Composition of CD40 binding molecules and medical application

Abstract

The invention relates to a composition of CD40 binding molecules and medical application. In particular, the composition comprises an anti-CD40 antibody, or an antigen-binding fragment thereof, or an antibody-drug conjugate, and a buffer.

Description

Composition and medical use of CD40 binding molecules

[0001] This application claims priority to Chinese patent application No. 202211698820.3 filed on December 28, 2022. Technical Field

[0002] The present disclosure relates to the field of pharmaceutical preparations, and in particular to a pharmaceutical composition comprising a CD40 binding molecule, and pharmaceutical uses thereof. Background Art

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

[0004] After CD40L binds to CD40, it recruits TRAFs and mediates downstream signaling through the NF-κB, JNK, and MAPK pathways, exerting multiple cell type-dependent activation outcomes, including immune cell activation and proliferation, inflammatory cytokine and chemokine secretion (Vonderheide RH et al., 2007). For example, signaling through this pathway is essential 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 its effects on B cells, CD40 pathway activation provides important signals for DC maturation and function, as well as monocyte and macrophage survival and cytokine secretion (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: activated epithelial cells from sites such as the kidney, salivary glands, and skin that secrete chemokines are able to respond to CD40. Furthermore, CD40 or CD40L expression is elevated in lesions of patients with atherosclerosis and preclinical atherosclerosis models. CD40 can stimulate the expression of matrix-degrading enzymes and promote the expression of tissue factor in cell types associated with atherosclerosis pathogenesis, such as endothelial cells, smooth muscle cells, and macrophages (Michel NA et al., 2017). The CD40 pathway upregulates the production of inflammatory cytokines such as IL-1, IL-6, and IL-8, as well as adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule (VCAM). The CD40 / CD40L interaction has also been used to prevent transplant rejection, and in rhesus monkey renal allograft studies using the chimeric anti-CD40 antagonist ch5D12, CD40 antagonism was sufficient to improve disease progression and extend median survival to over 100 days. When ch5D12 was combined with an anti-CD86 antibody and given only at the start of the allograft study, followed by extended treatment with cyclosporine, median survival of over 4 years was achieved, suggesting that this combination could potentially induce immune tolerance (Haanstra et al., 2005).

[0006] Numerous preclinical studies have provided evidence for the key role of the CD40 / CD40L interaction in promoting T cell-dependent immune responses. Therefore, blocking CD40 signaling is considered a suitable and necessary therapeutic strategy for suppressing 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 an urgent need in the art for therapeutic agents that can intervene in the CD40-CD40L interaction and block CD40 signaling.

[0007] The applicant's patent application WO2023274201 provides an anti-CD40 antibody with a new structure, which can specifically bind to CD40 and can be used to intervene in or treat diseases related to the CD40 signaling pathway.

[0008] Antibody drugs are an important class of biopharmaceuticals. Their large molecular weight and complex structure make them susceptible to physical or chemical degradation and aggregation during production, storage, and use, leading to reduced activity or even ineffectiveness. Therefore, developing excellent antibody preparations is crucial.

[0009] Summary of the Invention

[0010] The present disclosure provides a pharmaceutical composition containing a CD40 binding molecule, wherein the composition has excellent stability.

[0011] The present disclosure provides a pharmaceutical composition comprising a CD40 binding molecule and a buffer, wherein the buffer is selected from tris (hydroxymethylaminomethane) buffer, acetate buffer, succinate buffer, phosphate buffer, histidine salt buffer, acetate buffer, citrate buffer, tartrate buffer, fumarate buffer, glycylglycine buffer, citrate buffer. In certain embodiments, the buffer is selected from acetate buffer, citrate buffer, histidine salt buffer and phosphate buffer. In certain embodiments, the buffer is a histidine salt buffer. In certain embodiments, the histidine salt buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer. In certain embodiments, the buffer is a histidine-hydrochloride buffer. In certain embodiments, the buffer is a histidine-histidine hydrochloride buffer.

[0012] Anti-CD40 antibody or antigen-binding fragment thereof

[0013] In one aspect, the CD40 binding molecules of the present disclosure are anti-CD40 antibodies or antigen-binding fragments thereof.

[0014] In one aspect, the present disclosure provides a pharmaceutical composition comprising an anti-CD40 antibody or an antigen-binding fragment thereof, wherein the composition has excellent stability.

[0015] The present disclosure provides a pharmaceutical composition comprising an anti-CD40 antibody or an antigen-binding fragment thereof and a buffer, wherein the buffer is selected from tris (hydroxymethylaminomethane) buffer, acetate buffer, succinate buffer, phosphate buffer, histidine salt buffer, acetate buffer, citrate buffer, tartrate buffer, fumarate buffer, glycylglycine buffer, citrate buffer. In certain embodiments, wherein the buffer is selected from acetate buffer, citrate buffer, histidine salt buffer and phosphate buffer. In certain embodiments, the buffer is a histidine salt buffer. In certain embodiments, the histidine salt buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer. In certain embodiments, the buffer is a histidine-hydrochloride buffer. In certain embodiments, the buffer is a histidine-histidine hydrochloride buffer.

[0016] The present disclosure discloses the aforementioned pharmaceutical composition, wherein the anti-CD40 antibody and antigen-binding fragment thereof comprises:

[0017] Heavy chain HCDR1, HCDR2, and HCDR3 comprise the sequences shown in SEQ ID NOs: 3, 4, and 5, respectively; and / or, light chain LCDR1 comprises the sequence shown in SEQ ID NO: 6; light chain LCDR2 comprises the sequence shown in SEQ ID NO: 7; light chain LCDR3 comprises the sequence shown in SEQ ID NO: QGGYWTSTSNFGX9X 10 (SEQ ID NO: 19), wherein X9 is selected from N, S, T or Q, X 10 Select from V or G.

[0018] In certain embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof comprises:

[0019] The heavy chain HCDR1, HCDR2, and HCDR3 comprise the sequences shown in SEQ ID NOs: 3, 4, and 5, respectively; the light chain LCDR1 comprises the sequence shown in SEQ ID NO: 6; the light chain LCDR2 comprises the sequence shown in SEQ ID NO: 7; and the light chain LCDR3 comprises the sequence shown in any one of SEQ ID NOs: 15-18.

[0020] In certain embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0021] The VH comprises HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 2;

[0022] Wherein, the CDR is defined according to the Kabat, IMGT, Chothia, Ab M or Contact numbering system. In certain embodiments, the CDR is defined according to the Kabat numbering system.

[0023] In certain embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof is a recombinant antibody.

[0024] In certain embodiments, the aforementioned 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.

[0025] In certain embodiments, when the aforementioned anti-CD40 antibodies or antigen-binding fragments thereof are humanized antibodies, the heavy chain framework region is derived from IGHV2-26*01, IGHV4-30-4*02, IGHV4-4*08, or IGHJ1*01; and / or the light chain framework region is derived from IGkV1-13*02, IGkV1-9*01, IGkV1-6*01, or IGKJ4*01. For example, FR1-FR3 of the heavy chain framework region are derived from IGHV2-26*01, IGHV4-30-4*02, or IGHV4-4*08, and FR4 of the heavy chain framework region is derived from IGHJ1*01; FR1-FR3 of the light chain framework region are derived from IGkV1-13*02, IGkV1-9*01, or IGkV1-6*01, and FR4 of the light chain framework region is derived from IGKJ4*01.

[0026] In certain embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof comprises VH and VL, wherein:

[0027] A-1) VH has an amino acid sequence as set forth in SEQ ID NO: 1, or at least 90% identical thereto, and VL has an amino acid sequence as set forth in SEQ ID NO: 2, or at least 90% identical thereto;

[0028] A-2) VH has an amino acid sequence as set forth in SEQ ID NO: 13, or at least 90% identical thereto, and VL has an amino acid sequence as set forth in any one of SEQ ID NOs: 9-12, or at least 90% identical thereto;

[0029] A-3) The amino acid sequence of VH is as shown in SEQ ID NO: 14, or is at least 90% identical thereto, and the amino acid sequence of VL is as shown in any one of SEQ ID NOs: 9-12, or is at least 90% identical thereto.

[0030] In certain embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof is an IgG antibody or an antigen-binding fragment thereof, for example, an IgG1, IgG2, IgG2, IgG4 antibody or an antigen-binding fragment thereof, for example, an IgG1 antibody or an antigen-binding fragment thereof having an N297A mutation, for example, an IgG1 antibody or an antigen-binding fragment thereof having one of L234A, L235A, M252Y, S254T and T256E or any combination thereof.

[0031] In certain embodiments, the antigen-binding fragment of the aforementioned anti-CD40 antibody is 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 and tetrabody, tandem two-scFv, tandem three-scFv), for example, scFv, Fv, Fab or Fab' fragment.

[0032] In certain embodiments, the amino acid sequence of the full-length heavy chain of the antigen-binding fragment of the aforementioned anti-CD40 antibody is as shown in SEQ ID NO: 20 or 22, or is at least 90% identical thereto; and the amino acid sequence of the full-length light chain is as shown in SEQ ID NO: 21, or is at least 90% identical thereto.

[0033] As mentioned 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.

[0034] In certain embodiments, the heavy chain variable region of the aforementioned anti-CD40 antibodies or antigen-binding fragments thereof has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes; the light chain variable region has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes. In some specific embodiments, the amino acid changes are conservative substitutions, replacements, or modifications, and / or deletions or additions that do not affect function.

[0035] Antibody-drug conjugates

[0036] In another aspect, the CD40 binding molecule in the pharmaceutical composition of the present disclosure is an antibody-drug conjugate comprising an anti-CD40 antibody or an antigen-binding fragment thereof, wherein the anti-CD40 antibody or antigen-binding fragment thereof is any anti-CD40 antibody or antigen-binding fragment thereof disclosed herein.

[0037] The present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate and a buffer, wherein the antibody-drug conjugate comprises any anti-CD40 antibody or antigen-binding fragment thereof of the present disclosure; wherein the buffer is selected from tris (hydroxymethylaminomethane) buffer, acetate buffer, succinate buffer, phosphate buffer, histidine salt buffer, acetate buffer, citrate buffer, tartrate buffer, fumarate buffer, glycylglycine buffer, citrate buffer. In certain embodiments, the buffer is selected from acetate buffer, citrate buffer, histidine salt buffer and phosphate buffer. In certain embodiments, the buffer is a histidine salt buffer. In certain embodiments, the histidine salt buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer. In certain embodiments, the buffer is a histidine-hydrochloride buffer. In certain embodiments, the buffer is a histidine-histidine hydrochloride buffer.

[0038] In certain embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof comprises

[0039] Heavy chain HCDR1, HCDR2, and HCDR3 comprise the sequences shown in SEQ ID NOs: 3, 4, and 5, respectively; and, light chain LCDR1 comprises the sequence shown in SEQ ID NO: 6; light chain LCDR2 comprises the sequence shown in SEQ ID NO: 7; light chain LCDR3 comprises the sequence shown in SEQ ID NO: QGGYWTSTSNFGX9X 10 (SEQ ID NO: 19), wherein X9 is selected from N, S, T or Q, X 10 Select from V or G.

[0040] In certain embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof comprises:

[0041] The heavy chain HCDR1, HCDR2, and HCDR3 comprise the sequences shown in SEQ ID NOs: 3, 4, and 5, respectively; the light chain LCDR1 comprises the sequence shown in SEQ ID NO: 6; the light chain LCDR2 comprises the sequence shown in SEQ ID NO: 7; and the light chain LCDR3 comprises the sequence shown in any one of SEQ ID NOs: 15-18.

[0042] In certain embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0043] The VH comprises HCDR1, HCDR2, and HCDR3 of the VH as shown in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2, and LCDR3 of the VL as shown in SEQ ID NO: 2;

[0044] Wherein, the CDR is defined according to the Kabat, IMGT, Chothia, Ab M or Contact numbering system. In certain embodiments, the CDR is defined according to the Kabat numbering system.

[0045] In certain embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof is a recombinant antibody.

[0046] In certain embodiments, the aforementioned 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.

[0047] In certain embodiments, when the aforementioned anti-CD40 antibodies or antigen-binding fragments thereof are humanized antibodies, the heavy chain framework region is derived from IGHV2-26*01, IGHV4-30-4*02, IGHV4-4*08, or IGHJ1*01; and / or the light chain framework region is derived from IGkV1-13*02, IGkV1-9*01, IGkV1-6*01, or IGKJ4*01. For example, FR1-FR3 of the heavy chain framework region are derived from IGHV2-26*01, IGHV4-30-4*02, or IGHV4-4*08, and FR4 of the heavy chain framework region is derived from IGHJ1*01; FR1-FR3 of the light chain framework region are derived from IGkV1-13*02, IGkV1-9*01, or IGkV1-6*01, and FR4 of the light chain framework region is derived from IGKJ4*01.

[0048] In certain embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof comprises VH and VL, wherein:

[0049] A-1) VH has an amino acid sequence as set forth in SEQ ID NO: 1, or at least 90% identical thereto, and VL has an amino acid sequence as set forth in SEQ ID NO: 2, or at least 90% identical thereto;

[0050] A-2) VH has an amino acid sequence as set forth in SEQ ID NO: 13, or at least 90% identical thereto, and VL has an amino acid sequence as set forth in any one of SEQ ID NOs: 9-12, or at least 90% identical thereto;

[0051] A-3) The amino acid sequence of VH is as shown in SEQ ID NO: 14, or is at least 90% identical thereto, and the amino acid sequence of VL is as shown in any one of SEQ ID NOs: 9-12, or is at least 90% identical thereto.

[0052] In certain embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof is an IgG antibody or an antigen-binding fragment thereof, for example, an IgG1, IgG2, IgG2, IgG4 antibody or an antigen-binding fragment thereof, for example, an IgG1 antibody or an antigen-binding fragment thereof having an N297A mutation, for example, an IgG1 antibody or an antigen-binding fragment thereof having one of L234A, L235A, M252Y, S254T and T256E or any combination thereof.

[0053] In certain embodiments, the antigen-binding fragment of the aforementioned anti-CD40 antibody is 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 and tetrabody, tandem two-scFv, tandem three-scFv), for example, scFv, Fv, Fab or Fab' fragment.

[0054] In certain embodiments, the amino acid sequence of the full-length heavy chain of the antigen-binding fragment of the aforementioned anti-CD40 antibody is as shown in SEQ ID NO: 20 or 22, or is at least 90% identical thereto; and the amino acid sequence of the full-length light chain is as shown in SEQ ID NO: 21, or is at least 90% identical thereto.

[0055] As mentioned 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.

[0056] In certain embodiments, the heavy chain variable region of the aforementioned anti-CD40 antibodies or antigen-binding fragments thereof has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes; the light chain variable region has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes. In some specific embodiments, the amino acid changes are conservative substitutions, replacements, or modifications, and / or deletions or additions that do not affect function.

[0057] In certain embodiments, the disclosed antibody-drug conjugates have a structure as shown in formula (I):

[0058] Ab-(LD) k (I);

[0059] wherein Ab is any of the aforementioned anti-CD40 antibodies or antigen-binding fragments thereof;

[0060] D is selected from a glucocorticoid receptor agonist, a glucocorticoid, a calcineurin inhibitor, or a mTOR kinase inhibitor;

[0061] L is a linker that covalently links Ab to D,

[0062] k is an integer or decimal 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 decimal or integer between any two of the foregoing values). In certain embodiments, k is 4±0.4, 4±0.5, 4±0.6, 4±0.8. In certain 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, 5.0.

[0063] In certain embodiments, D is a glucocorticoid receptor agonist, for example, a glucocorticoid receptor agonist selected from any structure disclosed in WO2022166779A1.

[0064] In certain embodiments, D is a glucocorticoid, such as budesonide, ciclesonide.

[0065] In certain embodiments, D is a calcineurin inhibitor (CAI), such as tacrolimus, voclosporin, or cyclosporine A (CsA).

[0066] In certain embodiments, D is a mTOR kinase inhibitor, such as rapamycin, everolimus, and temsirolimus.

[0067] WO2022166779A1 discloses an antibody-drug conjugate of a glucocorticoid receptor agonist, and the present disclosure incorporates its contents in their entirety, including the structure and preparation method of the antibody-drug conjugate.

[0068] In certain embodiments, the disclosed antibody-drug conjugates have a structure as shown in formula (I): Ab-(LD) k (I);

[0069] wherein Ab is any of the aforementioned anti-CD40 antibodies or antigen-binding fragments thereof;

[0070] D is glucocorticoid;

[0071] L is a linker that covalently links Ab to D,

[0072] k is an integer or decimal selected from 1-20 (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).

[0073] WO2023143351A1 discloses a glucocorticoid drug conjugate, and the present disclosure incorporates its contents in their entirety, including the structure and preparation method of the antibody-drug conjugate.

[0074] In certain embodiments, D is represented by the formula:

[0075] In certain embodiments, k is any value between 1 and 10. k can be an integer or a decimal.

[0076] In certain embodiments, the present disclosure provides an antibody-drug conjugate (ADC) represented by the following formula:

[0077] wherein k is selected from 1 to 10 and can 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;

[0078] In certain embodiments, the disclosed antibody-drug conjugate (ADC) is selected from:

[0079] Wherein k is an integer or decimal from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or any decimal or integer between any two of the foregoing values). In some embodiments, k is an integer or decimal from 3 to 5. In some embodiments, k is 4±0.4, 4±0.5, 4±0.6, 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, 5.0. In certain embodiments, k is any value between 2 and 5.

[0080] In certain embodiments, the present invention discloses an antibody-drug conjugate (ADC) as follows:

[0081] wherein k is an integer or decimal from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any decimal or integer between any two of the foregoing values). In some embodiments, k is an integer or decimal 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 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, 5.0

[0082] In certain embodiments, the present invention discloses an antibody-drug conjugate (ADC) as follows:

[0083] Among them, 9E6-L4H2 is an anti-CD40 antibody, which comprises: a heavy chain and a light chain having amino acid sequences as shown in SEQ ID NOs: 20 and 21;

[0084] wherein k is an integer or decimal selected from 1 to 10. In certain embodiments, k is an integer or decimal selected from 2 to 5. In certain embodiments, k is 4±0.4, 4±0.5, 4±0.6, or 4±0.8. In certain 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.

[0085] In certain embodiments, the pH of the buffer in the pharmaceutical composition is 4-8, for example, 4.5-7.5, 4.5-7, 4.5-6.8, 4.5-6.5, 4.5-6.3, 4.5-6, 5-7.2, 5-7.1, 5-7, 5-6.9, 5-6.8, 5-6.7, 5-6.6, 5-6.5, 5-6.4, 5-6.3, 5-6.2, 5-6.1, 5-6, 5.5-7, 5.5-6 .9, 5.5-6.8, 5.5-6.7, 5.5-6.6, 5.5-6.5, 5.5-6.4, 5.5-6.3, 5.5-6.2, 5.5-6.1, 5.5-6, 5.6-7, 5.6-6.9, 5.6-6.8, 5.6-6.7, 5.6-6.6, 5.6-6.5, 5.6-6.4, 5.6-6.3, 5.6-6.2, 5.6-6.1 or 6-6.8.

[0086] In certain embodiments, the pH of the buffer in the pharmaceutical composition is 4.5-7.5. In certain embodiments, the pH of the buffer in the pharmaceutical composition is 5-7.2. In certain embodiments, the pH of the buffer in the pharmaceutical composition is 5.5-7. In certain embodiments, the pH of the buffer is 5.6-6.6. In certain 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, about 7.0. In certain 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.

[0087] In certain embodiments, the pH of the pharmaceutical composition differs by no more than ±0.5 from the pH of the buffer it contains.

[0088] In certain embodiments, the pH of the pharmaceutical composition is 4-8, for example, 4.5-7.5, 4.5-7, 4.5-6.8, 4.5-6.5, 4.5-6.3, 4.5-6, 5-7.2, 5-7.1, 5-7, 5-6.9, 5-6.8, 5-6.7, 5-6.6, 5-6.5, 5-6.4, 5-6.3, 5-6.2, 5-6.1, 5-6, 5.5-7, 5.5-6.9 , 5.5-6.8, 5.5-6.7, 5.5-6.6, 5.5-6.5, 5.5-6.4, 5.5-6.3, 5.5-6.2, 5.5-6.1, 5.5-6, 5.6-7, 5.6-6.9, 5.6-6.8, 5.6-6.7, 5.6-6.6, 5.6-6.5, 5.6-6.4, 5.6-6.3, 5.6-6.2, 5.6-6.1 or 6-6.8. In certain embodiments, the pH of the pharmaceutical composition is 4.5-7.5. In certain embodiments, the pH of the pharmaceutical composition is 5-7.2. In certain embodiments, the pH of the pharmaceutical composition is 5.5-7. In certain embodiments, the pH of the buffer is 5.6-6.6. In certain 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 certain 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 certain 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.

[0089] In certain embodiments, the buffer concentration in the pharmaceutical composition is 1-50 mM, for example, 5-45 mM, 5-40 mM, 5-35 mM, 5-30 mM, 5-25 mM, 5-20 mM, 10-40 mM, 10-35 mM, 10-30 mM, 10-25 mM, 10-20 mM, 15-35 mM, 15-30 mM, 15-25 mM, 15-20 mM. In certain embodiments, the buffer concentration in the pharmaceutical composition is 5-40 mM. In certain embodiments, the buffer concentration in the pharmaceutical composition is 10-30 mM. In certain embodiments, the buffer concentration in the pharmaceutical composition is 15-25 mM. In certain embodiments, the concentration of the buffer 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 certain embodiments, the concentration of the buffer in the pharmaceutical composition is about 20 mM.

[0090] In certain embodiments, the pharmaceutical composition further comprises a stabilizer selected from one or more of an amino acid or a pharmaceutically acceptable salt thereof and a sugar. In certain embodiments, the amino acid is selected from one or more of proline, aspartic acid, glutamic acid, lysine, arginine, glycine and histidine. In certain embodiments, the amino acid is selected from one or more of proline and arginine or a pharmaceutically acceptable salt thereof. In certain embodiments, the sugar is selected from one or more of glucose, sucrose, maltose and trehalose. In certain embodiments, the stabilizer is selected from one or more of arginine or a pharmaceutically acceptable salt thereof, proline or a pharmaceutically acceptable salt thereof, trehalose and sucrose. In certain embodiments, the stabilizer is selected from one or more of sucrose and trehalose. In certain embodiments, the stabilizer is proline or a pharmaceutically acceptable salt thereof.

[0091] In certain embodiments, the concentration of sucrose in the pharmaceutical composition is 0.1%-30% w / v, such as 0.5%-25% w / v, 0.5%-20% w / v, 0.5%-15% w / v, 0.5%-10% w / v, 1%-20% w / v, 1%-15% w / v, 1%-10% w / v, 5%-15% w / v, 5%-10% w / v. In certain embodiments, the concentration of sucrose in the pharmaceutical composition is 0.5%-20% w / v. In certain embodiments, the concentration of sucrose in the pharmaceutical composition is 1%-15% w / v. In certain embodiments, the concentration of sucrose in the pharmaceutical composition is 5%-10% w / v. In certain 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 certain embodiments, the concentration of sucrose in the pharmaceutical composition is about 7.5% w / v, about 8% w / v, or about 9% w / v.

[0092] In certain embodiments, the concentration of trehalose in the pharmaceutical composition is 0.1%-30% w / v, such as 0.5%-25% w / v, 0.5%-20% w / v, 0.5%-15% w / v, 0.5%-10% w / v, 1%-20% w / v, 1%-15% w / v, 1%-10% w / v, 5%-15% w / v, 5%-10% w / v. In certain embodiments, the concentration of trehalose in the pharmaceutical composition is 0.5%-20% w / v. In certain embodiments, the concentration of trehalose in the pharmaceutical composition is 1%-15% w / v. In certain embodiments, the concentration of sucrose in the pharmaceutical composition is 5%-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.

[0093] In certain embodiments, the concentration of proline or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.01%-20% w / v, e.g., 0.05%-20% w / v, 0.05%-15% w / v, 0.05%-10% w / v, 0.05%-5% w / v, 0.1%-20% w / v, 0.1%-15% w / v, 0.1%-10% w / v, 0.1%-5% w / v, 0.5%-20% w / v, 0.5%-15% w / v, 0.5%-10% w / v, 0.5%-5% w / v, 1%-20% w / v, 1%-15% w / v, 1%-10% w / v, or 1%-5% w / v. In certain embodiments, the concentration of proline or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.01%-20% w / v. In certain embodiments, the concentration of proline or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.1%-15% w / v. In certain embodiments, the concentration of proline or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.5%-10% w / v. In certain embodiments, the concentration of proline or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 1%-5% w / v. In certain embodiments, the concentration of proline or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is about 0.5% w / v, 1% w / v, 1.5% w / v, 2% w / v, 2.5% w / v, 3% w / v, 3.5% w / v, 4% w / v, 4.5% w / v, 5% w / v, 5.5% w / v, 6% w / v, 6.5% w / v, 7% w / v, 7.5% w / v, 8% w / v, 8.5% w / v, 9% w / v, 9.5% w / v, or 10% w / v. In certain embodiments, the concentration of proline or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is about 1% w / v, 1.5% w / v, 2% w / v, 2.5% w / v, 3% w / v, 3.5% w / v, 4% w / v, 4.5% w / v or 5% w / v. In certain embodiments, the concentration of proline or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is about 2.5% w / v.

[0094] In certain embodiments, the concentration of arginine or its salt in the pharmaceutical composition is 1-100nM, for example 10-90nM, 10-80nM, 10-70nM, 10-60nM, 10-55nM, 20-80nM, 20-70nM, 20-60nM, 20-55nM, 30-70nM, 30-60nM, 30-55nM, 40-60nM, 40-55nM. In certain embodiments, the concentration of arginine or its salt in the pharmaceutical composition is 20-80nM. In certain embodiments, the concentration of arginine or its salt in the pharmaceutical composition is 30-70nM. In certain embodiments, the concentration of arginine or its salt in the pharmaceutical composition is 40-60nM. In certain 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 certain embodiments, the concentration of arginine or a salt thereof in the pharmaceutical composition is 50 nM.

[0095] In certain embodiments, the stabilizer in the pharmaceutical composition is sucrose and arginine or a pharmaceutically acceptable salt thereof, wherein the concentration of sucrose is 0.5%-20% w / v and the concentration of arginine or a salt thereof is 20-80 nM. In certain embodiments, the stabilizer in the pharmaceutical composition is sucrose and arginine or a pharmaceutically acceptable salt thereof, wherein the concentration of sucrose is 1%-15% w / v and the concentration of arginine or a salt thereof is 30-70 nM. In certain embodiments, the stabilizer in the pharmaceutical composition is sucrose and arginine or a pharmaceutically acceptable salt thereof, wherein the concentration of sucrose is 5%-10% w / v and the concentration of arginine or a salt thereof is 40-60 nM. In certain embodiments, the stabilizer in the pharmaceutical composition is sucrose and arginine or a pharmaceutically acceptable salt thereof, wherein the concentration of sucrose is about 7.5% w / v or about 8% w / v and the concentration of arginine or a salt thereof is 50 nM. In certain embodiments, the arginine or a pharmaceutically acceptable salt thereof is arginine hydrochloride.

[0096] In certain embodiments, the pharmaceutical composition of the present disclosure further comprises a surfactant. In certain embodiments, the surfactant is an ionic or nonionic surfactant. In certain embodiments, the surfactant is selected from polysorbate, polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroyl One or more of aminopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoyl sodium, methyl oleyl taurate sodium, polyethylene glycol, polypropylene glycol and the copolymer of ethylene and propylene glycol. In certain embodiments, the surfactant is polysorbate 20 or polysorbate 80. In certain embodiments, the surfactant is polysorbate 80.

[0097] In certain embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.001%-1% w / v, for example, 0.005%-1% w / v, 0.005%-0.8% w / v, 0.005%-0.6% w / v, 0.005%-0.4% w / v, 0.005%-0.2% w / v, 0.005%-0.1% w / v, 0.005%-0.09% w / v, 0.005%-0.08% w / v, 0.005%-0.07% w / v, 0.005%-0.06% w / v, 0.005%-0.05% w / v, 0.008%-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 certain embodiments, the concentration of surfactant in the pharmaceutical composition is 0.005%-1% w / v. In certain embodiments, the concentration of surfactant in the pharmaceutical composition is 0.008%-0.2% w / v. In certain embodiments, the concentration of surfactant in the pharmaceutical composition is 0.01%-0.1% w / v. In certain 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 certain embodiments, the concentration of arginine or a salt thereof in the pharmaceutical composition is about 0.02% w / v.

[0098] In certain embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof in the pharmaceutical composition is 0.1-500 mg / mL, for example, 0.1-400 mg / mL, 0.1-350 mg / mL, 0.1-300 mg / mL, 0.1-250 mg / mL, 0.1-200 mg / mL, 0.1-190 mg / mL, 0.1-180 mg / mL, 0.1-170 mg / mL, 0.1-160 mg / mL, 0.1-150 mg / mL, 0.1-145 mg / mL, 0.1-140 mg / mL, 0.1-135 mg / mL, 0.1-130 mg / mL, 0.1-125 mg / 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, 1-300mg / mL, 1-2 50mg / 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 / m L, 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 certain embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 1-200 mg / mL. In certain embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 10-150 mg / mL. In certain embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 20-100 mg / mL. In certain embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 1-300 mg / mL. In certain embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 10-250 mg / mL. In certain embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 20-200 mg / mL. In certain embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 15-130 mg / mL or 15-132 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, 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 certain 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. In certain embodiments, the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is about 50 mg / mL, about 80 mg / mL, or about 150 mg / mL.

[0099] In certain embodiments, the concentration of the antibody-drug conjugate in the pharmaceutical composition is 0.1-300 mg / mL, for example, 1-200 mg / mL, 1-190 mg / mL, 1-180 mg / mL, 1-170 mg / mL, 1-160 mg / mL, 1-150 mg / mL, 1-145 mg / mL, 1-140 mg / mL, 1-135 mg / mL, 1-130 mg / mL, 1-120 mg / mL, 1-110 mg / mL, 1-100 mg / mL, 1-90 mg / mL, 1-80 mg / mL, 1-70 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate is 1-150 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate is 5-120 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate is about 20-100 mg / mL. In certain embodiments, the concentration of the antibody-drug conjugate is 10-100 mg / mL. In certain 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 certain 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.

[0100] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-CD40 antibody or an antigen-binding fragment thereof (e.g., 9E6 (L4H2) whose heavy chain and light chain sequences are SEQ ID NOs: 20 and 21), which comprises or is any one of the following groups A) to F):

[0101] A) an anti-CD40 antibody or an antigen-binding fragment thereof;

[0102] Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

[0103] Polysorbates, such as polysorbate 80;

[0104] sucrose, trehalose and / or arginine or pharmaceutically acceptable salts thereof;

[0105] B) an anti-CD40 antibody or an antigen-binding fragment thereof;

[0106] Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

[0107] Polysorbates, such as polysorbate 80;

[0108] Proline or a pharmaceutically acceptable salt thereof;

[0109] C) an anti-CD40 antibody or an antigen-binding fragment thereof;

[0110] Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

[0111] Polysorbates, such as polysorbate 80;

[0112] sucrose;

[0113] D) any one of A) to C) above, wherein the buffer has a pH of 4.5-7.5, such as a pH of 5-7.2, 5.5-7 or 5.6-6.6;

[0114] E) Any of the above A)-D), wherein the pharmaceutical composition further comprises water for injection;

[0115] F) Any one of A) to E) above, wherein the pH of the pharmaceutical composition is 4.5-7.5, such as 5-7.2, 5.5-7 or 5.6-6.6.

[0116] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate, which comprises or is any one of the following groups A) to H):

[0117] A) Antibody-drug conjugates;

[0118] Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

[0119] Polysorbates, such as polysorbate 80;

[0120] sucrose, trehalose and / or arginine or pharmaceutically acceptable salts thereof;

[0121] B) Antibody-drug conjugates;

[0122] Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

[0123] Polysorbates, such as polysorbate 80;

[0124] Proline or a pharmaceutically acceptable salt thereof;

[0125] C) Antibody-drug conjugates;

[0126] Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

[0127] Polysorbates, such as polysorbate 80;

[0128] sucrose;

[0129] D) Antibody-drug conjugates;

[0130] Histidine-hydrochloride buffers, such as histidine-histidine hydrochloride;

[0131] Polysorbates, such as polysorbate 80;

[0132] Trehalose;

[0133] E) Any one of A) to D) above, wherein the antibody-drug conjugate has the structure shown below:

[0134] Among them, 9E6-L4H2 is an anti-CD40 antibody, which includes a heavy chain and a light chain having amino acid sequences as shown in SEQ ID NOs: 20 and 21;

[0135] wherein k is an integer or decimal selected from 1 to 10, for example, k is an integer or decimal selected from 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;

[0136] F) any one of A) to E) above, wherein the buffer has a pH of 4.5-7.5, such as a pH of 5-7.2, 5.5-7 or 5.6-6.6;

[0137] G) Any of the above A)-F), wherein the pharmaceutical composition further comprises water for injection;

[0138] H) Any one of A) to G) above, wherein the pharmaceutical composition has a pH of 4.5-7.5, such as a pH of 5-7.2, 5.5-7 or 5.6-6.6.

[0139] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-CD40 antibody or an antigen-binding fragment thereof (e.g., 9E6 (L4H2) whose heavy chain and light chain sequences are SEQ ID NOs: 20 and 21), which comprises or is any one of the following groups 1) to 21):

[0140] 1) 0.1-300 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0141] 1-50 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0142] 0.001%-1% w / v polysorbate, such as polysorbate 80;

[0143] 0.1%-30% w / v sucrose;

[0144] and the pH of the composition is 4.5-7.5;

[0145] 2) 1-200 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0146] 5-40 mM histidine-HCl buffer, such as histidine-histidine hydrochloride;

[0147] 0.005%-1% w / v polysorbate, such as polysorbate 80;

[0148] 0.5%-20% w / v sucrose;

[0149] and the pH of the composition is 5-7.2;

[0150] 3) 10-150 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0151] 10-30 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0152] 0.008%-0.2% w / v polysorbate, such as polysorbate 80;

[0153] 1%-15% w / v sucrose;

[0154] and the pH of the composition is 5.5-7;

[0155] 4) 15-132 mg / mL or 20-100 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0156] 15-25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0157] 0.01%-0.1% w / v polysorbate, such as polysorbate 80;

[0158] 5%-10% w / v sucrose;

[0159] and the pH of the composition is 5.6-6.6;

[0160] 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;

[0161] 15-25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0162] 0.01%-0.1% w / v polysorbate, such as polysorbate 80;

[0163] 5%-10% w / v sucrose;

[0164] and the pH of the composition is 5.6-6.6;

[0165] 6) 20-100 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0166] about 15 mM, about 20 mM, or about 25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0167] 0.01%-0.1% w / v polysorbate, such as polysorbate 80;

[0168] 5%-10% w / v sucrose;

[0169] and the pH of the composition is 5.6-6.6;

[0170] 7) 20-100 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0171] 15-25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0172] 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 polysorbate, such as polysorbate 80;

[0173] 5%-10% w / v sucrose;

[0174] and the pH of the composition is 5.6-6.6;

[0175] 8) 20-100 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0176] 15-25 mM histidine-HCl buffer, such as histidine-histidine hydrochloride;

[0177] 0.01%-0.1% w / v polysorbate, such as polysorbate 80;

[0178] 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 sucrose;

[0179] and the pH of the composition is 5.6-6.6;

[0180] 9) 20-100 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0181] 15-25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0182] 0.01%-0.1% w / v polysorbate, such as polysorbate 80;

[0183] 5%-10% w / v sucrose;

[0184] 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;

[0185] 10) 0.1-500 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0186] 1-50 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0187] 0.001%-1% w / v polysorbate, such as polysorbate 80;

[0188] 0.01%-20% w / v proline or a pharmaceutically acceptable salt thereof;

[0189] and the pH of the composition is 4.5-7.5;

[0190] 11) 1-300 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0191] 5-40 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0192] 0.005%-1% w / v polysorbate, such as polysorbate 80;

[0193] 0.1%-15% w / v proline or a pharmaceutically acceptable salt thereof;

[0194] and the pH of the composition is 5-7.2;

[0195] 12) 10-250 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0196] 10-30 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0197] 0.008%-0.2% w / v polysorbate, such as polysorbate 80;

[0198] 0.5%-10% w / v proline or a pharmaceutically acceptable salt thereof;

[0199] and the pH of the composition is 5.5-7;

[0200] 13) 20-200 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0201] 15-25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0202] 0.01%-0.1% w / v polysorbate, such as polysorbate 80;

[0203] 1%-5% w / v proline or a pharmaceutically acceptable salt thereof;

[0204] and the pH of the composition is 5.6-6.6;

[0205] 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;

[0206] 15-25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0207] 0.01%-0.1% w / v polysorbate, such as polysorbate 80;

[0208] 1%-5% w / v proline or a pharmaceutically acceptable salt thereof;

[0209] and the pH of the composition is 5.6-6.6;

[0210] 15) 20-200 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0211] about 15 mM, about 20 mM, or about 25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0212] 0.01%-0.1% w / v polysorbate, such as polysorbate 80;

[0213] 1%-5% w / v proline or a pharmaceutically acceptable salt thereof;

[0214] and the pH of the composition is 5.6-6.6;

[0215] 16) 20-200 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0216] 15-25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0217] 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 polysorbates, such as polysorbate 80;

[0218] 1%-5% w / v proline or a pharmaceutically acceptable salt thereof;

[0219] and the pH of the composition is 5.6-6.6;

[0220] 17) 20-200 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0221] 15-25 mM histidine-HCl buffer, such as histidine-histidine hydrochloride;

[0222] 0.01%-0.1% w / v polysorbate, such as polysorbate 80;

[0223] 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 proline or a pharmaceutically acceptable salt thereof;

[0224] and the pH of the composition is 5.6-6.6;

[0225] 18) 20-200 mg / mL anti-CD40 antibody or antigen-binding fragment thereof;

[0226] 15-25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0227] 0.01%-0.1% w / v polysorbate, such as polysorbate 80;

[0228] 1%-5% w / v proline or a pharmaceutically acceptable salt thereof;

[0229] and 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;

[0230] 19) The pharmaceutical composition of any one of 6) to 9), wherein the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 15-130 mg / mL or 15-132 mg / mL;

[0231] 20) The pharmaceutical composition of any one of 15) to 18) above, wherein the concentration of the anti-CD40 antibody or antigen-binding fragment thereof is 30-190 mg / mL;

[0232] 21) The pharmaceutical composition according to any one of 1) to 20) above, further comprising water for injection.

[0233] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate, which comprises or is any one of the following 1) to 6):

[0234] 1) 0.1-300 mg / mL antibody-drug conjugate;

[0235] 1-50 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0236] 0.001%-1% w / v polysorbate, such as polysorbate 80;

[0237] 0.1%-30% w / v sucrose or trehalose;

[0238] and the pH of the composition is 4.5-7.5;

[0239] 2) 1-150 mg / mL antibody-drug conjugate;

[0240] 5-40 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0241] 0.005%-1% w / v polysorbate, such as polysorbate 80;

[0242] 0.5%-20% w / v sucrose or trehalose;

[0243] and the pH of the composition is 5-7.2;

[0244] 3) 5-120 mg / mL antibody-drug conjugate;

[0245] 10-30 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0246] 0.008%-0.2% w / v polysorbate, such as polysorbate 80;

[0247] 1%-15% w / v sucrose or trehalose;

[0248] and the pH of the composition is 5.5-7;

[0249] 4) 10-100 mg / mL antibody-drug conjugate;

[0250] 15-25 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0251] 0.01%-0.1% w / v polysorbate, such as polysorbate 80;

[0252] 5%-10% w / v sucrose or trehalose;

[0253] and the pH of the composition is 5.6-6.6;

[0254] 5) Any one of the above 1) to 4), wherein the antibody-drug conjugate has the structure shown below:

[0255] Among them, 9E6-L4H2 is an anti-CD40 antibody, which includes a heavy chain and a light chain having amino acid sequences as shown in SEQ ID NOs: 20 and 21;

[0256] wherein k is selected from an integer or decimal from 1 to 10, for example, k is an integer or decimal from 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.

[0257] 6) The pharmaceutical composition according to any one of 1) to 5) above, further comprising water for injection.

[0258] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-CD40 antibody or an antigen-binding fragment thereof (e.g., 9E6 (L4H2) whose heavy chain and light chain sequences are SEQ ID NOs: 20 and 21), which comprises or is any one of the following groups 1) to 19):

[0259] 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;

[0260] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0261] About 0.02% w / v polysorbate 80;

[0262] about 7.5% w / v sucrose;

[0263] and the pH of the composition is about 6.0;

[0264] 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;

[0265] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0266] About 0.02% w / v polysorbate 80;

[0267] about 7.5% w / v sucrose;

[0268] and the pH of the composition is about 6.1;

[0269] 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;

[0270] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0271] About 0.02% w / v polysorbate 80;

[0272] about 7.5% w / v sucrose;

[0273] and the pH of the composition is about 6.2;

[0274] 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;

[0275] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0276] About 0.02% w / v polysorbate 80;

[0277] about 7.5% w / v sucrose;

[0278] and the pH of the composition is about 6.3;

[0279] 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;

[0280] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0281] About 0.02% w / v polysorbate 80;

[0282] about 7.5% w / v sucrose;

[0283] and the pH of the composition is about 6.5;

[0284] 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;

[0285] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0286] About 0.02% w / v polysorbate 80;

[0287] about 7.5% w / v sucrose;

[0288] and the pH of the composition is about 6.6;

[0289] 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;

[0290] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0291] About 0.02% w / v polysorbate 80;

[0292] About 8% w / v sucrose;

[0293] and the pH of the composition is about 6.0;

[0294] 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;

[0295] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0296] About 0.02% w / v polysorbate 80;

[0297] About 8% w / v sucrose;

[0298] and the pH of the composition is about 6.1;

[0299] 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;

[0300] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0301] About 0.02% w / v polysorbate 80;

[0302] About 8% w / v sucrose

[0303] and the pH of the composition is about 6.2;

[0304] 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;

[0305] About 20 mM histidine-HCl buffer, such as histidine-histidine HCl;

[0306] About 0.02% w / v polysorbate 80;

[0307] About 8% w / v sucrose;

[0308] and the pH of the composition is about 6.3;

[0309] 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;

[0310] About 20 mM histidine-HCl buffer, such as histidine-histidine HCl;

[0311] About 0.02% w / v polysorbate 80;

[0312] About 8% w / v sucrose;

[0313] and the pH of the composition is about 6.5;

[0314] 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;

[0315] About 20 mM histidine-HCl buffer, such as histidine-histidine HCl;

[0316] About 0.02% w / v polysorbate 80;

[0317] About 8% w / v sucrose;

[0318] and the pH of the composition is about 6.6;

[0319] 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;

[0320] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0321] About 0.02% w / v polysorbate 80;

[0322] about 2.5% w / v proline;

[0323] and the pH of the composition is about 6.0;

[0324] 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;

[0325] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0326] About 0.02% w / v polysorbate 80;

[0327] about 2.5% w / v proline;

[0328] and the pH of the composition is about 6.1;

[0329] 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;

[0330] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0331] About 0.02% w / v polysorbate 80;

[0332] about 2.5% w / v proline;

[0333] and the pH of the composition is about 6.2;

[0334] 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;

[0335] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0336] About 0.02% w / v polysorbate 80;

[0337] about 2.5% w / v proline;

[0338] and the pH of the composition is about 6.3;

[0339] 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;

[0340] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0341] About 0.02% w / v polysorbate 80;

[0342] about 2.5% w / v proline;

[0343] and the pH of the composition is about 6.5;

[0344] 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;

[0345] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0346] About 0.02% w / v polysorbate 80;

[0347] about 2.5% w / v proline;

[0348] and the pH of the composition is about 6.6;

[0349] 19) The pharmaceutical composition according to any one of 1) to 18) above, further comprising water for injection.

[0350] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate, which comprises or is any one of the following groups 1) to 8):

[0351] 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 antibody-drug conjugate;

[0352] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0353] About 0.02% w / v polysorbate 80;

[0354] about 7.5% w / v or about 9% w / v sucrose;

[0355] and the pH of the composition is about 6.0;

[0356] 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 antibody-drug conjugate;

[0357] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0358] About 0.02% w / v polysorbate 80;

[0359] about 7.5% w / v or about 9% w / v sucrose;

[0360] and the pH of the composition is about 6.3;

[0361] 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 antibody-drug conjugate;

[0362] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0363] About 0.02% w / v polysorbate 80;

[0364] about 7.5% w / v or about 9% w / v sucrose;

[0365] and the pH of the composition is about 6.6;

[0366] 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 antibody-drug conjugate;

[0367] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0368] About 0.02% w / v polysorbate 80;

[0369] About 7.5% w / v trehalose;

[0370] and the pH of the composition is about 6.0;

[0371] 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 antibody-drug conjugate;

[0372] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0373] About 0.02% w / v polysorbate 80;

[0374] About 7.5% w / v trehalose;

[0375] and the pH of the composition is about 6.3;

[0376] 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 antibody-drug conjugate;

[0377] About 20 mM histidine-hydrochloride buffer, such as histidine-histidine hydrochloride;

[0378] About 0.02% w / v polysorbate 80;

[0379] About 7.5% w / v trehalose

[0380] and the pH of the composition is about 6.6;

[0381] 7) Any one of the above 1) to 6), wherein the antibody-drug conjugate has the structure shown below:

[0382] Among them, 9E6-L4H2 is an anti-CD40 antibody, which includes a heavy chain and a light chain having amino acid sequences as shown in SEQ ID NOs: 20 and 21;

[0383] wherein k is selected from an integer or decimal from 1 to 10, for example, k is an integer or decimal from 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.

[0384] 8) The pharmaceutical composition according to any one of 1) to 7) above, further comprising water for injection.

[0385] In certain embodiments, the pharmaceutical composition of the present disclosure has a viscosity of 30 cp or less, such as a viscosity of 25 cp or less, 20 cp or less. In certain embodiments, the viscosity of the pharmaceutical composition of the present disclosure is suitable for injection.

[0386] The present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of mixing an anti-CD40 antibody or antigen-binding fragment thereof with a buffer. In certain embodiments, the buffer is a histidine-hydrochloride buffer (eg, histidine-histidine hydrochloride).

[0387] In certain embodiments, the pharmaceutical composition as described in any one of the above items is a liquid preparation. In some embodiments, the solvent of the liquid preparation is water, normal saline or glucose solution.

[0388] The present disclosure also provides a lyophilized preparation, characterized in that the lyophilized preparation can form any of the above pharmaceutical compositions after reconstitution.

[0389] The present disclosure also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition as described above.

[0390] The present disclosure provides a reconstitution solution, wherein the reconstitution solution is prepared by reconstitution of the aforementioned lyophilized preparation. In certain embodiments, the reconstitution solution is selected from but not limited to water for injection, physiological saline or glucose solution.

[0391] The present disclosure also provides a product, comprising a container containing the aforementioned pharmaceutical composition, the aforementioned lyophilized formulation, or the aforementioned reconstituted solution. In certain embodiments, the container is a neutral borosilicate glass injection vial. In certain embodiments, the product includes a package insert.

[0392] The present disclosure provides uses of the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized preparation, or the aforementioned reconstituted solution in medicines for treating or alleviating autoimmune diseases, graft-versus-host disease, or alleviating transplant rejection.

[0393] In certain embodiments, methods for improving or treating autoimmune diseases and inflammatory diseases and related pharmaceutical uses are provided, comprising administering the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized preparation, or the aforementioned reconstituted solution to a subject.

[0394] In certain embodiments, methods for treating CD40-related disorders and related pharmaceutical uses are provided; in certain embodiments, methods for inhibiting the growth or differentiation of cells with CD40-related disorders 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; in certain embodiments, methods for treating patients with immune disorders and related pharmaceutical uses are provided. The above methods all comprise administering the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized formulation, or the aforementioned reconstituted solution to the subject or cell.

[0395] In certain embodiments, methods for inducing peripheral B cell depletion and related pharmaceutical uses are provided, comprising administering to a subject the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized preparation, or the aforementioned reconstituted solution.

[0396] In certain embodiments, methods for treating or alleviating diseases or conditions and related pharmaceutical uses are provided, comprising administering the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized formulation, or the aforementioned reconstituted solution to a subject in need thereof. The diseases or conditions may be CD40-related or CD40-independent, including: 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), allergies, allergic reactions, Sjögren's syndrome, type I diabetes, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, inflammatory myositis, multiple endocrine failure, Schmidt's syndrome, me), 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), menon), esophageal dysmotility, sclerodactyly and telangiectasias), autoimmune infertility in men and women, ankylosing spondylitis, 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 miscarriage, antiphospholipid syndrome, farmer's pneumoconiosis, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird fan's lung, toxic epidermal necrolysis, Alport's syndrome me), 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, Sumpter's syndromesyndrome), eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue fever, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevate perstans, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, metachronous cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft-versus-host disease, transplant rejection, cardiomyopathy, Eaton-Lambert syndrome me), relapsing polychondritis, cryoglobulinemia, Waldenstrom macroglobulinemia, Evan's syndrome, acute respiratory distress syndrome, lung inflammation, osteoporosis, delayed-type hypersensitivity reactions, and autoimmune glandular failure. For example, Sjögren's syndrome, multiple sclerosis, and systemic lupus erythematosus.

[0397] In certain embodiments, methods and related pharmaceutical uses are provided for treating diseases related to 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, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft-versus-host disease), or Th2 lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft-versus-host disease), comprising administering the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized preparation, or the aforementioned reconstituted solution to a subject in need thereof.

[0398] In certain embodiments, methods for treating tumors or cancers and related pharmaceutical uses are provided, comprising administering an effective amount of the aforementioned anti-CD40 antibody or antigen-binding fragment thereof to a subject in need thereof, wherein the tumor or cancer may be related to or unrelated to CD40 expression.

[0399] In certain embodiments, a method for treating or alleviating graft-versus-host disease or transplant rejection is provided, comprising administering the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized preparation, or the aforementioned reconstituted solution and tacrolimus to a subject in need thereof.

[0400] In certain embodiments, the use of the aforementioned pharmaceutical composition, the pharmaceutical composition obtained by the aforementioned method, the aforementioned lyophilized formulation, or the aforementioned reconstituted solution for preparing a medicament for treating or alleviating graft-versus-host disease or transplant rejection is provided, including use in combination with tacrolimus. In certain embodiments, the use of tacrolimus for preparing a medicament for treating or alleviating graft-versus-host disease or transplant rejection is provided, including use in combination with the aforementioned pharmaceutical composition, the pharmaceutical composition obtained by the aforementioned method, the aforementioned lyophilized formulation, or the aforementioned reconstituted solution.

[0401] In certain embodiments, provided are methods for treating or alleviating graft-versus-host disease or transplant rejection using the aforementioned anti-CD40 antibodies or antigen-binding fragments thereof in combination with tacrolimus, as well as their use in preparing a medicament for treating or alleviating graft-versus-host disease or transplant rejection.

[0402] In certain embodiments, the transplant is a solid organ transplant, such as a kidney transplant, a liver transplant, a heart transplant, a lung transplant, a pancreas transplant, a small intestine transplant, or a composite tissue transplant.

[0403] In certain embodiments, the transplantation refers to transplantation of one selected from the group consisting of allogeneic cells, xenogeneic cells, allogeneic tissues, xenogeneic tissues, allogeneic organs, and xenogeneic organs.

[0404] In certain embodiments, the aforementioned pharmaceutical composition, the pharmaceutical composition prepared by the aforementioned method, the aforementioned lyophilized preparation or the aforementioned reconstituted solution inhibits or reverses the rejection reaction of the tissue transplant based on the transplant recipient, or prolongs or preserves the function of the tissue transplanted to the transplant recipient, or restores the function of the damaged transplanted tissue in the transplant recipient.

[0405] Definition of terms

[0406] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0407] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be construed to have an inclusive sense rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to."

[0408] "CD40" and "CD40 antigen" refer to an approximately 48 kD glycoprotein expressed on the surface of normal and neoplastic B cells that 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 can be found in Table 2 of the present 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.

[0409] "Antibody" is used in the broadest sense to encompass various 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), as long as they exhibit the desired antigen-binding activity. An antibody may refer to an immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to the amino acid composition and arrangement order of their heavy chain constant regions. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class of Ig, subclasses can be further divided 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 classified as either kappa or lambda chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of both the heavy and light chains of antibodies vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region consists of three hypervariable regions (CDRs) and four framework regions (FRs) whose sequences are relatively conserved. These three hypervariable regions determine the specificity of the antibody and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0410] For the determination or definition of CDRs, the deterministic depiction of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled 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 IMGT numbering system, contact definitions, and conformational definitions.

[0411] 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 positions 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 produced by the Oxford Molecular Group for modeling antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "Ab MT M, 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 primary sequence (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on analysis of available complex crystal structures (see, for example, 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, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used herein, CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art, and is exemplified by the following Table 1.

[0412] Table 1. Relationships between CDR numbering systems

[0413] The CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments of the present disclosure conform in number and position to the well-known Kabat numbering system.

[0414] "Monoclonal antibody" or "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies contained in the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic of an antibody as obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method.

[0415] The term "rabbit-derived antibody" as used herein refers to a monoclonal antibody against human CD40 or an epitope thereof, prepared according to the knowledge and skill in the art. These antibodies are prepared by injecting test rabbits with the CD40 antigen, and then isolating the antibodies expressing the desired sequence or functional properties. In a specific embodiment of the present disclosure, the rabbit-derived anti-human anti-CD40 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a rabbit-derived kappa or lambda chain, or variants thereof, or a heavy chain constant region of a rabbit-derived IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0416] The term "fully human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as 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 (such as a rabbit) have been transplanted onto human framework sequences (i.e., "humanized antibodies").

[0417] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by transplanting non-human CDR sequences into the human antibody variable region framework. This overcomes the strong immune response induced by chimeric antibodies due to the large number of non-human protein components they carry. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the fully human antibody variable region to maintain activity.

[0418] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of an antibody of a first species with the constant region of an antibody of a second species, which can reduce the immune response induced by the antibody of the first species. As an example, to establish a chimeric antibody, it is necessary to first establish a rabbit that secretes rabbit-derived specific monoclonal antibodies, isolate the antibodies, clone the constant region genes of the fully human antibodies as needed, connect the rabbit variable region genes to the human constant region genes into a chimeric gene, and then insert it into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of the fully human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgG1 or IgG4 heavy chain constant region, or using an IgG1 that is free of ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

[0419] 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 or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any 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 generating and preparing these antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009 / 040562, and its disulfide-stabilized form, Fab-dsFv, was first disclosed in WO2010 / 035012. Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. Multivalent antibodies may comprise multiple specificities, such as bispecifics, or may be monospecific (see, for example, WO92 / 22583 and WO05 / 113605), an example of the latter being Tri-Fab (or TFM) described in WO 92 / 22583.

[0420] The term "binds to CD40" refers to the ability to interact with CD40 or its epitope, which may be human. The term "antigen binding site" of the present disclosure refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.

[0421] The term "antigen" refers to a molecule used to immunize an immunocompetent vertebrate to generate antibodies that recognize the antigen, or to screen an expression library (e.g., particularly a phage, yeast, or ribosome display library). In the present disclosure, antigen is defined more broadly to include a target molecule specifically recognized by an antibody, as well as a portion or mimetic of a molecule used in an immunization process for generating antibodies or in screening libraries for selecting antibodies. For antibodies that bind to human CD40 of the present disclosure, monomers and multimers (e.g., dimers, trimers, etc.) of human CD40, as well as truncated and other variants of human CD40, are referred to as antigens.

[0422] The term "epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope generally comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitope is bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0423] The terms "specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when human CD40 or its epitope is used as an analyte and an antibody is used as a ligand, the antibody binds to the antigen with a specific binding affinity of less than 10 μg / L when measured by surface plasmon resonance (SPR) technology in an instrument. -7 M or even smaller equilibrium dissociation constant (K D ) binds to a predetermined antigen or an epitope thereof, and its affinity for binding to the predetermined antigen or an epitope thereof is at least twice that of its affinity for binding to a nonspecific antigen (such as BSA, etc.) other than the predetermined antigen (or its epitope) or a closely related antigen. The term "antibody that recognizes an antigen" can be used interchangeably with the term "specifically binding antibody" in this disclosure.

[0424] "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 can be determined by determining the dissociation constant (K D) can be quantified. KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, the surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and dissociation of the monovalent complex are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. K D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that 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 exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K values ​​were compared with those of non-target interactions (e.g., control antibodies known not to bind CD40). D The value is evaluated.

[0425] The term "conservative substitution" refers to a substitution with another amino acid residue having properties similar 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. In addition, 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 non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0426] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. Inhibition / blocking of CD40 preferably reduces or alters the normal level or type of activity that would occur if CD40 binding occurred in the absence of inhibition or blocking. Inhibition and blocking are also intended to encompass any measurable decrease in CD40 binding affinity when contacted with an anti-CD40 antibody, compared to CD40 not contacted with the anti-CD40 antibody.

[0427] "Inhibiting growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0428] "Agonist activity," "agonist activity," or "agonism" refers to the ability to function as an agonist. Binding of an agonist to a cellular receptor elicits a response or activity that is similar or identical to that elicited by the natural ligand for the receptor. For example, a CD40 agonist can induce any or all of the following responses: cell proliferation and / or differentiation; upregulation of cell-to-cell adhesion through molecules such as ICA M-1, E-selectin, VCA M, etc.; secretion of proinflammatory cytokines, such as IL-1, IL-6, IL-8, IL-12, TNF, etc.; signal transduction through the CD40 receptor through the following pathways, such as TRAF (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 MEK / ERK pathway, PI3K / Akt pathway, P38MAPK pathway, etc.; transduction of anti-apoptotic signals through molecules such as XIAP, Mcl-1, BCLx, etc.; generation of B and / or T cell memory; B cell antibody production; B cell isotype switching; upregulation of cell surface expression of class II MHC and CD80 / 86, etc. "Antagonist activity," "antagonist activity," or "antagonism" refers to the function of a substance that can act as an antagonist. For example, an antagonist of CD40 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 determining binding specificity and antagonistic activity of anti-CD40 antibodies and CD40 ligands are known to those skilled in the art and include, but are not limited to, standard competitive binding assays, assays for monitoring immunoglobulin secretion by B cells, B cell proliferation assays, Banchereau-like B cell proliferation assays, T cell helper assays for antibody production, co-stimulatory assays for B cell proliferation, and assays for upregulation of B cell activation markers.

[0429] Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the Cold Spring Harbor Laboratory Manual (Chapters 5-8 and 15). For example, mice or rabbits can be immunized with human CD40 or fragments 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 herein are engineered using genetic engineering methods to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website.

[0430] Antibodies can be competitively screened for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be conducted to obtain antibodies that compete or cross-compete with each other for antigen binding. A high-throughput method for obtaining antibodies that bind to the same epitope based on their cross-competition is described in International Patent Publication No. WO 03 / 48731. Thus, antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present disclosure for binding to the same epitope on CD40 can be obtained using conventional techniques known to those skilled in the art.

[0431] The term "disorder" is any condition that would benefit from treatment with the humanized anti-CD40 antibodies of the present disclosure. This includes both chronic and acute disorders or diseases. Non-limiting examples of disorders to be treated by the present disclosure include cancer, hematological malignancies, benign and malignant tumors, leukemias and lymphoid malignancies, as well as inflammatory disorders, angiogenic disorders, autoimmune disorders, and immunological disorders.

[0432] The term "CD40-associated disorder" or "CD40-associated disease" refers to a condition in which modification or elimination of cells expressing CD40 is indicated. These cells include CD40-expressing cells that exhibit abnormal proliferation or CD40-expressing cells that are associated with cancerous or malignant growth. More specific examples of cancers that exhibit abnormal expression of the CD40 antigen include B lymphoblasts, 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 kidney cancer. Such disorders include, but are not limited to, leukemias, lymphomas (including B-cell lymphomas and non-Hodgkin's lymphomas), multiple myeloma, Waldenstrom's macroglobulinemia; solid tumors, including sarcomas, such as osteosarcoma, Ewing's sarcoma, malignant melanoma, adenocarcinomas (including ovarian adenocarcinoma), Kaposi's sarcoma / Kaposi's tumor, and squamous cell carcinoma. "CD40-related disorders" also include immune system diseases and disorders, such as autoimmune disorders and inflammatory disorders. Such conditions include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), lung inflammation, asthma, and idiopathic thrombocytopenic purpura (ITP).

[0433] The term "arresting 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, for example, significantly reduces the percentage of neoplastic cells in the S phase.

[0434] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the 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, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0435] "Cell," "cell line," and "cell culture" are 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 deliberate or unintentional mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0436] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the specified sequence may but need not be present.

[0437] The "CD40 binding molecules" of the present disclosure are interpreted in a broad sense and include the anti-CD40 antibodies or antigen-binding fragments thereof of the present disclosure. As long as the protein can bind to CD40, it is within the scope of this term. For example, the CD40 binding protein can contain one or more effector molecules, for example, in a conjugated manner. The "effector molecule" can have therapeutic activity (for example, anti-tumor activity or immune activation or inhibition activity) or detection function alone, and can be in any form, such as biologically active proteins (for example, enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, polynucleotides and fragments thereof such as DNA, RNA and fragments thereof, radionuclides (especially radioiodides), radioisotopes, chelated metals, nanoparticles and reporter groups (for example, fluorescent compounds), or compounds that can be detected by NMR or ESR spectroscopy. The conjugation of effector molecules to the anti-CD40 antibodies or antigen-binding fragments thereof of the present disclosure can be achieved by conventional methods.

[0438] The term "antibody-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker. In this disclosure, an "antibody-drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment linked to a drug molecule via a stable linker. The antibody or antibody fragment can be bound to the drug molecule containing a linker via specific groups therein (e.g., interchain disulfide bonds).

[0439] The term "drug loading" refers to the average number of drugs carried by each antibody-drug conjugate molecule in an antibody-drug conjugate population, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading range can be 1-20, preferably 1-10 drug molecules (D) connected to each antibody (Ab). In an embodiment of the present disclosure, the drug loading is expressed as k, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean of any two values. Preferably 1-10, more preferably 1-8, or 2-8, or 2-7, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or 4-5 mean. The average amount of drug per ADC molecule after the conjugation reaction can be determined by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, mAb size variant determination (CE-SDS), and HPLC characterization.

[0440] The disclosed monoclonal antibody size variant determination method (CE-SDS) can employ capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) ultraviolet detection to quantitatively determine the purity of recombinant monoclonal antibody products based on molecular weight under reducing and non-reducing conditions according to capillary electrophoresis (Chinese Pharmacopoeia 2015 edition, 0542).

[0441] In one embodiment of the present disclosure, the drug molecules are coupled to the N-terminal amino group and / or the ε-amino group of the lysine residue of the ligand via a linker. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.

[0442] The loading capacity of the antibody-drug conjugate can be controlled by the following non-limiting methods, including:

[0443] (1) Control the molar ratio of the linker and the monoclonal antibody,

[0444] (2) Control reaction time and temperature,

[0445] (3) Select different reaction reagents.

[0446] As used herein, the term "about" or "approximately" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation per practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to ±20%, for example, a pH of about 5.5 means pH 5.5 ± 1.1. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times a value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0447] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include tris (Tris), acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0448] "Citrate buffer" is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate buffer.

[0449] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. Preferred are 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.

[0450] "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, and the like. Preferably, the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0451] "Tris buffer" is a buffer containing tris (Tris). Examples of the Tris buffer include Tris-hydrochloric acid buffer (Tris-HCl), Tris-acetic acid buffer (Tris-AA), Tris-succinic acid buffer (Tris-SA), and Tris-citrate buffer (Tris-CA).

[0452] "Citrate buffer" is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

[0453] A "succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate. For example, the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0454] An "acetate buffer" is a buffer comprising 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, and the like. A preferred acetate buffer is acetic acid-sodium acetate.

[0455] A "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the active ingredient, facilitate administration to an organism, and promote absorption of the active ingredient to exert its biological activity.

[0456] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0457] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described in the present disclosure is water.

[0458] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0459] The pharmaceutical compositions disclosed herein can achieve a stable effect: the anti-CD40 antibody or antigen-binding fragment thereof substantially retains its physical stability and / or chemical stability and / or biological activity after storage. For example, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Various analytical techniques are currently available for measuring protein stability after storage at a selected temperature for a selected period of time.

[0460] A stable pharmaceutical formulation is one in which no significant changes are observed after storage at refrigerated temperatures (2-8°C) for at least 3 months, at least 6 months, at least 1 year, at least 2 years, or up to 2 years. In addition, a stable liquid formulation includes a liquid formulation that exhibits desired characteristics after storage at 25°C for 1 month, 3 months, 6 months, or at 40°C for 1 month. Typical acceptable standards for stability are as follows: as measured by SEC-HPLC, typically no more than about 10%, for example, no more than about 5%, of the antibody monomers degrade. By visual analysis, the pharmaceutical formulation is colorless or clear to slightly milky white. The concentration, pH, and weight-gram molecular osmotic pressure concentration of the formulation vary by no more than ±10%. Typically, no more than about 10%, for example, no more than about 5%, of truncation is observed, and typically no more than about 10%, for example, no more than about 5%, of aggregation is formed.

[0461] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured 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 can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0462] An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0463] A GLP-1 analogue "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared. The biological activity of the antibody can be determined, for example, by antigen binding assays.

[0464] "Administer," "give," and "treat," as they apply to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as it applies to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0465] "Treatment" means administering an internal or external therapeutic agent, such as a pharmaceutical composition comprising any of the present disclosure, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, a therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent. The amount of a therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0466] The equipment and methods used in the testing process are as follows:

[0467] Appearance:

[0468] Using the visual method, wipe the sample bottle clean and observe the sample color, clarity and visible foreign matter on a clarity tester with a white background and a black background under a light intensity of 1000-1500lx.

[0469] Appearance inspection instrument: Jingtuo Instrument YB-2A clarity tester.

[0470] pH value:

[0471] Using the potentiometric method, take 100 μL of sample and measure the pH value using a micro pH meter calibrated with standard solution.

[0472] pH detection instrument: Mettler Toledo, model S210.

[0473] SEC size exclusion chromatography:

[0474] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0475] SEC monomer content percentage = A monomer / A total * 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas.)

[0476] SEC instrument: Agilent 1260-Bio; chromatographic columns: Waters, XBrige SEC (300×7.8mm 3.5μm)

[0477] NR-CE capillary gel electrophoresis:

[0478] A method of electrophoresis in which gel is transferred to a capillary tube as a supporting medium and samples are separated according to their molecular weight at a certain voltage.

[0479] Non-reduced CE purity percentage = A main peak / A total * 100% (A main peak is the peak area of ​​the main peak in the sample, and A total is the sum of all peak areas.

[0480] CE measurement instrument: Sciex model PA800plus

[0481] icIEF imaging capillary isoelectric focusing electrophoresis:

[0482] A technique for separating proteins based on their isoelectric points (pI).

[0483] icIEF main peak content percentage = main peak area / total area*100% (total area is the sum of the areas of acidic peak, main peak and basic peak).

[0484] The instrument used for icIEF determination was manufactured by Protein Simple, model number: Muarice.

[0485] Protein concentration determination:

[0486] Protein concentration was determined using a UV-visible spectrophotometer (Nano Drop 2000) with a pathlength of 1 mm.

[0487] Melting temperature (Tm) and aggregation temperature (Tagg):

[0488] Tm is the temperature at which 50% of the protein components are denatured during heating; Tagg is the temperature at which protein aggregation occurs during heating. Load the sample into a Unitube and run the measurement at a 25-95°C heating ramp. Tm and Taag measurements were performed by Uncle Instruments, manufactured by Unchained.

[0489] Viscosity determination:

[0490] The rheometer measures sample viscosity. 35 μL of sample is placed in a test cell and inserted into the instrument. Pressure is then applied to push the sample through microfluidic channels. Software tracks the sample's speed in each channel, providing a measure of its viscosity.

[0491] Testing instrument: Honybun, instrument manufacturer is Unchained.

[0492] Exemplary Antibody Pharmaceutical Composition (Formulation) Preparation Process

[0493] Step 1: Anti-CD40 antibodies are obtained through cell culture and purification. The obtained anti-CD40 antibodies are subjected to ultrafiltration / concentration (UF / DF) and formulated with a buffer, a stabilizer and / or a surfactant to form a preparation solution containing anti-CD40 antibodies. The solution is sterilized and filtered through a 0.22 μm filter element, and the filtrate is collected.

[0494] Step 2: Adjust the filling volume (target filling volume is 2.835 mL / bottle), select penicillin bottles for filling, and take samples at the beginning, middle, and end of filling to detect the difference in filling volume.

[0495] Step 3: Add the stopper, start the capping machine, add the aluminum cap, and start capping.

[0496] Step 4: Visual inspection to confirm that the product has no defects such as inaccurate filling quantity and poor appearance. Print carton labels, fold cartons, pack cartons, and apply carton labels. BRIEF DESCRIPTION OF THE DRAWINGS

[0497] Figure 1A to Figure 1B: Activity results of CD40 antagonist antibodies in a reporter gene system. Figure 1A and Figure 1B are the relevant results for 9E6-L4H2 and 2F12-L4H2, respectively. Both Figure 1A and Figure 1B use human IgG1 isotype as a negative control and CFZ533 as a positive control.

[0498] Figures 2A to 2B: Inhibitory activity results of CD40 antagonist antibodies in a B cell activation experimental system. Figure 2A shows the inhibition results of CD19+CD69+ cell percentages of 9E6-L4H2 and 2F12-L4H2. Figure 2B shows the inhibition results of CD19+CD69+ cells MFI of 9E6-L4H2 and 2F12-L4H2. Both Figures 2A and 2B use human IgG1 isotype as a negative control and CFZ533 as a positive control.

[0499] Figures 3A to 3D: The inhibitory activity results of CD40 antagonist antibodies in the DC cell activation experimental system. Figure 3A is the CD11C+CD80+ cell MFI inhibition result of 9E6-L4H2 and 2F12-L4H2, Figure 3B is the CD11C+CD86+ cell MFI inhibition result of 9E6-L4H2 and 2F12-L4H2, Figure 3C is the IL-12 / 23p40 inhibition result of 9E6-L4H2 and 2F12-L4H2, Figure 3D is the TNFα inhibition result of 9E6-L4H2 and 2F12-L4H2, and Figures 3A-3D all use human IgG1 isotype as negative control and CFZ533 as positive control.

[0500] Figure 4: Results of endogenous agonist activity of CD40 antagonist antibodies 9E6-L4H2 and 2F12-L4H2 in a B cell activation assay, using human IgG1 isotype, CFZ533, and agonist anti-CD40 antibody 9E5-SELFNS as controls.

[0501] Figures 5A to 5B show the activity results of CD40 antagonist antibodies in a mouse T cell-dependent humoral immune response model. Figure 5A is a flow chart, and Figure 5B is a graph showing the test results on days 7, 14, 21, and 28.

[0502] Figures 6A to 6B show the activity of CD40 antagonist antibodies in a mouse skin transplant rejection model. Figure 6A is a flow chart, and Figure 6B shows the skin transplant survival rate (%) and skin transplant score results.

[0503] Figure 7: Activity results of CD40 antagonist antibodies combined with tacrolimus (FK506) in a mouse skin transplant rejection model. Figure 7 A shows the skin graft survival rate (%) of 9E6-L4H2 (10 mpk) and its combination with tacrolimus (FK506). Figure 7 B shows the skin graft survival rate (%) of 2F12-L4H2 (10 mpk) and its combination with tacrolimus (FK506). Figure 7 C shows the skin graft score results of 9E6-L4H2 (10 mpk) and its combination with tacrolimus (FK506). Figure 7 D shows the skin graft score results of 2F12-L4H2 (10 mpk) and its combination with tacrolimus (FK506).

[0504] FIG8 : PK test results of CD40 antagonist antibodies 9E6-L4H2 and 2F12-L4H2 in human CD40 transgenic mice, using CFZ533 as a control.

[0505] FIG9A : The inhibitory activity results of Fc-mutated CD40 antagonist antibodies in a B cell activation experimental system. FIG9A shows the CD19+CD69+ cell MFI inhibition results of 9E6-L4H2 and 9E6-L4H2-AAYTE.

[0506] Figures 10A to 10B: The inhibitory activity results of Fc-mutated CD40 antagonists in the DC cell activation experimental system. Figure 10A shows the results of 9E6-L4H2 and 9E6-L4H2-AAYTE inhibiting IL-12 / 23p40, and Figure 10B shows the results of 9E6-L4H2 and 9E6-L4H2-AAYTE inhibiting TNFα.

[0507] Figure 11: Flowchart of anti-CD40-ADC skin transplant rejection experiment in mice DETAILED DESCRIPTION

[0508] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present disclosure.

[0509] Experimental methods in the disclosed embodiments or test examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0510] The preparation and purification methods of the anti-CD40 antibodies in this application have been described in international patent application WO2023274201, and the entire contents of the aforementioned application documents can be incorporated into the present disclosure.

[0511] Example 1. CD40 immune antigen, sequence and preparation of screening antigen

[0512] His-tagged human CD40 (h-CD40-his) recombinant protein (Cat. No. CD0-H5228), mouse Fc-tagged human CD40 (h-CD40-mFc) recombinant protein (Cat. No. CD0-H525a), his-tagged and biotin-tagged human CD40 (hCD40-his-avi) recombinant protein (Cat. No. CD0-H82E8), and his-tagged cynomolgus monkey CD40 (cyno-CD40-his) recombinant protein (Cat. No. CD0-C52H6) were all purified commercial protein reagents purchased from Acrobiosystems. The sequence sources of each are shown in Table 2. These protein reagents can be used in the experiments in the following examples.

[0513] Table 2. Sources of recombinant protein amino acid sequences

[0514] Example 2. Screening of anti-CD40 rabbit monoclonal antibodies and preparation of human-rabbit chimeric antibodies

[0515] Anti-human CD40 monoclonal antibodies were produced by immunizing two New Zealand White rabbits. The immunizing antigen was a His-tagged recombinant human CD40 protein (h-CD40-his, prepared at 1 μg / μl in phosphate-buffered saline). The antigen was emulsified with Freund's complete adjuvant (CFA) for the first immunization and incomplete Freund's adjuvant (IFA) for subsequent boosters. Each immunization involved multiple subcutaneous injections of 400 μg of antigen. Immunizations occurred on days 0, 7, 20, and 41. Blood samples were collected on days 27 and 48, and antibody titers in rabbit sera were determined by ELISA and FACS. Rabbits with high serum antibody titers that plateaued were selected for a booster immunization on day 63, with an intravenous injection of 400 μg / rabbit of the antigen solution in phosphate-buffered saline. On day 67, spleens from these rabbits were harvested, biotin-tagged CD40 antigen was added, and labeled monoclonal B cells were sorted by flow cytometry and transferred to 96-well plates. After 14 days of culture, supernatants were collected and screened for clones capable of binding to human CD40, cynomolgus macaque CD40, and Raji cells (a tumor cell line expressing human CD40) using ELISA and FACS. A total of 28 B cell monoclonal clones were obtained. RNA was extracted from these monoclonal cells, reverse transcribed, amplified by PCR, and then sent to a sequencing company for sequencing. The sequences of 28 rabbit-derived antibodies were ultimately obtained. Affinity and activity assays were performed (see Examples 2-3 for methods), and monoclonal antibodies were screened. The heavy and light chain variable region sequences are shown in Table 3, and the CDR sequences are shown in Table 4.

[0516] Table 3. Anti-CD40 rabbit monoclonal antibody variable region sequences

[0517] (Note: The underlined CDR regions of the heavy and light chain variable regions are determined using the Kabat numbering rules)

[0518] Table 4. CDR regions of anti-CD40 rabbit monoclonal antibodies (Kabat numbering convention)

[0519] The obtained variable region sequence is connected to the human antibody IgG1 constant region sequence (with N297A mutation, Eu numbering system) and the human kappa chain constant region sequence to obtain a human-rabbit chimeric antibody sequence. Using molecular cloning technology, the chimeric antibody sequence is inserted into an expression vector, and the human-rabbit chimeric antibody can be obtained using the HEK293 cell expression system.

[0520] Example 3. Human-rabbit chimeric anti-CD40 antibody Raji cell binding experiment

[0521] Raji cells are a tumor cell line that overexpresses human CD40. 2E5 Raji cells were seeded onto a 96-well plate. 100 μL of the antibody to be tested was added, with the highest final concentration being 100 nM, diluted 5-fold, for a total of 8 concentrations, and incubated at 4°C for 1 hour. Wash once with washing solution, add anti-human IgG antibody conjugated to AF647 (Jackson Immunoresearch Laboratories, catalog number 205-609-088) at a dilution of 1:500, and incubate at 4°C for 30 minutes. Wash once with washing solution, and read the fluorescence intensity by flow cytometry. Calculate the binding EC value of the anti-CD40 antibody to CD40 50 The anti-CD40 antagonist antibody CFZ533 (ie, Iscali mab, Nova) was used as a positive control, and the heavy chain and light chain variable region sequences were derived from sequence 5 and sequence 2 of US8277810B, respectively.

[0522] The results in Table 5 show that 9E6 has a stronger binding EC than CFZ533. 50 .

[0523] Table 5. FACS binding of human-rabbit chimeric anti-CD40 antibodies to Raji cells EC 50 (n M)

[0524] Example 4 Human-rabbit chimeric anti-CD40 antibody reporter gene cell activity experiment

[0525] HEK-Blue CD40L cells were purchased from Invivogen (Cat#hkb-cd40). The cells were stably transfected with the human CD40 gene and the NF-kB-mediated SEAP genome. The SEAP substrate QUANTI-Blue was used to detect the SEAP secreted in the supernatant to characterize the activation level of the CD40 signaling pathway. This experiment detected the activation of HEK-Blue CD40L cells by CD40L. According to IC 50 To evaluate the in vitro antagonist activity of anti-CD40 antibodies, HEK-Blue CD40L cells were cultured in DMEM medium supplemented with 10% FBS, 100 μg / mL Zeocin, and 30 μg / mL Blasticidin. The cells were passaged two to three times a week at a 1:5 or 1:10 ratio. During passage, the medium was aspirated, and the cell layer was rinsed with 5 mL of 0.25% trypsin. The trypsin was then aspirated, and the cells were incubated in an incubator for 3 to 5 minutes before being resuspended in fresh medium. 100 μL of the cell suspension at a density of 5 × 10^5 cells / mL was added to a 96-well cell culture plate in DMEM supplemented with 10% FBS, 100 μg / mL Zeocin, and 30 μg / mL Blasticidin. Only 100 μL of sterile water was added to the periphery of the 96-well plate. The plates were incubated in an incubator for 24 hours (37°C, 5% CO2). After the cells adhere, add 100 μl of serially diluted test antibodies to each well and incubate at 37°C for 30 minutes. Add 25 ng / mL CD40L (R&D, Catalog No. 2706-CL) and 2 μg / mL anti-His antibody (R&D, Catalog No. MAB050), and incubate the culture plate in an incubator for 20-24 hours (37°C, 5% CO2). Transfer 20 μl of cell supernatant to each well of a new 96-well flat-bottom plate, add 180 μl of QUANTI-Blue substrate solution, and incubate the culture plate in a dark incubator for 1-3 hours. Measure the absorbance at 630 nm using a microplate reader (Thermo MμltiSkanFc) to calculate the IC 50 The values ​​were used to evaluate the in vitro cellular activity of anti-CD40 antibodies.

[0526] See Table 6. The results show that 9E6 has comparable or slightly stronger reporter gene cell inhibitory activity IC than CFZ533. 50 .

[0527] Table 6. IC of anti-CD40 antibody reporter gene cell activity 50 (nM)

[0528] Example 5. Humanization of anti-CD40 antibodies

[0529] The heavy and light chain variable region sequences of the rabbit antibody 9E6 were compared with the GermLine database to obtain human germline templates with high homology. The human germline template information for the final humanized antibody is shown in Table 7.

[0530] Table 7. Human germline template information for antibody humanization process

[0531] The CDR region of the rabbit antibody is transplanted onto the selected human germline template, replacing the human germline variable region, and then recombined with the corresponding human IgG constant region (preferably the heavy chain is IgG1 with an N297A mutation, and the light chain is κ). Then, based on the three-dimensional structure of the rabbit antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important influence on the conformation of VL and VH are reverse mutated, and the potential post-translational modification risk points are mutated to obtain the final humanized molecule. The humanized light and heavy chain variable region sequences corresponding to 9E6 are shown as an example (see Table 8), where L represents the light chain, H represents the heavy chain, and the numbers after L and H represent different versions of the humanized sequence containing different reverse mutations.

[0532] Table 8. Sequences of the heavy and light chain variable regions of humanized 9E6

[0533] (The CDRs are underlined according to the Kabat numbering convention.)

[0534] During the humanization process of 9E6, the following LCDR3 was obtained:

[0535] >9E6-L1 LCDR3

[0536] QGGYWTSTSNFGNV (SEQ ID NO: 15)

[0537] >9E6-L2 LCDR3

[0538] QGGYWTSTSNFGSG (SEQ ID NO: 16)

[0539] >9E6-L3 LCDR3

[0540] QGGYWTSTSNFGTG (SEQ ID NO: 17)

[0541] >9E6-L4 LCDR3

[0542] QGGYWTSTSNFGQG (SEQ ID NO: 18)

[0543] >LCDR3 general formula of 9E6-L

[0544] QGGYWTSTSNFGX9X 10 (SEQ ID NO: 19), wherein X9 is selected from N, S, T or Q, X 10 Select from V or G.

[0545] Each humanized version of the rabbit antibody was paired, expressed, and purified for each light and heavy chain. The naming convention is a combination of the heavy and light chain variable region numbers. For example, "9E6-L1H2" represents an antibody with SEQ ID NO: 9 for the VH and SEQ ID NO: 13 for the VL. The light and heavy chain constant regions were based on the sequences of the human IgG1 constant region (with the N297A mutation, Eu numbering system) and the human kappa chain constant region, respectively.

[0546] The sequence of an exemplary humanized molecule 9E6-L4H2 (using the 9E6-L4 light chain and the 9E6-H2 heavy chain) is as follows. The light and heavy chain constant regions thereof adopt the sequence of the human IgG1 constant region (with N297A mutation, Eu numbering system) and the sequence of the human kappa chain constant region, respectively.

[0547] >9E6-L4H2 HC:

[0548] >9E6-L4H2 LC:

[0549] The heavy chain constant region of the above-mentioned antibody was modified to use a human IgG1 constant region with five point mutations: L234A, L235A, M252Y, S254T, and T256E (Eu numbering system). The light chain constant region and the light and heavy chain variable regions remained unchanged. The newly generated antibody is 9E6-L4H2-AAYTE, and its complete heavy chain sequence is shown below:

[0550] >9E6-L4H2-AAYTE HC:

[0551] The underlined regions are the heavy or light chain constant regions.

[0552] An exemplary IgG Fc is shown below:

[0553] >IgG1-Fc(N297A)

[0554] >IgG1-Fc(AAYTE)

[0555] Example 6. Humanized anti-CD40 antibody Raji cell binding assay

[0556] Raji cells highly express human CD40 on their surface, which can be used to detect the binding characteristics of CD40 antagonist antibodies to human CD40 on the cell surface.

[0557] Raji cells were plated at 1.5E5 per well and incubated with various concentrations of CD40 antagonist antibodies at 4°C for 1 hour. After washing twice with FACS buffer (PBS + 2% FBS), secondary antibodies (Alexa Flour 488-conjugated anti-human IgG (H+L) antibodies) were added and incubated at 4°C for 0.5 hour. After washing twice with FACS buffer, cell surface fluorescence intensity was measured using a flow cytometer (BD FACS Celesta).

[0558] The results are shown in Table 9, in which 9E6-L4H2 and CFZ533 have similar binding abilities to human CD40 on the surface of Raji.

[0559] Table 9. FACS binding of humanized anti-CD40 antibodies to Raji cells 50 result

[0560] Example 7. Determination of the affinity of anti-CD40 antibodies to human CD40 and cynomolgus monkey CD40

[0561] The test antibody was affinity-captured on an anti-human Fc chip. A His-tagged human or cynomolgus macaque CD40 antigen was then passed over the chip surface at a concentration gradient. The reaction signal was monitored in real time using a Biacore instrument to generate binding and dissociation curves. The buffer used in the experiment was HBS-EP + 10× buffer (Cat.# BR-1006-69, GE), diluted to 1× (pH 7.4) with DI Water. The data were fitted with a (1:1) binding model to obtain affinity values, as shown in Table 10.

[0562] Compared with CFZ533, 9E6-L4H2 has a similar binding constant to human CD40, and 9E6-L4H2 has a stronger binding affinity to cynomolgus monkey CD40.

[0563] Table 10. SPR affinity of CD40 antagonistic antibodies to different CD40 strains

[0564] Example 8. Anti-CD40 antibody reporter gene cell activity experiment

[0565] HEK-Blue CD40L cells were purchased from Invivogen (Cat#hkb-cd40). The cells were stably transfected with the human CD40 gene and the NF-kB-mediated SEAP genome. The SEAP substrate QUANTI-Blue was used to detect the secreted SEAP content in the supernatant to characterize the activation level of the CD40 signaling pathway. This experiment was conducted to detect the inhibitory effect of CD40 antagonist antibodies on CD40L-induced HEK-Blue CD40L cell activation. According to the IC 50 The in vitro cellular activity of CD40 antagonistic antibodies was evaluated.

[0566] HEK-Blue CD40L cells were cultured in DMEM medium supplemented with 10% FBS, 100 μg / mL Normocin, 100 μg / mL Zeocin, and 30 pg / mL Blasticidin, and passaged two to three times a week. HEK-Blue CD40L cells were plated at 5E4 cells / well in a 96-well cell culture plate in DMEM medium supplemented with 10% FBS and 100 μg / mL Normocin and cultured overnight. After cell attachment, 100 μL of serially diluted test antibodies was added to each well and incubated at 37°C for 1 hour. 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 incubated in the dark for 15 minutes. The absorbance at 620 nm was measured using an Envision microplate reader to calculate the IC 50 The values ​​were used to evaluate the in vitro cellular activity of CD40 antagonistic antibodies.

[0567] As shown in Table 11 and Figures 1A and 1B, 9E6-L4H2 and CFZ533 had similar inhibitory activity against the reporter gene system, while 2F12-L4H2 had superior inhibitory activity to CFZ533. 2F12-L4H2 is another anti-CD40 antibody screened in this application.

[0568] Table 11. IC inhibition of CD40 reporter cell activity by anti-CD40 antibodies 50

[0569] Example 9. Inhibitory activity of anti-CD40 antibodies in a B cell activation experimental system

[0570] CD40 is highly expressed on B cells. Binding of CD40L to CD40 can induce B cell activation and upregulate the expression of a series of activation markers. CD40 antagonist antibodies block the binding of CD40L to CD40, thereby disrupting B cell immune activation.

[0571] Human PBMCs were plated at 2E5 cells / well in a 96-well cell culture plate (using RPMI-1640 medium, 10% FBS, and 1% penicillin-streptomycin). 50 μL of serially diluted test antibodies were added to each well and incubated at 37°C, 5% CO₂ for 0.5 hours. CD40L-his and anti-His antibodies were added to each well and stimulated overnight. The next day, the supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer and stained with 100 μL of a 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 a 1:200 dilution of human Fc blocker (BD, 564220) for 10 minutes at room temperature. After centrifugation, cells were added with 1:200 dilution of flow cytometry antibodies (PerCP / Cyanine 5.5 anti-human CD19 (Biolegend, 302230) and APC anti-human CD69 (Biolegend, 310910)) and incubated at 4°C for 0.5 h. The supernatant was removed by centrifugation, and cells were washed twice with FACS buffer and resuspended in 200 μL of PBS. Cell surface fluorescence intensity was measured using a flow cytometer (BD FACS Celesta).

[0572] As shown in Table 12 and Figures 2A and 2B, 9E6-L4H2 exhibited similar B cell activation inhibitory activity compared to CFZ533. 2F12-L4H2 exhibited stronger B cell inhibitory activity than CFZ533. 2F12-L4H2 is another anti-CD40 antibody screened in this application.

[0573] Table 12 Inhibitory activity of humanized anti-CD40 antibodies in B cell activation experimental system

[0574] After adding the antibody, the CD19+CD69+ signals were lower than the background value, which may be due to the inhibition of background activation of B cells to a certain extent (in addition to the inhibition of CD40L-induced signals).

[0575] Example 10. Inhibitory activity of anti-CD40 antibodies in DC cell activation experimental system

[0576] CD40 is highly expressed on dendritic cells (DCs). CD40L binding to CD40 can induce DC activation, upregulating the expression of multiple activation markers on the DC cell surface and promoting the secretion of various inflammatory factors, further amplifying the immune response. CD40 antagonistic antibodies block the binding of CD40L to CD40, thereby disrupting the immune activation process of DC cells.

[0577] Monocytes were isolated and enriched from fresh primary human peripheral blood mononuclear cells (PBMCs) using the EsaySepTM Human CD14 Isolation Kit (Stemcell, 19359). The cells were differentiated for 6 days using 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 DCs were plated at 1E5 cells / well in a 96-well cell culture plate. Serial dilutions of the test antibody were added to each well and incubated for 0.5 hours at 37°C, 5% CO2. CD40L-his and anti-His antibodies were then added to each well at a final concentration of 1:1. After 48 hours of culture, the activation level of DC cells was detected by flow cytometry: the supernatant was removed by centrifugation, the cells were washed twice with FACS buffer (PBS + 2% FBS), and 100 μL of 1:1000 diluted Fixable viability dye EF780 (Invitrogen, 65086514) was added and stained at room temperature for 15 minutes. After washing the cells twice, 100 μL of 1:200 diluted human Fc blocking (BD, 564220) was added and blocked at room temperature for 10 minutes. After centrifugation, the flow cytometry antibody (Alexa Fluor 5000) was added at 1:200 diluted. 700 anti-hu man CD11c (Biolegend, 337220), Brilliant Violet 421 TM Anti-human CD80 (Biolegend, 305221) and APC anti-human CD86 (Biolegend, 305412) were added and incubated at 4°C for 0.5 h. The supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer and resuspended in 200 μL of PBS. Cell surface fluorescence intensity was measured using a flow cytometer (BD FACS Celesta).

[0578] In addition, the secretion levels of cytokines in the supernatant were detected after 24 hours (TNFα, Cisbio, 62HTNFAPEG) and 48 hours (IL-12 / 23p40, Novus, VAL121) of culture.

[0579] The experimental results are shown in Table 13 and Figures 3A to 3D. 9E6-L4H2 and 2F12-L4H2 have similar DC cell inhibitory activity as the control antibody CFZ533. 2F12-L4H2 is another anti-CD40 antibody screened in this application.

[0580] Table 13. Inhibitory activity of humanized anti-CD40 antibodies in DC cell activation experimental system

[0581] Example 11. Agonistic activity of humanized anti-CD40 antibodies in a B cell activation experimental system

[0582] CD40 belongs to the TNF superfamily receptor. After binding to the ligand CD40L or being cross-linked by antibodies, it can mediate specific and nonspecific activation of downstream signaling pathways. Therefore, the background agonist activity of CD40 antagonistic antibodies on B cells can be detected without the addition of CD40L.

[0583] Human PBMCs were plated at 2E5 cells / well in a 96-well cell culture plate (100 μL per well in RPMI-1640 medium, 10% FBS, 1% penicillin-streptomycin). 100 μL of serially diluted test antibodies were added to each well and incubated overnight at 37°C in 5% CO2. The next day, the supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer. The cells were then stained with 100 μL of a 1:1000 dilution of Fixable Viability Dye EF780 for 15 minutes at room temperature. The cells were washed twice and blocked with 100 μL of a 1:200 dilution of human Fc blocking solution for 10 minutes at room temperature. After centrifugation, flow cytometry antibodies (PerCP / Cyanine 5.5 anti-human CD19 (Biolegend, 302230) and APC anti-human CD69 (Biolegend, 310910)) were added at a 1:200 dilution and incubated at 4°C for 0.5 hours. The supernatant was removed by centrifugation, the cells were washed twice with FACS buffer, and then resuspended in 200 μL PBS. The fluorescence intensity of the cell surface was detected by flow cytometer (BD FACS Celesta).

[0584] The results are shown in Figure 4. The CD40 agonistic antibody 9E5-SELFNS (WO2020108611A1) activated B cells in a dose-dependent manner, while 9E6-L4H2 and 2F12-L4H2 still did not show obvious B cell agonistic activity at 2.5 nM.

[0585] Example 12. Mouse T cell-dependent humoral immune response (TDAR) model

[0586] Female human CD40 transgenic mice, 6-7 weeks old, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. Housing: SPF; Production License: SCXK(Su)-2016-0004; Human CD40 Transgenic Mouse Qualification Certificate Number: 320726200100167773. Animals were acclimated for 7 days upon arrival and randomly assigned to groups. On day 0, one mouse in each group was bled and then administered intraperitoneally. On day 1, mice were immunized with 50 μg of KLH (KLH: Freund's complete adjuvant (CFA) emulsified immune complex, 1:1) per mouse. On day 15, mice were immunized with a second intraperitoneal injection of 50 μg of KLH (KLH: Freund's incomplete adjuvant (IFA) emulsified immune complex, 1:1) per mouse. Each group received intraperitoneal administration of drug twice weekly, with ~150 μL of blood collected via the orbital venous plexus on days 7, 14, 21, and 28. The whole blood was placed at room temperature for 1-4 hours, centrifuged at 7000 rpm for 10 minutes at 4°C to separate the serum, and stored at -80°C until use. The specific experimental process is shown in Figure 5A.

[0587] The specific dosing regimen is shown in Table 14. Mouse serum was separated every week and the level of anti-KLH specific IgG was detected by ELISA.

[0588] Table 14. Dosing regimen for mouse TDAR model experiment

[0589] As shown in Figure 5B and Table 15, 1 mg / kg of the CD40 antagonist antibody significantly inhibited the production of anti-KLH-specific IgG after two immunizations. Low doses of 0.3 mg / kg of 9E6-L4H2 and 2F12-L4H2 exhibited superior inhibitory effects on anti-KLH IgG production after two immunizations compared to the same dose of CFZ533. 2F12-L4H2 is another anti-CD40 antibody screened and identified in this application.

[0590] Table 15. Inhibitory effect of anti-CD40 antibodies on IgG production after immunization (0.3 mpk)

[0591] Example 13. Mouse skin transplant rejection model

[0592] Male Balb / c mice, 6 weeks old, were purchased from the Experimental Animal Management Department of the Shanghai Institute of Family Planning Science. Housing environment: SPF; Certificate Number: 20180006023393.

[0593] Female human CD40 transgenic mice, 6-7 weeks old, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. Housing environment: SPF; Certificate No.: 320726200100179778.

[0594] After arrival, the animals were acclimated for 7 days and randomly divided into groups. On day -2 of the experiment, mice were intraperitoneally injected with tacrolimus FK506 or CD40 antagonist antibodies. On day 0 of the experiment, donor Balb / c and C57BL6 / J mice were anesthetized with 4% chloral hydrate. The tails of the donor mice were removed, and a 1 cm long circle of tail skin was separated. The recipient mice were depilated, and an incision was made along the skin layer. While retaining the dorsal fat and connective tissue, an equal area of ​​skin was removed. The donor skin was placed on the incision of the recipient mouse, and the skin edges were sutured with glue. The recipient mice were placed in a cage to recover. The specific experimental process is shown in Figure 6A.

[0595] Mice were dosed according to the dosing regimen shown in Table 16. After 7 days of recovery, the survival of the skin was observed daily, and the rejection score was recorded based on the following scoring system: 3: smooth skin with no redness; 2: partially red, dull, and dry skin; 1: mostly red, without streaks, and shrunken skin; 0: transplant rejection resulting in 80% skin necrosis.

[0596] Table 16. Dosage regimen for mouse skin transplant rejection model

[0597] The results, as shown in Figure 6B and Tables 17-19, show that CD40 antagonist antibodies significantly improved mouse skin graft scores and prolonged skin graft survival compared to the model group. During skin graft scoring, since rejection could not be detected beforehand, scoring began on day 8. 9E6-L4H2 and 2F12-L4H2 demonstrated superior anti-graft rejection activity compared to the control antibody CFZ533. 2F12-L4H2 is another anti-CD40 antibody screened and obtained in this application.

[0598] Table 17. Skin graft survival rate on day 15 after anti-CD40 antibody (%)

[0599] Table 18. Anti-CD40 Antibody Skin Graft Scores (3 mpk)

[0600] Table 19. Anti-CD40 Antibody Skin Graft Scores (10 mpk)

[0601] In addition, as shown in FIG7 and Tables 20-22, the combination of 9E6-L4H2 and 2F12-L4H2 with tacrolimus further enhanced the efficacy against skin transplant rejection in mice.

[0602] Table 20. Skin graft survival on day 15 after combination of anti-CD40 antibody and tacrolimus

[0603] Table 21. Skin graft scores for the combination of anti-CD40 antibodies and tacrolimus

[0604] Table 22. Skin Graft Scores for Combination of Anti-CD40 Antibodies and Tacrolimus

[0605] Example 14. PK testing of humanized anti-CD40 antibodies in human CD40 transgenic mice

[0606] Female human CD40 transgenic mice, 6-7 weeks old, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. Housing: SPF; Production License: SCXK(Su)-2016-0004; Human CD40 Transgenic Mouse Qualification Certificate Number: 320726200100154632. Animals were acclimated for 7 days upon arrival and then randomly divided into groups. On day 0, mice in each group received an intraperitoneal injection of 10 mg / kg of anti-CD40 antibody. Blood samples (100-150 μL) were collected 15 minutes, 4 hours, and 8 hours after administration, and on days 1, 2, 4, 7, 10, and 14. Blood was anticoagulated with 10 μL of 0.1 M EDTA-K2 and stored on ice. Antibody concentrations in mouse plasma at different time points were determined by ELISA. The specific detection method is as follows: Dilute goat anti-human IgG Fc antibody (Rockland, Cat#609-101-017) to 2.5 μg / mL in PBS, add 50 μL / well to a 96-well plate, and incubate overnight at 4°C. After washing three times with the wash buffer, add 50 μL of blocking buffer to each well and incubate at 37°C for 1 hour. Add mouse plasma and a standard curve of the antibody to be tested, and incubate at 37°C for 2 hours. Wash three times with the wash buffer, add 50 μL / well of anti-hIgG Fab-HRP (Sigma, Cat#A0293, 1:10,000), and incubate at room temperature for 1 hour. Wash three times with the wash buffer. Add 100 μL of TMB to each well and react in the dark for 5 minutes. Add 100 μL of 0.16 M sulfuric acid to each well. Read the OD value at 450 nm using an Envision microplate reader to calculate the concentration of the CD40 antagonist antibody.

[0607] The results are shown in Table 23 and Figure 8. 9E6-L4H2, 2F12-L4H2, and the control molecule CFZ533 have similar PK characteristics in human CD40 transgenic mice. 2F12-L4H2 is another anti-CD40 antibody screened and obtained in this application.

[0608] Table 23. PK data of CD40 antagonist antibodies in hCD40 transgenic mice

[0609] Example 15. Determination of the affinity of humanized anti-CD40 antibodies to human FcRn

[0610] The test antibody was affinity-captured onto an anti-human Fab chip. A series of concentration gradients of human FcRn antigen (purchased from AcroBiosystem) were then passed over the chip surface. The signal was detected in real time using a Biacore instrument as the reaction reached steady-state. The buffer used in the experiment was HBS-EP + 10× buffer (Cat.# BR-1006-69, GE), diluted to 1× with DI Water (pH 7.4). The data were fitted to a steady-state binding model to generate affinity values, as shown in Table 24. Compared to the parent antibody, the anti-CD40 antibody carrying the AAYTE mutation exhibited higher binding affinity to human FcRn.

[0611] Table 24. Affinity determination of humanized anti-CD40 antibodies to human FcRn

[0612] Example 16. Inhibitory activity of anti-CD40 antibodies carrying AAYTE mutations in Fc in a B cell activation assay

[0613] The inhibitory activity of the anti-CD40 antibody 9E6-L4H2-AAYTE, carrying the AAYTE mutation on the Fc region, was tested in a B cell activation assay using the method described in Example 10. As shown in Figure 9A , the Fc-mutated anti-CD40 antibody exhibited similar B cell inhibitory activity to the parent anti-CD40 antibody.

[0614] Example 17. Inhibitory activity of anti-CD40 antibodies carrying AAYTE mutations in Fc in a DC cell activation experimental system

[0615] The inhibitory activity of the anti-CD40 antibody 9E6-L4H2-AAYTE, carrying the AAYTE mutation on the Fc region, was tested in a DC cell activation assay using the method described in Example 11. As shown in Figures 10A and 10B , the Fc-mutated anti-CD40 antibody exhibited similar DC cell inhibitory activity as the parent anti-CD40 antibody.

[0616] Example 18: Preparation of Antibody Drug Conjugate ADC-2 (9E6-L4H2-Compound 2)

[0617] 1. Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine tert-butyl ester (Compound 1b)

[0618] Place raw material 1a (5.14 g, 20.0 mmol, 1.0 eq) and L-phenylalanine tert-butyl ester hydrochloride (7.08 g, 20.0 mmol, 1.0 eq) in a 250 mL three-necked flask and add anhydrous DMF (60 mL) to dissolve. Cool in an ice bath, then add HATU (9.12 g, 24.0 mmol, 1.2 eq) and DIEA (7.74 g, 60.0 mmol, 3.0 eq). Maintain the ice bath and react for 2 hours. Add water to the reaction mixture, extract with ethyl acetate, and wash the organic phase with saturated brine, dry, and concentrate under reduced pressure to obtain product 1b (11.5 g, 100% yield) as a white solid. The crude product can be used directly in the next step.

[0619] MS (ESI): m / z 580.3 [M+Na] + .

[0620] 1 H NMR(400MHz,DMSO-d6)δ8.21-8.16(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).

[0621] 2. Preparation of Glycylglycyl-L-phenylalanine tert-butyl ester (Compound 1c)

[0622] Raw material 1b (5.57 g, 10.0 mmol, 1.0 eq) was placed in a 250 mL three-necked flask, and anhydrous DCM (60 mL) was added to dissolve the mixture. The mixture was then cooled in an ice bath. Piperidine (8.5 g, 100.0 mmol, 10.0 eq) was slowly added. After the addition, the mixture was stirred at room temperature for approximately 2 hours. The reaction solution was directly concentrated to dryness, and the crude product was purified by column chromatography to obtain product 1c as a light yellow slurry (2.70 g, 81% yield). MS (ESI): m / z 358.3 [M+H] + .

[0623] 3. Preparation of (1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentaoxo-4-tetraethylene glycol-19-oxy)glycylglycyl-L-phenylalanine tert-butyl ester (Compound 1d)

[0624] To a 100 mL three-necked flask, add 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentahydro-4-tetraethylene glycol-19-oleic acid (1.0 g, 2.05 mmol, 1.0 eq; source: Tonglai Biochemical, batch number: P110114) and 1c (0.69 g, 2.05 mmol, 1.0 eq). Dissolve in anhydrous DMF (25 mL) and cool in an ice bath. Add HATU (1.01 g, 2.67 mmol, 1.3 eq) and DIEA (529 mg, 4.10 mmol, 2.0 eq) and maintain the reaction in an ice bath for 1 hour. Add water to the reaction mixture, extract with ethyl acetate, wash with saturated brine, dry, and concentrate under reduced pressure to afford 1d as a light yellow oil (1.60 g, 97% yield).

[0625] MS (ESI): m / z 805.3 [M+H] + .

[0626] 4. Preparation of (1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentaoxo-4-tetraethylene glycol-19-oxy)glycylglycyl-L-phenylalanine (Compound 1e)

[0627] To a 100 mL single-necked flask, add raw material 1d (1.60 g, 1.99 mmol, 1.0 eq) and dissolve in anhydrous dichloromethane (16 mL). Add trifluoroacetic acid (8 mL) at room temperature and maintain stirring for 2 hours. The reaction solution is concentrated directly to dryness to obtain a yellow solid, which is dissolved in ethyl acetate, washed with saturated brine, dried, and concentrated under reduced pressure to obtain 1e as a yellow oil (1.23 g, 83% yield).

[0628] MS (ESI): m / z 749.3 [M+H] + .

[0629] 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]dioxolan-8b-yl)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (Compound 1f)

[0630] To a 100 mL three-necked flask, raw materials: budesonide (1.29 g, 3.0 mmol, 1.0 eq), methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (1.16 g, 3.15 mmol, 1.05 eq; prepared according to the reference “Tetrahedron, 2018, 74(15), 1951–1956”), and pyridinium p-toluenesulfonate (75 mg, 0.30 mmol, 0.1 eq) were added. Anhydrous tetrahydrofuran (20 mL) was added at room temperature, and the mixture was heated under reflux for 4 hours. The reaction solution was directly concentrated to dryness, and the crude product was purified by column chromatography to obtain product 1f as a white solid (0.54 g, yield 24%).

[0631] MS (ESI): m / z 739.4 [M+H] + .

[0632] 1 H NMR(400MHz,DMSO-d6)δ8.76-8.69(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=7.5Hz,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).

[0633] 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]dioxolan-8b-yl)-2-oxoethoxy)methyl)acetamide (Compound 1g)

[0634] Place raw material 1f (540 mg, 0.73 mmol, 1.0 eq) in a 25 mL single-necked flask and dissolve in anhydrous dichloromethane (10 mL). Cool in an ice bath, add DBU (167 mg, 1.10 mmol, 1.5 eq), and stir for 30 minutes. Add water to separate the mixture, extract the aqueous phase twice with dichloromethane, wash with saturated brine, and concentrate under reduced pressure to yield 1 g of a light yellow semisolid (450 mg of crude product containing fluorene, 100% yield). The crude product can be used directly in the next reaction.

[0635] MS (ESI): m / z 517.4 [M+H] + .

[0636] 7. Preparation of (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-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolan-8b-yl)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30-pentaoxo-5,8,11,14,17-pentahydrotriacont-32-yl)carbamate (Compound 1h)

[0637] In a 100 mL three-necked flask, 1 g (0.45 g crude product, equivalent to 0.73 mmol, 1.0 eq) and 1e (0.55 g, 0.73 mmol, 1.0 eq) of the raw materials were placed and dissolved in anhydrous DMF (9 mL). 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 sequentially and stirred for 1 hour. Water was added to the reaction mixture, and the mixture was extracted twice with dichloromethane. The organic phases were combined, washed once with saturated brine, dried, and concentrated under reduced pressure to obtain the crude yellow oily solid. The crude solid was purified by slurrying in a mixed solvent of petroleum ether and ethyl acetate to obtain the product 1h as a light yellow solid (710 mg, 78% yield).

[0638] MS (ESI): m / z 1269.7 [M+Na] + .

[0639] 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-oxo-5,8,11,14-polytetraethylene glycol-16-yl-3,6,9,12-tetraoxopentadecane-15-amide (Compound 1i)

[0640] In a 25 mL single-necked flask, place the raw material 1h (350 mg, 0.28 mmol, 1.0 eq) and dissolve in anhydrous dichloromethane (10 mL). Cool in an ice bath, add DBU (64 mg, 0.42 mmol, 1.5 eq), and stir for 1 hour. The reaction solution is concentrated to dryness to obtain a crude oil, which is then purified by column chromatography to afford the product 1i as a light yellow foamy solid (289 mg, 89% yield).

[0641] MS (ESI): m / z 1025.6 [M+H] + .

[0642] 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-pentahydro-3-oxo-5,8,11,14-tetraethylene glycol-16-yl)-1-(2-bromoacetylamino)-3,6,9,12-tetraoxopentadecane-15-amide (Compound 2)

[0643] Place solid bromoacetic acid (35.2 mg, 0.25 mmol, 2.0 eq) and EEDQ (63 mg, 0.25 mmol, 2.0 eq) in a 25 mL Schlenk tube and dissolve in anhydrous DMF (5 mL). Stir and react at room temperature for 1 hour. Starting material 1i (130 mg, 0.127 mmol, 1.0 eq) was dissolved in DMF (2 mL) and added dropwise to the activated ester solution prepared above. After addition, the reaction was maintained at room temperature for an additional 2 hours.

[0644] Water was added to the reaction system, extracted twice with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried and concentrated under reduced pressure to obtain a crude oil, which was then purified by column chromatography to obtain compound 2 as a light yellow solid (39 mg, yield 27%).

[0645] MS(ESI):m / z 1145.5 / 1147.6[M+1] + .

[0646] 1 H NMR (400MHz, CDCl3) δ8.62-8.57(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=9.6Hz,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),3.06-2.8 4(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).

[0647] 10. Preparation of ADC-2 (9E6-L4H2-Compound 2)

[0648] To a solution of antibody 9E6-L4H2 in Buffer A (0.05 M buffer, pH 6.3; 15.0 mg / ml, 1.5 mL, 0.10 mmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (2.5 mM, 170.3 μL, 0.42 mmol) at 37°C. The mixture was shaken in a water bath at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath. 1.0 M Tris buffer, pH 8.30 (210 μL) was added.

[0649] Compound 2 (1.16 mg, 1.0 mmol) was dissolved in 75 μl of DMSO and added to the reaction mixture. The mixture was shaken in a water bath at 25°C for 3 hours, and the reaction was stopped. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: buffer A) and concentrated using an ultrafiltration tube to obtain the title product ADC-2 (9E6-L4H2-compound 2) in buffer A (5.14 mg / mL, 2.48 mL) with a yield of 85%. The product was stored frozen at 4°C. The theoretical DAR value was 4.0, and the measured DAR value was 3.8.

[0650] Example 19. Affinity determination of anti-CD40-ADC to human CD40 and cynomolgus monkey CD40

[0651] The test antibody and anti-CD40 ADC were affinity-captured on an anti-human Fc chip. A His-tagged human or cynomolgus macaque CD40 antigen was then passed over the chip surface at a concentration gradient. The reaction signals were monitored in real time using a Biacore instrument to generate binding and dissociation curves. The buffer used in the experiment was HBS-EP + 10× buffer (Cat.# BR-1006-69, GE), diluted to 1× (pH 7.4) with DI Water. The data were fitted with a (1:1) binding model to obtain affinity values, as shown in Table 25.

[0652] Table 25. SPR affinity of anti-CD40-ADCs to different germline CD40s

[0653] Example 20. Inhibitory activity of anti-CD40-ADC in a B cell activation experimental system

[0654] CD40 is highly expressed on B cells. Binding of CD40L to CD40 can induce B cell activation and upregulate the expression of a series of activation markers. CD40 antagonist antibodies block the binding of CD40L to CD40, thereby disrupting B cell immune activation.

[0655] Human PBMCs were plated at 2E5 per well in a 96-well cell culture plate (RPMI-1640 medium, 10% FBS, 1% penicillin-streptomycin). 50 μL of serially diluted test antibodies or ADCs were added to each well and incubated at 37°C, 5% CO₂ for 0.5 hours. 50 μL of pre-incubated CD40L-his and anti-His antibodies were added to each well and stimulated overnight. The next day, the supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer and stained with 100 μL of a 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 a 1:200 dilution of human Fc blocker (BD, 564220) for 10 minutes at room temperature. After centrifugation, the cells were added with 100 μL of flow cytometry antibodies (PerCP / Cyanine 5.5 anti-human CD19 (Biolegend, 302230, 1:100 dilution) and APC anti-human CD69 (Biolegend, 310910, 1:200 dilution)) and incubated at 4°C for 0.5 h. The supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer and resuspended in 200 μL of FACS buffer. Cell surface fluorescence intensity was measured using a flow cytometer (BD FACS Celesta).

[0656] The results are shown in Table 26. ADC-1 and ADC-2 have similar B cell inhibitory activities to the monoclonal antibody 9E6-L4H2.

[0657] Table 26. Inhibitory activity of anti-CD40-ADC in B cell activation experimental system

[0658] After adding antibodies or ADCs, the CD19+CD69+ signals are lower than the background value, which may be due to a certain degree of inhibition of the background activation of B cells (in addition to the inhibition of CD40L-induced signals).

[0659] Example 21. Mouse skin transplant rejection model

[0660] Male Balb / c mice, 6 weeks old, were purchased from the Laboratory Animal Management Department of the Shanghai Institute of Family Planning Sciences. Housing environment: SPF; production license: SCXK (Shanghai) 2018-0006; Balb / c mouse qualification certificate number: 20180006023393.

[0661] Female human CD40 transgenic mice, 6–7 weeks old, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. Housing environment: SPF; Production license: SCXK(Su)-2016-0004; Human CD40 Transgenic Mouse Certificate Number: 320726200100179778.

[0662] After arrival, the animals were adaptively raised for 7 days and randomly divided into groups. On the second day of the experiment, mice were intraperitoneally injected with CD40 antagonist antibodies, anti-CD40-ADC or glucocorticoids. On the 0th day of the experiment, the donor Balb / c mice and C57BL6 / J mice were anesthetized with 4% chloral hydrate, the tails of the donor mice were removed, and the skin of the tail with a length of 1 cm was separated. The hair on the back of the recipient mouse was removed, and an incision was made along the skin layer. While retaining the back fat and connective tissue, an equal area of ​​skin was removed. The donor skin was placed on the incision of the recipient mouse, the edge of the skin was sutured with glue, and the mouse was placed in a cage to recover. The specific experimental process is shown in Figure 13A.

[0663] Mice were dosed according to the dosing regimen shown in Table 27. After 7 days of recovery, the skin was observed daily for survival and the rejection score was recorded. The scoring system is as follows: 3: smooth skin with no redness; 2: partially red, dull, and dry skin; 1: mostly red, without streaks, and shrunken skin; 0: graft rejection resulting in 80% skin necrosis.

[0664] Table 27. Dosage regimen for mouse skin transplant rejection model

[0665] The results are shown in Tables 28-29. ADC-2 showed significantly better anti-transplant rejection activity than the CD40 antagonist monoclonal antibody 9E6-L4H2 and monoclonal antibody combined with glucocorticoids, and was slightly better than high-dose dexamethasone.

[0666] Table 28. Anti-CD40-ADC skin graft survival rate (%)

[0667] Table 29. Anti-CD40-ADC Skin Graft Scores

[0668] Example 22. pH and buffer screening of anti-CD40 antibody formulations

[0669] Twelve different pH and buffer combinations (pH 4.5, 5.0, 5.5, 6.0, 6.5, 6.6, 7.0, and 7.4) were used to prepare 12 formulations of anti-CD40 antibody (antibody number 9E6 (L4H2), heavy and light chains as shown in SEQ ID NOs: 20 and 21, hereinafter the same) at a concentration of 20.0 mg / mL. The melting temperature (Tm) and aggregation temperature (Tag) of the antibodies in these formulations were determined. The formulations were also tested for purity by SEC, NRCE, and icIEF. The stability of the 12 formulations was investigated after 2 weeks at 2-8°C, 1 and 2 weeks at 25°C, 1 and 2 weeks at 40°C, and 5 freeze-thaw cycles (-35°C / room temperature). The following are formulations 1)-12):

[0670] 1) 20 mM acetic acid-sodium acetate, pH 4.5

[0671] 2) 20 mM acetic acid-sodium acetate, pH 5.0

[0672] 3) 20 mM acetic acid-sodium acetate, pH 5.5

[0673] 4) 20 mM citric acid-sodium citrate, pH 5.0

[0674] 5) 20 mM citric acid-sodium citrate, pH 5.5

[0675] 6) 20 mM citric acid-sodium citrate, pH 6.0

[0676] 7) 20 mM histidine-histidine hydrochloride, pH 5.5

[0677] 8) 20 mM histidine-histidine hydrochloride, pH 6.0

[0678] 9) 20 mM histidine-histidine hydrochloride, pH 6.5

[0679] 10) 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 6.6

[0680] 11) 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 7.0

[0681] 12) 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 7.4

[0682] During the research process, it was found that the solubility of anti-CD40 antibody in the buffer systems of Formulation 4 and Formulation 5 was poor, so the stability of these two buffer systems was not investigated.

[0683] Table 30. Anti-CD40 Antibody Formulation pH and Buffer Screening Results 1

[0684]

[0685]

[0686]

[0687] The results showed that the anti-CD40 antibody had higher Tm and Tagg values ​​in histidine buffer systems (Formulations 7 to 9), indicating that the antibody exhibited good conformational and colloidal stability in the histidine buffer system. After two weeks of observation at high temperature (40°C), the SEC-HPLC, NRCE, and icIEF purity of the anti-CD40 antibody in histidine pH 6.0 and pH 6.5 systems were higher than those in other systems, indicating that the anti-CD40 antibody exhibited better stability in histidine pH 6.0 and pH 6.5 systems. Therefore, a histidine system with a pH range of pH 6.0-pH 6.6 was selected for subsequent studies.

[0688] Example 23. Screening of anti-CD40 antibody concentration, excipient type and concentration

[0689] Six formulations were prepared using two buffer systems, 20 mM histidine-histidine hydrochloride, pH 6.0 and pH 6.6, with 8% (w / v) sucrose and 5% (w / v) sucrose + 50 mM arginine hydrochloride as stabilizers, 0.02% (w / v) polysorbate 80 as a surfactant, and anti-CD40 antibody concentrations of 50 mg / mL and 80 mg / mL. The stability of these six formulations was investigated by testing sample appearance, pH, SEC-HPLC, NRCE, and iCIEF purity. The stability of these six formulations was evaluated under conditions including exposure to high temperature (40°C) for 2 and 4 weeks, exposure to light (5000 ± 500 lx, 25°C) for 5 and 10 days, shaking (300 rpm / room temperature) for 2 days, and repeated freeze-thaw cycles (-35°C and room temperature) for 3 and 5 times.

[0690] 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

[0691] 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

[0692] 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

[0693] 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

[0694] 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

[0695] 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

[0696] Table 34. Anti-CD40 antibody protein concentration and excipient screening results 1

[0697]

[0698]

[0699]

[0700] Results showed that, in terms of appearance, the arginine hydrochloride-containing formulations exhibited a more pronounced opalescence. Regarding purity, all formulations showed only a slight decrease in SEC purity under both high temperature and light conditions, with the increase in aggregates exceeding 2% compared to T0, indicating minimal overall differences. Under high temperature, NRCE fragmentation increased slightly, with no significant differences between groups. iCIEF purity also varied under both high temperature and light conditions. Under high temperature, the acidic peak content in formulation 1 was slightly higher than that in formulation 2, and in formulation 4 than in formulation 5, but the differences were not significant. Furthermore, varying protein concentrations had no significant effect on purity, indicating that the stability of anti-CD40 antibodies at concentrations of 50 mg / mL to 80 mg / mL was excellent in a 10 mM histidine-histidine hydrochloride buffer system, pH 6.0-pH 6.6, with 8% (w / v) sucrose as a stabilizer and 0.02% (w / v) polysorbate 80 as a surfactant.

[0701] Example 24 Confirmation of Stabilizer Concentration in Anti-CD40 Antibody Preparations

[0702] An antibody formulation with an anti-CD40 antibody content of 80 mg / mL and a pH of 6.3 was prepared using 7.5% (w / v) sucrose, 20 mM histidine-histidine hydrochloride, and 0.02% (w / v) polysorbate 80. Stability studies were conducted under conditions of high temperature (40°C) and repeated freeze-thaw cycles (-35°C / room temperature) for five times.

[0703] 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

[0704] Table 38 Anti-CD40 Antibody Formulation Target pH and Stabilizer Concentration Confirmation Results 1

[0705] Table 39. Anti-CD40 Antibody Formulation Target pH and Stabilizer Concentration Confirmation Results 2 Note: NT means not tested. The repeated freeze-thaw cycles were performed at lower temperatures and for shorter periods of time, which had little effect on the purity of NRCE and iCIEF, and therefore were not tested. T0 means time 0.

[0706] Results showed that the formulation's appearance remained unchanged after five freeze-thaw cycles, with clarity changing from clear to slightly opalescent at elevated temperatures. The pH remained unchanged under all conditions. Regarding purity, SEC-HPLC purity remained unchanged after five freeze-thaw cycles. At elevated temperatures, SEC-HPLC aggregates increased slightly, with NRCE fragmentation increasing by approximately 2.8% compared to T0. The increase in iCIEF acidic peaks was within an acceptable range, demonstrating good freeze-thaw and thermal stability.

[0707] Example 25. Confirmation of pH of anti-CD40 antibody preparation

[0708] An antibody preparation with an anti-CD40 antibody content of 80 mg / mL 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. Stability studies were conducted under conditions including high temperature (40°C) and repeated freeze-thaw cycles (-35°C / room temperature) for 5 times.

[0709] 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

[0710] Table 40. Anti-CD40 Antibody Preparation pH Confirmation Results 1

[0711] Table 41. Anti-CD40 Antibody Preparation pH Confirmation Results 2 Note: NT means not tested, T0 means time 0.

[0712] Results showed no significant changes in pH or appearance after five freeze-thaw cycles and one month of high-temperature storage. Purity remained unchanged after five freeze-thaw cycles. After four weeks of high-temperature storage, the SEC-HPLC aggregates increased slightly compared to T0, while the NRCE purity was above 91%, and the iCIEF main peak remained above 40%, demonstrating good freeze-thaw and thermal stability for this pH formulation.

[0713] Example 26. Anti-CD40 antibody concentration screening

[0714] 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 anti-CD40 antibody preparations of varying concentrations for viscosity testing.

[0715] Table 42. Viscosity results of anti-CD40 antibodies Note: NT means not tested

[0716] The results showed that the higher the protein concentration, the higher the viscosity of the formulation. Using sucrose as a stabilizer, when the protein concentration reached 131.2 mg / mL, the viscosity was close to 20 cp.

[0717] Example 27. Anti-CD40 Antibody Concentration Screening

[0718] Anti-CD40 antibody preparations of different concentrations were prepared using a 20 mM histidine-histidine hydrochloride, pH 6.1 buffer system, 2.5% (w / v) proline, and 0.02% (w / v) polysorbate 80, and viscosity was tested.

[0719] Table 43. Anti-CD40 Antibody Viscosity Results

[0720] The results showed that the higher the protein concentration, the higher the viscosity of the formulation. Using proline as a stabilizer, when the protein concentration reached 187 mg / mL, the viscosity still did not exceed 20 cp.

[0721] Example 28. Screening of excipient types

[0722] Antibody preparations with anti-CD40 antibody contents of 80, 120, and 150 mg / mL were prepared using a 20 mM histidine-histidine hydrochloride, pH 6.1 buffer system, 0.02% (w / v) polysorbate 80, and 7.5% (w / v) sucrose or 2.5% (w / v) proline as stabilizers. The stability of the preparations was investigated under conditions of high temperature (40°C) for 2 and 4 weeks, illumination (4500±500lx / 25°C) for 5 days, shaking (300rpm / 25°C) for 2 days, freeze-thaw (-35°C / room temperature) for 5 times, storage at 2-8°C for 4 weeks, and frozen storage (-35°C) for 4 weeks.

[0723] 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

[0724] 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

[0725] 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

[0726] Table 44. Anti-CD40 antibody excipient screening results 1

[0727] Table 45. Anti-CD40 antibody excipient screening results 2

[0728] Table 46. Anti-CD40 antibody excipient screening results 3

[0729] Table 47. Anti-CD40 antibody excipient screening results 4

[0730] Table 48. Anti-CD40 antibody excipient screening results 5

[0731] Table 49. Anti-CD40 antibody excipient screening results 6

[0732] Table 50 Anti-CD40 Antibody Excipient Screening Results 7

[0733] The results showed that all formulations had a light yellow, slightly opalescent appearance under all conditions, with no visible protein particles, and binding activity ranging from 60% to 140%. There were no significant differences between the groups in terms of insoluble particulate matter, SEC purity, NRCE, and RCE purity. Regarding iCIEF purity, under high-temperature conditions, formulation No. 28 performed best, followed by formulations No. 27 and No. 26, with no significant differences among the three formulations. This indicates that the three formulations exhibited good appearance and stability under conditions of high temperature, light, shaking, freeze-thaw, and frozen storage.

[0734] Example 29. Anti-CD40 Antibody Optional Formulations

[0735] The present disclosure provides an anti-CD40 antibody pharmaceutical preparation having a formulation of “50-150 mg / mL anti-CD40 antibody, 7.5%-8% (w / v) sucrose or 2.5% (w / v) proline, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 5.6-6.6”, including but not limited to:

[0736] (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;

[0737] (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;

[0738] (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;

[0739] (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;

[0740] (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;

[0741] (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;

[0742] (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;

[0743] (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;

[0744] (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;

[0745] (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;

[0746] (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;

[0747] (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;

[0748] (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;

[0749] (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;

[0750] (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;

[0751] (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.

[0752] (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.

[0753] (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.

[0754] (19) 120 mg / mL anti-CD40 antibody, 7.5% (w / v) sucrose, 0.02% (w / v) polysorbate 80, 20 mM histidine-histidine hydrochloride pH 6.3.

[0755] (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.

[0756] (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.

[0757] (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.

[0758] The experimental results show that the anti-CD40 antibody preparations of the above formulations have good stability and can be used in the preparation of anti-CD40 antibody drugs.

[0759] Example 30: Investigation of the prescription of anti-CD40-ADC preparation

[0760] 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 a formulation of 50 mg / mL CD40-ADC (ADC-2 of Example 18 of the present disclosure, the same as in the following examples). The stability of the liquid formulation was investigated at different temperatures (-35°C, 2-8°C, 25°C, 40°C), illumination (5000±500lx, 25°C), shaking (300 rpm, 25°C), and repeated freeze-thaw (-35°C / room temperature). The formulation was then freeze-dried, and the stability of the lyophilized powder was investigated at 50°C.

[0761] 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

[0762] Table 51. Anti-CD40-ADC prescription investigation results 1 Note: Lyo T0 means the sample at time 0 after freeze-drying.

[0763] Table 52. Anti-CD40-ADC Prescription Investigation Results 2

[0764] Table 53. Anti-CD40-ADC prescription investigation results 3 Note: Lyo T0 means the sample at time 0 after freeze-drying.

[0765] Table 54. Anti-CD40-ADC Prescription Investigation Results 4 Note: Lyo T0 means the sample at time 0 after freeze-drying.

[0766] Table 55. Anti-CD40 ADC prescription investigation results 5

[0767] The results showed that the solution and lyophilized powder dosage forms of the anti-CD40-ADC formulation had good stability under different stability test conditions.

[0768] Example 31. Anti-CD40-ADC optional formulations

[0769] The present disclosure provides an anti-CD40-ADC formulation having a formulation of "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, 20 mM histidine-histidine hydrochloride pH 6.0-6.6", including but not limited to:

[0770] (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;

[0771] (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;

[0772] (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;

[0773] (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;

[0774] (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;

[0775] (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;

[0776] The experimental results show that the anti-CD40 ADC preparations of the above formulations have good stability and can be used in the preparation of anti-CD40-ADC drugs.

[0777] Although specific embodiments of the present disclosure are described above, those skilled in the art will appreciate that these are merely examples and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present disclosure.

Claims

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

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

3. The pharmaceutical composition of claim 2, wherein 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 as shown in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2, and LCDR3 in the VL as shown in SEQ ID NO: 2; Wherein, the CDR is defined according to the Kabat, IMGT, Chothia, Ab M or Contact numbering system, preferably defined according to the Kabat numbering system.

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

5. The pharmaceutical composition according to any one of claims 2 to 4, wherein 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.

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

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

8. The pharmaceutical composition according to any one of claims 2 to 7, wherein the anti-CD40 antibody or antigen-binding fragment thereof further contains an Fc region of an IgG antibody; preferably, the IgG antibody is an IgG1, IgG2 or IgG4 antibody; more preferably, the Fc region is an Fc region of an IgG1 containing an N297A mutation, or an Fc region of an IgG1 having any one or more mutations of L234A, L235A, M252Y, S254T, T256E.

9. The pharmaceutical composition according to any one of claims 2 to 8, wherein the antigen binding fragment is a scFv, Fv, Fab or Fab' fragment.

10. The pharmaceutical composition according to any one of claims 2 to 9, wherein the anti-CD40 antibody or antigen-binding fragment thereof comprises: The full-length amino acid sequence of the heavy chain is shown in SEQ ID NO: 20 or 22, or is at least 90% identical thereto; the full-length amino acid sequence of the light chain is shown in SEQ ID NO: 21, or is at least 90% identical thereto. 11 . The pharmaceutical composition according to claim 1 , wherein 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 .

12. The pharmaceutical composition according to claim 11, wherein the antibody-drug conjugate has a structure as shown in formula (I): Ab-(LD) k (I); in, Ab is an anti-CD40 antibody or an 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 rapamycin target protein (mTOR) kinase inhibitor; L is a linker that covalently connects Ab to D, and k is selected from an integer or decimal of 1-20.

13. The pharmaceutical composition according to claim 12, wherein D in the antibody-drug conjugate is:

14. The pharmaceutical composition according to claims 11 to 13, wherein the antibody-drug conjugate is represented by the following structure: in, k is an integer or decimal 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 has the following structure: Wherein, k is an integer or decimal selected from 1-10; More preferably, the antibody-drug conjugate has the following structure: Among them, 9E6-L4H2 is an anti-CD40 antibody, which includes a heavy chain and a light chain having amino acid sequences as shown in SEQ ID NOs: 20 and 21; k is an integer or decimal selected from 1-10.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the pH of the composition is 4.5-7.5, preferably 5-7.2, more preferably 5.5-7, most preferably 5.6-6.

6.

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

17. The pharmaceutical composition according to any one of claims 1 to 16, further comprising a stabilizer selected from one or more of amino acids or pharmaceutically acceptable salts thereof and sugars; preferably, the amino acids are selected from one or more of arginine and proline or pharmaceutically acceptable salts thereof, and the sugars are selected from one or more of sucrose and trehalose; more preferably, the stabilizer is selected from one or more of sucrose, trehalose, arginine or pharmaceutically acceptable salts thereof and proline or pharmaceutically acceptable salts thereof; most preferably, the stabilizer is sucrose, trehalose or proline or pharmaceutically acceptable salts thereof.

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

19. The pharmaceutical composition according to any one of claims 1 to 18, further comprising a surfactant, wherein the surfactant is preferably a polysorbate, more preferably one or more of polysorbate 20 and polysorbate 80, most preferably polysorbate 80.

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

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

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

23. A pharmaceutical composition comprising any one of the following: A) an anti-CD40 antibody or an antigen-binding fragment thereof according to any one of claims 2 to 10, or The 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 a pharmaceutically acceptable salt 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 pharmaceutically acceptable 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.

24. A pharmaceutical composition comprising any one of the following: 1) 0.1-300 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%-1% w / v polysorbate, preferably polysorbate 80; 0.1%-30% w / v sucrose; and the pH of the composition is 4.5-7.5; 2) 1-200 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%-1% w / v polysorbate, preferably polysorbate 80; 0.5%-20% w / v sucrose; and the pH of the composition is 5-7.2; 3) 10-150 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%-0.2% w / v polysorbate, preferably polysorbate 80; 1%-15% w / v sucrose; and the pH of the composition is 5.5-7; 4) 15-132 mg / mL or 20-100 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%-0.1% w / v polysorbate, preferably polysorbate 80; 5%-10% w / v sucrose; and the pH of the composition is 5.6-6.6; 5) 0.1-500 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%-1% w / v polysorbate, preferably polysorbate 80; 0.01%-20% w / v proline or a pharmaceutically acceptable salt thereof; and the pH of the composition is 4.5-7.5; 6) 1-300 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%-1% w / v polysorbate, preferably polysorbate 80; 0.1%-15% w / v proline or a pharmaceutically acceptable salt thereof; and the pH of the composition is 5-7.2; 7) 10-250 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%-0.2% w / v polysorbate, preferably polysorbate 80; 0.5%-10% w / v proline or a pharmaceutically acceptable salt thereof; and the pH of the composition is 5.5-7; 8) 20-200 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%-0.1% w / v polysorbate, preferably polysorbate 80; 1%-5% w / v proline or a pharmaceutically acceptable salt thereof; And the pH of the composition is 5.6-6.

6.

25. A pharmaceutical composition comprising any one of the following: 1) 0.1-300 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%-1% w / v polysorbate, preferably polysorbate 80; 0.1%-30% w / v sucrose or trehalose; and the pH of the composition is 4.5-7.5; 2) 1-150 mg / mL of the antibody-drug conjugate of any one of claims 11 to 14; 5-40 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.005%-1% w / v polysorbate, preferably polysorbate 80; 0.5%-20% w / v sucrose or trehalose; and the pH of the composition is 5-7.2; 3) 5-120 mg / mL of the antibody-drug conjugate of any one of claims 11 to 14; 10-30 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.008%-0.2% w / v polysorbate, preferably polysorbate 80; 1%-15% w / v sucrose or trehalose; and the pH of the composition is 5.5-7; 4) 10-100 mg / mL of the antibody-drug conjugate of any one of claims 11 to 14; 15-25 mM histidine-hydrochloride buffer, preferably histidine-histidine hydrochloride; 0.01%-0.1% w / v polysorbate, preferably polysorbate 80; 5%-10% w / v sucrose or trehalose; And the pH of the composition is 5.6-6.

6.

26. The pharmaceutical composition according to claim 23 or 24, comprising any one of the following: 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 according to any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof according to any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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) 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 the anti-CD40 antibody or antigen-binding fragment thereof of any one of claims 2 to 10; 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.

27. The pharmaceutical composition according to claim 23 or 25, comprising any one of the following: 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 of any one of claims 11 to 14; 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) 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 of any one of claims 11 to 14; 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) 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 of any one of claims 11 to 14; 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) 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 of any one of claims 11 to 14; 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) 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 of any one of claims 11 to 14; 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) 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 of any one of claims 11 to 14; 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.

28. A lyophilized preparation, which can form the pharmaceutical composition of any one of claims 1 to 27 after being reconstituted, or which is obtained by freeze-drying the pharmaceutical composition of any one of claims 1 to 27.

29. A reconstituted solution, wherein the reconstituted solution is prepared by reconstituted the lyophilized preparation according to claim 28.

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

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

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

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