequence of VL is set forth in SEQ ID NO: 2 or has at least 90% identity thereto; A-2) The amino acid sequence of VH is set forth in SEQ ID NO: 13 or has at least 90% identity thereto, and the amino acid sequence of VL is set forth in any one of SEQ ID NOs: 9 to 12 or has at least 90% identity thereto; A-3) The amino acid sequence of VH is set forth in SEQ ID NO: 14 or has at least 90% identity thereto, and the amino acid sequence of VL is set forth in any one of SEQ ID NOs: 9 to 12 or has at least 90% identity thereto.

[0021] In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof, for example, an IgG1, IgG2, IgG2, or IgG4 antibody or antigen-binding fragment thereof, for example, an IgG1 antibody or antigen-binding fragment thereof having an N297A mutation, for example, an IgG1 antibody or antigen-binding fragment thereof having any one of L234A, L235A, M252Y, S254T, and T256E, or any combination thereof.

[0022] In one embodiment, the antigen-binding fragment of the anti-CD40 antibody is a Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody, and multispecific antibody (bispecific antibody, diabody, triabody, tetrabody, tandem di-scFv, tandem tri-scFv), such as an scFv, Fv, Fab, or Fab' fragment.

[0023] In one embodiment, the amino acid sequence of the full-length heavy chain of the antigen-binding fragment of the anti-CD40 antibody is set forth in SEQ ID NO: 20 or 22 or has at least 90% identity thereto, and the amino acid sequence of the full-length light chain is set forth in SEQ ID NO: 21 or has at least 90% identity thereto.

[0024] The above "at least 90% identity" 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%, at least 99% identity.

[0025] In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid changes in the heavy chain variable region and 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid changes in the light chain variable region. In some specific embodiments, the amino acid changes are conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function.

[0026] antibody-drug conjugates In another embodiment, the CD40 binding molecule in the pharmaceutical composition of the present disclosure is an antibody-drug conjugate comprising an anti-CD40 antibody or antigen-binding fragment thereof, wherein the anti-CD40 antibody or antigen-binding fragment thereof is any anti-CD40 antibody or antigen-binding fragment thereof according to the present disclosure.

[0027] The present disclosure provides pharmaceutical compositions comprising an antibody-drug conjugate comprising any anti-CD40 antibody or antigen-binding fragment thereof according to the present disclosure and a buffer, wherein the buffer is selected from trishydroxymethylaminomethane (Tris) buffer, acetate buffer, succinate buffer, phosphate buffer, histidine salt buffer, acetate buffer, citrate buffer, tartrate buffer, fumarate buffer, glycylglycine buffer, and citrate buffer. In some embodiments, the buffer is selected from acetate buffer, citrate buffer, histidine salt buffer, and phosphate buffer. In some embodiments, the buffer is a histidine salt buffer. In some embodiments, the histidine salt buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer. In some embodiments, the buffer is a histidine-hydrochloride buffer. In one embodiment, the buffer is a histidine-histidine hydrochloride buffer.

[0028] In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof is heavy chain HCDR1, HCDR2, and HCDR3 containing the sequences shown in SEQ ID NOs: 3, 4, and 5, respectively; light chain LCDR1 containing the sequence shown in SEQ ID NO: 6; light chain LCDR2 containing the sequence shown in SEQ ID NO: 7; and QGGYWTSTSNFGX9X 10 and a light chain LCDR3 comprising the sequence set forth in (SEQ ID NO: 19), wherein X9 is selected from N, S, T, or Q; 10 is selected from V or G.

[0029] In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof is It comprises heavy chain HCDR1, HCDR2, and HCDR3 having the sequences shown in SEQ ID NOs: 3, 4, and 5, respectively; light chain LCDR1 having the sequence shown in SEQ ID NO: 6; light chain LCDR2 having the sequence shown in SEQ ID NO: 7; and light chain LCDR3 having the sequence shown in any one of SEQ ID NOs: 15 to 18.

[0030] In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises HCDR1, HCDR2, and HCDR3 in the VH shown in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2, and LCDR3 in the VL shown in SEQ ID NO: 2, wherein the CDRs are defined according to the Kabat, I MGT, Chothia, Ab M, or Contact numbering systems. In one embodiment, the CDRs are defined according to the Kabat numbering system.

[0031] In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof is a recombinant antibody.

[0032] In certain embodiments, the anti-CD40 antibody or antigen-binding fragment thereof is a rabbit antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antigen-binding fragment thereof.

[0033] In certain embodiments, when the anti-CD40 antibody or antigen-binding fragment thereof is a humanized antibody, the heavy chain framework regions are derived from IGHV2-26*01, IGHV4-30-4*02, IGHV4-4*08, or IGHJ1*01, and / or the light chain framework regions are derived from IGkV1-13*02, IGkV1-9*01, IGkV1-6*01, or IGKJ4*01. For example, FR1 to FR3 of the heavy chain framework region are derived from IGHV2-26*01, IGHV4-30-4*02, and IGHV4-4*08, FR4 of the heavy chain framework region is derived from IGHJ1*01, FR1 to FR3 of the light chain framework region are derived from IGkV1-13*02, IGkV1-9*01, and IGkV1-6*01, and FR4 of the light chain framework region is derived from IGKJ4*01.

[0034] In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein: A-1) The amino acid sequence of VH is set forth in SEQ ID NO: 1 or has at least 90% identity thereto, and the amino acid sequence of VL is set forth in SEQ ID NO: 2 or has at least 90% identity thereto; A-2) The amino acid sequence of VH is set forth in SEQ ID NO: 13 or has at least 90% identity thereto, and the amino acid sequence of VL is set forth in any one of SEQ ID NOs: 9 to 12 or has at least 90% identity thereto; A-3) The amino acid sequence of VH is set forth in SEQ ID NO: 14 or has at least 90% identity thereto, and the amino acid sequence of VL is set forth in any one of SEQ ID NOs: 9 to 12 or has at least 90% identity thereto.

[0035] In one embodiment, the anti-CD40 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof, for example, an IgG1, IgG2, IgG2, or IgG4 antibody or antigen-binding fragment thereof, for example, an IgG1 antibody or antigen-binding fragment thereof having an N297A mutation, for example, an IgG1 antibody or antigen-binding fragment thereof having any one of L234A, L235A, M252Y, S254T, and T256E, or any combination thereof.

[0036] In one embodiment, the antigen-binding fragment of the anti-CD40 antibody is a Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody, and multispecific antibody (bispecific antibody, diabody, triabody, tetrabody, tandem di-scFv, tandem tri-scFv), such as an scFv, Fv, Fab, or Fab' fragment.

[0037] In one embodiment, the amino acid sequence of the full-length heavy chain of the antigen-binding fragment of the anti-CD40 antibody is set forth in SEQ ID NO: 20 or 22 or has at least 90% identity thereto, and the amino acid sequence of the full-length light chain is set forth in SEQ ID NO: 21 or has at least 90% identity thereto.

[0038] The above "at least 90% identity" 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%, at least 99% identity.

[0039] In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid changes in the heavy chain variable region and 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid changes in the light chain variable region. In some specific embodiments, the amino acid changes are conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function.

[0040] In certain embodiments, the disclosed antibody-drug conjugate has the structure shown in formula (I): Ab-(LD) k (I), wherein Ab is any one of the above anti-CD40 antibodies or antigen-binding fragments thereof; D is selected from a glucocorticoid receptor agonist, a glucocorticoid, a calcineurin inhibitor, or a target of rapamycin (mTOR) kinase inhibitor; L is a linker covalently linking Ab to D; k is an integer or decimal number between 1 and 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any decimal or integer number between any two of the above numbers). In some embodiments, k is 4±0.4, 4±0.5, 4±0.6, or 4±0.8. In some embodiments, k is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0041] In certain embodiments, D is a glucocorticoid receptor agonist, for example, a glucocorticoid receptor agonist selected from any of the structures disclosed in WO2022166779A1.

[0042] In certain embodiments, D is a glucocorticoid, for example, budesonide, ciclesonide.

[0043] In certain embodiments, D is a calcineurin inhibitor (CAI), such as tacrolimus, voclosporin, or cyclosporin A (CsA).

[0044] In certain embodiments, D is a target of rapamycin (mTOR) kinase inhibitor, such as rapamycin, everolimus, and temsirolimus.

[0045] WO2022166779A1 discloses antibody-drug conjugates of glucocorticoid receptor agonists, and the contents thereof, including the structure and preparation method of the antibody-drug conjugates, are incorporated in their entirety into the present disclosure.

[0046] In certain embodiments, the disclosed antibody-drug conjugate has the structure shown in formula (I): Ab-(LD) k (I), wherein Ab is any one of the above anti-CD40 antibodies or antigen-binding fragments thereof; D is a glucocorticoid, L is a linker covalently linking Ab to D; k is an integer or decimal number selected from 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any number between any two numbers).

[0047] WO2023143351A1 discloses a glucocorticoid drug conjugate, and the contents thereof, including the structure and preparation method of the antibody-drug conjugate, are incorporated in their entirety into the present disclosure.

[0048] In one embodiment, D is represented by the formula: [ka]

[0049] In one embodiment, k is any number between 1 and 10. k may be an integer or a decimal number.

[0050] In one embodiment, the antibody-drug conjugate (ADC) of the present disclosure has the formula: [ka] wherein k is selected from 1 to 10 and may be an integer or a decimal; p1 is selected from 2, 4, 6, or 8; and p3 and p4 are each independently selected from 0, 1, or 2; In one embodiment, the antibody-drug conjugate (ADC) of the disclosure comprises: [ka] Selected from Here, k is an integer or decimal number between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any integer or decimal number between any two of the above numbers). In some embodiments, k is an integer or decimal number between 3 and 5. In some embodiments, k is 4±0.4, 4±0.5, 4±0.6, or 4±0.8. In some embodiments, k is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0. In some embodiments, k is any number between 2 and 5.

[0051] In one embodiment, the antibody-drug conjugate (ADC) of the disclosure has the structure shown below: [ka] Here, k is an integer or decimal number between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any integer or decimal number between any two of the above numbers). In some embodiments, k is an integer or decimal number between 2 and 5. In some embodiments, k is 4±0.4, 4±0.5, 4±0.6, or 4±0.8. In some embodiments, k is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0052] In one embodiment, the antibody-drug conjugate (ADC) of the disclosure has the structure shown below: [ka] Here, 9E6 to L4H2 are anti-CD40 antibodies comprising heavy and light chains containing the amino acid sequences shown in SEQ ID NOs: 20 and 21, Here, k is an integer or decimal point selected from 1 to 10. In some embodiments, k is an integer or decimal point from 2 to 5. In some embodiments, k is 4±0.4, 4±0.5, 4±0.6, or 4±0.8. In some embodiments, k is about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0.

[0053] In certain embodiments, the pH of the buffer in the pharmaceutical composition is 4 to 8, such as 4.5 to 7.5, 4.5 to 7, 4.5 to 6.8, 4.5 to 6.5, 4.5 to 6.3, 4.5 to 6, 5 to 7.2, 5 to 7.1, 5 to 7, 5 to 6.9, 5 to 6.8, 5 to 6.7, 5 to 6.6, 5 to 6.5, 5 to 6.4, 5 to 6.3, 5 to 6.2, 5 to 6.1, 5 to 6, 5.5 to 7, 5.6, 5.7, 5.8, 5.9, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 5.16, 5.17, 5.18, 5.19 ... 5 to 6.9, 5.5 to 6.8, 5.5 to 6.7, 5.5 to 6.6, 5.5 to 6.5, 5.5 to 6.4, 5.5 to 6.3, 5.5 to 6.2, 5.5 to 6.1, 5.5 to 6, 5.6 to 7, 5.6 to 6.9, 5.6 to 6.8, 5.6 to 6.7, 5.6 to 6.6, 5.6 to 6.5, 5.6 to 6.4, 5.6 to 6.3, 5.6 to 6.2, 5.6 to 6.1 or 6 to 6.8.

[0054] In some embodiments, the pH of the buffer in the pharmaceutical composition is 4.5 to 7.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is 5 to 7.2. In some embodiments, the pH of the buffer in the pharmaceutical composition is 5.5 to 7. In some embodiments, the pH of the buffer in the pharmaceutical composition is 5.6 to 6.6. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6. In certain embodiments, the pH of the buffer in the pharmaceutical composition is about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6.

[0055] In certain embodiments, the pH of the pharmaceutical composition differs from the pH of the buffer contained therein by no more than ±0.5.

[0056] In certain embodiments, the pH of the pharmaceutical composition is 4 to 8, e.g., 4.5 to 7.5, 4.5 to 7, 4.5 to 6.8, 4.5 to 6.5, 4.5 to 6.3, 4.5 to 6, 5 to 7.2, 5 to 7.1, 5 to 7, 5 to 6.9, 5 to 6.8, 5 to 6.7, 5 to 6.6, 5 to 6.5, 5 to 6.4, 5 to 6.3, 5 to 6.2, 5 to 6.1, 5 to 6, 5.5 to 7, 5.5 to 6. 5.6 to 6.9, 5.5 to 6.8, 5.5 to 6.7, 5.5 to 6.6, 5.5 to 6.5, 5.5 to 6.4, 5.5 to 6.3, 5.5 to 6.2, 5.5 to 6.1, 5.5 to 6, 5.6 to 7, 5.6 to 6.9, 5.6 to 6.8, 5.6 to 6.7, 5.6 to 6.6, 5.6 to 6.5, 5.6 to 6.4, 5.6 to 6.3, 5.6 to 6.2, 5.6 to 6.1, or 6 to 6.8. In some embodiments, the pH of the pharmaceutical composition is 4.5 to 7.5. In some embodiments, the pH of the pharmaceutical composition is 5 to 7.2. In some embodiments, the pH of the pharmaceutical composition is 5.5 to 7. In some embodiments, the pH of the buffer is 5.6 to 6.6. In some embodiments, the pH of the pharmaceutical composition is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0. In some embodiments, the pH of the pharmaceutical composition is about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6. In some embodiments, the pH of the pharmaceutical composition is about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6.

[0057] In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is 1 to 50 mM, for example, 5 to 45 mM, 5 to 40 mM, 5 to 35 mM, 5 to 30 mM, 5 to 25 mM, 5 to 20 mM, 10 to 40 mM, 10 to 35 mM, 10 to 30 mM, 10 to 25 mM, 10 to 20 mM, 15 to 35 mM, 15 to 30 mM, 15 to 25 mM, or 15 to 20 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is 5 to 40 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is 10 to 30 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is 15 to 25 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is about 20 mM.

[0058] In some embodiments, the pharmaceutical composition further comprises one or more stabilizers selected from an amino acid or a medicinal salt thereof and a sugar. In some embodiments, the amino acid is one or more selected from proline, aspartic acid, glutamic acid, lysine, arginine, glycine, and histidine. In some embodiments, the amino acid is one or more selected from proline and arginine or a medicinal salt thereof. In some embodiments, the sugar is one or more selected from glucose, sucrose, maltose, and trehalose. In some embodiments, the stabilizer is one or more selected from arginine or a medicinal salt thereof, proline or a medicinal salt thereof, trehalose, and sucrose. In some embodiments, the stabilizer is one or more selected from sucrose and trehalose. In some embodiments, the stabilizer is proline or a medicinal salt thereof.

[0059] In some embodiments, the concentration of sucrose in the pharmaceutical composition is 0.1% to 30% w / v, for example, 0.5% to 25% w / v, 0.5% to 20% w / v, 0.5% to 15% w / v, 0.5% to 10% w / v, 1% to 20% w / v, 1% to 15% w / v, 1% to 10% w / v, 5% to 15% w / v, or 5% to 10% w / v. In some embodiments, the concentration of sucrose in the pharmaceutical composition is 0.5% to 20% w / v. In some embodiments, the concentration of sucrose in the pharmaceutical composition is 1% to 15% w / v. In some embodiments, the concentration of sucrose in the pharmaceutical composition is 5% to 10% w / v. In some embodiments, the concentration of sucrose in the pharmaceutical composition is about 1% w / v, about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 5.5% w / v, about 6% w / v, about 6.5% w / v, about 7% w / v, about 7.5% w / v, about 8% w / v, about 8.5% w / v, about 9% w / v, about 9.5% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v, or about 15% w / v. In some embodiments, the concentration of sucrose in the pharmaceutical composition is about 7.5% w / v, about 8% w / v, or about 9% w / v.

[0060] In some embodiments, the concentration of trehalose in the pharmaceutical composition is 0.1% to 30% w / v, for example, 0.5% to 25% w / v, 0.5% to 20% w / v, 0.5% to 15% w / v, 0.5% to 10% w / v, 1% to 20% w / v, 1% to 15% w / v, 1% to 10% w / v, 5% to 15% w / v, or 5% to 10% w / v. In some embodiments, the concentration of trehalose in the pharmaceutical composition is 0.5% to 20% w / v. In some embodiments, the concentration of trehalose in the pharmaceutical composition is 1% to 15% w / v. In some embodiments, the concentration of sucrose in the pharmaceutical composition is 5% to 10% w / v. In certain embodiments, the concentration of trehalose in the pharmaceutical composition is about 1% w / v, about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 5.5% w / v, about 6% w / v, about 6.5% w / v, about 7% w / v, about 7.5% w / v, about 8% w / v, about 8.5% w / v, about 9% w / v, about 9.5% w / v, about 10% w / v, about 11% w / v, about 12% w / v, about 13% w / v, about 14% w / v, or about 15% w / v. In certain embodiments, the concentration of trehalose in the pharmaceutical composition is about 7.5% w / v.

[0061] In certain embodiments, the concentration of proline or a medicament salt thereof in the pharmaceutical composition is 0.01% to 20% w / v, for example, 0.05% to 20% w / v, 0.05% to 15% w / v, 0.05% to 10% w / v, 0.05% to 5% w / v, 0.1% to 20% w / v, 0.1% to 15% w / v, 0.1% to 10% w / v, 0.1% to 5% w / v, 0.5% to 20% w / v, 0.5% to 15% w / v, 0.5% to 10% w / v, 0.5% to 5% w / v, 1% to 20% w / v, 1% to 15% w / v, 1% to 10% w / v, or 1% to 5% w / v. In one embodiment, the concentration of proline or a medicinal salt thereof in the pharmaceutical composition is 0.01% to 20% w / v. In one embodiment, the concentration of proline or a medicinal salt thereof in the pharmaceutical composition is 0.1% to 15% w / v. In one embodiment, the concentration of proline or a medicinal salt thereof in the pharmaceutical composition is 0.5% to 10% w / v. In one embodiment, the concentration of proline or a medicinal salt thereof in the pharmaceutical composition is 1% to 5% w / v. In some embodiments, the concentration of proline or a medicinal salt thereof in the pharmaceutical composition is about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% w / v. In some embodiments, the concentration of proline or a medicinal salt thereof in the pharmaceutical composition is about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% w / v. In one embodiment, the concentration of proline or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is about 2.5% w / v.

[0062] In some embodiments, the concentration of arginine or a salt thereof in the pharmaceutical composition is 1 to 100 nM, for example, 10 to 90 nM, 10 to 80 nM, 10 to 70 nM, 10 to 60 nM, 10 to 55 nM, 20 to 80 nM, 20 to 70 nM, 20 to 60 nM, 20 to 55 nM, 30 to 70 nM, 30 to 60 nM, 30 to 55 nM, 40 to 60 nM, or 40 to 55 nM. In some embodiments, the concentration of arginine or a salt thereof in the pharmaceutical composition is 20 to 80 nM. In some embodiments, the concentration of arginine or a salt thereof in the pharmaceutical composition is 30 to 70 nM. In some embodiments, the concentration of arginine or a salt thereof in the pharmaceutical composition is 40 to 60 nM. In some embodiments, the concentration of arginine or a salt thereof in the pharmaceutical composition is about 40 nM, about 41 nM, about 42 nM, about 43 nM, about 44 nM, about 45 nM, about 46 nM, about 47 nM, about 48 nM, about 49 nM, about 50 nM, about 51 nM, about 52 nM, about 53 nM, about 54 nM, about 55 nM, about 56 nM, about 57 nM, about 58 nM, about 59 nM, or about 60 nM. In some embodiments, the concentration of arginine or a salt thereof in the pharmaceutical composition is 50 nM.

[0063] In one embodiment, the stabilizer in the pharmaceutical composition is sucrose and arginine or a medicinal salt thereof, wherein the sucrose concentration is 0.5% to 20% w / v and the arginine or its salt concentration is 20 to 80 nM. In one embodiment, the stabilizer in the pharmaceutical composition is sucrose and arginine or a medicinal salt thereof, wherein the sucrose concentration is 1% to 15% w / v and the arginine or its salt concentration is 30 to 70 nM. In one embodiment, the stabilizer in the pharmaceutical composition is sucrose and arginine or a medicinal salt thereof, wherein the sucrose concentration is 5% to 10% w / v and the arginine or its salt concentration is 40 to 60 nM. In one embodiment, the stabilizer in the pharmaceutical composition is sucrose and arginine or a medicinal salt thereof, wherein the sucrose concentration is about 7.5% w / v or about 8% w / v and the arginine or its salt concentration is 50 nM. In certain embodiments, the arginine or pharmaceutically acceptable salt thereof is arginine hydrochloride.

[0064] In some embodiments, the pharmaceutical composition of the present disclosure further comprises a surfactant. In some embodiments, the surfactant is an ionic or nonionic surfactant. In some embodiments, the surfactant is a polysorbate, poloxamer, Triton, sodium dodecylsulfonate, sodium laurylsulfonate, sodium octylglucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linole-sulfobetaine, stearic-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linole-sarcosine, stearic-sarcosine, linole-betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl be ... The surfactant is one or more selected from the group consisting of linoleamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, and a copolymer of ethylene and propylene glycol. In one embodiment, the surfactant is a polysorbate. In one embodiment, the surfactant is polysorbate 20 or polysorbate 80. In one embodiment, the surfactant is polysorbate 80.

[0065] In one embodiment, the concentration of the surfactant in the pharmaceutical composition is 0.001% to 1% w / v, for example, 0.005% to 1% w / v, 0.005% to 0.8% w / v, 0.005% to 0.6% w / v, 0.005% to 0.4% w / v, 0.005% to 0.2% w / v, 0.005% to 0.1% w / v, 0.005% to 0.09% w / v, 0.005% to 0.08% w / v, 0.005% to 0.07% w / v, 0.005% to 0.06% w / v, 0.005% to 0.05% w / v, or 0.008% to 0.5% w / v. , 0.008%~0.4%w / v, 0.008%~0.3%w / v, 0.008%~0.2%w / v, 0.008%~0.1%w / v, 0.008%~0.09%w / v, 0.008%~0.08%w / v, 0.008%~0.07%w / v, 0.008%~ 0.06%w / v, 0.008%~0.05%w / v, 0.01%~0.1%w / v, 0.01%~0.09%w / v, 0.01 %~0.08%w / v, 0.01%~0.07%w / v, 0.01%~0.06%w / v, 0.01%~0.05%w / v. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.005% to 1% w / v, 0.008% to 0.2% w / v, or 0.01% to 0.1% w / v. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.001% w / v, about 0.002% w / v, about 0.003% w / v, about 0.004% w / v, about 0.005% w / v, about 0.006% w / v, about 0.007% w / v, about 0.008% w / v, about 0.009% w / v, about 0.01% w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v, about 0.08% w / v, about 0.09% w / v, or about 0.1% w / v. In some embodiments, the concentration of arginine or a salt thereof in the pharmaceutical composition is about 0.02% w / v.

[0066] In one embodiment, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof in the pharmaceutical composition is 0.1 to 500 mg / mL, for example, 0.1 to 400 mg / mL, 0.1 to 350 mg / mL, 0.1 to 300 mg / mL, 0.1 to 250 mg / mL, 0.1 to 200 mg / mL, 0.1 to 190 mg / mL, 0.1 to 180 mg / mL, 0.1 to 170 mg / mL, 0.1 to 160 mg / mL, 0.1 to 150 mg / mL, 0.1 to 145 mg / mL, 0.1 to 140 mg / mL, 0.1 to 135 mg / mL, 0.1 to 130 mg / mL, 0.1 to 1 25mg / mL, 0.1~120mg / mL, 0.1~115mg / mL, 0.1~110mg / mL, 0.1~105mg / mL, 0.1~100mg / mL, 0.1~95mg / mL, 0.1~90mg / mL, 0.1~85mg / mL, 0.1~80mg / mL, 1~3 00mg / mL, 1~250mg / mL, 1~240mg / mL, 1~230mg / mL, 1~220mg / mL, 1~210mg / mL, 1~200mg / mL, 1~190mg / mL, 1~180mg / mL, 1~170mg / mL, 1~160mg / mL, 1~150mg / mL, 1~145mg / mL, 1~140mg / mL, 1~135mg / mL, 1~130mg / mL, 1~125mg / mL, 1~120mg / mL, 1~115mg / mL, 1~110mg / mL, 1~105mg / mL, 1~100mg / mL, 1~95mg / mL, 1~90mg / mL, 1~85mg / mL, 1~80mg / mL, 10~250mg / mL, 10~240mg / mL, 10~230mg / mL, 10~220mg / mL, 10~210mg / mL, 10~200mg / mL, 10~190mg / mL, 10~180mg / mL , 10~170mg / mL, 10~160mg / mL, 10~150mg / mL, 10~145mg / mL, 10~140mg / mL, 10~135mg / mL, 10~132mg / mL, 10~130mg / mL, 10~125mg / mL, 10~120mg / mL, 10~ 115mg / mL, 10~110mg / mL, 10~105mg / mL, 10~100mg / mL, 10~95mg / mL, 10~90mg / mL, 10~85mg / mL, 10~80mg / mL, 15~200mg / mL, 15~190mg / mL, 15~180mg / mL,15~170mg / mL、15~160mg / mL、15~150mg / mL、15~145mg / mL、15~140mg / mL、15~132mg / mL、15~135mg / mL、15~130mg / mL、15~125mg / mL、15~120mg / mL、15~115mg / mL、15~110mg / mL、15~105mg / mL、15~100mg / mL、15~95mg / mL、15~90mg / mL、15~85mg / mL、15~80mg / mL、20~200mg / mL、20~190mg / mL、20~180mg / mL、20~170mg / mL、20~160mg / mL、20~150mg / mL、20~145mg / mL、20~140mg / mL、20~135mg / mL、20~130mg / mL、20~125mg / mL、20~120mg / mL、20~115mg / mL、20~110mg / mL、20~105mg / mL、20~100mg / mL、20~95mg / mL、20~90mg / mL、20~85mg / mL、20~80mg / mL、30~200mg / mL、30~190mg / mL、30~180mg / mL、30~170mg / mL、30~160mg / mL、30~150mg / mL、30~145mg / mL、30~140mg / mL、30~135mg / mL、30~130mg / mL、30~125mg / mL、30~120mg / mL、30~115mg / mL、30~110mg / mL、30~105mg / mL、30~100mg / mL、30~95mg / mL、30~90mg / mL、30~85mg / mL、30~80mg / mL、40~90mg / mL、40~85mg / mL、40~80mg / mL、50~200mg / mL、50~190mg / mL、50~180mg / mL、50~170mg / mL、50~160mg / mL、50~150mg / mL、50~145mg / mL、50~140mg / mL、50~135mg / mL、50~130mg / mL、50~125mg / mL、50~120mg / mL、50~115mg / mL、50~110mg / mL、50~105mg / mL、50~100mg / mL、50~95mg / mL、50~90mg / mL、50~85mg / mL、50~80mg / mL、85~200mg / mL、85~190mg / mL、85~180mg / mL、85~170mg / mL、In some embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 85 to 160 mg / mL or 85 to 150 mg / mL. In some embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 1 to 200 mg / mL. In some embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 10 to 150 mg / mL. In some embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 20 to 100 mg / mL. In some embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 1 to 300 mg / mL. In some embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 10 to 250 mg / mL. In some embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 20 to 200 mg / mL. In some embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 15 to 130 mg / mL or 15 to 132 mg / mL. In certain embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, or about 75 mg / mL. , about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 105 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 165 mg / mL, about 170 mg / mL, about 175 mg / mL, about 180 mg / mL, about 185 mg / mL, 190 mg / mL or about 200 mg / mL. In some embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, or about 190 mg / mL.About 80 mg / mL or about 150 mg / mL.

[0067] In one embodiment, the concentration of the antibody-drug conjugate in the pharmaceutical composition is 0.1 to 300 mg / mL, for example, 1 to 200 mg / mL, 1 to 190 mg / mL, 1 to 180 mg / mL, 1 to 170 mg / mL, 1 to 160 mg / mL, 1 to 150 mg / mL, 1 to 145 mg / mL, 1 to 140 mg / mL, 1 to 135 mg / mL, 1 to 130 mg / mL, 1 to 120 mg / mL, 1 to 110 mg / mL, 1 to 100 mg / mL, 1 to 90 mg / mL, 1 to 80 mg / mL, 1 to 70 mg / mL, 1 to 80 mg / mL, 1 to 90 mg / mL, 1 to 100 mg / mL, 1 to 120 mg / mL, 1 to 110 mg / mL, 1 to 100 mg / mL, 1 to 140 mg / mL, 1 to 150 mg / mL, 1 to 160 mg / mL, 1 to 170 mg / mL, 1 to 180 mg / mL, 1 to 190 mg / mL, 1 to 200 mg / mL, 1 to 210 mg / mL, 1 to 220 mg / mL, 1 to 230 mg / mL, 1 to 240 mg / mL, 1 to 250 mg / mL, 1 to 260 mg / mL, 1 to 270 mg / mL, 1 to 280 mg / mL, 1 to 290 mg / mL, 1 to 300 mg / mL, 1 to 310 mg / mL, 1 to 320 mg / mL, 1 to 330 mg / mL, 1 to 340 mg / mL, 1 to 350 The antibody-drug conjugate has a concentration of 0 mg / mL, 1 to 60 mg / mL, 5 to 150 mg / mL, 5 to 140 mg / mL, 5 to 130 mg / mL, 5 to 120 mg / mL, 5 to 110 mg / mL, 5 to 100 mg / mL, 5 to 90 mg / mL, 5 to 80 mg / mL, 5 to 70 mg / mL, 5 to 60 mg / mL, 10 to 130 mg / mL, 10 to 120 mg / mL, 10 to 110 mg / mL, 10 to 100 mg / mL, 10 to 90 mg / mL, 10 to 80 mg / mL, 10 to 70 mg / mL, or 10 to 60 mg / mL. In some embodiments, the antibody-drug conjugate has a concentration of 1 to 150 mg / mL. In some embodiments, the antibody-drug conjugate has a concentration of 5 to 120 mg / mL. In some embodiments, the antibody-drug conjugate has a concentration of about 20 to 100 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate is 10 to 100 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate is about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, or about 100 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate is about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL. In certain embodiments, the concentration of the antibody-drug conjugate is about 30 mg / mL, about 40 mg / mL, or about 50 mg / mL.

[0068] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an anti-CD40 antibody or antigen-binding fragment thereof (e.g., 9E6(L4H2) whose heavy chain and light chain sequences are SEQ ID NOs: 20 and 21), the pharmaceutical composition comprising or being any one of the following groups A) to F): A) an anti-CD40 antibody or antigen-binding fragment thereof; histidine-hydrochloride buffers, such as histidine-histidine hydrochloride; Polysorbates, such as polysorbate 80, sucrose, trehalose and / or arginine or medicinal salts thereof; B) an anti-CD40 antibody or an antigen-binding fragment thereof; histidine-hydrochloride buffers, such as histidine-histidine hydrochloride; Polysorbates, such as polysorbate 80, proline or a medicinal salt thereof, C) an anti-CD40 antibody or an antigen-binding fragment thereof; histidine-hydrochloride buffers, such as histidine-histidine hydrochloride; Polysorbates, such as polysorbate 80, sucrose, D) In ​​any one of A) to C), the pH of the buffer is 4.5 to 7.5, for example, 5 to 7.2, 5.5 to 7, or 5.6 to 6.6; E) In any one of the above A) to D), the pharmaceutical composition further comprises water for injection; F) In any one of the above A) to E), the pH of the pharmaceutical composition is 4.5 to 7.5, for example, 5 to 7.2, 5.5 to 7, or 5.6 to 6.6.

[0069] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate, the pharmaceutical composition comprising or being any one of the following groups A) to H): A) antibody-drug conjugates; histidine-hydrochloride buffers, such as histidine-histidine hydrochloride; Polysorbates, such as polysorbate 80, sucrose, trehalose and / or arginine or medicinal salts thereof; B) antibody-drug conjugates; histidine-hydrochloride buffers, such as histidine-histidine hydrochloride; Polysorbates, such as polysorbate 80, proline or a medicinal salt thereof, C) antibody-drug conjugates; histidine-hydrochloride buffers, such as histidine-histidine hydrochloride; Polysorbates, such as polysorbate 80, sucrose, D) antibody-drug conjugates; histidine-hydrochloride buffers, such as histidine-histidine hydrochloride; Polysorbates, such as polysorbate 80, trehalose, E) Any one of A) to D) above, wherein the antibody-drug conjugate has the structure shown below: [ka] wherein 9E6-L4H2 is an anti-CD40 antibody comprising a heavy chain and a light chain comprising the amino acid sequences shown in SEQ ID NOs: 20 and 21, Here, k is an integer or decimal number selected from 1 to 10, for example, k is an integer or decimal number of 2 to 5, for example, k is 4±0.4, 4±0.5, 4±0.6, 4±0.8, for example, k is about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9 or about 5.0, F) In any one of A) to E), the pH of the buffer is 4.5 to 7.5, for example, 5 to 7.2, 5.5 to 7, or 5.6 to 6.6; G) In any one of the above A) to F), the pharmaceutical composition further comprises water for injection; H) In any one of the above A) to G), the pharmaceutical composition has a pH of 4.5 to 7.5, for example, a pH of 5 to 7.2, 5.5 to 7, or 5.6 to 6.6.

[0070] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-CD40 antibody or antigen-binding fragment thereof (e.g., 9E6(L4H2) whose heavy chain and light chain sequences are SEQ ID NOs: 20 and 21), the pharmaceutical composition comprising or being any one of the following groups 1) to 21): 1) 0.1 to 300 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 1-50 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride; 0.001% to 1% w / v of a polysorbate, e.g., polysorbate 80; 0.1% to 30% w / v sucrose, and the pH of the composition is 4.5 to 7.5; 2) 1 to 200 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 5-40 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride; 0.005% to 1% w / v of a polysorbate, e.g., polysorbate 80; 0.5% to 20% w / v sucrose, and the pH of the composition is 5 to 7.2; 3) 10 to 150 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 10-30 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride; 0.008% to 0.2% w / v of a polysorbate, e.g., polysorbate 80; 1% to 15% w / v sucrose, and the pH of the composition is 5.5 to 7. 4) 15 to 132 mg / mL or 20 to 100 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 15-25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, e.g., polysorbate 80; 5% to 10% w / v sucrose, and the pH of the composition is 5.6 to 6.6; 5) about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; 15-25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, e.g., polysorbate 80; 5% to 10% w / v sucrose, and the pH of the composition is 5.6 to 6.6; 6) 20 to 100 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; about 15 mM, about 20 mM, or about 25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, e.g., polysorbate 80; 5% to 10% w / v sucrose, and the pH of the composition is 5.6 to 6.6; 7) 20 to 100 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 15-25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; about 0.01% w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, or about 0.07% w / v of a polysorbate, e.g., polysorbate 80; 5% to 10% w / v sucrose, and the pH of the composition is 5.6 to 6.6; 8) 20 to 100 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 15 to 25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, e.g., polysorbate 80; about 5% w / v, about 5.5% w / v, about 6% w / v, about 6.5% w / v, about 7% w / v, about 7.5% w / v, about 8% w / v, about 8.5% w / v, about 9% w / v, about 9.5% w / v, or about 10% w / v of sucrose; and the pH of the composition is 5.6 to 6.6; 9) 20 to 100 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 15-25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, e.g., polysorbate 80; 5% to 10% w / v sucrose, and the pH of the composition is about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6; 10) 0.1 to 500 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 1-50 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride; 0.001% to 1% w / v of a polysorbate, e.g., polysorbate 80; 0.01% to 20% w / v proline or a medicinal salt thereof; and the pH of the composition is 4.5 to 7.5; 11) 1 to 300 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 5-40 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride; 0.005% to 1% w / v of a polysorbate, e.g., polysorbate 80; 0.1% to 15% w / v proline or a medicinal salt thereof; and the pH of the composition is 5 to 7.2; 12) 10 to 250 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 10-30 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride; 0.008% to 0.2% w / v of a polysorbate, e.g., polysorbate 80; 0.5% to 10% w / v proline or a medicinal salt thereof; and the pH of the composition is 5.5 to 7. 13) 20 to 200 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 15-25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, e.g., polysorbate 80; 1% to 5% w / v proline or a medicinal salt thereof; and the pH of the composition is 5.6 to 6.6; 14) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; 15-25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, e.g., polysorbate 80; 1% to 5% w / v proline or a medicinal salt thereof; and the pH of the composition is 5.6 to 6.6; 15) 20 to 200 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; about 15 mM, about 20 mM, or about 25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, e.g., polysorbate 80; 1% to 5% w / v proline or a medicinal salt thereof; and the pH of the composition is 5.6 to 6.6; 16) 20 to 200 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 15-25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; about 0.01% w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v of a polysorbate, e.g., polysorbate 80; 1% to 5% w / v proline or a medicinal salt thereof; and the pH of the composition is 5.6 to 6.6; 17) 20 to 200 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 15-25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, e.g., polysorbate 80; about 1% w / v, about 1.5% w / v, about 2% w / v, about 2.5% w / v, about 3% w / v, about 3.5% w / v, about 4% w / v, about 4.5% w / v, about 5% w / v of proline or a medicinal salt thereof; and the pH of the composition is 5.6 to 6.6; 18) 20 to 200 mg / mL of an anti-CD40 antibody or an antigen-binding fragment thereof; 15-25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, e.g., polysorbate 80; 1% to 5% w / v proline or a medicinal salt thereof; and the pH of the composition is about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.6; 19) The pharmaceutical composition according to any one of 6) to 9) above, wherein the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 15 to 130 mg / mL or 15 to 132 mg / mL; 20) The pharmaceutical composition according to any one of 15) to 18) above, wherein the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 30 to 190 mg / mL; 21) The pharmaceutical composition according to any one of 1) to 20) above, which further comprises water for injection.

[0071] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate, the pharmaceutical composition comprising or being any one of the following groups 1) to 6): 1) 0.1 to 300 mg / mL of antibody-drug conjugate; 1-50 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride; 0.001% to 1% w / v of a polysorbate, e.g., polysorbate 80; 0.1% to 30% w / v sucrose or trehalose, and the pH of the composition is 4.5 to 7.5; 2) 1 to 150 mg / mL of antibody-drug conjugate; 5-40 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride; 0.005% to 1% w / v of a polysorbate, e.g., polysorbate 80; 0.5% to 20% w / v sucrose or trehalose, and the pH of the composition is 5 to 7.2; 3) 5 to 120 mg / mL antibody-drug conjugate; 10-30 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride; 0.008% to 0.2% w / v of a polysorbate, e.g., polysorbate 80; 1% to 15% w / v sucrose or trehalose, and the pH of the composition is 5.5 to 7. 4) 10 to 100 mg / mL antibody-drug conjugate; 15-25 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, e.g., polysorbate 80; 5% to 10% w / v sucrose or trehalose, and the pH of the composition is 5.6 to 6.6; 5) In any one of the above 1) to 4), the antibody-drug conjugate has the following structure: [ka] wherein 9E6-L4H2 is an anti-CD40 antibody comprising a heavy chain and a light chain comprising the amino acid sequences shown in SEQ ID NOs: 20 and 21, Here, k is an integer or decimal number selected from 1 to 10, for example, k is an integer or decimal number of 3 to 5, for example, k is 4±0.4, 4±0.5, 4±0.6, 4±0.8, for example, k is about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9 or about 5.0.

[0072] 6) The pharmaceutical composition according to any one of the above 1) to 5), which further contains water for injection.

[0073] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-CD40 antibody or antigen-binding fragment thereof (e.g., 9E6(L4H2) whose heavy chain and light chain sequences are SEQ ID NOs: 20 and 21), the pharmaceutical composition comprising or being any one of the following groups 1) to 19): 1) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 7.5% w / v sucrose, and the pH of the composition is about 6.0; 2) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 7.5% w / v sucrose, and the pH of the composition is about 6.1; 3) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 7.5% w / v sucrose, and the pH of the composition is about 6.2; 4) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 7.5% w / v sucrose, and the pH of the composition is about 6.3; 5) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 7.5% w / v sucrose, and the pH of the composition is about 6.5; 6) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 7.5% w / v sucrose, and the pH of the composition is about 6.6; 7) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; Approximately 8% w / v sucrose, and the pH of the composition is about 6.0; 8) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; Approximately 8% w / v sucrose, and the pH of the composition is about 6.1; 9) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; Approximately 8% w / v sucrose, and the pH of the composition is about 6.2; 10) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-HCl buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; Approximately 8% w / v sucrose, and the pH of the composition is about 6.3; 11) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-HCl buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; Approximately 8% w / v sucrose, and the pH of the composition is about 6.5; 12) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-HCl buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; Approximately 8% w / v sucrose, and the pH of the composition is about 6.6; 13) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 2.5% w / v proline, and the pH of the composition is about 6.0; 14) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 2.5% w / v proline, and the pH of the composition is about 6.1; 15) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 2.5% w / v proline, and the pH of the composition is about 6.2; 16) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 2.5% w / v proline, and the pH of the composition is about 6.3; 17) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 2.5% w / v proline, and the pH of the composition is about 6.5; 18) about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL of an anti-CD40 antibody or antigen-binding fragment thereof; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 2.5% w / v proline, and the pH of the composition is about 6.6; 19) The pharmaceutical composition according to any one of the above 1) to 18), which further contains water for injection.

[0074] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate, the pharmaceutical composition comprising or being any one of the following groups 1) to 8): 1) about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL of the antibody-drug conjugate; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; about 7.5% w / v or about 9% w / v sucrose; and the pH of the composition is about 6.0; 2) about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL of the antibody-drug conjugate; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; about 7.5% w / v or about 9% w / v sucrose; and the pH of the composition is about 6.3; 3) about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL of the antibody-drug conjugate; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; about 7.5% w / v or about 9% w / v sucrose; and the pH of the composition is about 6.6; 4) about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL of the antibody-drug conjugate; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 7.5% w / v trehalose, and the pH of the composition is about 6.0; 5) about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL of the antibody-drug conjugate; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 7.5% w / v trehalose, and the pH of the composition is about 6.3; 6) about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL of the antibody-drug conjugate; about 20 mM histidine-hydrochloride buffer, e.g., histidine-histidine hydrochloride; approximately 0.02% w / v polysorbate 80; approximately 7.5% w / v trehalose, and the pH of the composition is about 6.6; 7) In any one of the above 1) to 6), the antibody-drug conjugate has the structure shown below: [ka] wherein 9E6-L4H2 is an anti-CD40 antibody comprising a heavy chain and a light chain comprising the amino acid sequences shown in SEQ ID NOs: 20 and 21, Here, k is an integer or decimal number selected from 1 to 10, for example, k is an integer or decimal number of 3 to 5, for example, k is 4±0.4, 4±0.5, 4±0.6, 4±0.8, for example, k is about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9 or about 5.0.

[0075] 8) The pharmaceutical composition according to any one of the above 1) to 7), which further contains water for injection.

[0076] In certain embodiments, the pharmaceutical composition of the present disclosure has a viscosity of 30 cp or less, e.g., 25 cp, 20 cp or less. In certain embodiments, the viscosity of the pharmaceutical composition of the present disclosure is suitable for injection.

[0077] The present disclosure further provides a method of preparing the pharmaceutical composition, the method comprising mixing an anti-CD40 antibody or antigen-binding fragment thereof with a buffer. In some embodiments, the buffer is a histidine-hydrochloride buffer (e.g., histidine-histidine hydrochloride).

[0078] In some embodiments, the pharmaceutical composition is a liquid formulation, and in some embodiments, the solvent of the liquid formulation is water, saline, or a glucose solution.

[0079] The present disclosure further provides a lyophilized formulation, which, after being reconstituted, can form a pharmaceutical composition as described above.

[0080] The present disclosure further provides a lyophilized formulation obtained by lyophilizing any one of the pharmaceutical compositions described above.

[0081] The present disclosure further provides a reconstituted solution prepared by reconstituting the lyophilized formulation. In some embodiments, the reconstituted solution is selected from, but not limited to, water for injection, saline, or glucose solution.

[0082] The present disclosure further provides a product comprising a container containing the pharmaceutical composition, the lyophilized formulation, or the reconstituted solution. In some embodiments, the container is an injection vial made of neutral borosilicate glass tubing. In some embodiments, the product includes a pharmaceutical package insert.

[0083] The present disclosure provides use of the above pharmaceutical composition, the pharmaceutical composition prepared by the above method, the above lyophilized preparation, or the above reconstituted solution in a medicament for treating or alleviating autoimmune diseases, graft-versus-host disease, or alleviating transplant rejection.

[0084] In one embodiment, there is provided a method for ameliorating or treating an autoimmune disease or an inflammatory disease, and related pharmaceutical uses, which include administering to a subject the pharmaceutical composition, the pharmaceutical composition prepared by the method, the lyophilized preparation, or the reconstituted solution.

[0085] In certain embodiments, methods for treating CD40-associated disorders and related pharmaceutical uses are provided, in certain embodiments, methods for inhibiting the growth or differentiation of CD40-associated disorder cells and related pharmaceutical uses are provided, in certain embodiments, methods for inhibiting the growth and / or differentiation of cells expressing the human CD40 antigen and related pharmaceutical uses are provided, in certain embodiments, methods for inhibiting antibody production by B cells in a subject and related pharmaceutical uses are provided, and in certain embodiments, methods for treating immune disorder diseases and related pharmaceutical uses are provided. All of the above methods include administering the above pharmaceutical composition, the pharmaceutical composition prepared by the above method, the lyophilized preparation, or the reconstituted solution to a subject or cells.

[0086] In one embodiment, there is provided a method for inducing depletion of peripheral B cells and related pharmaceutical uses, which comprises administering to a subject the pharmaceutical composition, the pharmaceutical composition prepared by the method, the lyophilized preparation, or the reconstituted solution.

[0087] In certain embodiments, methods for treating or alleviating a disease or condition and related pharmaceutical uses are provided, which comprise administering to a subject in need thereof the pharmaceutical composition, the pharmaceutical composition prepared by the method, the lyophilized formulation, or the reconstituted solution, wherein the disease or condition may or may not be CD40-related, 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 diseases, inflammatory bowel diseases (e.g., Crohn's disease or ulcerative colitis), hypersensitivity reactions, anaphylactic reactions, Sjogren's syndrome, type 1 diabetes, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, inflammatory myositis, polyendocrine deficiency, Schmidt's syndrome, and the like. syndrome), autoimmune uveitis, Addison's disease, adrenalitis, 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, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal peristalsis disorders, digital sclerosis and telangiectasia), male and female autoimmune infertility, ankylosing spondylitis spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, antiphospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird breeder's lung, toxic epidermal necrolysis, Alport's syndromesyndrome, alveolitis, allergic alveolitis, fibrosing 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, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue fever, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum elevans, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome syndrome, filariasis, cyclitis, chronic cyclitis, heterochromic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft-versus-host disease, transplant rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenström's macroglobulinemia, Evan's syndrome, acute respiratory distress syndrome, pulmonary inflammation, osteoporosis, delayed hypersensitivity, and autoimmune gonadal dysfunction, such as Sjögren's syndrome, multiple sclerosis, and systemic lupus erythematosus.

[0088] In certain embodiments, there is provided a method and related pharmaceutical use for treating a disease associated with B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I 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), which comprises administering to a subject in need thereof the pharmaceutical composition, the pharmaceutical composition prepared by the method, the lyophilized formulation, or the reconstituted solution.

[0089] In certain embodiments, methods and related pharmaceutical uses for treating tumors or cancers, which may or may not be associated with expression of CD40, are provided, comprising administering to a subject in need thereof an effective amount of the above-described anti-CD40 antibody or antigen-binding fragment thereof.

[0090] In one embodiment, a method for treating or alleviating graft-versus-host disease or transplant rejection is provided, comprising administering to a subject in need thereof the pharmaceutical composition, the resulting pharmaceutical composition prepared by the method, the lyophilized formulation, or the reconstituted solution and tacrolimus.

[0091] In one embodiment, there is provided use of the above pharmaceutical composition, the pharmaceutical composition prepared by the above method, the lyophilized formulation, or the reconstituted solution for the preparation of a medicament for treating or alleviating graft-versus-host disease or transplant rejection, including in combination with tacrolimus. In one embodiment, there is provided use of tacrolimus for the preparation of a medicament for treating or alleviating graft-versus-host disease or transplant rejection, including in combination with the above pharmaceutical composition, the pharmaceutical composition prepared by the above method, the lyophilized formulation, or the reconstituted solution.

[0092] In certain embodiments, methods for treating or alleviating graft-versus-host disease or transplant rejection using the above-described anti-CD40 antibody or antigen-binding fragment thereof in combination with tacrolimus, and uses for preparing a medicament for treating or alleviating graft-versus-host disease or transplant rejection in combination are provided.

[0093] In certain 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.

[0094] In one embodiment, the transplantation refers to transplantation of one selected from the group consisting of allogeneic cells, xenogeneic cells, allogeneic tissue, xenogeneic tissue, allogeneic organ, and xenogeneic organ.

[0095] In certain embodiments, the pharmaceutical composition, the pharmaceutical composition prepared by the method, the lyophilized preparation, or the reconstituted solution inhibits or reverses the rejection of a tissue graft in a transplant recipient, or prolongs or preserves the function of a tissue transplanted in a transplant recipient, or restores the function of a damaged tissue in a transplant recipient. Definition of Terms

[0096] In order that this disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0097] Unless the context clearly indicates otherwise, throughout the specification and claims, the words "comprises," "having," "including," and the like are to be understood to have an inclusive meaning, i.e., "including but not limited to," rather than an exclusive or exhaustive meaning.

[0098] "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). A cDNA molecule encoding CD40 was isolated from a library prepared from the Burkitt's lymphoma cell line Raji (Stamenkovic et al., 1989, EMBO J. 8:1403). Sequence information may be found in Table 2 of this disclosure. Cells that endogenously express CD40 are any cells characterized by surface expression of CD40, including, but not limited to, normal and neoplastic B cells, interdigitating cells, basal epithelial cells, cancer cells, macrophages, endothelial cells, follicular dendritic cells, tonsillar cells, and bone marrow-derived plasma cells.

[0099] The term "antibody" is used in the broadest sense to refer to any antibody that exhibits the desired antigen-binding activity, and covers 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). Antibodies may also refer to immunoglobulins, which have a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in the amino acid composition and sequence of the heavy chain constant regions, resulting in different antigenicities. Therefore, immunoglobulins can be divided into five types, or immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are 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 the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains and λ chains based on differences in the constant region. Each of the five types of Ig may have either κ chains or λ chains. In antibody heavy and light chains, the sequence of approximately 110 amino acids near the N-terminus is highly variable and forms the variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable and forms the constant region (C region). The variable region contains three hypervariable regions (CDRs) and four framework regions (FRs) whose sequences are relatively conserved. 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 in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0100] The determination or definition of CDRs can be achieved by solving the structure of the antibody and / or the structure of the antibody-ligand complex, thereby accurately delineating the CDRs and identifying the residues that comprise the antibody's binding site. This can be accomplished by any one of a variety of techniques known to those of skill in the art, such as X-ray crystallography. Various analytical methods can be used to identify CDRs, including, but not limited to, the Kabat numbering system, the Chothia numbering system, the Ab M numbering system, the I MGT numbering system, contact definitions, and conformational definitions.

[0101] 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 takes into account the location of certain structural loop regions (see, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The Ab M numbering system uses an integrated suite of computer programs from the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "Ab MTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.). The Ab M numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from the base sequence (see, e.g., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in Sa mudrala et al., 1999, PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In conformational definitions, CDR positions can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).Additionally, the definition of the boundaries of other CDRs may not strictly follow one of the above methods, but may be shortened or extended depending on predictions or experimental results that show that a particular residue or group of residues does not significantly affect antigen binding, while still overlapping with at least a portion of the Kabat CDRs. As used in this disclosure, CDR can refer to a CDR defined by any method (including a combination of methods) known in the art. The correspondence between each numbering system is well known to those skilled in the art and is illustratively shown in Table 1 below.

[0102] [Table 1]

[0103] The CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments of the disclosure conform in number and position to the known Kabat numbering system.

[0104] A "monoclonal antibody" or "monoclonal antibody" is an antibody obtained from a substantially homogeneous antibody population; i.e., each antibody within the population is identical except for minor, naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal," for example, indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method.

[0105] The term "rabbit antibody" as used herein refers to a monoclonal antibody against human CD40 or an epitope thereof, prepared using knowledge and techniques within the art. During preparation, CD40 antigen is injected into test rabbits, and antibodies expressing the desired sequence or functional characteristics are isolated. In a specific embodiment of the present disclosure, the rabbit anti-human anti-CD40 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a rabbit kappa or lambda chain or a variant thereof, or may further comprise a heavy chain constant region of a rabbit IgG1, IgG2, IgG3, or IgG4 chain or a variant thereof.

[0106] The term "fully human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "fully human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., rabbit) have been grafted onto human framework sequences (i.e., "humanized antibodies").

[0107] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting non-human CDR sequences onto a human antibody variable region framework. This can overcome the strong immune response induced by chimeric antibodies containing large amounts of non-human protein components. To avoid a decrease in activity associated with reduced immunogenicity, minimal back mutations can be performed on the fully human antibody variable region to maintain activity.

[0108] The term "chimeric antibody" refers to an antibody in which the variable region of an antibody of one species is fused with the constant region of an antibody of a second species, and can reduce the immune response elicited by the antibody of the first species. For example, to create a chimeric antibody, first, rabbits secreting rabbit-specific monoclonal antibodies are generated, the antibodies are isolated, and then, if necessary, the constant region genes of a fully human antibody are cloned. The rabbit variable region genes and human constant region genes are ligated to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell system. 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 variants thereof. Preferably, the constant region of the fully human antibody contains a human IgG1 or IgG4 heavy chain constant region, or an IgG1 that lacks ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation is used.

[0109] 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 or VL or VHH), scFv, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any one of the above (see, e.g., 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 the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv form was first disclosed in WO 2009 / 040562, and its disulfide bond stabilized form, Fab-dsFv, was first disclosed in WO 2010 / 035012. Antigen-binding fragments of the present disclosure further include the Fab and Fab' fragments described in WO 2005 / 003169, WO 2005 / 003170, and WO 2005 / 003171. Multivalent antibodies may be multispecific, e.g., bispecific, or monospecific (see, e.g., WO 92 / 22583 and WO 05 / 113605), an example of the latter being Tri-Fab (or TFM) described in WO 92 / 22583.

[0110] The term "binds to CD40" refers to the ability to interact with CD40 or an epitope thereof, which may be of human origin. The term "antigen-binding site" of the present disclosure refers to a non-contiguous portion of an antigen in three dimensions that is recognized by an antibody or antigen-binding fragment of the present disclosure.

[0111] The term "antigen" refers to a molecule used to immunize an immunocompetent vertebrate to produce antibodies that recognize the antigen, or to screen an expression library (e.g., phage, Saccharomyces, or ribosome display libraries, among others). In the present disclosure, antigen is defined more broadly to include a target molecule that is specifically recognized by an antibody, and also includes portions or mimetics of molecules used in immunization processes to produce antibodies or library screening to select antibodies. With respect to the human CD40-binding antibodies of the present disclosure, both monomers and multimers (e.g., dimers, trimers, etc.) of human CD40, and deletion and other variants of human CD40, are referred to as antigens.

[0112] The term "epitope" refers to the site on an antigen that binds to an immunoglobulin or antibody. Epitopes may be formed from contiguous amino acids or from juxtaposed non-contiguous amino acids formed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically contain at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody are well known in the art and include Western blotting and immunoprecipitation assays. Methods for determining the spatial conformation of epitopes include techniques known in the art and described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0113] 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 an epitope thereof is used as an analyte and an antibody is used as a ligand, the antibody binds to an epitope on an instrument using surface plasmon resonance (SPR) technology at a binding affinity of about 10 -7 The equilibrium dissociation constant (K D) and the binding affinity for the predetermined antigen or its epitope is at least twice its binding affinity for a non-specific antigen other than the predetermined antigen (or its epitope) or a closely related antigen (e.g., BSA, etc.). The term "antibody that recognizes an antigen" may be used interchangeably with the term "antibody that specifically binds" in the present disclosure.

[0114] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules is determined by the dissociation constant (K D The KD can be determined by measuring the kinetics of complex formation and dissociation, for example, by surface plasmon resonance (SPR) (Biacore). The rate constants corresponding to the binding and dissociation of monovalent complexes are called the binding rate constant ka (or k) and the dissociation rate constant kd (or koff), respectively. K D is K D The dissociation constants ka and kd are related by the equation ka = kd / ka. The value of the dissociation constant can be determined directly 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). For example, the K can be calculated by double filtration nitrocellulose filter binding assays such as those disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays mentioned elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can be determined by standard assays known in the art, such as surface plasmon resonance (SPR), e.g., Biacore. TM The K of each antibody / antigen complex may be evaluated by the KinExA system or KinExA. DBy comparing values, one can compare the binding affinities associated with interactions with different molecules, for example, comparing the binding affinities of different antibodies to a given antigen. Similarly, the specificity of an interaction can be determined by the K D value and the K of a non-target interaction (e.g., a known control antibody that does not bind CD40). D It can be evaluated by determining and comparing values.

[0115] The term "conservative substitution" refers to a substitution of an amino acid residue with another amino acid residue having similar properties to the original amino acid residue. 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 uncharged polar side chains, 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 is clear to those skilled in the art that even when amino acid residues in the above-mentioned group showing similar properties are substituted, it does not show a specific change in properties.

[0116] "Inhibition" or "blocking" may be used interchangeably and cover both partial and complete inhibition / blocking. Preferably, inhibition / blocking of CD40 reduces or alters the normal level or type of activity that occurs when CD40 binding occurs in the absence of inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in CD40 binding affinity when contacted with an anti-CD40 antibody compared to CD40 not contacted with an anti-CD40 antibody.

[0117] "Inhibition of growth" (eg, of a cell) is intended to include any measurable decrease in cell growth.

[0118] "Agonist activity," "agonist activity," or "agonistic" refers to the function of an agonist, where the binding of an agonist to a cellular receptor elicits a response or activity similar to or identical to that of the receptor's natural ligand. For example, a CD40 agonist can induce any or all of the following responses: cell proliferation and / or differentiation; upregulation of cell-cell adhesion via 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; transduction of signals via the CD40 receptor through TRAFs (e.g., TRAF2 and / or TRAF3), MAP kinases such as NIK (NF-κB-inducing kinase), 1-κB kinase (IKKα / β), transcription factor NF-κB, Ras, and pathways such as the MEK / ERK pathway, the PI3K / Akt pathway, and the p38 MAPK pathway; transduction of anti-apoptotic signals via molecules such as XIAP, Mcl-1, and BCLx; production of B and / or T cell memory; production of B cell antibodies; B cell isotype switching; and upregulation of cell surface expression of class II MHC and CD80 / 86. "Antagonist activity," "antagonist activity," or "antagonism" refers to the ability of a substance to act as an antagonist. For example, a CD40 antagonist can prevent or reduce any response induced by binding of the CD40 receptor to an agonistic 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 ligand are known to those of skill in the art and include, but are not limited to, standard competitive binding assays, assays monitoring immunoglobulin secretion by B cells, B cell proliferation assays, Banchereau-like B cell proliferation assays, T cell helper assays for antibody production, costimulation assays for B cell proliferation, and assays that upregulate B cell activation markers.

[0119] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in Reizei Port's Antibody Laboratory Techniques Manual (Chapters 5-8 and 15). For example, mice can be immunized with human CD40 or a fragment thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this disclosure have one or more human-derived FR regions added to non-human-derived CDR regions by genetic engineering methods. Human FR germline sequences are available from the Im MunoGeneTics (IMGT) website.

[0120] Conventional techniques known to those skilled in the art can be used to competitively screen antibodies for binding to the same epitope. For example, competition and cross-competition studies can be performed to obtain antibodies that bind to an antigen by competing with or cross-competing with each other. A high-throughput method for obtaining antibodies that bind to the same epitope by cross-competition is described in International Patent Publication WO 03 / 48731. Therefore, conventional techniques known to those skilled in the art can be used to obtain antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present disclosure and bind to the same epitope on CD40.

[0121] The term "disorder" refers to any condition that would benefit from treatment with a humanized anti-CD40 antibody of the present disclosure. It includes chronic and acute disorders or diseases. Non-limiting examples of disorders to be treated in the present 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.

[0122] The term "CD40-associated disorder" or "CD40-associated disease" refers to a condition characterized by the modification or elimination of cells expressing CD40. These cells include CD40-expressing cells that exhibit abnormal proliferation or that are associated with cancerous or malignant growth. More specific examples of cancers that exhibit aberrant expression of the CD40 antigen include B-lymphoblastic neoplasms, Burkitt's 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 renal cancer. Such disorders include, but are not limited to, leukemia, lymphoma (including B-cell lymphoma and non-Hodgkin's lymphoma), multiple myeloma, Waldenstrom's macroglobulinemia, sarcomas such as osteosarcoma and Ewing's sarcoma, malignant melanoma, adenocarcinoma (including ovarian adenocarcinoma), Kaposi's sarcoma / tumor, and solid tumors, including squamous cell carcinoma. "CD40-associated disorders" also include diseases and disorders of the immune system, such as autoimmune and inflammatory disorders. Such conditions include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), pulmonary inflammation, asthma, and idiopathic thrombocytopenic purpura (ITP).

[0123] The term "preventing the growth of" or "growth inhibition" refers to inhibiting the growth or proliferation of cells, particularly neoplastic cell types that express the CD40 antigen. Thus, growth inhibition would, for example, significantly reduce the percentage of neoplastic cells in S phase.

[0124] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. If every position in two compared sequences is occupied by the same nucleotide or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by the same nucleotide, the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences, divided by the number of positions compared, multiplied by 100%. For example, if 6 out of 10 positions in two sequences are matched or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Typically, two sequences are compared when aligned to obtain the maximum percentage of homology.

[0125] The terms "cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to intentional or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for from the originally transformed cell are included.

[0126] The words "optionally" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and the description includes both cases where the event or circumstance occurs and cases where it does not. For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may be present, but are not necessarily present.

[0127] The term "CD40 binding molecule" of the present disclosure, in its broadest sense, encompasses any protein capable of binding to CD40, including an anti-CD40 antibody or antigen-binding fragment thereof of the present disclosure. For example, a CD40 binding protein may comprise one or more effector molecules, e.g., conjugated. The "effector molecule" may have therapeutic activity alone (e.g., anti-tumor activity or immune stimulatory or inhibitory activity) or a detectable function, and may take any form, such as a biologically active protein (e.g., an enzyme), another antibody or antibody fragment, a synthetic or naturally occurring polymer, a polynucleotide and its fragment, e.g., DNA, RNA and its fragment, a radionuclide (particularly radioactive iodide), a radioisotope, a chelated metal, a nanoparticle, and a reporter group (e.g., a fluorescent compound), or a compound detectable by NMR or ESR spectroscopy. Conjugation of an effector molecule to an anti-CD40 antibody or antigen-binding fragment thereof of the present disclosure can be achieved by conventional methods.

[0128] The term "antibody-drug conjugate" refers to a ligand linked to a biologically active drug by a stable linking unit. In the present disclosure, "antibody drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment linked to a drug molecule by a stable linking unit, where the antibody or antibody fragment can be bound to a drug molecule containing a linker via a specific group therein (e.g., an interchain disulfide bond).

[0129] The term "drug loading" refers to the average amount of drug loaded on each antibody-drug conjugate molecule in a group of antibody-drug conjugates, and may be expressed as the ratio of the amount of drug to the amount of antibody. As for the range of drug loading, each antibody (Ab) may be linked to 1 to 20, preferably 1 to 10, drug molecules (D). In an embodiment of the present disclosure, the drug loading is represented by k, which may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or an average value between any two numbers. It is preferably 1 to 10, and more preferably an average value of 1 to 8, 2 to 8, 2 to 7, 3 to 8, 3 to 7, 3 to 6, 4 to 7, 4 to 6, or 4 to 5. Conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA test, monoclonal antibody molecular size variant assay (CE-SDS) and HPLC can specifically identify the average drug quantity in each ADC molecule after the coupling reaction.

[0130] The monoclonal antibody molecular size variant assay (CE-SDS) disclosed herein can quantitatively measure the purity of recombinant monoclonal antibody products according to the molecular size of the product under reducing and non-reducing conditions using sodium dodecyl sulfate capillary electrophoresis (CE-SDS) with ultraviolet detection, in accordance with the capillary electrophoresis method (2015 edition of the Chinese Pharmacopoeia 0542).

[0131] In one embodiment of the present disclosure, a drug molecule is coupled to the N-terminal amino group and / or the ε-amino group of a lysine residue of a ligand via a linking unit, and typically, the number of drug molecules that can be coupled to an antibody in a coupling reaction is less than the theoretical maximum.

[0132] The amount of antibody-drug conjugate loaded is (1) controlling the molar ratio of the linking reagent to the monoclonal antibody; (2) controlling the reaction time and temperature; (3) selecting different reaction reagents; The amount of oxygen can be controlled by a number of methods, including but not limited to:

[0133] As used herein, the terms "about" or "approximately" refer to a numerical value that is within an acceptable error range of the specified value as determined by one of ordinary skill in the art, the numerical portion being determined by how it is measured or determined (i.e., the limitations of the measurement system). For example, in the practice of the art, "about" may mean within or more than 1 standard deviation. Alternatively, "about" or "essentially comprising" may mean a range of at most ±20%, e.g., a pH of about 5.5 means pH 5.5 ± 1.1. Furthermore, particularly with respect to biological systems or processes, the terms may mean at most one order of magnitude or at most 5 times the numerical value. Unless otherwise indicated, when specific values ​​appear in this application and claims, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range of the specified value.

[0134] "Buffer" refers to a buffer that resists changes in pH by the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include tris(hydroxymethyl)aminomethane (Tris), acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0135] A "citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, etc. A preferred citrate buffer is citric acid-sodium citrate buffer.

[0136] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, and are preferably histidine hydrochloride buffers or histidine acetate buffers. Histidine acetate buffers are prepared from histidine and acetic acid, and histidine hydrochloride buffers are prepared from histidine and histidine hydrochloride or histidine and hydrochloric acid.

[0137] A "phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, etc. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0138] A "trishydroxymethylaminomethane buffer" is a buffer containing trishydroxymethylaminomethane (Tris). Examples of trishydroxymethylaminomethane buffers include buffers such as trishydroxymethylaminomethane-hydrochloric acid buffer (Tris-HCl), trishydroxymethylaminomethane-acetic acid buffer (Tris-AA), trishydroxymethylaminomethane-succinic acid buffer (Tris-SA), or trishydroxymethylaminomethane-citric acid buffer (Tris-CA).

[0139] A "citric acid salt buffer" is a buffer containing citric acid ions. Examples of citric acid salt buffers include citric acid-sodium citric acid, citric acid-potassium citric acid, citric acid-calcium citric acid, citric acid-magnesium citric acid, etc. A preferred citric acid salt buffer is citric acid-sodium citric acid.

[0140] A "succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid-sodium salt, succinic acid-potassium succinate, succinic acid-calcium salt, etc. A preferred succinate buffer is succinic acid-sodium salt. Illustratively, the succinic acid-sodium succinate may be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate salt.

[0141] An "acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine-histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. A preferred acetate buffer is acetic acid-sodium acetate.

[0142] A "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients, that maintain the stability of the active ingredient, facilitate administration to the body, and contribute to the absorption of the active ingredient to further exert its biological activity.

[0143] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0144] Unless otherwise specified, the solvent in any solution form of the pharmaceutical composition described in the present disclosure is water.

[0145] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a freeze-drying step is performed on a liquid or solution formulation.

[0146] The pharmaceutical compositions described herein can achieve stable efficacy, i.e., the anti-CD40 antibody or antigen-binding fragment thereof therein essentially retains its physical stability and / or chemical stability and / or biological activity after storage, e.g., the pharmaceutical composition essentially retains its physical and chemical stability and biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, several analytical techniques are available for measuring protein stability, which can be used to measure stability after storage at a specified temperature for a specified period of time.

[0147] A stable drug formulation is one that exhibits no significant changes when stored at refrigerated temperatures (2°C-8°C) for at least 3 months, at least 6 months, at least 1 year, at least 2 years, or even up to 2 years. Stable liquid formulations also include liquid formulations that exhibit desired characteristics after storage at temperatures including 25°C for periods including 1 month, 3 months, or 6 months, or at 40°C for periods including 1 month. A typical acceptable standard for stability is degradation of antibody monomers, typically about 10%, e.g., about 5%, as measured by SEC-HPLC. Upon visual analysis, the drug formulation is colorless or clear to slightly opalescent. The concentration, pH, and osmolality of the formulation vary by no more than ±10%. Typically, no more than about 10%, e.g., no more than about 5%, of the antibody is cleaved, and no more than about 10%, e.g., no more than about 5%, of the antibody is aggregated.

[0148] An antibody "retains its physical stability" in a drug formulation if it does not exhibit significant increased aggregation, precipitation, and / or denaturation as determined by visual inspection of color and / or clarity or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation may also be assessed by fluorescence spectroscopy (which determines the tertiary structure of proteins) and by FTIR spectroscopy (which determines the secondary structure of proteins).

[0149] An antibody "retains its chemical stability" in a drug formulation if it does not undergo significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that constantly alter the chemical structure of proteins include hydrolysis or cleavage (assessed by methods such as size-exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping coupled with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion-exchange chromatography, capillary isoelectric focusing, peptide mapping, and isoaspartic acid measurement), and isomerization (assessed by isoaspartic acid content measurement, peptide mapping, etc.).

[0150] A GLP-1 analog is said to "retain its biological activity" in a drug formulation if the antibody's biological activity over a given period of time is within a given range of biological activity exhibited when the drug formulation was prepared. Antibody biological activity can be determined, for example, by antigen binding assays.

[0151] "Administration," "giving," and "treatment," when used on an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with a cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Administration," "giving," and "treatment" also refer to ex vivo and in vitro treatment, e.g., of cells, with a reagent, diagnostic, binding composition, or through another cell. "Treatment," when used on a human, veterinary, or research subject, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic uses.

[0152] "Treatment" refers to providing an internal or external therapeutic agent, including, for example, any one of the pharmaceutical compositions disclosed herein, to a patient having one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the patient or population being treated is provided with a therapeutic agent in an amount that effectively alleviates one or more disease symptoms, thereby inducing the resolution of those symptoms or inhibiting those symptoms from progressing to any clinically measurable extent. The amount of therapeutic agent that effectively alleviates any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on various factors, including the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional health care providers to assess the severity or progression of the condition. Although embodiments of the present disclosure (e.g., methods of treatment or products) may be ineffective in alleviating the respective target disease symptoms, they should reduce the target disease symptoms in a statistically significant number of patients, as determined by any statistical testing method known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0153] The instruments and methods used in the detection process are as follows.

[0154] exterior: The sample bottle was wiped clean, and the color, transparency, and visible foreign matter of the sample were visually observed under a white background and a black background of a transparency meter at an illumination intensity of 1000 to 1500 lx, respectively.

[0155] Appearance detection equipment: Seitakuki YB-2A transparency detector.

[0156] pH value: 100 μL of sample was collected by potentiometry, and the pH value was measured using a micro pH meter calibrated with a standard solution.

[0157] pH detection instrument: Mettler Toledo, type S210.

[0158] SEC molecular exclusion chromatography: This is an analytical method that separates solutes based on the correlation between the pore size of gel pores and the coil size of polymer sample molecules.

[0159] SEC monomer content percentage=Amonomer / Atotal*100% (Amonomer is the peak area of ​​the main peak monomer in the sample, and Atotal is the sum of all peak areas).

[0160] SEC instrument: Agilent 1260-Bio; column: Waters, XBrige BEH200Å SEC (300 × 7.8 mm, 3.5 μm) NR-CE Capillary Gel Electrophoresis: This is electrophoresis, which is carried out by moving a gel as a support medium in a capillary, and is a method of separating samples according to their molecular weight at a constant voltage.

[0161] Percent purity of non-reduced CE = A Main / A Total * 100% (A Main is the peak area of ​​the main peak in the sample, and A Total is the sum of all peak areas).

[0162] CE measuring equipment: Sciex model number PA800 plus icIEF Imaging Capillary Isoelectric Focusing: This is a technique for separating proteins based on their isoelectric points (pI).

[0163] icIEF main peak content percentage = peak area of ​​main peak / total area * 100% (total area is the sum of acidic peak, main peak and basic peak areas).

[0164] icIEF measurement equipment manufacturer: Protein Simple, model number Muarice.

[0165] Protein concentration determination: Protein concentration measurement equipment: UV-visible spectrophotometer, model number: Nano Drop 2000, light path 1 mm.

[0166] Melting temperature (Tm) and aggregation temperature (Tagg): Tm is the temperature at which 50% of the protein components are denatured during the temperature rise process, and Tagg is the temperature at which the protein aggregates during the temperature rise process. The sample was loaded into a Unitube and measured by heating from 25 to 95°C. Tm and Tagg measurement equipment: Uncle, the equipment manufacturer is Unchained.

[0167] Viscosity measurement: The rheometer was used to measure the sample viscosity. A 35 μL sample was placed in a detection cell and inserted into the instrument. Pressure was then applied, pushing the sample through the microfluidic channels. Software tracked the sample's velocity within each channel to obtain its viscosity.

[0168] Detection equipment: Honybun, equipment manufacturer is Unchained.

[0169] Exemplary antibody pharmaceutical composition (formulation) preparation process Step 1: Anti-CD40 antibody was obtained by cell culture and purification, and the obtained anti-CD40 antibody was subjected to ultrafiltration / concentration (UF / DF) to prepare a stock formulation containing anti-CD40 antibody together with a buffer, stabilizer, and / or surfactant. The solution was then sterilized and filtered through a 0.22 μm filter cartridge, and the filtrate was collected.

[0170] Step 2: The filling volume was adjusted (the target filling volume was 2.835 mL / bottle), and vials were used for filling. Sampling was carried out at the start, during, and end of filling to detect any differences in the filling volume.

[0171] Step 3: The plugs were inserted, the capping machine was started, and the aluminum lids were applied and capped.

[0172] Step 4: Visually inspect the product to ensure there are no defects such as incorrect filling or poor appearance. Print carton labels, fold the cartons, box them, and apply carton labels. [Brief explanation of the drawings]

[0173] [Figure 1] Figures 1A and 1B show the activity results of CD40 antagonistic antibodies in a reporter gene system for 9E6-L4H2 and 2F12-L4H2, respectively. In both Figures 1A and 1B, human IgG1 isotype was used as a negative control and CFZ533 as a positive control. [Figure 2] These figures show the inhibitory activity of CD40 antagonistic antibodies in a B cell activation experiment. Figure 2A shows the percentage inhibition of CD19+CD69+ cells by 9E6-L4H2 and 2F12-L4H2, and Figure 2B shows the MFI inhibition of CD19+CD69+ cells by 9E6-L4H2 and 2F12-L4H2. In both Figures 2A and 2B, human IgG1 isotype was used as a negative control, and CFZ533 was used as a positive control. [Figure 3] These figures show the inhibitory activity of CD40 antagonistic antibodies in a DC cell activation experiment. Figure 3A shows the results of 9E6-L4H2 and 2F12-L4H2 inhibiting CD11C+CD80+ cell MFI. Figure 3B shows the results of 9E6-L4H2 and 2F12-L4H2 inhibiting CD11C+CD86+ cell MFI. Figure 3C shows the results of 9E6-L4H2 and 2F12-L4H2 inhibiting IL-12 / 23 p40. Figure 3D shows the results of 9E6-L4H2 and 2F12-L4H2 inhibiting TNFα. In all Figures 3A to 3D, human IgG1 isotype was used as a negative control, and CFZ533 was used as a positive control. [Figure 4] These are the results of the intrinsic agonist activity of the CD40 antagonistic antibodies 9E6-L4H2 and 2F12-L4H2 in a B cell activation experimental system, using the human IgG1 isotype, CFZ533, and the agonist anti-CD40 antibody 9E5-SELFNS as controls. [Figure 5]FIG. 5A shows a flow chart of the activity of CD40 antagonistic antibodies in a mouse T cell-dependent humoral immune response model, and FIG. 5B shows the detection results on days 7, 14, 21, and 28. [Figure 6] FIG. 6 shows the activity of CD40 antagonistic antibodies in a mouse skin graft rejection model. FIG. 6A is a flow chart, and FIG. 6B shows the skin graft survival rate (%) and the results of scoring the skin grafts. [Figure 7] Figure 7 shows the activity results of a combination of a CD40 antagonistic antibody and tacrolimus (FK506) in a mouse skin graft rejection model. Figure 7A shows the skin graft survival rate (%) for 9E6-L4H2 (10mpk) and its combination with tacrolimus (FK506). Figure 7B shows the skin graft survival rate (%) for 2F12-L4H2 (10mpk) and its combination with tacrolimus (FK506). Figure 7C shows the scoring results for skin grafts for 9E6-L4H2 (10mpk) and its combination with tacrolimus (FK506). Figure 7D shows the scoring results for skin grafts for 2F12-L4H2 (10mpk) and its combination with tacrolimus (FK506). [Figure 8] This shows the results of PK detection of CD40 antagonistic antibodies 9E6-L4H2 and 2F12-L4H2 in human CD40 transgenic mice, using CFZ533 as a control. [Figure 9A] FIG. 9A shows the results of the inhibitory activity of CD40 antagonistic antibodies with Fc mutations in a B cell activation experimental system, and FIG. 9B shows the results of 9E6-L4H2 and 9E6-L4H2-AAYTE inhibiting cell MFI by CD19+CD69+. [Figure 10] Figure 10 shows the results of the inhibitory activity of CD40 antagonists with Fc mutations in a DC cell activation experimental system. Figure 10A shows the results of inhibition of IL-12 / 23p40 by 9E6-L4H2 and 9E6-L4H2-AAYTE, and Figure 10B shows the results of inhibition of TNFα by 9E6-L4H2 and 9E6-L4H2-AAYTE. [Figure 11] 1 is a flow chart of anti-CD40 ADC in a mouse skin graft rejection experiment. DETAILED DESCRIPTION OF THE INVENTION

[0174] The present disclosure will be further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure.

[0175] Experimental methods for which specific conditions are not specified in the examples or experimental examples of this disclosure generally follow conventional conditions or conditions recommended by the manufacturers of materials or products. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Modern Molecular Biology Methods, Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which specific sources are not specified are conventional commercially available reagents.

[0176] The methods for preparing and purifying the anti-CD40 antibody in the present application are described in International Patent Application WO2023274201, the entire contents of which are incorporated herein by reference.

[0177] Example 1. CD40 Immunizing Antigen, Sequence and Preparation of Screening Antigen His-tagged human CD40 (h-CD40-his) recombinant protein (product number CD0-H5228), mouse Fc-tagged human CD40 (h-CD40-mFc) recombinant protein (product number CD0-H525a), his-tagged and biotin-tagged human CD40 (hCD40-his-avi) recombinant protein (product number CD0-H82E8), and his-tagged cynomolgus monkey CD40 (cyno-CD40-his) recombinant protein (product number CD0-C52H6) were all purified commercially available protein reagents purchased from Acrobiosystems, and the origins of their respective sequences are shown in Table 2. The above protein reagents can be used in the experiments in the following Examples.

[0178] [Table 2]

[0179] 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 immunizing antigen was a His-tagged human CD40 recombinant protein (h-CD40-his, prepared at 1 μg / μL in phosphate buffer). The antigen was emulsified with Freund's adjuvant: complete Freund's adjuvant (CFA) was used for the first immunization, and incomplete Freund's adjuvant (IFA) was used for the subsequent booster immunizations. Each immunization consisted of multiple subcutaneous injections of 400 μg of antigen. The immunization injection times were days 0, 7, 20, and 41. Blood samples were collected on days 27 and 48 for detection, and antibody titers in the rabbit sera were determined by ELISA and FACS. Rabbits with high and stable serum antibody titers were selected. On day 63, a booster immunization was administered intravenously with 400 μg / rabbit of an antigen solution prepared from phosphate buffer. On day 67, the rabbits' spleens were harvested and biotin-labeled CD40 antigen was added. The labeled monoclonal B cells were selected using a flow cytometer and plated in 96-well plates. After 14 days of culture, the supernatants were collected and screened for clones capable of binding to human CD40, cynomolgus monkey CD40, and Raji cells (a tumor cell line expressing human CD40). A total of 28 B cell monoclonal lines were obtained. RNA was extracted from these monoclonal cells, reverse transcribed, amplified by PCR, and sent to a sequencing company for sequencing. The sequences of the 28 rabbit antibody lines were obtained and screened in affinity and activity identification experiments (see Examples 2-3 for methods). The heavy and light chain variable region sequences are shown in Table 3, and the CDR sequences are shown in Table 4.

[0180] [Table 3] [Table 4-1] [Table 4-2]

[0181] The obtained variable region sequences were linked to the sequence of a human antibody IgG1 constant region (with the N297A mutation, EU numbering system) and the sequence of a human kappa chain constant region to obtain a human-rabbit chimeric antibody sequence. The chimeric antibody sequence was then inserted into an expression vector using molecular cloning technology, and a human-rabbit chimeric antibody could be obtained using the HEK293 cell expression system.

[0182] Example 3. Human-rabbit chimeric anti-CD40 antibody Raji cell binding experiment Raji cells are a tumor cell line that overexpresses human CD40. 2E5 Raji cells were seeded into a 96-well plate. 100 μL of ready-to-measure antibody was added, with a final concentration of 100 nM and 5-fold dilutions (8 concentrations total), and incubated for 1 hour at 4°C. After washing once with washing buffer, 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 for 30 minutes at 4°C. After washing once with washing buffer, the fluorescence intensity was read on a flow cytometer. Binding EC of anti-CD40 antibody to CD40 was measured. 50 The values ​​were calculated. The anti-CD40 antagonistic antibody CFZ533 (i.e., Iscalimab, Nova) was used as a positive control, and the heavy and light chain variable region sequences were derived from sequence 5 and sequence 2 of US8277810B, respectively.

[0183] The results in Table 5 show that 9E6 has stronger binding EC than CFZ533. 50 It was shown that

[0184] [Table 5] Example 4. Reporter gene cellular activity experiment of human-rabbit chimeric anti-CD40 antibody HEK-Blue CD40L cells were purchased from Invivogen (Cat# hkb-cd40). These cells were stably transfected with the human CD40 gene and the NF-kB-mediated SEAP genome. The activation level of the CD40 signaling pathway can be expressed by detecting SEAP secreted in the supernatant using QUANTI-Blue, a SEAP substrate. This experiment was carried out by detecting the activation of HEK-Blue CD40L cells by CD40L. 50 The in vitro antagonist activity of anti-CD40 antibodies was evaluated based on the size of the cells. 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. At the time of passage, the medium was aspirated, the cell layer was rinsed with 5 mL of 0.25% pancreatin, the pancreatin was aspirated, and the cells were placed in an incubator for 3–5 minutes to digest. Fresh medium was then added and the cells were resuspended. 100 μL of the cell suspension was added to a 96-well cell culture plate at a density of 5 × 10^5 cells / mL in DMEM medium containing 10% FBS, 100 μg / mL Zeocin, and 30 μg / mL Blasticidin. 100 μL of sterile water was added to the periphery of the 96-well plate. The culture plate was incubated in an incubator for 24 hours (37°C, 5% CO2). After the cells adhered to the wall, 100 μL of gradient-diluted antibody was added to each well and incubated at 37°C for 30 minutes. 25 ng / mL CD40L (R&D, Product No. 2706-CL) and 2 μg / mL anti-His antibody (R&D, Product No. MAB050) were added, and the culture plate was 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 plate was incubated in the dark in an incubator for 1-3 hours. The absorbance at 630 nm was measured using a plate reader (Thermo MultiSkanFc), and the IC was determined. 50 The values ​​were calculated to evaluate the in vitro cellular activity of anti-CD40 antibodies.

[0185] Referring to Table 6, the results show that 9E6 has an IC inhibitory activity equivalent to or slightly stronger than that of CFZ533. 50 It was shown that

[0186] [Table 6]

[0187] Example 5. Humanization of anti-CD40 antibodies The heavy and light chain variable region sequences of rabbit antibody 9E6 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 antibody.

[0188] [Table 7]

[0189] The CDR regions of a rabbit antibody were grafted onto a selected human germline template to replace the human germline variable regions, followed by recombination with the corresponding human IgG constant region (preferably, an IgG1 heavy chain with an N297A mutation and a κ light chain). Based on the three-dimensional structure of the rabbit antibody, buried residues, residues directly interacting with the CDR regions, and residues critical to the conformation of the VL and VH were backmutated, and potential risk sites for post-translational modification were mutated to obtain the final humanized molecule. As an example, the humanized light and heavy chain variable region sequences corresponding to 9E6 are shown in Table 8, where L represents the light chain, H represents the heavy chain, and the numbers following L and H represent different versions of the humanized sequence containing different backmutations.

[0190] [Table 8-1] [Table 8-2]

[0191] During the humanization process, 9E6 obtained the following LCDR3: >9E6-L1 LCDR3 [ka] >9E6-L2 LCDR3 [ka] >9E6-L3 LCDR3 [ka] >9E6-L4 LCDR3 [ka] LCDR3 general formula for 9E6-L [ka] where X9 is selected from N, S, T or Q, and X 10 is selected from V or G.

[0192] Two pairs of light and heavy chains from each humanized version of each rabbit antibody were combined and expressed and purified. The naming convention is based on the combination of the heavy and light chain variable region numbers. For example, "9E6-L1H2" is an antibody with SEQ ID NO: 9 for VH and SEQ ID NO: 13 for VL. The light and heavy chain constant regions were derived from the human IgG1 constant region (with N297A mutation, EU numbering system) and human kappa chain constant region sequences, respectively.

[0193] The sequence of an exemplary humanized molecule, 9E6-L4H2 (using the 9E6-L4 light chain and the 9E6-H2 heavy chain), is as follows, and its light and heavy chain constant regions are the sequences of a human IgG1 constant region (with the N297A mutation, EU numbering system) and a human kappa chain constant region, respectively.

[0194] >9E6-L4H2 HC: [ka] >9E6-L4H2 LC: [ka] The heavy chain constant region of the above antibody was modified to contain a human IgG1 constant region with five mutations: L234A, L235A, M252Y, S254T, and T256E (Eu numbering system). The light chain constant region and the light and heavy chain variable regions were left unchanged. The newly generated antibody, 9E6-L4H2-AAYTE, has the following complete heavy chain sequence:

[0195] >9E6-L4H2-AAYTE HC: [ka] The underlined regions above are the heavy or light chain constant regions.

[0196] An exemplary IgG Fc is as follows:

[0197] > IgG1-Fc(N297A) [ka] > IgG1-Fc(AAYTE) [ka]

[0198] Example 6. Raji cell binding experiment of humanized anti-CD40 antibodies The high expression of human CD40 on the surface of Raji cells can be used to detect the binding properties of CD40 antagonistic antibodies to cell surface human CD40.

[0199] Raji cells were plated at 1.5E5 / well, and various concentrations of CD40 antagonistic antibodies were added and incubated for 1 hour at 4°C. After washing twice with FACS buffer (PBS + 2% FBS), secondary antibodies (anti-human IgG (H+L) antibody conjugated to Alexa Flour 488) were added and incubated for 0.5 hours at 4°C. After washing twice with FACS buffer, the cell surface fluorescence intensity was detected using a flow cytometer (BD FACS Celesta).

[0200] The results are shown in Table 9, where 9E6-L4H2 and CFZ533 have similar binding ability to human CD40 on the Raji surface.

[0201] [Table 9]

[0202] Example 7. Affinity assay of anti-CD40 antibodies to human CD40 and cynomolgus monkey CD40 After affinity capture of the antibody to be measured onto an anti-human Fc chip, a series of His-tagged human or cynomolgus monkey CD40 antigens at a concentration gradient was applied to the chip surface, and the reaction signal was detected in real time using a Biacore instrument to obtain binding / dissociation curves. The buffer used in the experiment was HBS-EP + 10x buffer solution (Cat. #BR-1006-69, GE) diluted to 1x (pH 7.4) with DI Water. The data obtained from the experiment were fitted with a (1:1) binding model to obtain affinity values, as shown in Table 10.

[0203] Compared with CFZ533, 9E6-L4H2 has a similar binding constant to human CD40, and the binding affinity of 9E6-L4H2 to cynomolgus monkey CD40 is relatively strong.

[0204] [Table 10]

[0205] Example 8. Reporter gene cellular activity experiment of 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-kB-mediated SEAP genome. The activation level of the CD40 signaling pathway can be characterized by detecting the content of SEAP secreted in the supernatant using QUANTI-Blue, a SEAP substrate. This experiment was conducted to detect the inhibitory effect of CD40 antagonistic antibodies on CD40L-induced activation of HEK-Blue CD40L cells, thereby determining the level of IC. 50 The in vitro cellular activity of CD40 antagonistic antibodies was evaluated based on the size of the antibody.

[0206] 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 (DMEM medium containing 10% FBS and 100 μg / mL Normocin) and cultured overnight. After the cells adhered to the plate, 100 μL of gradient-diluted antibodies to be tested was added to each well and incubated for 1 hour at 37°C. CD40L-his (R&D, 2706-CL-025) and anti-His antibodies (R&D, MAB050-500) were added and cultured overnight. The cells were centrifuged, and 20 μL of cell supernatant was transferred to a new 96-well white plate. 180 μL of QUANTI-Blue substrate solution was added, and the mixture was incubated in the dark for 15 minutes. The absorbance at 620 nm was measured using an Envision plate reader, and the IC was calculated. 50 The values ​​were calculated to evaluate the in vitro cellular activity of CD40 antagonistic antibodies.

[0207] As shown in Table 11 and Figures 1A and 1B, 9E6-L4H2 and CFZ533 have similar inhibitory activity against the reporter gene system, but 2F12-L4H2 has superior inhibitory activity to CFZ533. 2F12-L4H2 is another anti-CD40 antibody obtained in the screening of this application.

[0208] [Table 11]

[0209] Example 9. Inhibitory activity of anti-CD40 antibodies in B cell activation experiments CD40 is highly expressed on 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 antagonistic antibodies block the binding of CD40L to CD40, thereby canceling the immune activation process of B cells.

[0210] Human PBMCs were seeded at 2E5 / well, 50 μL / well, into a 96-well cell culture plate (RPMI-1640 medium, 10% FBS, 1% penicillin-streptomycin). 50 μL of gradient-diluted antibodies were added to each well and co-incubated for 0.5 hours at 37°C, 5% CO2. CD40L-his and anti-His antibodies were added to each well and stimulated overnight. The next day, the cells were centrifuged and the supernatant removed. The cells were washed twice with FACS buffer and stained with 100 μL of 1:1000 dilution of fixable viability dye EF780 (Invitrogen, 65086514) for 15 minutes at room temperature. After washing twice, the cells were blocked with 100 μL of 1:200 dilution of human Fc blocking agent (BD, 564220) for 10 minutes at room temperature. After centrifugation, the cells were incubated for 0.5 hours at 4°C with flow antibodies (PerCP / Cyanine5.5 anti-human CD19 (Biolegend, 302230) and APC anti-human CD69 (Biolegend, 310910)) diluted 1:200. 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 cell surface fluorescence intensity was detected using a flow cytometer (BD FACS Celesta).

[0211] As shown in Table 12 and Figures 2A and 2B, 9E6-L4H2 has similar inhibitory activity against B cell activation as CFZ533. 2F12-L4H2 has stronger inhibitory activity against B cells than CFZ533. 2F12-L4H2 is another anti-CD40 antibody obtained in the screening of the present application.

[0212] [Table 12]

[0213] After addition of the antibody, the CD19+CD69+ signal fell below background levels, likely due to some inhibition of background B cell activation (other than inhibition of CD40L-induced signals).

[0214] Example 10. Inhibitory activity of anti-CD40 antibodies in DC cell activation experiments CD40 is highly expressed on dendritic cells (DCs), and when CD40L binds to CD40, it induces DC activation, upregulating the expression of multiple activation markers on the DC cell surface and promoting DCs to secrete multiple inflammatory factors, further amplifying the immune response. CD40 antagonistic antibodies block the binding of CD40L to CD40, thereby canceling the immune activation process of DC cells.

[0215] Mononuclear cells were enriched from fresh primary human peripheral blood PBMCs using the EsaySep™ Human CD14 Selection Reagent Kit (Stemcell, 19359) and 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. A gradient of dilutions of the antibody to be tested 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 also added to each well at the final concentrations. After 48 hours of culture, the activation level of DC cells was detected by flow cytometry: the supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer (PBS + 2% FBS). Then, 100 μL of Fixable viability dye EF780 (Invitrogen, 65086514) diluted 1:1000 was added and stained for 15 minutes at room temperature. After washing twice, 100 μL of human Fc blocking agent (BD, 564220) diluted 1:200 was added and blocked for 10 minutes at room temperature. After centrifugation, the cells were stained with flow antibodies (Alexa Fluor® 700 anti-human CD11c (Biolegend, 337220), Brilliant Violet 421) diluted 1:200. TM Anti-human CD80 (Biolegend, 305221) and APC anti-human CD86 (Biolegend, 305412) were added and incubated for 0.5 hours at 4°C. The supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer. They were then resuspended in 200 μL of PBS and the cell surface fluorescence intensity was detected using a flow cytometer (BD FACS Celesta).

[0216] Furthermore, the secretion levels of cytokines in the supernatants were detected after 24 hours (TNFα, Cisbio, 62HTNFAPEG) and 48 hours (IL-12 / 23 p40, Novus, VAL121) of culture, respectively.

[0217] The experimental results, as shown in Table 13 and Figures 3A to 3D, demonstrate that 9E6-L4H2 and 2F12-L4H2 have DC cell inhibitory activity similar to that of the control antibody CFZ533. 2F12-L4H2 is another anti-CD40 antibody obtained in the screening of this application.

[0218] [Table 13]

[0219] Example 11. Agonist activity of humanized anti-CD40 antibodies in B cell activation experiments CD40 belongs to the TNF superfamily of receptors, and when bound to its ligand CD40L or cross-linked by antibodies, it can mediate specific and nonspecific activation of downstream signaling pathways. Therefore, background agonistic activity of CD40 antagonistic antibodies on B cells can be detected in the absence of CD40L.

[0220] Human PBMCs were seeded at 2E5 / well, 100μL / well, into a 96-well cell culture plate (RPMI-1640 medium, 10% FBS, 1% penicillin-streptomycin). 100μL of gradient diluted antibody was added to each well and incubated overnight at 37℃, 5% CO2. The next day, the supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer and stained with 100μL of Fixable viability dye EF780 diluted 1:1000 for 15 minutes at room temperature. After washing twice, the cells were blocked with 100μL of human Fc blocking agent diluted 1:200 for 10 minutes at room temperature. After centrifugation, the cells were incubated for 0.5 hours at 4°C with flow antibodies (PerCP / Cyanine5.5 anti-human CD19 (Biolegend, 302230) and APC anti-human CD69 (Biolegend, 310910)) diluted 1:200. 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 cell surface fluorescence intensity was detected using a flow cytometer (BD FACS Celesta).

[0221] As shown in Figure 4, the CD40 agonist antibody 9E5-SELFNS (WO2020108611A1) activated B cells in a dose-dependent manner, whereas 9E6-L4H2 and 2F12-L4H2 did not show any clear B cell agonist activity even at 2.5 nM.

[0222] Example 12. Mouse T cell-dependent humoral immune response (TDAR) model Female human CD40 transgenic mice, 6-7 weeks old, were purchased from Baiaosaito Jiangsu Gene Biotechnology Co., Ltd. (Baiaosaito Jiangsu Gene Biotechnology Co., Ltd.). The breeding environment was SPF, the production license was SCXK(Su)-2016-0004, and the human CD40 transgenic mouse qualification certificate number was 320726200100167773. After arrival, the animals were acclimated for 7 days and then randomly assigned to groups. On day 0 of the experiment, one mouse from each group was bled and then injected intraperitoneally. On day 1, mice were immunized intraperitoneally with 50 μg of KLH (KLH: complete Freund's adjuvant CFA = 1:1 emulsified immunocomplex) per mouse. On day 15 of the experiment, secondary immunizations were performed intraperitoneally with 50 μg of KLH (KLH: Incomplete Freund's Adjuvant IFA = 1:1 emulsified immunocomplex) per animal. Each group received intraperitoneal injections twice weekly, and approximately 150 μL of blood was collected from the orbital venous plexus on days 7, 14, 21, and 28. The whole blood was allowed to stand at room temperature for 1-4 hours, then centrifuged at 7000 rpm for 10 minutes at 4°C to separate the serum, which was then stored at -80°C for further use. The specific experimental process is shown in Figure 5.

[0223] The specific administration schedule is shown in Table 14. Mouse serum was separated weekly and anti-KLH specific IgG levels were detected by ELISA.

[0224] [Table 14]

[0225] As shown in Figure 5B and Table 15, the results show that 1 mg / kg of the CD40 antagonistic antibody significantly inhibited the production of anti-KLH-specific IgG after two immunizations. The lower doses of 0.3 mg / kg of 9E6-L4H2 and 2F12-L4H2 had a more potent inhibitory effect on anti-KLH IgG production after two immunizations than the same dose of CFZ533. Here, 2F12-L4H2 is another anti-CD40 antibody obtained in the screening of the present application.

[0226] [Table 15]

[0227] Example 13. Mouse skin graft rejection model Male BALB / c mice, 6 weeks old, were purchased from the Laboratory Animal Management Department of the Shanghai Institute of Family Planning. The breeding environment was SPF, and the certification number was 20180006023393.

[0228] Female human CD40 transgenic mice, 6-7 weeks old, were purchased from Jiangsu Gene Biotechnology Co., Ltd. The breeding environment was SPF, and the certification number was 320726200100179778.

[0229] After arrival, the animals were housed for 7 days and then randomly assigned to groups. On day -2 of the experiment, mice were intraperitoneally injected with tacrolimus FK506 or a CD40 antagonist antibody. On day 0 of the experiment, donor Balb / C and C57BL6 / J mice were anesthetized with 4% chloral hydrate. The donor mice's tails were removed and a 1 cm circumference of tail skin was separated. The recipient mice's backs were shaved, an incision was made along the dermal layer, and an equal area of ​​skin was removed while preserving the dorsal fat and connective tissue. The donor skin was then placed over the incision, the marginal skin was sutured with adhesive, and the recipient mice were allowed to recover. The specific experimental process is shown in Figure 6A.

[0230] The mice were administered the dosage regimen shown in Table 16, and allowed to recover for 7 days. The survival status of the mouse skin was then observed daily, and a rejection score was recorded according to the rejection scoring system. The scoring system was as follows: 3: The skin was smooth with no redness; 2: Part of the skin was red, had lost its luster, and was dry; 1: Most of the skin was red, had no stripes, and was shrunken; 0: 80% of the skin was necrotic due to graft rejection.

[0231] [Table 16-1] [Table 16-2]

[0232] As shown in Figure 6B and Tables 17 to 19, the CD40 antagonistic antibodies significantly improved the scoring of mouse skin grafts and prolonged the survival of the skin grafts compared to the model group. Because rejection was not observed up to that point during the scoring process, scoring began on day 8. 9E6-L4H2 and 2F12-L4H2 exhibited superior anti-graft rejection activity to the control antibody CFZ533. 2F12-L4H2 is another anti-CD40 antibody obtained in the screening of this application.

[0233] [Table 17]

[0234] [Table 18]

[0235] [Table 19]

[0236] Furthermore, as shown in FIG. 7 and Tables 20 to 22, the combined use of 9E6-L4H2 and 2F12-L4H2 with tacrolimus further improved the efficacy against mouse skin graft rejection.

[0237] [Table 20]

[0238] [Table 21]

[0239] [Table 22]

[0240] Example 14. PK detection of humanized anti-CD40 antibody in human CD40 transgenic mice Female human CD40 transgenic mice, 6–7 weeks old, were purchased from Baiaosaito Jiangsu Gene Biotechnology Co., Ltd. (Baiaosaito Jiangsu Gene Biotechnology Co., Ltd.). The breeding environment was SPF, the production license was SCXK(Su)-2016-0004, and the human CD40 transgenic mouse certification number was 320726200100154632. After arrival, the animals were housed for 7 days and then randomly assigned to groups. On day 0 of the experiment, mice in each group were intraperitoneally injected with 10 mg / kg anti-CD40 antibody. Blood samples (100–150 μL) were collected at 15 min, 4 h, 8 h, 1, 2, 4, 7, 10, and 14 days after administration. The samples were anticoagulated with 10 μL of EDTA-K2 (0.1 M) and stored on ice. Antibody concentrations in mouse plasma at different time points were measured 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 at 50 μL per well to a 96-well plate and incubated overnight at 4°C. After washing three times with washing buffer, 50 μL of blocking solution was added to each well and incubated at 37°C for 1 hour. A standard curve of mouse plasma and the antibody to be detected was added and incubated at 37°C for 2 hours. After washing three times with washing buffer, 50 μL of anti-hlgG Fab-HRP (Sigma, Cat# A0293, 1:10000) was added at 50 μL per well and incubated at room temperature for 1 hour. After washing three times with washing buffer, 100 μL of TMB was added to each well and incubated for 5 minutes in the dark. 100 μL of 0.16 M sulfuric acid was added to each well. The OD at 450 nm was read using an Envision plate reader, and the concentration of the CD40 antagonistic antibody was calculated.

[0241] The results, as shown in Table 23 and Figure 8, show that 9E6-L4H2, 2F12-L4H2, and the control molecule CFZ533 have similar PK properties in human CD40 transgenic mice, where 2F12-L4H2 is another anti-CD40 antibody obtained in the screening of this application.

[0242] [Table 23]

[0243] Example 15. Measurement of affinity of humanized anti-CD40 antibodies to human FcRn After affinity capture of the antibody to be measured onto an anti-human Fab chip, a gradient series of human FcRn antigen (purchased from AcroBiosystem) was flowed over the chip surface, and the signal was detected in real time by the Biacore instrument when the reaction reached steady state. The buffer used in the experiment was HBS-EP + 10x buffer solution (Cat. #BR-1006-69, GE) diluted to 1x (pH 7.4) with DI Water. The data obtained from the experiment were fitted with a steady-state binding model to obtain the affinity values ​​shown in Table 24. Compared to the parent antibody, the anti-CD40 antibody with the AAYTE mutation has higher binding affinity to human FcRn.

[0244] [Table 24]

[0245] Example 16. Inhibitory activity of anti-CD40 antibodies with AAYTE mutation in Fc in a B cell activation experiment system The inhibitory activity of the anti-CD40 antibody 9E6-L4H2-AAYTE, which has the AAYTE mutation in its Fc region, in a B cell activation assay was examined using the method described in Example 10. The results, shown in Figure 9A, indicate that the Fc-mutated anti-CD40 antibody has similar B cell inhibitory activity to the parent anti-CD40 antibody.

[0246] Example 17. Inhibitory activity of anti-CD40 antibodies with AAYTE mutation in Fc in DC cell activation experiments The inhibitory activity of the anti-CD40 antibody 9E6-L4H2-AAYTE, which has the AAYTE mutation in its Fc region, in a DC cell activation experiment was examined using the method described in Example 11. The results, shown in Figures 10A and 10B, show that the Fc-mutated anti-CD40 antibody has DC cell inhibitory activity similar to that of the parent anti-CD40 antibody.

[0247] Example 18: Preparation of antibody drug conjugate ADC-2 (9E6-L4H2-Compound 2) 1. Preparation of tert-butyl(((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalaninate (Compound 1b) JPEG2026502840000046.jpg41143 Starting material 1a (5.14 g, 20.0 mmol, 1.0 eq) and L-phenylalanine tert-butyl hydrochloride (7.08 g, 20.0 mmol, 1.0 eq) were placed in a 250 mL three-neck flask and clarified with anhydrous DMF (60 mL). After cooling in an ice bath, HATU (9.12 g, 24.0 mmol, 1.2 eq) and DIEA (7.74 g, 60.0 mmol, 3.0 eq) were added and the mixture was allowed to react for 2 h in the ice bath. Water was added to the reaction mixture, which was then extracted with ethyl acetate. The organic phase was washed with saturated brine, dried, and concentrated under reduced pressure to give product 1b (11.5 g, 100% yield), a white solid that could be used directly in the next step. MS (ESI): m / z 580.3 [M+Na] + . 1 H NMR (400 MHz, DMSO-d6) δ 8.21-8.16(m, 1H), 8.12-8.05 (m, 1H), 7.91-7.87 (m, 2H), 7.72-7.68 (m, 2H), 7.64-7.59 (m, 1H), 7.44-7.38 (m, 2H), 7.36-7.25 (m, 4H), 7.23-7.18 (m, 3H), 4.41-4.18 (m, 4H), 3.76-3.72 (m, 2H), 3.68-3.61 (m, 2H), 2.98-2.89 (m, 2H), 2.69 (s, 2H), 1.30 (s, 9H).

[0248] 2. Preparation of tert-butyl glycylglycyl-L-phenylalanine (Compound 1c) JPEG2026502840000047.jpg29146 The starting material 1b (5.57 g, 10.0 mmol, 1.0 eq) was placed in a 250 mL three-neck flask, clarified with anhydrous DCM (60 mL), and cooled in an ice bath. Piperidine (8.5 g, 100.0 mmol, 10.0 eq) was slowly added, and after the addition was complete, the reaction was allowed to proceed with stirring at room temperature for approximately 2 hours. The reaction mixture was directly concentrated to dryness, and the crude product was purified by column chromatography to obtain product 1c (2.70 g, 81% yield) as a pale yellow slurry. MS (ESI): m / z 358.3 [M+H] + .

[0249] 3. Preparation of (1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentoxy-4-tetrapolyethylene glycol-19-oxy)glycylglycyl-L-phenylalanine tert-butyl ester (Compound 1d) JPEG2026502840000048.jpg45150 The starting materials, 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentaoxy-4-tetrapolyethylene glycol-19-oleic acid (1.0 g, 2.05 mmol, 1.0 eq, Tongli Biochemical, lot number P110114) and 1c (0.69 g, 2.05 mmol, 1.0 eq) were added to a 100 mL three-neck flask, clarified with anhydrous DMF (25 mL), and cooled in an ice bath. HATU (1.01 g, 2.67 mmol, 1.3 eq) and DIEA (529 mg, 4.10 mmol, 2.0 eq) were added and reacted for 1 h in the ice bath. The reaction mixture was added with water, extracted with ethyl acetate, washed with saturated brine, dried, and concentrated under reduced pressure to give 1d (1.60 g, 97% yield) as a pale yellow oil. MS (ESI): m / z 805.3 [M+H] + .

[0250] 4. Preparation of (1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentaoxy-4-tetrapolyethyleneglycol-19-oxy)glycylglycyl-L-phenylalanine (Compound 1e) JPEG2026502840000049.jpg34150 Compound 1d (1.60 g, 1.99 mmol, 1.0 eq) was placed in a 100 mL single-neck flask and dissolved in anhydrous dichloromethane (16 mL). Trifluoroacetic acid (8 mL) was added at room temperature, and the mixture was stirred for 2 hours. The reaction mixture was directly concentrated to dryness to obtain a yellow slurry, which was dissolved in ethyl acetate, washed with saturated brine, dried, and concentrated under reduced pressure to obtain a yellow oily product, 1e (1.23 g, 83% yield). MS (ESI): m / z 749.3 [M+H] + .

[0251] 5. Preparation of (9H-fluoren-9-yl)methyl (2-((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (Compound 1f) JPEG2026502840000050.jpg47150 The raw materials budesonide (1.29 g, 3.0 mmol, 1.0 eq) and methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (1.16 g, 3.15 mmol, 1.05 eq, prepared according to the method of reference "Tetrahedron, 2018, 74(15), 1951-1956") and p-toluenesulfonic acid pyridine salt (75 mg, 0.30 mmol, 0.1 eq) were added to a 100 mL three-neck flask. Anhydrous tetrahydrofuran (20 mL) was added and the mixture was refluxed for 4 hours. The reaction mixture was directly concentrated to dryness, and the crude product was purified by column chromatography to obtain product 1f (0.54 g, 24% yield) as a white solid. MS (ESI): m / z 739.4 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 8.76-8.69 (m, 1H), 7.91-7.84 (m, 2H), 7.73-7.67 (m, 2H), 7.63-7.57 (m, 1H), 7.43-7.58 (m, 2H), 7.35-7.23 (m, 3H), 6.13 (d, J = 7.5 Hz, 1H), 5.91 (s, 2H), 5.15-4.97 (m, 1H), 4.73-4.45 (m, 5H), 4.30-4.09 (m, 5H), 3.65-3.59 (m, 2H), 2.29-2.21 (m, 1H), 2.11-1.87 (m, 2H), 1.74-1.21 (m, 11H), 1.09-0.77 (m, 8H).

[0252] 6. Preparation of 2-amino-N-((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)methyl)acetamide (Compound 1g) JPEG2026502840000051.jpg54148 A 25 mL single-neck flask was charged with starting material 1f (540 mg, 0.73 mmol, 1.0 eq) and dissolved in anhydrous dichloromethane (10 mL). After cooling in an ice bath, DBU (167 mg, 1.10 mmol, 1.5 eq) was added and the mixture was stirred for 30 minutes. Water was added and the mixture was separated. The aqueous phase was extracted twice with dichloromethane, washed with saturated brine, and concentrated under reduced pressure to give 1 g of a pale yellow semi-solid (450 mg of crude fluorene-containing product, 100% yield). The crude product could be used directly in the next step. MS (ESI): m / z 517.4 [M+H] + .

[0253] 7. (9H-Fluoren-9-yl)methyl ((10S)~10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodeca Preparation of hydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30-pentaoxa-5,8,11,14,17-pentaazadotriacontan-32-yl)carbamate (compound 1h) JPEG2026502840000052.jpg47148 A 100 mL three-neck flask was charged with starting material 1g (0.45 g crude product, equivalent to 0.73 mmol, 1.0 eq) and 1e (0.55 g, 0.73 mmol, 1.0 eq), and anhydrous DMF (9 mL) was added for clarification. After cooling in an ice bath, HATU (361 mg, 0.95 mmol, 1.3 eq) and DIEA (189 mg, 1.46 mmol, 2.0 eq) were added and the mixture was stirred for 1 h. Water was added to the reaction mixture, which was then extracted twice with dichloromethane. The combined organic phases were washed once with saturated brine, dried, and concentrated under reduced pressure to give the crude product as a yellow oily solid. The solid crude product was purified by slurrying in a mixture of petroleum ether and ethyl acetate to give product 1h (710 mg, 78% yield) as a pale yellow solid. MS (ESI): m / z 1269.7 [M+Na] + .

[0254] 8. Preparation of 1-amino-N-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl-3,6,9,12-tetraoxapentadecan-15-amide (Compound 1i) JPEG2026502840000053.jpg40140 A 25 mL single-neck flask was charged with starting material 1h (350 mg, 0.28 mmol, 1.0 eq) and clarified with anhydrous dichloromethane (10 mL). After cooling in an ice bath, DBU (64 mg, 0.42 mmol, 1.5 eq) was added and the mixture was stirred for 1 hour. The reaction mixture was directly concentrated to dryness to obtain the crude oil. This crude oil was further purified by column chromatography to obtain product 1i (289 mg, 89% yield) as a pale yellow foamy solid. MS (ESI): m / z 1025.6 [M+H]+ .

[0255] 9. Preparation of N-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxy-3-oxo-5,8,11,14-tetraazahexadecan-16-yl)-1-(2-bromoacetamido)-3,6,9,12-tetraoxapentadecan-15-amide (Compound 2) JPEG2026502840000054.jpg42146 Bromoacetic acid solid (35.2 mg, 0.25 mmol, 2.0 eq) and EEDQ (63 mg, 0.25 mmol, 2.0 eq) were placed in a 25 mL Schlenk tube, and anhydrous DMF (5 mL) was added to clarify the mixture. The mixture was allowed to react at room temperature for 1 hour with stirring. Starting material 1i (130 mg, 0.127 mmol, 1.0 eq) was dissolved in DMF (2 mL), and this solution was added dropwise to the activated ester solution prepared above. After the addition was complete, the mixture was allowed to react at room temperature for an additional 2 hours. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated under reduced pressure to obtain a crude oil. The crude oil was further purified by column chromatography to obtain compound 2 (39 mg, 27% yield) as a pale yellow solid. MS (ESI): m / z 1145.5 / 1147.6 [M+1] + . 1H NMR (400 MHz, CDCl3) δ 8.62-8.57 (m, 1H), 8.34-8.28 (m, 2H), 8.19-8.11(m, 2H), 8.03-7.99(m, 1H), 7.30-7.17(m, 6H), 6.15 (d, J = 9.6 Hz, 1H), 5.92 (s, 2H), 5.18-5.03 (m, 1H), 4.72-4.47 (m, 5H), 4.29-4.15 (m, 2H), 3.78-3.42 (m, 27H), 3.27-3.21 (m, 2H), 3.06-2.84 (m, 2H), 2.41-2.28 (m, 3H), 2.06-1.73 (m, 2H), 1.60-1.24 (m, 10H), 1.01-0.82 (m, 8H).

[0256] 10. Preparation of ADC-2 (9E6-L4H2-Compound 2) [ka] At 37°C, an aqueous solution of antibody 9E6-L4H2 in buffer A (0.05 M buffer solution, pH 6.3, 15.0 mg / mL, 1.5 mL, 0.10 mmol) was added to the prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (2.5 mM, 170.3 μL, 0.42 mmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath. 210 μL of 1.0 M Tris buffer, pH 8.30, was added.

[0257] Compound 2 (1.16 mg, 1.0 mmol) was dissolved in 75 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: buffer A) and concentrated using an ultrafiltration tube. The title product ADC-2 (9E6-L4H2-compound 2) was obtained in buffer A buffer (5.14 mg / mL, 2.48 mL) with a yield of 85% and stored frozen at 4 °C. The theoretical DAR was 4.0, and the measured DAR was 3.8.

[0258] Example 19. Affinity assay of anti-CD40-ADCs for human CD40 and cynomolgus monkey CD40 After affinity capture of the target antibody and anti-CD40 ADC onto an anti-human Fc chip, a gradient series of His-tagged human or cynomolgus monkey CD40 antigen was applied to the chip surface. The reaction signal was detected in real time using a Biacore instrument to obtain binding / dissociation curves. The buffer used in the experiment was HBS-EP + 10x buffer solution (Cat. #BR-1006-69, GE) diluted to 1x (pH 7.4) with DI Water. The data obtained from the experiment were fitted with a (1:1) binding model to obtain affinity values, as shown in Table 25.

[0259] [Table 25]

[0260] Example 20. Inhibitory activity of anti-CD40-ADC antibodies in B cell activation experiments CD40 is highly expressed on 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 antagonistic antibodies block the binding of CD40L to CD40, thereby canceling the immune activation process of B cells.

[0261] Human PBMCs were seeded at 2E5 / well, 50 μL / well, into a 96-well cell culture plate (RPMI-1640 medium, 10% FBS, 1% penicillin-streptomycin). 50 μL of gradient-diluted ready-to-measure antibodies or ADCs were added to each well and co-incubated for 0.5 hours at 37°C and 5% CO2. 50 μL of pre-incubated CD40L-his and anti-His antibodies were added to each well for overnight stimulation. The next day, the supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer and stained with 100 μL of 1:1000 dilution of fixable viability dye EF780 (Invitrogen, 65086514) for 15 minutes at room temperature. After washing twice, the cells were blocked with 100 μL of 1:200 dilution of human Fc blocker (BD, 564220) for 10 minutes at room temperature. After centrifugation, 100 μL of flow antibodies (PerCP / Cyanine5.5 anti-human CD19 (Biolegend, 302230, diluted 1:100) and APC anti-human CD69 (Biolegend, 310910, diluted 1:200)) were added and incubated at 4°C for 0.5 hours. After centrifugation, the supernatant was removed and the cells were washed twice with FACS buffer. They were then resuspended in 200 μL of FACS buffer and the cell surface fluorescence intensity was detected using a flow cytometer (BD FACS Celesta).

[0262] The results, as shown in Table 26, showed that ADC-1 and ADC-2 had similar B cell inhibitory activity to monoclonal anti-9E6-L4H2.

[0263] [Table 26]

[0264] After addition of antibody or ADC, the CD19+CD69+ signal fell below background levels, likely due to some inhibition of background B cell activation (other than inhibition of CD40L-induced signals).

[0265] Example 21. Mouse skin graft rejection model Male BALB / c mice, 6 weeks old, were purchased from the Laboratory Animal Management Department of the Shanghai Institute of Family Planning. The breeding environment was SPF, the production license was SCXK(Hu)2018-0006, and the BALB / c mouse certification number was 20180006023393.

[0266] Female human CD40 transgenic mice, 6–7 weeks old, were purchased from Jiangsu Gene Biotechnology Co., Ltd. The breeding environment was SPF, production license number was SCXK(Su)-2016-0004, and the human CD40 transgenic mouse certification number was 320726200100179778.

[0267] After arrival, the animals were housed for 7 days and randomly assigned to groups. On day -2 of the experiment, mice were intraperitoneally injected with CD40 antagonistic antibodies, anti-CD40 ADCs, or glucocorticoids. On day 0 of the experiment, donor Balb / C and C57BL6 / J mice were anesthetized with 4% chloral hydrate. The donor mice's tails were removed and a 1 cm circumference of tail skin was isolated. The recipient mice's backs were shaved, an incision was made along the dermal layer, and an equal area of ​​skin was removed while preserving the dorsal fat and connective tissue. The donor skin was then placed on the incision, the marginal skin was sutured with adhesive, and the recipient mice were allowed to recover. The specific experimental process is shown in Figure 13A.

[0268] The mice were administered the dosage regimen shown in Table 27, and allowed to recover for 7 days. The survival status of the mouse skin was then observed daily and a rejection score was recorded according to the rejection scoring system. The scoring system was as follows: 3: The skin was smooth with no redness; 2: Part of the skin was red, had lost its luster, and was dry; 1: Most of the skin was red, had no stripes, and was shrunken; 0: 80% of the skin was necrotic due to graft rejection.

[0269] [Table 27]

[0270] The results, as shown in Tables 28-29, show that the anti-transplant rejection activity of ADC-2 was significantly superior to that of the CD40 antagonist monoclonal antibody 9E6-L4H2, the monoclonal antibody in combination with glucocorticoids, and slightly superior to that of high-dose dexamethasone.

[0271] [Table 28]

[0272] [Table 29]

[0273] Example 22. pH and buffer screening of anti-CD40 antibody formulations Twelve combinations of 20 mM acetate, 20 mM citrate, 20 mM histidine, and 20 mM phosphate buffers, as well as pH values ​​of 4.5, 5.0, 5.5, 6.0, 6.5, 6.6, 7.0, and 7.4, were selected to prepare a total of 12 20.0 mg / mL formulations of anti-CD40 antibody (antibody number 9E6(L4H2); heavy and light chains are set forth in SEQ ID NOs: 20 and 21, respectively). The melting temperature (Tm) and aggregation temperature (Tagg) of the antibody in these formulations were measured. The SEC purity, NRCE purity, and icIEF purity of the formulations were also examined to assess the sample stability of the 12 formulations after storage at 2-8°C for 2 weeks, at 25°C for 1 and 2 weeks, at 40°C for 1 and 2 weeks, and after five freeze-thaw cycles (-35°C / room temperature). Prescriptions 1) to 12) are as follows: 1) 20 mM acetic acid-sodium acetate, pH 4.5 2) 20 mM acetic acid-sodium acetate, pH 5.0 3) 20 mM acetic acid-sodium acetate, pH 5.5 4) 20 mM citric acid-sodium citrate, pH 5.0 5) 20 mM citric acid-sodium citrate, pH 5.5 6) 20 mM citric acid-sodium citrate, pH 6.0 7) 20 mM histidine-histidine hydrochloride, pH 5.5 8) 20 mM histidine-histidine hydrochloride, pH 6.0 9) 20 mM histidine-histidine hydrochloride, pH 6.5 10) 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 6.6 11) 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 7.0 12) 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 7.4 During the course of the study, the anti-CD40 antibody showed relatively low solubility in the buffer systems of Formulation 4 and Formulation 5, so the stability of these two buffer systems was not considered.

[0274] [Table 30]

[0275] [Table 31]

[0276] [Table 32]

[0277] [Table 33]

[0278] The results showed that the anti-CD40 antibody had relatively high Tm and Tag values ​​in the histidine salt buffer systems (Formulations 7 to 9), indicating relatively high conformational and colloidal stability of the antibody in the histidine buffer system. After two weeks of high-temperature (40°C) testing, the purity of the anti-CD40 antibody in the histidine pH 6.0 and pH 6.5 systems was higher than in the other systems, as measured by SEC-HPLC, NRCE, and icIEF. The stability of the anti-CD40 antibody in the histidine pH 6.0 and pH 6.5 systems was also better. Therefore, the histidine systems with a pH range of 6.0 to 6.6 were selected for further studies.

[0279] Example 23. Screening of anti-CD40 antibody concentration, type and concentration of additives Six formulations were prepared using two buffer systems (20 mM histidine-histidine hydrochloride, pH 6.0 and pH 6.6), 8% (w / v) sucrose, 5% (w / v) sucrose + 50 mM arginine hydrochloride as stabilizers, and 0.02% (w / v) polysorbate 80 as surfactant. The anti-CD40 antibody concentrations were 50 mg / mL and 80 mg / mL. The stability of the six formulations was evaluated by analyzing the sample appearance, pH, SEC-HPLC, NRCE, and iCIEF purity. The formulations were then stored at high temperature (40°C) for 2 and 4 weeks, exposed to light (5000 ± 500 lx, 25°C) for 5 and 10 days, shaken at 300 rpm / room temperature for 2 days, and repeatedly frozen and thawed at -35°C and room temperature for 3 and 5 times.

[0280] 13) 20 mM histidine-histidine hydrochloride, pH 6.0, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 50 mg / mL anti-CD40 antibody 14) 20 mM histidine-histidine hydrochloride, pH 6.0, 5% (w / v) sucrose, 50 mM arginine hydrochloride, 0.02% (w / v) polysorbate 80, 50 mg / mL anti-CD40 antibody 15) 20 mM histidine-histidine hydrochloride, pH 6.0, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 80 mg / mL anti-CD40 antibody 16) 20 mM histidine-histidine hydrochloride, pH 6.6, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 50 mg / mL anti-CD40 antibody 17) 20 mM histidine-histidine hydrochloride, pH 6.6, 5% (w / v) sucrose, 50 mM arginine hydrochloride, 0.02% (w / v) polysorbate 80, 50 mg / mL anti-CD40 antibody 18) 20 mM histidine-histidine hydrochloride, pH 6.6, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 80 mg / mL anti-CD40 antibody

[0281] [Table 34]

[0282] [Table 35]

[0283] [Table 36]

[0284] [Table 37]

[0285] The results showed that the opalescence of the formulation containing arginine hydrochloride was relatively clear in appearance.In terms of purity, the SEC purity of all formulations decreased slightly only under high temperature and light irradiation conditions, and the increase in the polymer ratio compared to TO did not exceed 2%, and the overall difference was relatively small.Under high temperature and light irradiation conditions, the NRCE fragments increased slightly, and the difference between the groups was not significant.Under high temperature and light irradiation conditions, the purity of iCIEF changed.Under high temperature conditions, the acid peak content of formulation 1 was slightly higher than that of formulation 2, and the acid peak content of formulation 4 was slightly higher than that of formulation 5, but the difference was not significant. Moreover, different protein concentrations did not have an obvious effect on purity, and anti-CD40 antibodies at 50 mg / mL to 80 mg / mL showed relatively high stability in formulations containing 10 mM histidine-histidine hydrochloride, a pH 6.0 to pH 6.6 buffer system, 8% (w / v) sucrose as a stabilizer, and 0.02% (w / v) polysorbate 80 as a surfactant.

[0286] Example 24. Confirmation of stabilizer concentration in anti-CD40 antibody formulations An antibody formulation containing 7.5% (w / v) sucrose, 20 mM histidine-histidine hydrochloride, and 0.02% (w / v) polysorbate 80 was prepared with an anti-CD40 antibody content of 80 mg / mL and a pH of 6.3. Stability studies were conducted under the conditions investigated, including a high temperature of 40°C and repeated freezing and thawing (-35°C / room temperature) five times.

[0287] 19) 20 mM histidine-histidine hydrochloride, pH 6.3, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 80 mg / mL anti-CD40 antibody [Table 38]

[0288] [Table 39]

[0289] NOTE: NT indicates not detected, and T0 indicates 0 time, because the repeated freeze-thaw temperature is relatively low and the time is relatively short, which has a relatively small effect on the purity of NRCE and iCIEF.

[0290] The results showed that the appearance of the formulation did not change significantly after five freeze-thaw cycles, and the clarity changed from clear to slightly opalescent at high temperature. There was no significant change in pH under any condition. Regarding purity, there was no significant change in SEC-HPLC purity after five freeze-thaw cycles. At high temperature, the SEC-HPLC polymer slightly increased, and the NRCE fragments increased by about 2.8% compared to TO. The increase in the iCIEF acidic peak was within an acceptable range, indicating that the formulation had good freeze-thaw and thermal stability.

[0291] Example 25. Confirmation of pH of anti-CD40 antibody formulation A 20 mM histidine-histidine hydrochloride, pH 6.1 buffer system, 7.5% (w / v) sucrose, and 0.02% (w / v) polysorbate 80 were selected to prepare an antibody formulation with an anti-CD40 antibody content of 80 mg / mL, and stability studies were conducted under the conditions considered, including a high temperature of 40°C and repeated freezing and thawing (-35°C / room temperature) five times.

[0292] 20) 20 mM histidine-histidine hydrochloride, pH 6.1, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 80 mg / mL anti-CD40 antibody [Table 40]

[0293] [Table 41]

[0294] NOTE: NT indicates not detected and T0 indicates 0 time.

[0295] The results showed that the formulation showed no significant changes in pH or appearance after five freeze-thaw cycles and one month at high temperature. After five freeze-thaw cycles, there was no significant change in purity. After four weeks at high temperature, the SEC-HPLC polymer showed a slight increase compared to TO, with the NRCE purity at 91% or higher and the iCIEF main peak still at 40% or higher, indicating that the formulation at this pH had good freeze-thaw and thermal stability.

[0296] Example 26. Screening of anti-CD40 antibody concentration A 20 mM histidine-histidine hydrochloride, pH 6.1 buffer system, 7.5% (w / v) sucrose, and 0.02% (w / v) polysorbate 80 were selected, and anti-CD40 antibody formulations with different concentrations were prepared and viscosity was detected.

[0297] [Table 42] The results showed that the higher the protein concentration, the higher the formulation viscosity, and with sucrose as a stabilizer, the viscosity was close to 20 cp when the protein concentration reached 131.2 mg / mL.

[0298] Example 27. Screening of anti-CD40 antibody concentration A 20 mM histidine-histidine hydrochloride, pH 6.1 buffer system, 2.5% (w / v) proline, and 0.02% (w / v) polysorbate 80 were selected to prepare anti-CD40 antibody formulations with different concentrations and perform viscosity detection.

[0299] [Table 43]

[0300] The results showed that the higher the protein concentration, the higher the formulation viscosity, and with proline as a stabilizer, the viscosity still did not exceed 20 cp when the protein concentration reached 187 mg / mL.

[0301] Example 28. Screening of additive types Antibody formulations containing 80, 120, and 150 mg / mL of anti-CD40 antibody were prepared using a 20 mM histidine-histidine hydrochloride, pH 6.1 buffer system with 0.02% (w / v) polysorbate 80 and stabilized with 7.5% (w / v) sucrose or 2.5% (w / v) proline. The formulations were then stored at high temperature (40°C) for 2 and 4 weeks, exposed to light (4500±500 lx / 25°C) for 5 days, shaken at 300 rpm / 25°C for 2 days, freeze-thawed (-35°C / room temperature) five times, stored at 2-8°C for 4 weeks, and frozen at -35°C for 4 weeks.

[0302] 26) 20 mM histidine-histidine hydrochloride, pH 6.1, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 80 mg / mL anti-CD40 antibody 27) 20 mM histidine-histidine hydrochloride, pH 6.1, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 120 mg / mL anti-CD40 antibody 28) 20 mM histidine-histidine hydrochloride, pH 6.1, 2.5% (w / v) proline, 0.02% (w / v) polysorbate 80, 150 mg / mL anti-CD40 antibody

[0303] [Table 44]

[0304] [Table 45]

[0305] [Table 46]

[0306] [Table 47]

[0307] [Table 48]

[0308] [Table 49]

[0309] [Table 50]

[0310] The results showed that all formulations had a pale yellow appearance, slight opalescence, and no visible protein particles under all conditions, and binding activity was within the range of 60% to 140%. There were no significant differences between the groups in insoluble particles, SEC purity, NRCE, and RCE purity. For iCIEF purity, formulation 28 was the best under high-temperature conditions, followed by formulations 27 and 26, with no significant differences among the three formulations. The above three formulations had good appearance and demonstrated good stability under high-temperature, light exposure, shaking, freeze-thaw, and frozen storage conditions.

[0311] Example 29. Alternative formulations of anti-CD40 antibodies The present disclosure provides pharmaceutical formulations of anti-CD40 antibodies containing "50 to 150 mg / mL anti-CD40 antibody, 7.5% to 8% (w / v) sucrose or 2.5% (w / v) proline, 0.02% (w / v) polysorbate 80, and 20 mM histidine-histidine hydrochloride, pH 5.6 to 6.6," including, but not limited to, the following: (1) 80 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.3. (2) 50 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.3. (3) 80 mg / mL anti-CD40 antibody, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.3. (4) 50 mg / mL anti-CD40 antibody, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.3. (5) 80 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.0. (6) 80 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.6. (7) 50 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.0. (8) 50 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.6. (9) 80 mg / mL anti-CD40 antibody, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.0. (10) 80 mg / mL anti-CD40 antibody, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.6. (11) 50 mg / mL anti-CD40 antibody, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.0. (12) 50 mg / mL anti-CD40 antibody, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.6. (13) 80 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.1. (14) 50 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.1. (15) 80 mg / mL anti-CD40 antibody, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.1. (16) 50 mg / mL anti-CD40 antibody, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.1. (17) 120 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.1. (18) 120 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.2. (19) 120 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-HCl histidine hydrochloride pH 6.3. (20) 150 mg / mL anti-CD40 antibody, 2.5% (w / v) proline, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.1. (21) 150 mg / mL anti-CD40 antibody, 2.5% (w / v) proline, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.2. (22) 150 mg / mL anti-CD40 antibody, 2.5% (w / v) proline, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.3.

[0312] The experimental results showed that all of the anti-CD40 antibody formulations prepared using the above formulations had good stability and could be used to prepare anti-CD40 antibody drugs.

[0313] Example 30. Formulation considerations for anti-CD40-ADC formulations A 50 mg / mL CD40-ADC formulation (ADC-2 in Example 18 of the present disclosure, the same applies to the following Examples) was prepared using a 20 mM histidine-histidine hydrochloride, pH 6.1 buffer system, 7.5% (w / v) sucrose, and 0.02% (w / v) polysorbate 80. The stability of the liquid formulation was examined at different temperatures (-35°C, 2-8°C, 25°C, 40°C), under light irradiation (5000±500 lx, 25°C), shaking (300 rpm, 25°C), and repeated freeze-thawing (-35°C / room temperature). The formulation was then lyophilized, and the stability of the lyophilized powder was examined at 50°C.

[0314] 29) 20 mM histidine-histidine hydrochloride, pH 6.1 buffer system, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 50 mg / mL anti-CD40 ADC [Table 51]

[0315] NOTE: Lyo T0 indicates the sample at 0 hours after lyophilization.

[0316] [Table 52] [Table 53]

[0317] NOTE: Lyo T0 indicates the sample at 0 hours after lyophilization.

[0318] [Table 54]

[0319] NOTE: Lyo T0 indicates the sample at 0 hours after lyophilization.

[0320] [Table 55]

[0321] The results showed that the solution and lyophilized powder formulations of the anti-CD40-ADC formulations had good stability under different stability considerations.

[0322] Example 31. Alternative formulations of anti-CD40 ADCs The present disclosure provides anti-CD40-ADC formulations containing "30-50 mg / mL anti-CD40-ADC, 7.5%-9% (w / v) sucrose or 7.5% (w / v) trehalose, 0.02% (w / v) polysorbate 80, and 20 mM histidine-histidine hydrochloride, pH 6.0-6.6," including, but not limited to, the following: (1) 50 mg / mL anti-CD40 ADC, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.3. (2) 50 mg / mL anti-CD40 ADC, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.0. (3) 50 mg / mL anti-CD40 ADC, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.6. (4) 30 mg / mL anti-CD40 ADC, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.3. (5) 50 mg / mL anti-CD40 ADC, 9% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.3.

[0323] (6) 50 mg / mL anti-CD40 ADC, 7.5% (w / v) trehalose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.3.

[0324] The experimental results showed that all of the anti-CD40 ADC formulations prepared using the above formulations had good stability and could be used to prepare anti-CD40-ADC drugs.

[0325] While specific embodiments of the present disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples and that various changes and modifications can be made to these embodiments without departing from the principles and spirit of the present disclosure.

Claims

1. A pharmaceutical composition comprising a CD40 binding molecule and a buffer, wherein the buffer is selected from acetate buffers, citrate buffers, histidine buffers, and phosphate buffers, preferably a histidine buffer, more preferably a histidine-hydrochloride buffer or a histidine-acetate buffer, and most preferably a histidine-histidine hydrochloride buffer. Pharmaceutical compositions.

2. the CD40 binding molecule is an anti-CD40 antibody or an antigen-binding fragment thereof; The pharmaceutical composition of claim 1.

3. The anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises HCDR1, HCDR2, and HCDR3 in the VH shown in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2, and LCDR3 in the VL shown in SEQ ID NO: 2, wherein the CDRs are defined according to the Kabat, I MGT, Chothia, Ab M or Contact numbering system, preferably according to the Kabat numbering system. The pharmaceutical composition of claim 2.

4. The anti-CD40 antibody or antigen-binding fragment thereof is heavy chain HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 3, 4, 5, respectively, and light chain LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 6, 7, 19, respectively; Preferably, the anti-CD40 antibody or antigen-binding fragment thereof is The antibody comprises heavy chain HCDR1, HCDR2, and HCDR3 having the sequences shown in SEQ ID NOs: 3, 4, and 5, respectively; light chain LCDR1 having the sequence shown in SEQ ID NO: 6; light chain LCDR2 having the sequence shown in SEQ ID NO: 7; and light chain LCDR3 having the sequence shown in any one of SEQ ID NOs: 15 to 18. The pharmaceutical composition according to claim 2 or 3.

5. The anti-CD40 antibody or antigen-binding fragment thereof is a recombinant antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. The pharmaceutical composition according to any one of claims 2 to 4.

6. the heavy chain framework regions of the humanized antibody or antigen-binding fragment thereof are derived from IGHV2-26*01, IGHV4-30-4*02, IGHV4-4*08, or IGHJ1*01, and / or the light chain framework regions are derived from IGkV1-13*02, IGkV1-9*01, IGkV1-6*01, or IGKJ4*01; Preferably, FR1 to FR3 of the heavy chain framework regions are derived from IGHV2-26*01, IGHV4-30-4*02, or IGHV4-4*08, FR4 of the heavy chain framework regions is derived from IGHJ1*01, FR1 to FR3 of the light chain framework regions are derived from IGkV1-13*02, IGkV1-9*01, or IGkV1-6*01, and FR4 of the light chain framework regions is derived from IGKJ4*01. The pharmaceutical composition according to claim 5.

7. The anti-CD40 antibody or antigen-binding fragment thereof is A-1) VH comprising an amino acid sequence shown in SEQ ID NO: 13 or having at least 90% identity thereto, and VL comprising an amino acid sequence shown in any one of SEQ ID NOs: 9 to 12 or having at least 90% identity thereto; A-2) VH comprising an amino acid sequence shown in SEQ ID NO: 14 or having at least 90% identity thereto, and VL comprising an amino acid sequence shown in any one of SEQ ID NOs: 9 to 12 or having at least 90% identity thereto; A-3) VH comprising an amino acid sequence shown in SEQ ID NO: 1 or having at least 90% identity thereto, VL comprising an amino acid sequence shown in SEQ ID NO: 2 or having at least 90% identity thereto; Including, The pharmaceutical composition according to any one of claims 2 to 6.

8. The anti-CD40 antibody or antigen-binding fragment thereof further comprises 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 IgG1 Fc region comprising an N297A mutation, or an IgG1 Fc region comprising any one or more of the following mutations: L234A, L235A, M252Y, S254T, and T256E. The pharmaceutical composition according to any one of claims 2 to 7.

9. the antigen-binding fragment is an scFv, Fv, Fab, or Fab′ fragment; The pharmaceutical composition according to any one of claims 2 to 8.

10. The anti-CD40 antibody or antigen-binding fragment thereof is a full-length heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 20 or 22 or having at least 90% identity thereto, and a full-length light chain comprising the amino acid sequence set forth in SEQ ID NO: 21 or having at least 90% identity thereto; The pharmaceutical composition according to any one of claims 2 to 9.

11. The CD40 binding molecule is an antibody-drug conjugate comprising the anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10. The pharmaceutical composition of claim 1.

12. The antibody-drug conjugate has the structure shown in formula (I): Ab-(L-D) ｋ (I)、 wherein Ab is the anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 8, D is selected from a glucocorticoid receptor agonist, a glucocorticoid, a calcineurin inhibitor, or a target of rapamycin (mTOR) kinase inhibitor, L is a linker covalently linking Ab to D, and k is an integer or decimal number selected from 1 to 20. The pharmaceutical composition of claim 11.

13. D in the antibody-drug conjugate is 【Chemistry 1】 That is, The pharmaceutical composition of claim 12.

14. The antibody-drug conjugate is shown in the structure below: 【Chemistry 2】 wherein k is an integer or decimal number selected from 1 to 10, p1 is selected from 2, 4, 6, or 8, and p3 and p4 are each independently selected from 0, 1, or 2; Preferably, the antibody-drug conjugate is shown in the following structure: 【Transformation 3】 where k is an integer or decimal number selected from 1 to 10, More preferably, the antibody-drug conjugate is shown in the structure below: 【Chemistry 4】 wherein 9E6-L4H2 is an anti-CD40 antibody comprising a heavy chain and a light chain comprising the amino acid sequences shown in SEQ ID NOs: 20 and 21, k is an integer or decimal number selected from 1 to 10; The pharmaceutical composition according to any one of claims 11 to 13.

15. the pH of the composition is 4.5 to 7.5, preferably 5 to 7.2, more preferably 5.5 to 7, and most preferably 5.6 to 6.6; The pharmaceutical composition according to any one of claims 1 to 14.

16. The concentration of the buffer is 1 to 50 mM, preferably 5 to 40 mM, more preferably 10 to 30 mM, and most preferably 15 to 25 mM. The pharmaceutical composition according to any one of claims 1 to 15.

17. The composition further comprises one or more stabilizers selected from an amino acid or a medicinal salt thereof and a sugar, wherein preferably the amino acid is one or more selected from arginine and proline or a medicinal salt thereof, and the sugar is one or more selected from sucrose and trehalose, more preferably the stabilizer is one or more selected from sucrose, trehalose, arginine or a medicinal salt thereof, and proline or a medicinal salt thereof, and most preferably the stabilizer is sucrose, trehalose, or proline or a medicinal salt thereof. The pharmaceutical composition according to any one of claims 1 to 16.

18. the concentration of said sucrose or trehalose is 0.1% to 30% w / v, preferably 0.5% to 20% w / v, more preferably 1% to 15% w / v, and most preferably 5% to 10% w / v, or the concentration of said proline or a medicament salt thereof is 0.01% to 20% w / v, preferably 0.1% to 15% w / v, more preferably 0.5% to 10% w / v, and most preferably 1% to 5% w / v; 18. The pharmaceutical composition of claim 17.

19. further comprising a surfactant, said surfactant being preferably a polysorbate, more preferably one or more of polysorbate 20 and polysorbate 80, most preferably polysorbate 80; A pharmaceutical composition according to any one of claims 1 to 18.

20. the concentration of the surfactant is 0.001% to 1% w / v, preferably 0.005% to 1% w / v, more preferably 0.008% to 0.2% w / v, and most preferably 0.01% to 0.1% w / v; 20. The pharmaceutical composition of claim 19.

21. the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 0.1 to 500 mg / mL, preferably 1 to 300 mg / mL, more preferably 10 to 250 mg / mL, and most preferably 20 to 200 mg / mL; The pharmaceutical composition according to any one of claims 2 to 10 and 15 to 20.

22. the concentration of the antibody-drug conjugate is 0.1 to 300 mg / mL, preferably 1 to 150 mg / mL, more preferably 5 to 120 mg / mL, and most preferably 10 to 100 mg / mL; The pharmaceutical composition according to any one of claims 11 to 20.

23. 1. A pharmaceutical composition comprising: A) an anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, or an antibody-drug conjugate according to any one of claims 11 to 14; histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Polysorbate, preferably Polysorbate 80, sucrose, trehalose and / or arginine or medicinal salts thereof; B) an anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, or an antibody-drug conjugate according to any one of claims 11 to 14; histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Polysorbate, preferably Polysorbate 80, proline or a medicinal salt thereof, C) an anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, or an antibody-drug conjugate according to any one of claims 11 to 14; histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Polysorbate, preferably Polysorbate 80, sucrose, D) The antibody-drug conjugate according to any one of claims 11 to 14; histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; Polysorbate, preferably Polysorbate 80, trehalose, Any one of the following groups is included: Pharmaceutical compositions.

24. 1. A pharmaceutical composition comprising: 1) 0.1 to 300 mg / mL of the anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10; 1-50 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.001% to 1% w / v of a polysorbate, preferably polysorbate 80; 0.1% to 30% w / v sucrose, and the pH of the composition is 4.5 to 7.5; 2) 1 to 200 mg / mL of the anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10; 5-40 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.005% to 1% w / v of a polysorbate, preferably polysorbate 80; 0.5% to 20% w / v sucrose, and the pH of the composition is 5 to 7.2; 3) 10 to 150 mg / mL of the anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10; 10-30 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.008% to 0.2% w / v of a polysorbate, preferably polysorbate 80; 1% to 15% w / v sucrose, and the pH of the composition is 5.5 to 7; 4) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of 15 to 132 mg / mL or 20 to 100 mg / mL. 15-25 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, preferably polysorbate 80; 5% to 10% w / v sucrose, and the pH of the composition is 5.6 to 6.6; 5) 0.1 to 500 mg / mL of the anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10; 1-50 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.001% to 1% w / v of a polysorbate, preferably polysorbate 80; 0.01% to 20% w / v proline or a medicinal salt thereof; and the pH of the composition is 4.5 to 7.5; 6) 1 to 300 mg / mL of the anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10; 5-40 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.005% to 1% w / v of a polysorbate, preferably polysorbate 80; 0.1% to 15% w / v proline or a medicinal salt thereof; and the pH of the composition is 5 to 7.2; 7) 10 to 250 mg / mL of the anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10; 10-30 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.008% to 0.2% w / v of a polysorbate, preferably polysorbate 80; 0.5% to 10% w / v proline or a medicinal salt thereof; and the pH of the composition is 5.5 to 7; 8) 20 to 200 mg / mL of the anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10; 15-25 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, preferably polysorbate 80; 1% to 5% w / v proline or a medicinal salt thereof; and the pH of the composition is 5.6 to 6.6; Any one of the following groups is included: Pharmaceutical compositions.

25. 1. A pharmaceutical composition comprising: 1) 0.1 to 300 mg / mL of the antibody-drug conjugate according to any one of claims 11 to 14; 1-50 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.001% to 1% w / v of a polysorbate, preferably polysorbate 80; 0.1% to 30% w / v sucrose or trehalose, and the pH of the composition is 4.5 to 7.5; 2) 1 to 150 mg / mL of the antibody-drug conjugate according to any one of claims 11 to 14; 5-40 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.005% to 1% w / v of a polysorbate, preferably polysorbate 80; 0.5% to 20% w / v sucrose or trehalose, and the pH of the composition is 5 to 7.2; 3) 5-120 mg / mL of the antibody-drug conjugate according to any one of claims 11 to 14; 10-30 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.008% to 0.2% w / v of a polysorbate, preferably polysorbate 80; 1% to 15% w / v sucrose or trehalose, and the pH of the composition is 5.5 to 7; 4) 10 to 100 mg / mL of the antibody-drug conjugate according to any one of claims 11 to 14; 15-25 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.01% to 0.1% w / v of a polysorbate, preferably polysorbate 80; 5% to 10% w / v sucrose or trehalose, and the pH of the composition is 5.6 to 6.6; Any one of the following groups is included: Pharmaceutical compositions.

26. 1) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL; about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v sucrose, and the pH of the composition is about 6.0; 2) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v sucrose, and the pH of the composition is about 6.1; 3) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v sucrose, and the pH of the composition is about 6.2; 4) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v sucrose, and the pH of the composition is about 6.3; 5) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v sucrose, and the pH of the composition is about 6.5; 6) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v sucrose, and the pH of the composition is about 6.6; 7) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 8% w / v sucrose, and the pH of the composition is about 6.0; 8) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 8% w / v sucrose, and the pH of the composition is about 6.1; 9) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 8% w / v sucrose, and the pH of the composition is about 6.2; 10) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 8% w / v sucrose, and the pH of the composition is about 6.3; 11) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 8% w / v sucrose, and the pH of the composition is about 6.5; 12) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or about 130 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 8% w / v sucrose, and the pH of the composition is about 6.6; 13) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 2.5% w / v proline, and the pH of the composition is about 6.0; 14) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 2.5% w / v proline, and the pH of the composition is about 6.1; 15) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 2.5% w / v proline, and the pH of the composition is about 6.2; 16) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 2.5% w / v proline, and the pH of the composition is about 6.3; 17) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 2.5% w / v proline, and the pH of the composition is about 6.5; 18) The anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10, having a concentration of about 50 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 2.5% w / v proline, and the pH of the composition is about 6.

6. Any one of the following groups is included:

25. The pharmaceutical composition of claim 23 or 24.

27. 1) the antibody-drug conjugate of any one of claims 11 to 14 at about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL; about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v or about 9% w / v sucrose; and the pH of the composition is about 6.0; 2) The antibody-drug conjugate of any one of claims 11 to 14, at a concentration of about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v or about 9% w / v sucrose; and the pH of the composition is about 6.3; 3) The antibody-drug conjugate of any one of claims 11 to 14, at a concentration of about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v or about 9% w / v sucrose; and the pH of the composition is about 6.6; 4) The antibody-drug conjugate of any one of claims 11 to 14, at a concentration of about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v trehalose, and the pH of the composition is about 6.0; 5) The antibody-drug conjugate of any one of claims 11 to 14, at a concentration of about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v trehalose, and the pH of the composition is about 6.3; 6) The antibody-drug conjugate of any one of claims 11 to 14, at a concentration of about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL. about 20 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; about 0.02% w / v polysorbate 80, about 7.5% w / v trehalose, and the pH of the composition is about 6.

6. Any one of the following groups is included:

26. The pharmaceutical composition of claim 23 or 25.

28. A lyophilized formulation, which can form the pharmaceutical composition of any one of claims 1 to 27 after reconstitution of said lyophilized formulation, or said lyophilized formulation is obtainable by lyophilizing the pharmaceutical composition of any one of claims 1 to 27. Lyophilized formulation.

29. 29. The freeze-dried formulation according to claim 28, which is prepared by reconstituting the freeze-dried formulation. Reconstitution solution.

30. A container containing the pharmaceutical composition according to any one of claims 1 to 27, the lyophilized formulation according to claim 28, or the reconstitution solution according to claim 29. product.

31. Use of the pharmaceutical composition according to any one of claims 1 to 27, the lyophilized formulation according to claim 28 or the reconstituted solution according to claim 29 in the preparation of a medicament for the treatment of an autoimmune disease, comprising: The autoimmune disease is preferably Sjögren's syndrome, multiple sclerosis or lupus erythematosus, more preferably systemic lupus erythematosus. use.

32. Use of the pharmaceutical composition according to any one of claims 1 to 27, the lyophilized formulation according to claim 28 or the reconstituted solution according to claim 29 in the preparation of a medicament for use in treating graft-versus-host disease or alleviating transplant rejection, comprising: The transplant is preferably a solid organ transplant, more preferably a liver, kidney, heart or lung transplant. use.

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