TW202430226A - Anti-cd40 antibody drug conjugates, preparation methods and pharmaceutical uses thereof - Google Patents

Abstract

The present disclosure relates to anti-CD40 antibody drug conjugates, preparation methods and pharmaceutical uses thereof. Specifically, the present disclosure relates to an antibody-drug conjugate comprising an anti-CD40 antibody or antigen-binding fragment thereof, pharmaceutical composition comprising the antibody-drug conjugate, and their use in the method for the treatment of autoimmune diseases, graft vsersus host disease and/or graft rejective reaction and their relateive pharmaceutical uses.

Description

Anti-CD40 antibody-drug conjugate, its preparation method and its medical use

This disclosure claims priority to the following patent application: Chinese patent application filed on November 28, 2022, application number CN202211699375.2, the entire contents of the aforementioned patent application are incorporated into this disclosure by reference.

The present disclosure relates to the field of biomedicine. Specifically, the present disclosure relates to an anti-CD40 antibody-drug conjugate, a pharmaceutical composition comprising the anti-CD40 antibody-drug conjugate, and a method for treating autoimmune diseases (including graft-versus-host disease and transplant rejection) and related pharmaceutical uses.

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

After CD40L binds to CD40, it can recruit TRAF and mediate downstream signaling through the NF-kB, JNK and MAPK pathways, exerting a variety of cell type-dependent activation results, including immune cell activation and proliferation, inflammatory factors and trend factor secretion, etc. (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 response (TDAR), B cell proliferation, germinal center (GC) formation, immunoglobulin (Ig) isotype switching, somatic cell 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).

Functional disorders of the CD40 signaling pathway can lead to autoimmune diseases (Karnell JL et al., 2018). The CD40-CD40L signaling pathway has been found to be involved in the function of parenchymal cells in inflammatory tissues: activated epithelial cells from the kidneys, salivary glands, and skin that can secrete cytokines can respond to CD40. In addition, the expression levels of CD40 or CD40L in lesions of patients with atherosclerosis and preclinical atherosclerosis models are increased. CD40 can stimulate the expression of matrix-degrading enzymes and promote the expression of tissue factors in cell types related to the pathogenesis of atherosclerosis, such as endothelial cells, smooth muscle cells, and macrophages (Michel NA et al., 2017). The CD40 pathway upregulates the production of inflammatory factors such as IL-1, IL-6, and IL-8, as well as adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule (VCAM). The CD40/CD40L interaction has also been used to prevent transplant rejection, and the use of the chimeric anti-CD40 antagonist ch5D12 in a kidney allograft study in Gangetic monkeys showed that CD40 antagonism was sufficient to improve disease and prolong median survival by more than 100 days. When ch5D12 was combined with an anti-CD86 antibody and given only at the beginning of an allogeneic transplant study, followed by extended treatment with cyclosporine, a median survival of greater than 4 years was achieved, suggesting that this combination could potentially induce immune tolerance (Haanstra et al., 2005).

Numerous preclinical studies have provided evidence for the critical role of CD40/CD40L interactions in promoting T cell-dependent immune responses. Therefore, blockade of CD40 signaling is considered an appropriate and desirable therapeutic strategy to inhibit pathogenic autoimmune responses. Currently, no anti-CD40 antibodies have been approved for the treatment of such diseases.

Antibody drug conjugate (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active drug via a stable chemical linker compound. Most ADCs in preclinical and clinical development are used for oncology indications, where the cytotoxic payload targets cancer cells expressing the antigen. However, modulation of pathogenic cell activity through ADC-mediated delivery of biologically active small molecules is also attractive for non-oncology indications, leading to the widespread application of this technology.

Currently, there is still an urgent need for therapeutic agents that can intervene in CD40-CD40L interactions and block CD40 signaling.

The present disclosure provides an antibody-drug conjugate, a pharmaceutical composition comprising the antibody-drug conjugate, a method for treating or intervening in autoimmune diseases (including graft-versus-host disease and transplant rejection), and related pharmaceutical uses.

Anti-CD40 antibodies, antigen-binding fragments thereof

On the one hand, the present disclosure provides anti-CD40 antibodies and antigen-binding fragments thereof, which comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein,

a-1) the VH comprises HCDR1, HCDR2 and HCDR3 in any of the VHs shown in SEQ ID NOs: 13-14, and the VL comprises LCDR1, LCDR2 and LCDR3 in any of the VLs shown in SEQ ID NOs: 9-12; or

a-2) 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.

In some embodiments, the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems, for example, according to the Kabat numbering system.

In some specific embodiments, the present disclosure provides an anti-CD40 antibody or an antigen-binding fragment thereof, which comprises:

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 _{QGGYWTSTSNFGX1X2} (SEQ ID NO: ₁₉ ), wherein _X1 is selected from N, S, T or Q, and _X2 is selected from V or G.

In some specific embodiments, the present disclosure provides an anti-CD40 antibody or an antigen-binding fragment thereof, which comprises:

Heavy chain HCDR1, HCDR2 and HCDR3, which respectively comprise the sequences shown in SEQ ID NO: 3, 4 and 5; light chain LCDR1, which comprises the sequence shown in SEQ ID NO: 6; light chain LCDR2, which comprises the sequence shown in SEQ ID NO: 7; light chain LCDR3, which comprises the sequence shown in any one of SEQ ID NO: 8, 15-18.

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

In some embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof is a recombinant antibody.

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

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

In some embodiments, the aforementioned anti-CD40 antibody or its antigen-binding fragment comprises VH and VL, wherein,

A-1) VH comprises an amino acid sequence as shown in SEQ ID NO: 13 or having at least 80% identity thereto, and VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 9-12 or having at least 80% identity thereto;

A-2) VH comprises an amino acid sequence as shown in SEQ ID NO: 14 or having at least 80% identity thereto, and VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 9-12 or having at least 80% identity thereto;

A-3) VH comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least 80% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO: 2 or having at least 80% identity thereto.

In some embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof, such as an IgG1, IgG2, IgG2, IgG4 antibody or antigen-binding fragment thereof, such as an IgG1 antibody or antigen-binding fragment thereof having a 297A mutation, such as an IgG1 antibody or antigen-binding fragment thereof having one of 234A, 235A, 252Y, 254T and 256E or any combination thereof. The mutation is numbered according to the EU numbering rules.

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

In some 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 has at least 80% identity thereto; the amino acid sequence of the full-length light chain is as shown in SEQ ID NO: 21 or has at least 80% identity thereto; or

As mentioned above, "at least 80% identity" includes, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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.

In some embodiments, the heavy chain variable region of the aforementioned anti-CD40 antibody or its antigen-binding fragment has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes; 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 the function.

In some embodiments, an anti-CD40 antibody or antigen-binding fragment is provided, which binds or competes for binding to the same epitope as the aforementioned anti-CD40 antibody or antigen-binding fragment.

In some embodiments, an anti-CD40 antibody or antigen-binding fragment is provided, which blocks the binding of the aforementioned anti-CD40 antibody or antigen-binding fragment thereof to CD40 (e.g., human CD40).

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

In some embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment has at least one of the following:

(i) binds human CD40 with a _KD of 10 nM or lower;

(ii) No obvious stimulatory activity.

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

In some embodiments, the aforementioned anti-CD40 antibody or antigen-binding fragment binds to human CD40 with a _KD of ^10-7 M, ^10-8 M, ^10-9 M, ^10-10 M, ^10-11 M or lower.

In some embodiments, a CD40 binding molecule is provided, comprising any of the aforementioned anti-CD40 antibodies or antigen-binding fragments thereof.

In some embodiments, the present disclosure provides an isolated polynucleotide encoding an anti-CD40 antibody or an antigen-binding fragment thereof disclosed herein. The isolated polynucleotide may be RNA, DNA or cDNA. According to some embodiments of the present disclosure, the polynucleotide disclosed herein is an isolated polynucleotide.

In some embodiments, the present disclosure also provides a DNA molecule encoding any anti-CD40 antibody or antigen-binding fragment thereof disclosed above.

The polynucleotides disclosed herein may also be in the form of a vector, may be present in a vector and/or may be part of a vector, such as a plasmid, cosmid, YAC or viral vector. The vector may in particular be an expression vector, i.e. a vector that can provide for the expression of a VEGF binding molecule or its complex in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression system). The expression vector generally comprises at least one polynucleotide disclosed herein, which is operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). The selection of the elements and their sequences for expression in a specific host is within the ordinary skill of the art. Regulatory elements and other elements useful or necessary for the expression of the anti-CD40 antibody or its antigen-binding fragment disclosed herein include, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

The polynucleotide disclosed herein can be prepared or obtained by known means (e.g., by automatic DNA synthesis and/or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptide disclosed herein, and/or can be isolated from a suitable natural source.

In some embodiments, the present disclosure provides a host cell expressing the anti-CD40 antibody or antigen-binding fragment thereof or containing the polynucleotide or vector of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell or a mammalian cell.

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

Fungal cells include, for example, cells of species of Trichoderma , Neurospora , and Aspergillus ; or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae ), Schizosaccharomyces (e.g., Schizosaccharomyces pombe ), Pichia (e.g., Pichia pastoris and Pichia methanolica ), and Hansenula .

Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

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

In one embodiment, the host cells used in the present disclosure are incapable of developing into finished plants or animal individuals.

Antibody-drug conjugates

The present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, which comprises an anti-CD40 antibody or an antigen-binding fragment thereof, wherein the anti-CD40 antibody or the antigen-binding fragment thereof is any anti-CD40 antibody or the antigen-binding fragment thereof disclosed herein.

In some embodiments of the antibody-drug conjugate, the anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein,

a-1) the VH comprises HCDR1, HCDR2 and HCDR3 in any of the VHs shown in SEQ ID NOs: 13-14, and the VL comprises LCDR1, LCDR2 and LCDR3 in any of the VLs shown in SEQ ID NOs: 9-12; or

a-2) 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;

In some embodiments, the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems.

In some embodiments of the antibody-drug conjugate, the anti-CD40 antibody or antigen-binding fragment thereof comprises:

Heavy chain HCDR1, HCDR2 and HCDR3, which respectively comprise the sequences shown in SEQ ID NO: 3, 4 and 5; and light chain LCDR1, LCDR2 and LCDR3, which respectively comprise the sequences shown in SEQ ID NO: 6, 7 and 19.

In some embodiments of the antibody-drug conjugate, the anti-CD40 antibody or antigen-binding fragment thereof comprises:

Heavy chain HCDR1, HCDR2 and HCDR3, which respectively comprise the sequences shown in SEQ ID NO: 3, 4, 5; light chain LCDR1, which comprises the sequence shown in SEQ ID NO: 6; light chain LCDR2, which comprises the sequence shown in SEQ ID NO: 7; and light chain LCDR3, which comprises the sequence shown in any one of SEQ ID NO: 8, 15-18.

In some embodiments of the antibody-drug conjugate, the anti-CD40 antibody or its antigen-binding fragment comprises VH and VL, wherein,

A-1) VH comprises an amino acid sequence as shown in SEQ ID NO: 13 or having at least 80% identity thereto, and VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 9-12 or having at least 80% identity thereto;

A-2) VH comprises an amino acid sequence as shown in SEQ ID NO: 14 or having at least 80% identity thereto, and VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 9-12 or having at least 80% identity thereto; or

A-3) VH comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least 80% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO: 2 or having at least 80% identity thereto.

In some embodiments of the antibody-drug conjugate, the antigen-binding fragment of the anti-CD40 antibody comprises a heavy chain and a light chain, wherein,

The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 20 or 22 or having at least 80% identity thereto; the light chain comprises an amino acid sequence as shown in SEQ ID NO: 21 or having at least 80% identity thereto.

In some embodiments of the antibody-drug conjugate, it is an antibody-drug conjugate with a structure shown in formula (I):

Ab-(LD) _k (I);

Wherein, Ab is any of the aforementioned anti-CD40 antibodies or antigen-binding fragments thereof;

D is an effector molecule with immunosuppressive activity.

L is a linker that covalently links Ab to D, and k is an integer or decimal of 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 decimal or integer between any two of the foregoing values). In some embodiments, k is 4±0.4, 4±0.5, 4±0.6, 4±0.8. For example, 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, etc.

In some embodiments, D is selected from glucocorticoid receptor agonists, calcitonin phosphatase inhibitors, or mTOR kinase inhibitors. In other embodiments, D can also be other compounds, polypeptides, proteins, nucleic acids, and other substances with immunosuppressive activity.

In some embodiments, D is a glucocorticoid receptor agonist, for example, a glucocorticoid receptor agonist of any structure selected from WO2022166779A1.

In some embodiments, D is a glucocorticoid, such as dexamethasone, prednisone, prednisolone, budesonide, mometasone, beclomethasone dipropionate, fluticasone, triamcinolone acetonide, and ciclesonide. In some embodiments, D is budesonide.

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

In some embodiments, D is a mTOR kinase inhibitor, such as rapamycin, everolimus, and temsirolimus.

Antibody-drug (glucocorticoid receptor agonist) conjugate

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

The present disclosure provides an antibody-drug conjugate having a structure shown in formula (I):

Ab-(LD) _k (I);

Wherein, Ab is the anti-CD40 antibody or its antigen-binding fragment disclosed herein;

D is glucocorticoid;

L is a linker that covalently links Ab to D, and 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 values).

In some embodiments, D is as follows:

或

or

in,

R _1a are each independently selected from hydrogen, alkyl and alkoxy, and the alkyl and alkoxy are optionally substituted with one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl and hydroxyalkyl;

Ring A is selected from aryl or heteroaryl groups which are optionally substituted by one or more substituents _Q1 ;

Ring B is selected from aryl or heteroaryl groups which are optionally substituted by one or more substituents _Q1 ;

X ₁ is selected from -(CR _5a R _5b ) m -, an aryl group or a heteroaryl group which is optionally substituted by one or more substituents Q ₁ ;

R _5a , R _5b are each independently selected from hydrogen, halogen, hydroxyl, alkyl, deuterium, oxo, thio, nitro, cyano or the following groups which are optionally substituted with one or more substituents Q ₁ : alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k , -S(O)R _k , -S(O)OR _k , -S(O)(O)R _k , -S(O)(O)OR _k , -C(S)R _k , alkoxy, alkylthio, alkenyl and alkynyl;

Ring C and Ring D are each independently selected from an aryl group, a heteroaryl group, a fused aryl group or a fused heteroaryl group which may be substituted with one or more substituents Q ₁ , and at least one of Ring C and Ring D is selected from a fused aryl group or a fused heteroaryl group which may be substituted with one or more substituents Q ₁ ;

_X2 is selected _from -( _CR6aR6b )n-, aryl or heteroaryl optionally substituted by one or more substituents _Q1 , -O-, -S-, -S(O)-, -S(O)(O)-, -NR6c- _{, -CH2S- , -CH2O-} , _-NHCR6dR6e- , _-CR6f = _CR6g- , -C≡C-, or X is absent;

R _6a , R _6b are each independently selected from hydrogen, halogen, hydroxyl, alkyl, deuterium, oxo, thio, nitro, cyano or the following groups which are optionally substituted with one or more substituents Q ₁ : alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k , -S(O)R _k , -S(O)OR _k , -S(O)(O)R _k , -S(O)(O)OR _k , -C(S)R _k , alkoxy, alkylthio, alkenyl and alkynyl, or R _6a , R _6b together with the adjacent carbon atoms form a 3- to 10-membered cycloalkyl group;

R _6c , R _6d , R _6e , R _6f , and R _6g are each independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ halogenalkyl, or C ₁ -C ₆ alkoxy;

_R1 is each independently selected from hydrogen, alkyl or alkoxy, wherein the alkyl and alkoxy are each independently optionally substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl and hydroxyalkyl;

R ₂ is each independently selected from -CH ₂ OH, -CH ₂ SH, -CH ₂ Cl, -SCH ₂ Cl, -SCH ₂ F, -SCH ₂ CF ₃ , -OH, -OCH ₂ CN, -OCH ₂ Cl. , -OCH ₂ F , -OCH ₃ , -OCH ₂ CH ₃ , -SCH ₂ CN,

R _2a are each independently selected from hydrogen or C ₁ -C ₆ alkyl;

R _2b are each independently selected from C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy;

R _2c are each independently selected from hydrogen, C ₁ -C ₆ alkyl, -CH ₂ OH or C ₁ -C ₆ alkoxy;

R _2d and R _2e are each independently selected from hydrogen or C ₁ -C ₆ alkyl;

_R3 are each independently selected from hydrogen or halogen;

_R4 are each independently selected from hydrogen, halogen or hydroxyl;

m and n are independently selected from integers between 1 and 6;

The substituent groups _Q1 are each independently selected from _C1 - _C6 alkyl, halogen, deuterium, hydroxyl, alkyl, _-NRiRj , _oxo , thio, -C(O) _Rk , -C(O) _ORk , -S(O) _Rk , -S(O) _ORk , -S(O)(O) _Rk , -S(O)(O) _ORk , -C(S) _Rk , nitro, cyano, C1- _C6 alkoxy, _C1 -C6 alkylthio, _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, 3- to 10 _- membered cycloalkyl, 3- to 10-membered heterocyclic group, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to ₁₂ -membered fused cycloaryl, and 5- to 12-membered fused heteroaryl;

R _i and R _j are each independently selected from a hydrogen atom, a hydroxyl group, a C ₁ -C ₆ alkyl group, and a C ₁ -C ₆ alkoxy group; R _{k is} independently selected from a hydrogen atom, a C ₁ -C ₆ alkyl group, a C ₁ -C ₆ halogenalkyl group, a C ₁ -C ₆ alkoxy group, a hydroxyl group, and -NR _i R _j , wherein the alkyl group, the alkoxy group, and the halogenalkyl group are optionally selected from C ₁ -C ₆ alkyl group, a halogen group, a hydroxyl group, a hydroxyl group, -NR _i R _j , a pendoxy group, a thio group, a carboxyl group, a nitro group, a cyano group, a C ₁ -C ₆ alkoxy group, a C ₁ -C ₆ alkylthio group, a C ₂ -C ₆ alkenyl group, a C ₂ -C The alkylene group is substituted by one or more substituents selected from the group consisting of _6- membered alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclic group, 6- to 10-membered aryl and 5- to 10-membered heteroaryl;

Provided that when _R5a is hydrogen or alkyl, _R5b is not hydrogen or alkyl.

In certain embodiments, R _1a is each independently selected from hydrogen, C ₁ -C ₆ alkyl and C ₁ -C ₆ alkoxy, and the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, deuterium, amino, cyano and hydroxyl.

In certain embodiments, each R _1a is independently selected from hydrogen, C ₁ -C ₆ alkyl, or C ₁ -C ₆ alkoxy.

In certain embodiments, Ring A is selected from a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group, which is optionally substituted with one or more substituents Q ₁ , and the heteroaryl group contains at least one nitrogen atom.

或

。 In certain embodiments, Ring A is selected from optionally substituted with one or more substituents Q ₁

or

.

In certain embodiments, Ring B is selected from a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group, optionally substituted with one or more substituents Q ₁ , wherein the heteroaryl group contains at least one nitrogen atom.

或

。 In certain embodiments, Ring B is selected from optionally substituted with one or more substituents Q ₁

or

.

In certain embodiments, X ₁ is selected from -(CR _5a R _5b ) m -, a 6- to 10-membered aryl group optionally substituted with one or more substituents Q ₁ , or a 5- to 10-membered heteroaryl group, wherein the heteroaryl group contains at least one nitrogen atom.

In certain embodiments, R _5a , R _5b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, oxo, thio, nitro, cyano, or the following groups, which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR ₁ R _j , -C(O)R _k , -C(O)OR _k , -S(O)R _k , -S(O)OR _k , -S(O)(O)R _k , -S(O)(O)OR _k , -C(S)R _k , C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl.

In certain embodiments, R _5a , R _5b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, oxo, thio, cyano, or the following groups, which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR ₁ R _j , -C(O)R _k , -C(O)OR _k , -S(O)R _k , -S(O)OR _k , -S(O)(O)R _k , -S(O)(O)OR _k , C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl.

In certain embodiments, R _5a , R _5b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, oxo, thio, cyano, or the following groups, which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k , -S(O)R _k , -S(O)(O)R _k , C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl.

In certain embodiments, R _5a , R _5b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, oxo, thio, cyano, or the following groups which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k or C ₁ -C ₆ alkoxy.

In certain embodiments, Ring C and Ring D are each independently selected from a 6- to 10-membered aryl group, a 5- to 10-membered heteroaryl group, an 8- to 12-membered fused ring aryl group or a 5- to 12-membered fused heteroaryl group, which is optionally substituted with one or more substituents _Q1 , wherein the heteroaryl group or the fused heteroaryl group contains at least one nitrogen atom.

In certain embodiments, Ring C and Ring D are each independently selected from the following groups which are optionally substituted with one or more substituents Q ₁ :

、

、

、

或

。 In certain embodiments, Ring C is selected from the group consisting of: optionally substituted with one or more substituents Q ₁ :

,

,

,

or

.

In certain embodiments, ring D is selected from a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group, optionally substituted with one or more substituents Q ₁ , wherein the heteroaryl group contains at least one nitrogen atom.

或

。 In certain embodiments, Ring D is selected from optionally substituted with one or more substituents Q ₁

or

.

In certain embodiments, X ₂ is selected from -(CR _6a R _6b )n-, -O-, -S-, -NR _6c -, -CH ₂ S-, -CH ₂ O-, -NHCR _6d R _6e -, or a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group which is optionally substituted with one or more substituents Q ₁ , wherein the heteroaryl group contains at least one nitrogen atom.

In certain embodiments, R _6a , R _6b are each independently selected from hydrogen, halogen, hydroxyl, alkyl, deuterium, oxo, thio, cyano, or the following groups, which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k , -S(O)R _k , -S(O)OR _k , -S(O)(O)R _k , -S(O)(O)OR _k , C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, or R _6a , R _6b together with their adjacent carbon atoms form a 3- to 10-membered cycloalkyl group.

In certain embodiments, R _6a , R _6b are each independently selected from hydrogen, halogen, hydroxyl, alkyl, deuterium, oxo, thio, cyano, or the following groups, which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k , -S(O)R _k , -S(O)(O)R _k , C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, or R _6a , R _6b together with their adjacent carbon atoms form a 3- to 10-membered cycloalkyl group.

In certain embodiments, R _6a , R _6b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, oxo, thio, cyano, or the following groups which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k or C ₁ -C ₆ alkoxy.

In certain embodiments, R ₁ is each independently selected from hydrogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy, preferably hydrogen.

In certain embodiments, each R ₄ is independently selected from hydrogen.

In certain embodiments, each substituent group Q ₁ is independently selected from halogen, hydroxyl, halogen, deuterium, oxo, sulfhydryl, cyano, amine, carboxyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, or halogen C ₁ -C ₆ alkoxy.

In certain embodiments, R _k is independently selected from a hydrogen atom, a C ₁ -C ₆ alkyl group, a C ₁ -C ₆ halogenalkyl group, a C ₁ -C ₆ alkoxy group, a hydroxyl group, and -NR _i R _j .

In some embodiments, -D is as follows:

或

or

in,

R _1a are each independently selected from hydrogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy;

或

，該環A視需要被一個或多個取代基Q₁所取代； Ring A selected from

or

, the ring A is optionally substituted by one or more substituents Q ₁ ;

或

，該環B視需要被一個或多個取代基Q₁所取代； Ring B selected from

or

, the ring B is optionally substituted by one or more substituents Q ₁ ;

X ₁ is selected from -(CR _5a R _5b ) m - or a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group which is optionally substituted by one or more substituents Q ₁ , wherein the heteroaryl group contains at least one nitrogen atom;

R _5a and R _5b are each independently selected from hydrogen, halogen, hydroxyl, alkyl, deuterium, oxo, thio, cyano or the following groups which are optionally substituted by one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k or C ₁ -C ₆ alkoxy;

、

、

、

或

，該環C視需要被一個或多個取代基Q₁所取代； Ring C selected from

,

,

,

or

, the ring C is optionally substituted by one or more substituents Q ₁ ;

或

，該環D視需要被一個或多個取代基Q₁所取代； Ring D selected from

or

, the ring D is optionally substituted by one or more substituents Q ₁ ;

_X2 is selected from -( _CR6aR6b )n-, -O-, -S-, _{-NR6c- ,} -CH2S-, _-CH2O- , _-NHCR6dR6e- , or _a 6- to 10-membered aryl group or a 5- to 10-membered _heteroaryl group which is optionally substituted by one or more substituents _Q1 ;

R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, oxo, thio, cyano or the following groups which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k or C ₁ -C ₆ alkoxy;

R _6c , R _6d , and R _6e are each independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ halogenalkyl, or C ₁ -C ₆ alkoxy;

R ₂ is each independently selected from -CH ₂ OH, -CH ₂ SH, -CH ₂ Cl, -SCH ₂ Cl, -SCH ₂ F, -SCH ₂ CF ₃ , -OH, -OCH ₂ CN, -OCH ₂ Cl. , -OCH ₂ F , -OCH ₃ , -OCH ₂ CH ₃ , -SCH ₂ CN,

R _2a are each independently selected from hydrogen or C ₁ -C ₆ alkyl;

R _2b are each independently selected from C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy;

R _2c are each independently selected from hydrogen, C ₁ -C ₆ alkyl, -CH ₂ OH or C ₁ -C ₆ alkoxy;

R _2d and R _2e are each independently selected from hydrogen or C ₁ -C ₆ alkyl;

_R3 are each independently selected from hydrogen or halogen;

m and n are independently selected from integers between 1 and 6;

The substituent groups _Q1 are each independently selected from halogen, hydroxyl, halogen, deuterium, oxo, sulfhydryl, cyano, amine, carboxyl, _C1 - _C6 alkyl, _C1 - _C6 alkoxy or halogen _C1 - _C6 alkoxy;

R _i and R _j are each independently selected from a hydrogen atom, a hydroxyl group, a C ₁ -C ₆ alkyl group, and a C ₁ -C ₆ alkoxy group; R _k is independently selected from a hydrogen atom, a C ₁ -C ₆ alkyl group, a C ₁ -C ₆ halogenalkyl group, a C ₁ -C ₆ alkoxy group, a hydroxyl group, and -NR _i R _j ;

Provided that when _R5a is hydrogen or alkyl, _R5b is not hydrogen or alkyl.

In certain embodiments, R _1a is hydrogen.

、

或

。 In certain embodiments, X ₁ is selected from -(CR _5a R _5b ) m - or optionally substituted with one or more substituents Q ₁ :

,

or

.

In certain embodiments, R _5a , R _5b are both fluoro.

In certain embodiments, R _5a is pendoxo or thiol, preferably pendoxo.

、

或

。 In certain embodiments, X ₂ is selected from -(CR _6a R _6b )n- or optionally substituted by one or more substituents Q ₁ :

,

or

.

In certain embodiments, R _6a , R _6b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, oxo, thio, cyano, C ₁ -C ₆ alkyl, —NR _i R _j , —C(O)OR _k or C ₁ -C ₆ alkoxy.

。 In certain embodiments, _R2 is each independently selected from _-CH2OH , _-CH2SH , -OH or

.

In certain embodiments, R ₃ is hydrogen.

In certain embodiments, R ₃ is fluoro.

In some implementations, k is any number between 1-10, preferably any number between 2-5. k can be an integer or a decimal.

In some specific implementation schemes, D is as shown in the following structure:

In certain embodiments, the linker is stable outside of the cell, allowing the ADC to remain intact when present in the extracellular environment, but to be cleaved when internalized in the cell. In certain embodiments, when the ADC enters a cell expressing an antigen specific for the antibody portion of the ADC, the glucocorticoid receptor agonist drug portion is cleaved from the antibody portion, and the cleavage releases the unmodified form of the glucocorticoid receptor agonist.

In some embodiments, the cleavable portion of the linker is a cleavable peptide portion. In some embodiments, ADCs comprising a cleavable peptide portion exhibit lower aggregation levels, improved antibody to drug ratios, relative to ADCs comprising other cleavable portions. In some embodiments, the addition of a cleavable portion increases cytotoxicity and/or potency relative to a non-cleavable linker. In some embodiments, the cleavable peptide portion is cleavable by an enzyme, and the linker is an enzyme-cleavable linker. In some embodiments, the enzyme is a cathepsin, and the linker is a cathepsin-cleavable linker. In some embodiments, an enzyme-cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the above-mentioned improved properties compared to other cleavage mechanisms.

(q為1-6的整數)的胺基酸構成的肽殘基，示例性胺基酸單元包括但不限於纈胺酸-瓜胺酸(Val-Cit)、丙胺酸-苯丙胺酸(Ala-Phe)；苯丙胺酸-賴胺酸(Phe-Lys)、苯丙胺酸-高賴胺酸(Phe-Homolys)、n-甲基-纈胺酸-瓜胺酸(Me-Val-Cit)、丙胺酸-丙胺酸(Ala-Ala)、甘胺酸-谷胺酸(Gly-Glu)、谷胺酸-丙胺酸-丙胺酸(Glu-Ala-Ala)和甘胺酸-賴胺酸(Gly- Lys)、甘胺酸-纈胺酸-瓜胺酸(Glv-Val-Cit)和甘胺酸-甘胺酸-甘胺酸(Gly-Gly-Gly)、

。 In certain embodiments, the linker comprises an amino acid unit _L2 , which preferably comprises 2 to 7 amino acid units selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamate, aspartic acid, homolysine, n-methyl-valine,

(q is an integer of 1-6), exemplary amino acid units include but are not limited to valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe); phenylalanine-lysine (Phe-Lys), phenylalanine-homolysine (Phe-Homolys), n-methyl-valine-citrulline (Me-Val-Cit), alanine-alanine (Ala-Ala), glycine-glutamate (Gly-Glu), glutamate-alanine-alanine (Glu-Ala-Ala) and glycine-lysine (Gly-Lys), glycine-valine-citrulline (Glv-Val-Cit) and glycine-glycine-glycine (Gly-Gly-Gly),

.

In certain embodiments, the linker comprises a stretching unit, a chemical structure fragment having one end covalently linked to the antibody via a carbon atom and the other end linked to an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptide chemical moiety. Exemplary stretching units include, but are not limited to,

In some embodiments, the stretching unit is selected from

、

、

，其中p各自獨立地選自1、2、3、4、5或6。

,

,

, wherein p is each independently selected from 1, 2, 3, 4, 5 or 6.

In some embodiments, the connector is selected from

In some embodiments, the connector is selected from

。 In some specific implementation schemes, the linker is:

.

In certain embodiments, the antibody-drug conjugate is selected from:

Wherein, Ab, D, and k are as described above, and p is independently selected from 1, 2, 3, 4, 5, or 6.

In certain embodiments, the antibody-drug conjugate is selected from

k is an integer or decimal selected from 1-10 (for example, 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 4±0.4, 4±0.5, 4±0.6, 4±0.8. For example, 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, etc.

In some specific embodiments, the antibody-drug conjugate is as follows:

Wherein, k is an integer or decimal of 1-10 (for example, 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 4±0.4, 4±0.5, 4±0.6, 4±0.8. For example, 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, etc.

In some embodiments, the antibody-drug conjugate is selected from any antibody-drug conjugate structure disclosed in WO2022166779A1.

In some embodiments, the antibody-drug conjugate has improved anti-transplant rejection activity. The anti-transplant rejection activity of the antibody-drug conjugate disclosed in the present invention is significantly better than anti-CD40 antibodies, anti-CD40 antibodies combined with glucocorticoids, and better than dexamethasone. In some embodiments, the antibody-drug conjugate has improved safety. For example, reduced toxic side effects. In some embodiments, the reduced toxic side effects include but are not limited to reduced effects on body weight, reduced effects on endogenous corticosterone release, and reduced effects on the bone formation indicator P1NP.

Antibody-drug (glucocorticoid) conjugates

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

The present disclosure provides an antibody-drug conjugate having a structure shown in formula (I):

Ab-(LD) _k (I);

Wherein, Ab is the anti-CD40 antibody or its antigen-binding fragment disclosed herein;

D is glucocorticoid;

L is a linker that covalently links Ab to D, and 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).

In some embodiments, D is as follows:

In some implementations, k is any number between 1-10, preferably any number between 2-5. k can be an integer or a decimal.

In certain embodiments, the linker is stable outside of the cell, allowing the ADC to remain intact when present in the extracellular environment, but to be cleaved when internalized in the cell. In some embodiments, when the ADC enters a cell expressing an antigen specific for the antibody portion of the ADC, the glucocorticoid drug portion is cleaved from the antibody portion, and the cleavage releases the unmodified form of the glucocorticoid.

In some embodiments, the cleavable portion of the linker is a cleavable peptide portion. In some embodiments, ADCs comprising a cleavable peptide portion exhibit lower aggregation levels, improved antibody to drug ratios, relative to ADCs comprising other cleavable portions. In some embodiments, the addition of a cleavable portion increases cytotoxicity and/or potency relative to a non-cleavable linker. In some embodiments, the cleavable peptide portion is cleavable by an enzyme, and the linker is an enzyme-cleavable linker. In some embodiments, the enzyme is a cathepsin, and the linker is a cathepsin-cleavable linker. In some embodiments, an enzyme-cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the above-mentioned improved properties compared to other cleavage mechanisms.

In some embodiments, the linker comprises a stretching unit, a chemical structure fragment having one end covalently linked to the antibody via a carbon atom and the other end linked to an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptide chemical moiety. Exemplary stretching units include, but are not limited to,

(q為1-6的整數)的胺基酸構成的肽殘基，示例性胺基酸單元包括但不限於纈胺酸-瓜胺酸(Val-Cit)、丙胺酸-苯丙胺酸(Ala-Phe)；苯丙胺酸-賴胺酸(Phe-Lys)、苯丙胺酸-高賴胺酸(Phe-Homolys)、n-甲基-纈胺酸-瓜胺酸(Me-Val-Cit)、丙胺酸-丙胺酸(Ala-Ala)、甘胺酸-谷胺酸(Gly-Glu)、谷胺酸-丙胺酸-丙胺酸(Glu-Ala-Ala)和甘胺酸-賴胺酸(Gly-Lys)、甘胺酸-纈胺酸-瓜胺酸(Glv-Val-Cit)和甘胺酸-甘胺酸-甘胺酸(Gly-Gly-Gly)、

。 In some embodiments, the linker comprises an amino acid unit, preferably comprising 2 to 7 amino acid units selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamate, aspartic acid, homolysine, n-methyl-valine,

(q is an integer of 1-6), exemplary amino acid units include but are not limited to valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe); phenylalanine-lysine (Phe-Lys), phenylalanine-homolysine (Phe-Homolys), n-methyl-valine-citrulline (Me-Val-Cit), alanine-alanine (Ala-Ala), glycine-glutamate (Gly-Glu), glutamate-alanine-alanine (Glu-Ala-Ala) and glycine-lysine (Gly-Lys), glycine-valine-citrulline (Glv-Val-Cit) and glycine-glycine-glycine (Gly-Gly-Gly),

.

，其中p1為整數1至20，例 如

，(PEG)₂；

，(PEG)₄；

，(PEG)₅。 In some embodiments, the linker can include at least one polyethylene glycol (PEG) moiety. The PEG moiety can, for example, include -(PEG) _p1 -,

, where p1 is an integer from 1 to 20, for example

, (PEG) ₂ ;

, (PEG) ₄ ;

, (PEG) ₅ .

In some embodiments, the linker comprises a spacer unit connected to D. In some embodiments, the spacer unit comprises p-aminobenzyloxycarbonyl (PAB),

In some embodiments, the spacer unit comprises a p-aminobenzoyl group,

In some embodiments, the spacer unit comprises:

-NH(CH ₂ ) _n1 -L ^a -L ^b -L ^c -, wherein n1 is selected from an integer between 0 and 6; ^La represents -C(═O)-NH-, -NR ⁷ -(CH ₂ ) _n2 -, -O- or a single bond, R ⁷ is selected from hydrogen, C _1-6 alkyl, -(CH ₂ ) _n3 -COOH, -(CH ₂ ) _n4 -OH, n3 is selected from an integer between 1 and 4, and n4 is selected from an integer between 1 and 6; L ^b represents -CR ⁸ (R ⁹ )-, -O-, -NR ¹⁰ - or a single bond, R ⁸ and R ⁹ are independently selected from hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, -(CH ₂ ) _n5 -NH ₂ , -(CH ₂ ) _n6 -COOH, -(CH ₂ ) _n7 -OH, R ¹⁰ is selected from hydrogen or C _1-6 alkyl, n5 is selected from an integer between 0-6, n6 is selected from an integer between 1-4, n7 is selected from an integer between 1-4, and when n5 is 0, R ⁸ and R ⁹ are different, or R ⁸ and R ⁹ together with the carbon atom to which they are connected form a C _3-6 cycloalkyl; L ^c represents -CH ₂ - or -C(=O)-.

In some embodiments, in -NH(CH ₂ ) _n1 -L ^a -L ^b -L ^c -, L ^c represents -NHCH ₂ -.

In some embodiments, R ⁸ and R ⁹ in -NH(CH ₂ ) _n1 -L ^a -L ^b -L ^c - are independently selected from hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, such as hydrogen, methyl, ethyl or cyclopropyl.

In some embodiments, in -NH(CH ₂ ) _n1 -L ^a -L ^b -L ^c -, ^La represents -O- or a single bond, L ^b represents -CR ⁸ (R ⁹ )- or a single bond, L ^c represents -C(=O)-, and R ⁸ and R ⁹ together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group.

In some embodiments, in -NH(CH ₂ ) _n1 -L ^a -L ^b -L ^c -, ^La represents -O- or a single bond, L ^b represents -CR ⁸ (R ⁹ )- or a single bond, L ^c represents -C(=O)-, R ⁸ and R ⁹ are independently selected from hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, -(CH ₂ ) _n5 -NH ₂ , -(CH ₂ ) _n6 -COOH, -(CH ₂ ) _n7 -OH, R ¹⁰ is selected from hydrogen or C _1-6 alkyl, n5 is selected from an integer between 0 and 6, n6 is selected from an integer between 1 and 4, n7 is selected from an integer between 1 and 4, and when n5 is 0, R ⁸ and R ⁹ are different.

In some embodiments, in -NH(CH ₂ ) _n1 -L ^a -L ^b -L ^c -, L ^a represents -O- or a single bond, L ^b represents -CR ⁸ (R ⁹ )- or a single bond, and L ^c represents -CH ₂ -.

In some embodiments, in -NH(CH ₂ ) _n1 -L ^a -L ^b -L ^c -, ^La represents -O- or a single bond, L ^b represents -CR ⁸ (R ⁹ )- or a single bond, L ^c represents -CH ₂ -, and R ⁸ and R ⁹ are independently selected from hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, preferably hydrogen, methyl, ethyl or cyclopropyl.

In some embodiments, in the antibody-drug conjugate, -NH(CH ₂ ) _n1 -L ^a -L ^b -L ^c - is: -NHCH ₂ -, -NHCH ₂ CH ₂ -, -NHCH ₂ CH ₂ CH ₂ -, -NHCH ₂ -O-CH ₂ -, -NHCH ₂ CH ₂ -O-CH ₂ -, -NH(CH ₂ ) ₃ -C(O)-, -NHCH ₂ -O-CH ₂ -C(O)-, -NH(CH ₂ ) ₂ -O-CH ₂ -C(O)-,

In some embodiments, -NH-(CH ₂ ) _n1 -L ^a -L ^b -L ^c - is -NHCH ₂ - or -NHCH ₂ CH ₂ CH ₂ -.

In some embodiments, the spacer unit in the linker comprises (PEG) ₄ . In some embodiments, despite the short linker length, ADCs comprising a shorter spacer unit (e.g., (PEG) ₄ ) exhibit lower aggregation levels and/or higher drug loading relative to ADCs comprising a longer spacer unit (e.g., (PEG) ₈ ).

On the other hand, in the disclosed antibody conjugate (ADC), L-D is a chemical moiety represented by the following formula:

-Str-Pep-Sp-D

Str is the stretching unit covalently linked to Ab.

Sp is the spacing unit,

Pep is selected from an amino acid unit, a disulfide moiety, a sulfonamide moiety, or the following non-peptide chemical moieties:

，

或

，其中，W是-NH-亞(伸)雜環烷基-或雜環烷基；Y是亞(伸)雜芳基、亞(伸)芳基、-C(O)C_1-6亞(伸)烷基、C_2-6亞(伸)烯基、C_1-6亞(伸)烷基或-C_1-6亞(伸)烷基-NH-；每個R¹⁶獨立選自C_1-6烷基、C_2-6烯基、-(C_1-6亞(伸)烷基)NHC(NH)NH₂或-(C_1-6亞(伸)烷基)NHC(O)NH₂；R¹⁷和R¹⁸各自獨立選自氫、C_1-6烷基、C_2-6烯基、芳基、雜芳基，或R¹⁷和R¹⁸一起可形成C_3-6環烷基；R¹⁹和R²⁰各自獨立選自C_1-6烷基、C_2-6烯基、芳基、雜芳基、(C_1-6烷基)OCH₂-，或R¹⁹和R²⁰一起可形成C_3-6環烷基環。

,

or

, wherein W is -NH-heterocycloalkylene- or heterocycloalkylene; Y is heteroarylene, arylene, -C(O)C 1-6 alkylene, C 2-6 alkenylene, C _1-6 alkylene or -C _1-6 alkylene-NH-; each R ¹⁶ is independently selected from C 1-6 alkyl, C _2-6 alkenyl, -(C _1-6 alkylene)NHC(NH)NH 2 or -(C _1-6 alkylene)NHC(O)NH ₂ ; R 17 and R ₁₈ are each independently selected from hydrogen, C _{1-6 alkyl, C 2-6 alkenyl, aryl, heteroaryl, or R 17 and R 18 together can form a C 3-6 cycloalkyl} ^; _{R 19 and R 19 are each independently selected from hydrogen, C 1-6} ^alkyl _, C _2-6 alkenyl, aryl, heteroaryl, or R ¹⁹ and R ¹⁹ together can form a C _3-6 cycloalkyl; R ¹⁹ and R ^{R 20} are each independently selected from C _1-6 alkyl, C _2-6 alkenyl, aryl, heteroaryl, (C _1-6 alkyl)OCH ₂ —, or R ¹⁹ and R ²⁰ together can form a C _3-6 cycloalkyl ring.

In some embodiments, Str in the antibody-drug conjugate (ADC) is selected from a chemical moiety represented by the following formula:

或

，其中R²¹選自-W1-C(O)-、-C(O)-W1-C(O)-、(CH₂CH₂O)_p1C(O)-、(CH₂CH₂O)_p1CH₂C(O)-、(CH₂CH₂O)_p1CH₂CH₂C(O)-，其中p1為1至20的整數(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)，W1選自C_1-6亞(伸)烷基、C_1-6亞(伸)烷基-環烷基或1至8個原子的直鏈雜烷基，該雜烷基包含1至3個選自N、O或S的雜原子，其中該烷基、環烷基和直鏈雜烷基各自獨立地視需要被一個或多個選自鹵素、氘、羥基、氰基、胺基、C_1-6烷基、鹵C_1-6烷基、氘代C_1-6烷基、C_1-6烷氧基和C_3-6環烷基所取代；

or

, wherein R ²¹ is selected from -W1-C(O)-, -C(O)-W1-C(O)-, (CH ₂ CH ₂ O) _p1 C(O)-, (CH ₂ CH ₂ O) _p1 CH ₂ C(O)-, (CH 2 CH 2 O) _p1 CH _{2 CH 2} C(O)-, wherein p1 is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, ₁₅ , 16, 17, 18, 19 or 20), and W1 is selected from C _1-6 alkylene, C _C1-6alkylene -cycloalkyl or a straight-chain heteroalkyl group of 1 to 8 atoms, the heteroalkyl group containing 1 to 3 heteroatoms selected from N, O or S, wherein the alkyl, cycloalkyl and straight-chain heteroalkyl group are each independently and optionally substituted by one or more selected from halogen, deuterium, hydroxyl, cyano, amino, _C1-6alkyl , halogenated _C1-6alkyl , deuterated _C1-6alkyl , _C1-6alkoxy and _{C3-6cycloalkyl} ;

^L1 is selected from ^-NR22 _{( CH2CH2O} ) _p1CH2CH2C (O _{) -} , -NR22( _{CH2CH2O )} p1CH2C ₍ O)-, -S( _CH2 ) _p1C (O)-, -( _CH2 ) _p1C (O)- or a single bond, preferably a single bond; _p1 is an integer from 1 to ²⁰ , and ^R22 is selected from a hydrogen atom, a _C1-6 alkyl group, a halogenated _C1-6 alkyl group or a deuterated _C1-6 alkyl group.

In some embodiments, R ²¹ in the antibody-drug conjugate Str is selected from C _1-6 alkylene C(O)-, -(CH ₂ -CH ₂ O) ₂ C(O)-, -(CH 2 -CH ₂ O) ₂ CH 2 C(O)-, -(CH ₂ -CH 2 O) 2 CH ₂ C(O)-, -(CH ₂ -CH ₂ O) ₂ CH ₂ CH ₂ C(O)-, -(CH ₂ -CH ₂ O) ₃ C(O)-, -(CH ₂ -CH ₂ O) ₄ C(O)-, -(CH ₂ -CH ₂ O) ₄ CH ₂ C(O)-, and -(CH ₂ -CH ₂ O) ₄ CH ₂ CH ₂ C(O)-.

In some embodiments, the Pep is selected from valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe), or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

、

、

、

、

和

。 In some embodiments, the Sp is selected from _-NHCH2- , -NHCH2CH2-, _{-NHCH2CH2CH2- , -NHCH2} - _{O -} CH2-, _{-NHCH2CH2 -} O- _CH2- , -NH(CH2)3- _C (O)-, _{-NHCH2 -} O _{- CH2 -} C(O)-, -NH(CH2) ₂ -O- _{CH2 -} C(O)-,

,

,

,

,

and

.

In some embodiments, the linker L in the antibody-drug conjugate comprises: cis-1-nitrilodiamide-(PEG) ₄ -CH ₂ CH ₂ C(O)-Gly-Gly-Phe-Gly, cis-1-nitrilodiamide-(PEG) ₂ -Val-Cit, cis-1-nitrilodiamide-(PEG) ₆ -Val-Cit, cis-1-nitrilodiamide-(PEG) ₈ -Val-Cit, cis-1-nitrilodiamide-(PEG) ₄ -CH ₂ CH ₂ C(O)-Val-lys, cis-1-nitrilodiamide-(CH ₂ ) ₅ -Val-Cit, cis-1-nitrilodiamide-(PEG _{) 5} -Val-lys, cis-1-nitrilodiamide-(CH ₂ ) ₅ -Gly-Gly-Phe-Gly, cis-1-imide-(PEG) ₂ -CH ₂ CH ₂ C(O)-Gly-Gly-Phe-Gly, cis-1-imide-(PEG) ₂ -Ala-Ala-Asn, cis-1-imide-(PEG) ₆ -Ala-Ala-Asn, cis-1-imide-(PEG) ₈ -Ala-Ala-Asn, cis-1-imide-(PEG) ₄ -triazole-(PEG) ₃ -sulfonamide, cis-1-imide-(PEG) _2- CH ₂ CH ₂ C(O)-Val-lys, cis-1-imide-(PEG) ₄ -triazole-(PEG) ₃ -sulfonamide, or Mal-(PEG) ₄ -Triazole-(PEG) ₃ -disulfide.

In some embodiments, the linker L in the antibody-drug conjugate comprises: _-CH2C (O)NH-(PEG) _{4 - CH2CH2C} (O ₎ -Gly-Gly-Phe-Gly, -CH2C ₍ O)NH-(PEG) ₂ -Val-Cit, -CH2C(O)NH-(PEG _{) 6} -Val-Cit, -CH2C(O ₎ NH-(PEG) _8- Val-Cit, -CH2C(O)NH-(PEG) ₄ - _CH2CH2C (O ₎ -Val-lys, _-CH2C (O)NH-( _CH2 ) ₅ -Val-Cit, _-CH2C (O)NH-( _CH2 ) ₅ -Val-lys, -CH2C ₍ O)NH-( _CH2 ) ₅ -Gly-Gly-Phe-Gly, _-CH2 C(O)NH-(PEG) ₂ -CH ₂ CH ₂ C(O)-Gly-Gly-Phe-Gly, -CH ₂ C(O)NH-(PEG) ₂ -Ala-Ala-Asn, -CH ₂ C(O)NH-(PEG) ₆ -Ala-Ala-Asn, -CH ₂ C(O)NH-(PEG) ₈ -Ala-Ala-Asn, -CH ₂ C(O)NH-(PEG) ₄ -triazole-(PEG) ₃ -sulfonamide, -CH ₂ C(O)NH-(PEG) ₂ -CH ₂ CH ₂ C(O)-Val-lys, -CH ₂ C(O)NH-(PEG) ₄ -triazole-(PEG) ₃ -sulfonamide, or Mal-(PEG) ₄ -triazole-(PEG) ₃ -disulfide.

In other embodiments, the present invention discloses an antibody-drug conjugate (ADC), which is represented by the following formula:

，其中k選自1至10，可以為整數，也可以為小數，p1選自2、4、6或8，p3、p4各自獨立地選自0、1或2；

, where 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 independently selected from 0, 1 or 2;

，其中k選自1至10，可以為整數，也可以為小數，p1選自2、4、6或8，p3、p4各自獨立地選自0、1或2；

, where 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 independently selected from 0, 1 or 2;

In other embodiments, the disclosed antibody-drug conjugate (ADC) is selected from:

Wherein k is an integer or decimal of 1-10 (for example, 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 4±0.4, 4±0.5, 4±0.6, 4±0.8. For example, 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, etc.

In other embodiments, the disclosed antibody-drug conjugate (ADC) is as follows:

其中k為1-10的整數或小數(例如，1、2、3、4、5、6、7、8、9、10或前述任意兩數值之間任意小數或整數)。在一些實施方案中，k為4±0.4、4±0.5、4±0.6、4±0.8。例如，k為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等。

Wherein k is an integer or decimal between 1 and 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 4±0.4, 4±0.5, 4±0.6, 4±0.8. For example, 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, etc.

The present disclosure also provides a compound represented by formula II-A or a pharmaceutically acceptable salt thereof,

Where p1 is selected from 2, 4, 6 or 8, and p3 and p4 are independently selected from 0, 1 or 2.

The present disclosure also provides a compound represented by formula II-B or a pharmaceutically acceptable salt thereof,

Where X is a halogen, p1 is selected from 2, 4, 6 or 8, and p3 and p4 are independently selected from 0, 1 or 2.

表示單鍵或雙鍵。 In the structure disclosed in the present disclosure,

Indicates a single key or two keys.

In some embodiments, the antibody-drug conjugate comprises any linker and/or drug moiety disclosed in WO2023143351A1.

In some embodiments, the antibody-drug conjugate has improved anti-transplant rejection activity. The anti-transplant rejection activity of the antibody-drug conjugate disclosed in the present invention is significantly better than anti-CD40 antibodies, anti-CD40 antibodies combined with glucocorticoids, and better than dexamethasone. In some embodiments, the antibody-drug conjugate has improved safety. For example, reduced toxic side effects. In some embodiments, the reduced toxic side effects include but are not limited to reduced effects on body weight, reduced effects on endogenous corticosterone release, and reduced effects on the bone formation indicator P1NP.

Composition

The present disclosure provides a composition comprising the aforementioned anti-CD40 antibody-drug conjugate. For example, a pharmaceutical composition is provided, which contains a therapeutically or palliatively effective amount of the aforementioned anti-CD40 antibody-drug conjugate and at least one pharmaceutically acceptable excipient, diluent or carrier.

In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the anti-CD40 antibody-drug conjugate in a unit dosage, or the amount of the anti-CD40 antibody-drug conjugate in a unit dosage of the pharmaceutical composition is 0.1-2000 mg, and in some specific embodiments, it is 1-1000 mg.

In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the antibody-drug conjugate in a unit dosage, or the amount of the antibody-drug conjugate in a unit dosage of the pharmaceutical composition is 0.1-2000 mg, and in some specific embodiments, it is 1-1000 mg.

In some embodiments, a combination or composition of the aforementioned anti-CD40 antibody or its antigen-binding fragment, antibody-drug conjugate and one or more additional immunosuppressants is provided. The composition is, for example, a pharmaceutical composition. Optionally, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, diluent or carrier.

In some embodiments, a product is provided, comprising the aforementioned anti-CD40 antibody or its antigen-binding fragment, or antibody-drug conjugate. Optionally, the product comprises a container and a label. The container is such as a bottle, a syringe, and a test tube. The container contains a composition effective for treating a disease. The label on or connected to the container indicates that the composition is used to treat the selected disease. The composition contains the aforementioned anti-CD40 antibody or its antigen-binding fragment, or antibody-drug conjugate.

Treatment methods and pharmaceutical uses

The present disclosure provides methods for treating, intervening, preventing, and diagnosing diseases or symptoms using the aforementioned anti-CD40 antibodies or their antigen-binding fragments, antibody-drug conjugates, or pharmaceutical compositions.

Specifically, in some embodiments, the present disclosure provides the use of the anti-CD40 antibody-drug conjugate or pharmaceutical composition described in the present disclosure in the preparation of a drug for treating or alleviating autoimmune diseases, graft-versus-host disease, or alleviating transplant rejection reactions.

In some embodiments, methods for improving or treating autoimmune diseases and related pharmaceutical uses are provided, including administering to a subject an effective amount of the aforementioned anti-CD40 antibody-drug conjugate or its pharmaceutical composition for improving or treating the disease.

In some embodiments, methods for treating CD40-related disorders and related pharmaceutical uses are provided; in some embodiments, methods for inhibiting the growth or differentiation of CD40-related disorder cells and related pharmaceutical uses are provided; in some embodiments, methods for inhibiting the growth and/or differentiation of cells expressing human CD40 antigen and related pharmaceutical uses are provided; in some embodiments, methods for inhibiting antibody production by B cells in subjects and related pharmaceutical uses are provided; in some embodiments, methods for treating patients with immune disorders and related pharmaceutical uses are provided. The above methods all include administering a therapeutic or inhibitory effective amount of the aforementioned anti-CD40 antibody-drug conjugate or its pharmaceutical composition to the subject or cell.

In some embodiments, methods for inducing peripheral B cell depletion and related pharmaceutical uses are provided, comprising administering to a subject an inducing effective amount of the aforementioned anti-CD40 antibody or its antigen-binding fragment, antibody-drug conjugate or its pharmaceutical composition.

In some embodiments, methods for treating or alleviating a disease or condition and related pharmaceutical uses are provided, comprising administering an effective amount of the aforementioned anti-CD40 antibody or its antigen-binding fragment, antibody-drug conjugate or pharmaceutical composition to a subject in need thereof, wherein the disease or condition may be CD40-related or CD40-unrelated. In some embodiments, the disease or condition is an immune disease. In some embodiments, the disease or condition is a transplant rejection reaction.

In some embodiments, methods for treating diseases related to B lymphocytes, Th1 lymphocytes or Th2 lymphocytes and related pharmaceutical uses are provided, comprising administering an effective amount of the aforementioned anti-CD40 antibody or its antigen-binding fragment, antibody-drug conjugate or pharmaceutical composition to a subject in need.

In some embodiments, a method for treating or alleviating graft-versus-host disease or transplant rejection is provided, comprising administering the aforementioned anti-CD40 antibody-drug conjugate or pharmaceutical composition to a subject in need thereof.

In some embodiments, an antibody-drug conjugate or pharmaceutical composition is provided for use in preparing a drug for treating or alleviating graft-versus-host disease or transplant rejection.

In some embodiments, the transplant is a solid organ and/or tissue transplant.

In some embodiments, the above-mentioned transplantation refers to transplantation of one selected from the group consisting of homologous cells, heterologous cells, homologous tissues, heterologous tissues, homologous organs and heterologous organs.

In some embodiments, the aforementioned anti-CD40 antibody or its antigen-binding fragment, antibody-drug conjugate or pharmaceutical composition 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.

In some embodiments, the antibody-drug conjugates provided by the present disclosure can be used to treat or alleviate autoimmune diseases, graft-versus-host disease and/or transplant rejection reactions.

Therefore, the present disclosure provides the use of the antibody-drug conjugates herein in the preparation of drugs for treating or alleviating autoimmune diseases, graft-versus-host disease and/or transplant rejection reactions.

On the other hand, the present disclosure provides a method for preventing or treating transplant rejection, graft-versus-host disease and/or autoimmune disease, which comprises administering a preventive or therapeutically effective amount of the antibody-drug conjugate described herein to a subject in need thereof.

Definition of terms

In order to make it easier to understand the present disclosure, 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 of ordinary skill in the art to which the present disclosure belongs.

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

"CD40" and "CD40 antigen" refer to an approximately 48 kD glycoprotein expressed on the surface of normal and proliferative B cells that functions 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 has been 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 this disclosure. Cells that endogenously express CD40 are any cells characterized by surface expression of CD40, including but not limited to normal and myogenic B cells, cross-linked cells, basal epithelial cells, cancer cells, macrophages, endothelial cells, follicular dendritic cells, tonsil cells, and bone marrow-derived plasma cells.

The term "linker", "linker unit", "linker unit", "linker" or "linker fragment" refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other linkers and then connected to a drug.

The linker may comprise one or more linker components. Exemplary linker components include 6-maleimidohexanoyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with linker reagents: N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC, also referred to herein as MCC) and N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB). The linker may include stretching units, spacer units, amino acid units, and extension units. It can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker can be a "cleavable linker" that facilitates release of the drug in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

The term "stretcher" refers to a chemical structural fragment that is covalently linked to the antibody via a carbon atom at one end and linked to an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptide chemical moiety at the other end.

The term "spacer unit" refers to a bifunctional chemical structural fragment that can be used to couple amino acid units and effector molecules to ultimately form antibody-drug conjugates. This coupling method can selectively link the effector molecule to the amino acid unit.

The term "amino acid" refers to an organic compound that contains an amine group and a carboxyl group in its molecular structure, and both the amine group and the carboxyl group are directly connected to the -CH- structure. The general formula is H2NCHRCOOH, where R is H, substituted or unsubstituted alkyl, etc. Depending on the position of the amine group connected to the carbon atom in the carboxylic acid, it can be divided into α, β, γ, δ, ε...-amino acids. In the biological world, the amino acids that make up natural proteins have specific structural characteristics, that is, their amine groups are directly connected to the α-carbon atom, namely α-amino acids, including glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, proline, etc. Non-natural amino acids such as citrulline. As is well known to those skilled in the art, non-natural amino acids do not constitute natural proteins and therefore do not participate in the synthesis of antibodies disclosed herein. The three-letter and one-letter amino acid codes used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

The term "antibody-drug conjugate" refers to an antibody connected to a biologically active drug via a stable linker. In the present disclosure, "antibody-drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment connected to an effector molecule with immunosuppressive activity via a stable linker. The antibody or antibody fragment can be bound to the effector molecule containing the linker via a specific group therein (such as an interchain disulfide bond).

In some embodiments, the effector molecule has anti-autoimmune disease activity. In some embodiments, the effector molecule has anti-transplant rejection activity.

In some embodiments, the effector molecule is selected from glucocorticoid receptor agonists, glucocorticoids, calcitonin inhibitors, or mTOR kinase inhibitors. In other embodiments, the effector molecule can also be a polypeptide, nucleic acid, protein, compound, etc. with immunosuppressive activity.

The term "drug loading" refers to the average amount of drug carried by each antibody-drug conjugate molecule in the 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 can range from 1-20, preferably 1-10 effector molecules (D) linked to each antibody (Ab). In the embodiments disclosed herein, 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 average 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. The average amount of drug per ADC molecule after the conjugation reaction can be determined using conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays, monoclonal antibody size variant assays (CE-SDS), and HPLC characterization.

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

In one embodiment of the present disclosure, the drug moiety is coupled to the N-terminal amine group and/or the ε-amine group of the lysine residue of the antibody via a linker unit. 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.

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

(1) Control the molar ratio of the linker reagent and the monoclonal antibody,

(2) Control the reaction time and temperature,

(3) Choose different reaction reagents.

The term "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into kappa chains or lambda chains according to the difference in the constant region. Each of the five types of Ig can have a kappa chain or a lambda chain.

The sequences of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region. The variable region includes 3 hypervariable regions (HVR) and 4 relatively conserved framework regions (FR). The 3 hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the order from the amino end to the carboxyl end is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

The antibodies disclosed herein include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.

The term "mouse antibody" in this disclosure refers to antibodies prepared in mice according to the knowledge and skills in the art. During the preparation, the test subject is injected with a specific antigen and then the fusion tumor expressing the antibody with the desired sequence or functional properties is isolated.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, you must first establish a fusion tumor that secretes mouse-specific monoclonal antibodies, then select variable region genes from the mouse fusion tumor cells, and then select human antibody constant region genes as needed. The mouse variable region gene and the human constant region gene are connected to form a chimeric gene and inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into human antibody variable region frameworks, i.e., different types of human germline antibody framework sequences. It can overcome the heterologous reactions induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the variable region framework sequence of the human antibody can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies after CDR affinity maturation by phage display. Literature further describing methods for humanization using mouse antibodies includes, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and the methods of Winter and colleagues [Jones et al., Nature, 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of the antibody. Examples of binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') ₂ fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; and (vi) isolated complementary determining regions (CDRs) or (vii) a combination of two or more isolated CDRs, which may be optionally linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker using recombinant methods, thereby enabling them to be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those of ordinary skill in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen binding moieties may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

Fab is an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.

F(ab') ₂ is an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin and comprising two Fab regions linked at the hinge position.

Fab' is an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, obtained by cleaving the disulfide bond of the hinge region of the F(ab') ₂ .

In addition, the Fab' can be produced by inserting a DNA encoding the Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or a eukaryotic organism to express the Fab'.

The term "single-chain antibody", "single-chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have the general structure: _NH2 -VL-linker-VH-COOH or _NH2 -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example variants with 1-4 repeats are used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of the CDRs may be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)). For example, for the classical format, following the Kabat rule, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following the Chothia rule, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). Following the IMGT rules, the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.

The term "antibody framework" refers to the part of the variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular B iology, Vol. 66, G.E.Morris, Ed. (1996)).

The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of less than about ^10-7 M, e.g., less than about ^10-8 M, ^10-9 M or ^10-10 M or less.

The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be linked. In another embodiment, the vector is a viral vector, in which additional DNA segments can be linked to the viral genome. The vectors disclosed herein are capable of autonomous replication in the host cell into which they have been introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimental Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared using conventional methods. The invention is to add one or more human FR regions to the non-human CDR region using genetic engineering methods. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by aligning the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN012441351.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Bacteria that are readily transformed include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Appropriate animal host cell lines include CHO (Chinese Hamster Ovary) and NS0 cells.

The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems will lead to glycosylation of antibodies, especially at the highly conserved N-terminal site in the Fc region. Positive strains are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes the antibodies can be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. Then use the pH gradient method to wash out the bound antibodies, use SDS-PAGE to detect the antibody fragments, and collect them. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular screening and ion exchange. The obtained product needs to be immediately frozen, such as -70℃, or freeze-dried.

Amino acid sequence "identity" refers to the percentage of amino acid residues in a first sequence that are identical to the amino acid residues in a second sequence after aligning the amino acid sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be achieved in a variety of ways that are within the skill of the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine the parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences.

1μM、

100nM、

10nM、

1nM或

0.1nM的解離常數(Kd)。 The term "anti-CD40 antibody" or "antibody that binds to CD40" refers to an antibody that is capable of binding to CD40, e.g., with sufficient affinity, such that the antibody can be used as a therapeutic agent targeting CD40. The extent of binding of the anti-CD40 antibody to unrelated, non-CD40 proteins can be less than about 10% of the binding of the antibody to CD40 as measured, e.g., by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CD40 has

1μM,

100nM,

10nM,

1nM or

The dissociation constant (Kd) is 0.1 nM.

The term "peptide" refers to a compound fragment between amino acids and proteins, which is composed of two or more amino acid molecules connected to each other by peptide bonds. It is a structural and functional fragment of protein, such as hormones and enzymes, which are essentially peptides.

The term "sugar" refers to biological macromolecules composed of three elements: C, H, and O, which can be divided into monosaccharides, disaccharides, and polysaccharides.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary butyl, di-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferred are alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary butyl, secondary butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, alkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, pendoxy, carboxyl or carboxylate.

The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of a parent alkane by removing two hydrogen atoms, and is a straight or branched chain group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (—CH ₂ —), 1,1-ethylene (—CH(CH ₃ )—), 1,2-ethylene (—CH ₂ CH ₂ )—, 1,1-propylene (—CH(CH ₂ CH ₃ )—), 1,2-propylene (—CH ₂ CH(CH ₃ )—), 1,3-propylene (—CH ₂ CH ₂ CH ₂ —), 1,4-butylene (—CH ₂ CH ₂ CH ₂ CH ₂ —), and the like. Alkylene groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment.

The term "alkenylene" refers to a linear alkenyl group having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms and having at least one double bond at any position, including, for example, vinylene, allylene, propenylene, butenylene, prenylene, butadienylene, pentenylene, pentadienylene, hexenylene, hexadienylene, etc.

The term "alkynylene" includes linear alkynylene groups having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms and having at least one triple bond at any position, including, for example, ethynylene, propynylene, butynylene, pentynylene, hexynylene, etc.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include spirocyclic, fused-ring, and bridged-ring cycloalkyls. "Carbocycle" refers to the ring system in the cycloalkyl.

The term "spirocycloalkyl" refers to a polycyclic group in which a carbon atom (called a spiro atom) is shared between single rings of 5 to 20 members, which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 members, and more preferably, it is 7 to 10 members. According to the number of spiro atoms shared between rings, spirocycloalkyl is divided into monospirocycloalkyl, dispirocycloalkyl or polyspirocycloalkyl, preferably monospirocycloalkyl and dispirocycloalkyl. More preferably, it is 4-member/4-member, 4-member/5-member, 4-member/6-member, 5-member/5-member or 5-member/6-member monospirocycloalkyl. "Spirocarbocycle" refers to the ring system in spirocycloalkyl. Non-limiting examples of spirocycloalkyl include:

The term "fused cycloalkyl" refers to a full-carbon polycyclic group with 5 to 20 members, each ring in the system shares a pair of adjacent carbon atoms with other rings in the system, one or more of which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 members, and more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl, preferably bicyclic or tricyclic, and more preferably 5-member/5-member or 5-member/6-member bicyclic alkyl. "Fused carbocyclic" refers to the ring system in the fused cycloalkyl. Non-limiting examples of fused cycloalkyl include:

The term "bridged cycloalkyl" refers to a 5-20-membered, all-carbon polycyclic group in which any two rings share two carbon atoms that are not directly connected. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. It is preferably 6-14 members, and more preferably 7-10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl include:

The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring connected to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, etc. The cycloalkyl may be substituted or unsubstituted as desired, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, oxirane, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, pendoxy, carboxyl or carboxylate.

The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), but excluding the ring part of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 6 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl and pyrrolidinyl. Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups. "Heterocyclic" refers to the ring system in the heterocyclic group.

The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group in which one atom (called a spiro atom) is shared between monocyclic rings of 5 to 20 members, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. According to the number of spiro atoms shared between rings, the spiroheterocyclic group is divided into a single spiroheterocyclic group, a double spiroheterocyclic group or a multi-spiroheterocyclic group, preferably a single spiroheterocyclic group and a double spiroheterocyclic group. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiroheterocyclic group. "Spiroheterocyclic" refers to the ring system in the spiroheterocyclic group. Non-limiting examples of spiroheterocyclic groups include:

The term "fused heterocyclic group" refers to a polycyclic heterocyclic group having 5 to 20 members, each ring in the system shares a pair of adjacent atoms with other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, the ring has 6 to 14 members, and more preferably, it has 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic groups. "Fused heterocyclic" refers to the ring system in the fused heterocyclic group. Non-limiting examples of fused heterocyclic groups include:

The term "bridged heterocyclic group" refers to a polycyclic heterocyclic group of 5 to 14 members, in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6 to 14 members, and more preferably, it is 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

The heterocyclic ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic ring, non-limiting examples of which include:

和

等。

and

wait.

The heterocyclic group may be substituted or unsubstituted as required. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, alkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, pendoxy, carboxyl or carboxylate.

The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group with a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring can be fused to a heteroaryl, heterocyclic or cycloalkyl ring, where the ring attached to the parent structure is the aryl ring. "Aryl ring" refers to the ring system in an aryl group. Non-limiting examples of aryl include:

The aryl group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, alkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate, preferably phenyl.

The term "fused aryl" can be an unsaturated aromatic fused ring structure containing 8-14 ring atoms formed by two or more cyclic structures sharing two adjacent atoms, preferably 8-12 ring atoms. For example, it includes all unsaturated fused aryl groups, such as naphthalene, phenanthrene, etc., and also includes partially saturated fused aryl groups, such as benzo 3-8 member saturated monocyclic cycloalkyl, benzo 3-8 member partially saturated monocyclic cycloalkyl. "Fused aromatic ring" refers to the ring system in the fused aryl group. Specific examples of fused aryl groups include 2,3-dihydro-1H-indenyl, IH-indenyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl, etc.

The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl is preferably 5 to 12 members, such as imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, pyrazolyl, pyrimidyl or thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring can be fused to an aryl, heterocyclo or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. "Heteroaryl ring" refers to the ring system in the heteroaryl. Non-limiting examples of heteroaryl include:

The heteroaryl group may be substituted or unsubstituted as desired. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, alkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

The term "fused heteroaryl" may be an unsaturated fused ring structure with aromaticity containing 5-14 ring atoms (including at least one heteroatom) formed by two or more ring structures sharing two adjacent atoms, and the carbon atoms, nitrogen atoms and sulfur atoms may be pendant oxygen, preferably "5-12 membered fused heteroaryl", "7-12 membered fused heteroaryl", "9-12 membered fused heteroaryl" or "12-membered fused heteroaryl". "Base", for example, benzofuranyl, benzoisofuranyl, benzothiophenyl, indolyl, isoindolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolyl, acridinyl, phenanthridinyl, benzoxazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, phenolazine, pteridinyl, purinyl, naphthyridinyl, phenazine, phenothiazine, etc. "Fused aromatic ring" refers to the ring system in the fused aromatic group.

The fused heteroaryl group may be substituted or unsubstituted as required. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, alkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy groups may be substituted or unsubstituted as desired, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, oxalyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

The term "alkylthio" refers to -S-(alkyl) and -S-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkylthio include: methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio. Alkylthio may be substituted or unsubstituted as desired, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, oxirane, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

The term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

The term "haloalkyl" refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.

The term "deuterated alkyl" refers to an alkyl group substituted with a deuterium atom, wherein alkyl is as defined above.

The term "hydroxy" refers to the -OH group.

The term "oxo" refers to a =O group. For example, a carbon atom is connected to an oxygen atom via a double bond, forming a ketone or aldehyde group.

The term "sulfenyl" refers to a =S group. For example, a carbon atom is connected to a sulfur atom via a double bond to form a sulfenylcarbonyl -C(S)-.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "amine" refers to _-NH2 .

The term "cyano" refers to -CN.

The term "nitro" refers to _-NO2 .

The term "carboxyl" refers to -C(O)OH.

The term "aldehyde group" refers to -CHO.

The term "carboxylate" refers to -C(O)O(alkyl) or -C(O)O(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

The term "acyl halides" refers to compounds containing a -C(O)-halogen group.

The term "sulfonyl" refers to -S(O)(O)-.

The term "sulfinyl" refers to -S(O)-.

The "amine protecting group" is a suitable group for protecting an amine group known in the art, see the amine protecting group in the literature ("Protective Groups in Organic Synthesis", 5 ^Th . Ed. TW Greene & PGM Wuts). Preferably, the amine protecting group can be a ( _C1-10 alkyl or aromatic) acyl group, such as a methyl group, an acetyl group, a benzoyl group, etc.; can be a ( _C1-6 alkyl or _C6-10 aromatic) sulfonyl group; can also be a ( _C1-6 alkoxy or _C6-10 aromatic) carbonyl group, such as Boc or Cbz; can also be a substituted or unsubstituted alkyl group, such as a trityl (Tr), a 2,4-dimethoxybenzyl (DMB), a p-methoxybenzyl (PMB) or a benzyl (Bn).

"Optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocycloalkyl group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and the description includes instances where the heterocycloalkyl group is substituted with an alkyl group and instances where the heterocycloalkyl group is not substituted with an alkyl group.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

The term "drug carrier" is used for the drugs disclosed herein and refers to a system that can change the way drugs enter the human body and their distribution in the body, control the release rate of drugs, and transport drugs to targeted organs. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure. Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, and can be used as excellent drug carriers.

The term "excipient" refers to the additives in drug preparations other than the main drug, which can also be called excipients. For example, adhesives, fillers, disintegrants, and lubricants in tablets; the base part in semisolid preparations such as ointments and creams; preservatives, antioxidants, flavor enhancers, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, coloring agents, etc. in liquid preparations can all be called excipients.

The term "diluent" is also called filler, and its main purpose is to increase the weight and volume of tablets. The addition of diluents not only ensures a certain volume size, but also reduces the dosage deviation of the main ingredients and improves the compressibility of the drug. When the drug in the tablet contains oily components, an absorbent needs to be added to absorb the oily substance to keep it in a "dry" state, which is conducive to making tablets.

Figure 1 shows the activity results of the CD40 antagonist antibody 9E6-L4H2 in the reporter gene system. Figure 1 uses the human IgG1 isotype as a negative control and CFZ533 as a positive control.

Figures 2A and 2B show the inhibitory activity results of CD40 antagonist antibodies in the B cell activation experimental system. Figure 2A shows the percentage inhibition results of CD19+CD69+ cells of 9E6-L4H2 and 2F12-L4H2, and Figure 2B shows the MFI inhibition results of CD19+CD69+ cells of 9E6-L4H2 and 2F12-L4H2. Both Figures 2A and 2B use human IgG1 isotype as negative control and CFZ533 as positive control.

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

Figure 4 shows the endogenous stimulatory activity results of CD40 antagonist antibodies 9E6-L4H2 and 2F12-L4H2 in the B cell activation experimental system, using human IgG1 isotype, CFZ533, and stimulatory anti-CD40 antibody 9E5-SELFNS as controls.

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

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

Figure 7 shows the activity results of CD40 antagonist antibody combined with tacrolimus (FK506) in the mouse skin transplant rejection model. Figure 7 A shows the results of skin transplant survival rate (%) of 9E6-L4H2 (10mpk) and its combination with tacrolimus (FK506), Figure 7 B shows the results of skin transplant survival rate (%) of 2F12-L4H2 (10mpk) and its combination with tacrolimus (FK506), Figure 7 C shows the results of skin transplant scoring of 9E6-L4H2 (10mpk) and its combination with tacrolimus (FK506), and Figure 7 D shows the results of skin transplant scoring of 2F12-L4H2 (10mpk) and its combination with tacrolimus (FK506).

Figure 8 shows the PK test results of CD40 antagonist antibodies 9E6-L4H2 and 2F12-L4H2 in human CD40 transgenic mice, using CFZ533 as a control.

Figures 9A and 9B show the inhibitory activity results of Fc-mutated CD40 antagonist antibodies in the B cell activation experimental system. Figure 9A shows the MFI inhibition results of CD19+CD69+ cells of 9E6-L4H2 and 9E6-L4H2-AAYTE, and Figure 9B shows the MFI inhibition results of CD19+CD69+ cells of 2F12-L4H2 and 2F12-L4H2-AAYTE.

Figures 10A to 10D show the inhibitory activity results of Fc mutant CD40 antagonists in the DC cell activation experimental system. Figure 10A shows the results of 9E6-L4H2 and 9E6-L4H2-AAYTE inhibiting IL-12/23 p40, Figure 10B shows the results of 9E6-L4H2 and 9E6-L4H2-AAYTE inhibiting TNFα, Figure 10C shows the results of 2F12-L4H2 and 2F12-L4H2-AAYTE inhibiting IL-12/23 p40, and Figure 10D shows the results of 2F12-L4H2 and 2F12-L4H2-AAYTE inhibiting TNFα.

Figures 11A and 11B show the activity of anti-CD40-ADC in the CD40-mediated GRE reporter gene system. Figures 11A and 11B are the relevant results of ADC-1 and ADC-2, respectively. The IgG1 isotype hIgG-compound 1 coupled to hormone was used as a negative control, and ref-ADC was used as a positive control.

Figures 12A and 12B show the results of the inhibitory activity of anti-CD40-ADC in the B cell activation experimental system. Figures 12A and 12B show the results of the percentage inhibition of CD19+CD69+ cells by 9E6-L4H2, ADC-1 and ADC-2 in PBMCs from donor 1 (Figure 12A) and donor 2 (Figure 12B). Both Figures 12A and 12B use human IgG1 isotype hIgG1 as a negative control.

Figures 13A to 13E show the activity results of anti-CD40-ADC in the mouse skin transplant rejection model. Figure 13A is a flow chart. Figure 13B shows the skin transplant survival rate (%) of different doses of ADC-1. Figure 13C shows the skin transplant scoring results of different doses of ADC-1. Figure 13D shows the skin transplant survival rate (%) of ADC-1. Figure 13E shows the skin transplant scoring results of ADC-1.

Figures 14A and 14B show the activity results of anti-CD40-ADC in the mouse skin transplant rejection model. Figure 14A shows the skin transplant survival rate (%) of ADC-1 and ADC-2, and Figure 14B shows the skin transplant scoring results of ADC-1 and ADC-2.

The following embodiments are used to further describe the present disclosure, but these embodiments do not limit the scope of the present disclosure.

The experimental methods disclosed in the embodiments or test examples without specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory; Contemporary Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents without specific sources are conventional reagents purchased on the market.

Example 1. Sequence and preparation of CD40 immune antigen and screening antigen

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) are all purified commercial protein reagents purchased from Acrobiosystems. The sources of their respective sequences are shown in Table 1. The protein reagents can be used in the experiments of the following examples.

Table 1. Sources of recombinant protein amino acid sequences

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

Anti-human CD40 monoclonal antibody was produced by immunizing 2 New Zealand white rabbits. The immunizing antigen was a His-tagged human CD40 recombinant protein (h-CD40-his, prepared at 1μg/μl in phosphate buffer). It was emulsified with Freund's adjuvant: Freund's complete adjuvant (CFA) was used for the first immunization, and Freund's incomplete adjuvant (IFA) was used for the remaining booster immunizations. Each immunization used a multi-point subcutaneous injection of 400μg of antigen. The immunization injection time was on days 0, 7, 20, and 41. Blood was collected for blood tests on days 27 and 48, and the antibody titer in the rabbit serum was detected by ELISA and FACS methods. Rabbits with high antibody titers in serum and stable titers were selected: booster immunization on day 63, intravenous injection of 400μg/animal antigen solution prepared with phosphate buffer; spleen of the rabbit was collected on day 67, biotin-labeled CD40 antigen was added, and labeled single B cells were sorted into 96-well plates by flow cytometry. After 14 days of culture, supernatants were collected, and supernatants were screened by ELISA and FACS for strains that could bind to human CD40, cynomolgus monkey CD40, and Raji cells (tumor cell line expressing human CD40), a total of 28 B cell strains. RNA was extracted from these individual cells, reverse transcribed, and sent to a sequencing company for sequencing after PCR amplification. Finally, the sequences of 28 rabbit-derived antibodies were obtained. After affinity and activity identification experiments (see Example 2-3 for methods), multiple monoclonal antibodies (9E6, 3F6, 8E1, 2F1, 2F12) were screened. The heavy chain and light chain variable region sequences of the 9E6 antibody are shown in Table 2, and the CDR sequence is shown in Table 3.

Table 2. Variable region sequences of anti-CD40 rabbit monoclonal antibodies

Table 3. CDR regions of anti-CD40 rabbit monoclonal antibodies (Kabat numbering rules)

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 the human-rabbit chimeric antibody sequence. The chimeric antibody sequence is inserted into the expression vector using molecular cloning technology, and the human-rabbit chimeric antibody is obtained using the HEK293 cell expression system.

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

Raji cells are tumor cell lines that overexpress human CD40. 2E5 Raji cells were inoculated into 96-well plates. 100 μL of the antibody to be tested was added, the highest final concentration was 100 nM, 5-fold dilution, 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 Laborotories, 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 on a flow cytometer. Calculate the EC ₅₀ value of the binding of the anti-CD40 antibody to CD40. The anti-CD40 antagonist antibody CFZ533 (i.e., Iscalimab, 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.

The results in Table 4 show that 3F6 and 9E6 have stronger binding EC ₅₀ than CFZ533, while the EC ₅₀ of other antibodies, such as 8E1 (sequence not shown), is weaker than CFZ533.

Table 4. FACS binding EC ₅₀ (nM) of human-rabbit chimeric anti-CD40 antibody to Raji cells

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

HEK-Blue CD40L cells were purchased from Invivogen (Cat#hkb-cd40). The cells were stably transfected with human CD40 gene and NF-kB-mediated SEAP gene group. SEAP secreted in the supernatant can be detected by SEAP substrate QUANTI-Blue to characterize the activation level of CD40 signaling pathway. This experiment evaluated the in vitro antagonist activity of anti-CD40 antibodies based on the IC ₅₀ size by detecting the activation of CD40L on HEK-Blue CD40L cells. HEK-Blue CD40L cells were cultured in DMEM medium containing 10% FBS, 100μg/mL Zeocin and 30μg/mL Blasticidin, and passaged 2-3 times a week, with a passage ratio of 1:5 or 1:10. When subculturing, remove the culture medium, rinse the cell layer with 5 mL of 0.25% trypsin, then remove the trypsin, place the cells in the incubator for digestion for 3-5 minutes, and resuspend the cells in fresh culture medium. Add 100 μL of cell suspension to a 96-well cell culture plate, with a density of 5×10^5 cells/mL. The culture medium is DMEM containing 10% FBS, 100 μg/mL Zeocin and 30 μg/mL Blasticidin. Only 100 μl of sterile water is added to the periphery of the 96-well plate. Incubate the culture plate in the incubator for 24 hours (37°C, 5% CO ₂ ). After the cells adhered, 100 μl of the test antibody was added to each well and incubated at 37°C for 30 minutes. 25 ng/mL CD40L (R&D, Catalog No. 2706-CL) and 2 μg/mL anti-His antibody (R&D, Catalog No. MAB050) were added, and the culture plate was incubated in an incubator for 20-24 hours (37°C, 5% CO2). 20 μl of cell supernatant was taken from each well into a new 96-well flat-bottom plate, 180 μl of QUANTI-Blue substrate solution was added, and the culture plate was incubated in an incubator in the dark for 1-3 hours. The absorbance at 630 nm was measured using an enzyme labeler (Thermo MμltiSkanFc), and the IC ₅₀ value was calculated to evaluate the in vitro cell activity of the anti-CD40 antibody. As shown in Table 5, the results show that 9E6 has a slightly stronger reporter gene cell inhibitory activity IC ₅₀ than CFZ533, while the IC ₅₀ of other antibodies such as 2F1 (sequence not shown) is weaker than that of CFZ533.

Table 5. Anti-CD40 antibody reporter gene cell activity IC ₅₀ (nM)

Example 5. Humanization of anti-CD40 antibodies

The heavy and light chain variable region sequences of rabbit antibodies 2F12 and 9E6 were compared with the antibody GermLine database to obtain human germline templates with high homology. The human germline template information for the final antibody humanization is shown in Table 6.

Table 6. Human germline template information for antibody humanization process

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 (the preferred heavy chain is IgG1 with 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 impact on the conformation of VL and VH are reversed and mutated at potential post-translation modification risk points 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 7), 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.

(下劃線為Kabat編碼規則的CDR。) Table 7. Humanized 9E6 heavy and light chain variable region sequences

(The underlined part is the CDR of Kabat coding rule.)

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

> LCDR3 of 9E6-L1

QGGYWTSTSNFGNV(SEQ ID NO:15)

> LCDR3 of 9E6-L2

QGGYWTSTSNFGSG(SEQ ID NO: 16)

>9E6-L3 LCDR3

QGGYWTSTSNFGTG (SEQ ID NO: 17)

> LCDR3 of 9E6-L4

QGGYWTSTSNFGQG(SEQ ID NO: 18)

>LCDR3 general formula of 9E6-L

QGGYWTSTSNFGX ₉ X ₁₀ (SEQ ID NO: 19), wherein X ₉ is selected from N, S, T or Q, and X ₁₀ is selected from V or G.

Each version of the humanized light and heavy chains of each rabbit antibody was combined, expressed and purified in pairs. The naming convention is a combination of the heavy and light chain variable region numbers, for example, "9E6-L1H1" is an antibody with VH selected SEQ ID NO: 13 and VL selected SEQ ID NO: 9. The light and heavy chain constant regions use the human IgG1 constant region (with N297A mutation, Eu numbering system) sequence and the human kappa chain constant region sequence, respectively.

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

>9E6-L4H2 HC:

SEQ ID NO：20

SEQ ID NO: 20

>9E6-L4H2 LC:

SEQ ID NO：21

SEQ ID NO: 21

>2F12-L4H2 HC:

SEQ ID NO：25

SEQ ID NO: 25

>2F12-L4H2 LC:

SEQ ID NO：26

SEQ ID NO: 26

The heavy chain constant region of the above antibodies was modified. The antibody used the human IgG1 constant region with 5 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 antibodies are 2F12-L4H2-AAYTE and 9E6-L4H2-AAYTE, and their complete heavy chain sequences are as follows:

>9E6-L4H2-AAYTE HC:

SEQ ID NO：22

SEQ ID NO: 22

>2F12-L4H2-AAYTE HC:

SEQ ID NO：27

SEQ ID NO: 27

The area with upper and lower lines is the heavy chain or light chain constant area.

An exemplary IgG Fc is shown below:

>IgG1-Fc(N297A)

SEQ ID NO：23

SEQ ID NO: 23

>IgG1-Fc(AAYTE)

SEQ ID NO：24

SEQ ID NO: 24

Example 6. Humanized anti-CD40 antibody Raji cell binding experiment

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.

Raji was plated at 1.5E5/well, and different concentrations of CD40 antagonist antibodies were added and incubated at 4 degrees for 1 hour. After washing twice with FACS buffer (PBS+2% FBS), secondary antibody (anti-human IgG (H+L) antibody conjugated with Alexa Flour488) was added and incubated at 4 degrees for 0.5 hours. After washing twice with FACS buffer, the fluorescence intensity on the cell surface was detected by flow cytometer (BD FACS Celesta).

The results are shown in Table 8, where 2F12-L4H2, 9E6-L4H2 and CFZ533 have similar binding abilities to human CD40 on the surface of Raji.

Table 8. FACS binding _EC50 results of humanized anti-CD40 antibodies to Raji cells

Example 7. Affinity determination of anti-CD40 antibodies to human CD40 and cynomolgus monkey CD40

The antibody to be tested was affinity captured on the anti-human Fc chip, and then a series of concentration gradients of human or cynomolgus monkey CD40 antigens with a His tag were passed through the chip surface. The reaction signal was detected in real time using a Biacore instrument to obtain the binding and dissociation curves. The buffer used in the experiment was HBS-EP + 10× buffer solution (Cat.# BR-1006-69, GE), which was diluted to 1× (pH 7.4) with D.I. Water. The data obtained in the experiment were fitted with the (1:1) Binding model to obtain the affinity values, see Table 9.

Compared with CFZ533, 9E6-L4H2 and 2F12-L4H2 have similar binding constants to human CD40. 9E6-L4H2 has a stronger binding affinity to cynomolgus monkey CD40, while 2F12-L4H2 has a slightly weaker binding affinity to cynomolgus monkey CD40.

Table 9. SPR affinity of CD40 antagonist antibodies to different CD40 strains

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

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 gene group. The SEAP content secreted in the supernatant can be detected by the SEAP substrate QUANTI-Blue to characterize the activation level of the CD40 signaling pathway. This experiment evaluated the in vitro cell activity of CD40 antagonist antibodies by detecting the inhibitory effect of CD40 antagonist antibodies on CD40L-induced HEK-Blue CD40L cell activation based on the IC ₅₀ size.

HEK-Blue CD40L cells were cultured in DMEM containing 10% FBS, 100μg/ml Normocin, 100μg/ml Zeocin and 30pg/ml Blasticidin, and passaged 2-3 times a week. HEK-Blue CD40L cells were plated at 5E4/well in a 96-well cell culture plate (culture medium: DMEM, 10% FBS, 100μg/ml Normocin) and cultured overnight. After the cells adhered, 100μL of gradient dilutions of the antibody to be tested were added to each well and incubated at 37℃ for 1 hour. CD40L-his (R&D, 2706-CL-025) and anti-His antibody (R&D, MAB050-500) were added and cultured overnight. The cells were centrifuged, 20 μL of cell supernatant was transferred to a new 96-well white plate, 180 μL of QUANTI-Blue substrate solution was added, and the cells were incubated in the dark for 15 minutes. The absorbance at 620 nm was measured using an Envision ELISA instrument, and the IC ₅₀ value was calculated to evaluate the in vitro cell activity of the CD40 antagonist antibody.

The results are shown in Table 10 and Figure 1. 9E6-L4H2 and CFZ533 have similar inhibitory activities against the reporter gene system, and the inhibitory activity of 2F12-L4H2 is better than that of CFZ533.

Table 10. IC ₅₀ of anti-CD40 antibodies inhibiting CD40 reporter gene cell activity

Example 9. Inhibitory activity of anti-CD40 antibodies in the B cell activation experimental system

CD40 is highly expressed on B cells. CD40L can induce B cell activation after binding to CD40, upregulating the expression of a series of activation markers. CD40 antagonist antibodies block the binding of CD40L to CD40, thereby eliminating the immune activation process of B cells.

Human PBMCs were plated at 2E5/well, 50 μL per well in a 96-well cell culture plate (medium: RPMI-1640, 10% FBS, 1% penicillin-streptomycin). 50 μL of gradient dilutions of the antibody to be tested were added to each well and incubated for 0.5 hours at 37°C, 5% CO _2. CD40L-his and anti-His antibodies were added to each well and stimulated overnight. The next day, the supernatant was removed by centrifugation, the cells were washed twice with FACS buffer, and 100 μL of 1:1000 diluted Fixable viability dye EF780 (Invitrogen, 65086514) was added for staining at room temperature for 15 minutes. After washing the cells twice, 100 μL of 1:200 diluted human Fc blocker (Fc blocker, BD, 564220) was added for 10 minutes at room temperature. After centrifugation, 1:200 diluted flow cytometry antibodies (PerCP/Cyanine5.5 anti-human CD19 (Biolegend, 302230), APC anti-human CD69 (Biolegend, 310910)) were added and incubated at 4°C for 0.5 hours. After centrifugation, the supernatant was removed, the cells were washed twice with FACS buffer, and the cells were resuspended with 200 μL PBS. The fluorescence intensity on the cell surface was detected by flow cytometer (BD FACS Celesta).

The results are shown in Table 11 and Figure 2A to Figure 2B. 9E6-L4H2 has similar B cell activation inhibitory activity compared to CFZ533. The B cell inhibitory activity of 2F12-L4H2 is stronger than that of CFZ533.

Table 11. Inhibitory activity of humanized anti-CD40 antibodies in the B cell activation experimental system

After adding the antibody, the CD19+CD69+ signal was lower than the background value, which may be because the background activation of B cells was inhibited to a certain extent (in addition to inhibiting the CD40L-induced signal).

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

CD40 is highly expressed on dendritic cells (DC). CD40L can induce DC activation after binding to CD40, upregulate the expression of multiple activation markers on the surface of DC cells, and promote DC to secrete a variety of inflammatory factors, further amplifying the immune response. CD40 antagonist antibodies block the binding of CD40L to CD40, thereby relieving the immune activation process of DC cells.

Use EsaySepTM human CD14 sorting kit (Stemcell, 19359) to sort and enrich monocytes from fresh primary human peripheral blood PBMCs, and differentiate them for 6 days with RPMI-1640 medium (10% FBS, 1% penicillin-streptomycin), 50ng/ml IL-4 (PeproTech, 200-04) and 50ng/ml GM-CSF (PeproTech, 300-03). On the 7th day, the differentiated DC cells were plated into 96-well cell culture plates at 1E5/well. Add gradient dilutions of the test antibody to each well and incubate for 0.5 hours at 37°C and 5% _CO2 . Add CD40L-his and anti-His antibodies to each well at a final concentration. 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 for staining at room temperature for 15 minutes. After washing the cells twice, 100 μL of 1:200 diluted human Fc blocking (BD, 564220) was added for blocking at room temperature for 10 minutes. After centrifugation, flow cytometry antibodies (Alexa Fluor® 700 anti-human CD11c (Biolegend, 337220), Brilliant Violet 421 ^TM anti-human CD80 (Biolegend, 305221), APC anti-human CD86 (Biolegend, 305412)) diluted 1:200 were added and incubated at 4°C for 0.5 hours. After centrifugation, the supernatant was removed, and the cells were washed twice with FACS buffer, and then resuspended in 200 μL PBS. The fluorescence intensity on the cell surface was detected by flow cytometer (BD FACS Celesta).

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

The experimental results are shown in Table 12 and Figures 3A to 3D. 9E6-L4H2 and 2F12-L4H2 have similar DC cell inhibitory activity as the control antibody CFZ533.

Table 12. Inhibitory activity of humanized anti-CD40 antibodies in DC cell activation experimental system

Example 11. Activation activity of humanized anti-CD40 antibodies in the B cell activation experimental system

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

Human PBMCs were plated at 2E5/well, 100 μL per well in a 96-well cell culture plate (medium: RPMI-1640, 10% FBS, 1% penicillin-streptomycin). 100 μL of gradient dilutions of the antibody to be tested were added to each well and cultured overnight at 37°C, 5% CO _2. The next day, the supernatant was removed by centrifugation, the cells were washed twice with FACS buffer, and 100 μL of 1:1000 diluted Fixable viability dye EF780 was added for staining at room temperature for 15 minutes. After washing the cells twice, 100 μL of 1:200 diluted human Fc blocking was added and blocked for 10 minutes at room temperature. After centrifugation, flow cytometry antibodies (PerCP/Cyanine5.5 anti-human CD19 (Biolegend, 302230), APC anti-human CD69 (Biolegend, 310910)) diluted 1:200 were added and incubated at 4°C for 0.5 hours. After centrifugation, the supernatant was removed, and the cells were washed twice with FACS buffer, and then resuspended with 200 μL PBS. The fluorescence intensity on the cell surface was detected by flow cytometer (BD FACS Celesta).

As shown in Figure 4, the CD40 stimulating 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 stimulating activity at 2.5nM.

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

Female human CD40 transgenic mice, 6-7 weeks old, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. Housing environment: SPF; Production license: SCXK (Suzhou)-2016-0004; Human CD40 transgenic mouse qualification certificate number: 320726200100167773. Animals were adaptively housed for 7 days after arrival and randomly divided into groups. On day 0 of the experiment, one mouse in each group was blooded and then intraperitoneally injected with drugs. On day 1, mice were immunized with 50 μg KLH (KLH: Freund's complete adjuvant CFA = 1:1 emulsified immune complex) per mouse by intraperitoneal injection. On the 15th day of the experiment, 50μg KLH (KLH: Freund's incomplete adjuvant IFA = 1:1 emulsified immune complex)/head was injected intraperitoneally for secondary immunization. Each group of drugs was injected intraperitoneally twice a week, and blood was collected through the orbital vein on the 7th, 14th, 21st and 28th days, respectively. The whole blood was placed at room temperature for 1-4 hours, centrifuged at 7000rpm at 4℃ for 10 minutes, and the serum was separated and stored at -80℃ for standby. The specific experimental process is shown in Figure 5A.

The specific dosing regimen is shown in Table 13. Mouse serum was separated every week and the anti-KLH specific IgG level was detected by ELISA.

Table 13. Drug administration scheme for mouse TDAR model experiment

The results are shown in Figure 5B and Table 14. 1 mg/kg CD40 antagonist antibody significantly inhibited the production of anti-KLH specific IgG after two immunizations. Low doses of 0.3 mg/kg 9E6-L4H2 and 2F12-L4H2 had a better inhibitory effect on the production of anti-KLH IgG after two immunizations than the same dose of CFZ533.

Table 14. Inhibitory effect of anti-CD40 antibody on IgG production after immunization (0.3 mpk)

Example 13. Mouse skin transplant rejection model

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

Female human CD40 transgenic mice, 6-7 weeks old, purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. Housing environment: SPF; Certificate number: 320726200100179778.

The animals were adaptively raised for 7 days after arrival and randomly divided into groups. On the second day of the experiment, mice were intraperitoneally injected with tacrolimus FK506 or CD40 antagonist antibodies. 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 total length of 1 cm was separated. The hair on the back of the recipient mice was removed, and an incision was made along the skin layer. While retaining the back fat and connective tissue, the skin of the same area was removed. The donor skin was placed on the incision of the recipient mice, the edge skin was sutured with glue, and the mice were placed in a cage to recover. The specific experimental process is shown in Figure 6A.

The mice were given the drug according to the dosing regimen in Table 15. After the mice recovered for 7 days, the survival of the mouse skin was observed every day, and the rejection score was recorded according to the rejection score. The scoring system is as follows: 3: The skin is not red and smooth; 2: Part of the skin is red, loses its luster, and is dry; 1: Most of the skin turns red, has no stripes, and shrinks; 0: Transplant rejection causes 80% skin necrosis.

Table 15. Drug administration scheme for mouse skin transplant rejection model

The results are shown in Figure 6B and Tables 16-18. Compared with the model group, CD40 antagonist antibodies can significantly improve the mouse skin graft score and prolong the survival time of skin grafts. In the process of scoring skin grafts, since it was not possible to see whether there was rejection before, scoring was started from the 8th day. 9E6-L4H2 and 2F12-L4H2 showed better anti-graft rejection activity than the control antibody CFZ533.

Table 16. Skin graft survival rate on day 15 with anti-CD40 antibody (%)

Table 17. Anti-CD40 antibody skin graft scores (3mpk)

Table 18. Skin graft scores of anti-CD40 antibodies (10 mpk)

In addition, as shown in Figure 7 and Tables 19-21, 9E6-L4H2 and 2F12-L4H2 combined with tacrolimus further enhanced the efficacy against mouse skin transplant rejection.

Table 19. Day 15 skin graft survival rate after combination therapy with anti-CD40 antibody and tamoxifen

Table 20. Skin graft scores of anti-CD40 antibody combined with tamoxifen

Table 21. Skin graft scores of anti-CD40 antibody combined with tamoxifen

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

Female human CD40 transgenic mice, 6-7 weeks old, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. Housing environment: SPF; Production license: SCXK (Suzhou)-2016-0004; Human CD40 transgenic mouse qualification certificate number: 320726200100154632. Animals were adaptively housed for 7 days after arrival and randomly divided into groups. On day 0 of the experiment, mice in each group were intraperitoneally injected with 10 mg/kg of anti-CD40 antibody. At 15 minutes, 4 hours, 8 hours, and on days 1, 2, 4, 7, 10, and 14 after administration, 100-150 μL of blood was collected, anticoagulated with 10 μL EDTA-K2 (0.1M), and stored on ice. ELISA was used to detect the antibody concentration in mouse plasma at different time points. The specific detection method is as follows: dilute goat anti-human IgG Fc antibody (Rockland, Cat#609-101-017) with PBS to 2.5μg/ml, add 50μL/well to a 96-well plate, and incubate overnight at 4 degrees. After washing three times with the washing solution, add 50μL of blocking solution to each well and incubate at 37°C for 1 hour. Add mouse plasma and the standard curve of the antibody to be detected, and incubate at 37°C for 2 hours. Wash three times with the washing solution, add anti-hlgG Fab-HRP (Sigma, Cat# A0293, 1:10000) at 50μL/well, and incubate at room temperature for 1 hour. Wash three times with the washing solution. Add 100μL TMB to each well and react for 5 minutes in the dark. Add 100 μL of 0.16 M sulfuric acid to each well. Read the OD value at 450 nm using the Envision enzyme marker to calculate the concentration of the CD40 antagonist antibody.

The results are shown in Table 22 and Figure 8. 9E6-L4H2, 2F12-L4H2 and the control molecule CFZ533 have similar PK characteristics in human CD40 transgenic mice.

Table 22. PK data of CD40 antagonist antibodies in hCD40 transgenic mice

Example 15. Affinity determination of humanized anti-CD40 antibody and human FcRn

The antibody to be tested was affinity captured on the anti-human Fab chip, and then a series of human FcRn antigens (purchased from AcroBiosystem) were passed through the chip surface at a concentration gradient. The signal when the reaction reached a steady state was detected in real time using a Biacore instrument. The buffer used in the experiment was HBS-EP + 10× buffer solution (Cat.# BR-1006-69, GE), which was diluted to 1× (pH 7.4) with D.I. Water. The data obtained from the experiment were fitted with a steady state binding model to obtain affinity values, see Table 23. Compared with the parent antibody, the anti-CD40 antibody carrying the AAYTE mutation has a higher binding affinity to human FcRn.

Table 23. Affinity determination of humanized anti-CD40 antibodies to human FcRn

Example 16. Inhibitory activity of anti-CD40 antibodies carrying AAYTE mutations in Fc in a B cell activation experimental system

Referring to the method in Example 10, the inhibitory activity of the anti-CD40 antibodies 2F12-L4H2-AAYTE and 9E6-L4H2-AAYTE carrying AAYTE mutation on Fc in the B cell activation experimental system was detected. The results are shown in Figures 9A to 9B. The anti-CD40 antibodies with Fc mutations have similar B cell inhibitory activity as the parent anti-CD40 antibodies.

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

Referring to the method in Example 11, the inhibitory activity of anti-CD40 antibodies 2F12-L4H2-AAYTE and 9E6-L4H2-AAYTE carrying AAYTE mutation on Fc in the DC cell activation experimental system was detected. The results are shown in Figures 10A to 10D. The Fc mutant anti-CD40 antibody has similar DC cell inhibitory activity as the parent anti-CD40 antibody.

Example 18. Preparation of Antibody Drug Conjugate ADC-1 (9E6-L4H2-Compound 1)

1. Synthesis of 781F02

In a 1000 mL single-necked bottle, raw material 781F01c (48 g, 176.4 mmol, 1.0 eq), pinacol diborate (71.7 g, 282.2 mmol, 1.6 eq), potassium acetate (34.6 g, 352.8 mmol, 2.0 eq), PdCl2(dppf) (6.4 g, 8.82 mmol, 0.05 eq) and dioxane (500 mL) were added, and the temperature was raised to 95 ° C and stirred for 2 hours under nitrogen protection. The raw material was sampled for LCMS detection. The reaction was stopped, the reaction was cooled, and then the raw material 781F01f (80.8g, 352.8mmol, 2.0eq), potassium carbonate (48.8g, 352.8mmol, 2.0eq), PdCl2(dppf) (6.4g, 8.82mmol, 0.05eq) were added, and water (100mL) was added and stirred. The mixture was heated to 80 degrees and stirred overnight under nitrogen protection. The raw material was sampled and the reaction was complete. After the reaction solution was cooled, ethyl acetate and water were added and stirred, the mixture was separated, dried over anhydrous sodium sulfate, and dried by rotation. The crude product was passed through a column to obtain about 54g of a white solid with a purity of 95% and a yield of 90%. Ms (ESI): m/z 342.1 [M+1] ⁺ .

2. Synthesis of 781F03

Add raw material 781F02 (7.0 g, 20.5 mmol, 1.0 eq), potassium permanganate (9.7 g, 61.6 mmol, 3.0 eq), tetra-tert-butylammonium bromide (20.0 g, 61.6 mmol, 3.0 eq) and dichloroethane (140 mL) to a 500 mL three-necked flask, and stir at room temperature for 48 hours under nitrogen protection. Take a sample for LCMS detection, the raw material reacts completely, stop the reaction, cool the reaction solution with ice water, add 10% sodium bisulfite and acetic acid and stir, the solid is completely dissolved, separate the liquid, add anhydrous sodium sulfate to dry the organic layer, filter, spin dry, and pass the crude product through a column to obtain about 6.2 g of a white solid with a purity of 95% and a yield of 85%. Ms(ESI): m/z 378.1[M+23] ⁺ .

3. Synthesis of 781F04

Add raw material 781F03 (20.0 g, 56.0 mmol, 1.0 eq), propylene glycol (12.1 g, 112.0 mmol, 2.0 eq), boron trifluoride etherate (23.8 g, 168.0 mmol, 3.0 eq) and chloroform (100 mL) into a 500 mL three-necked flask, and heat under reflux and stir for 4 hours under nitrogen protection. Take a sample for LCMS detection. The raw material reacts completely, stop the reaction, cool the reaction solution with ice water, add water and stir, the solid is completely dissolved, separate the liquid, add anhydrous sodium sulfate to dry the organic layer, filter, spin dry, add petroleum ether to stir the crude product, filter to obtain about 22 g of off-white solid, which is directly put into the next step. Ms (ESI): m/z 346.0 [M + 1] ⁺ .

4. Synthesis of 781F05

Add crude raw material 781F04 (22.0 g, 56.0 mmol, 1.0 eq), dibutyl dicarbonate (24.4 g, 112.0 mmol, 2.0 eq) and ethanol (60 mL) into a 100 mL three-necked flask, and heat at 50 degrees and stir for 4 hours under nitrogen protection. Sample LCMS detection, the raw material reaction is complete, stop the reaction, spin dry the reaction solution, pass through a column, and obtain about 18.5 g of white solid, purity 95%, yield 71%. Ms (ESI): m/z 468.1 [M + 23] ⁺ .

5. Synthesis of 781F06

Add crude raw material 781F05 (4.7 g, 13.6 mmol, 1.0 eq), DAST (6.6 g, 40.9 mmol, 3.0 eq) and dichloromethane (50 mL) into a 100 mL three-necked flask, heat at 50 degrees and stir for 4 hours under nitrogen protection. Take a sample for LCMS detection. The raw material reacts completely. Stop the reaction, add water to quench under ice water cooling, separate the liquids, dry the organic layer with anhydrous sodium sulfate, filter, spin dry, and pass through a column to obtain about 3.9 g of white solid, purity 95%, yield 76%. Ms (ESI): m/z 400.1 [M + 23] ⁺ .

6. Synthesis of 781F07

Add the crude raw material 781F06 (2.0 g, 5.3 mmol, 1.0 eq) to a 100 mL three-necked flask, and then add tetrahydrofuran (25 mL) to dissolve. Under nitrogen protection, cool to about 0 degrees, slowly add 1.0 M aluminum lithium hydroxide tetrahydrofuran solution (8.0 mL, 8.0 mmol, 1.5 eq), keep stirring at about 0 degrees for 1 hour. Sample LCMS detection, the raw material reaction is complete, stop the reaction, add water (0.8 mL) to quench under ice water cooling, add 3.0 M potassium hydroxide aqueous solution (0.8 mL), add water (1.6 mL) and stir for 15 minutes, filter, add anhydrous sodium sulfate to the filtrate to dry, filter, spin dry, and obtain about 2.2 g of crude product, which is directly put into the next step. Ms(ESI): m/z 372.1[M+23] ⁺ .

7. Synthesis of 781F08

Add the crude raw material 781F07 (2.2 g, 5.3 mmol, 1.0 eq) to a 100 mL three-necked flask, then add ethyl acetate (25 mL) to dissolve, cool to about 5 degrees under nitrogen protection, add Dess-Martin oxidant (6.7 g, 15.9 mmol, 3.0 eq), keep stirring at about 10 degrees for 1 hour. Take a sample for LCMS detection, the raw material reaction is complete, stop the reaction, filter, spin dry the filtrate, and purify the crude product through a column to obtain about 1.5 g of a white solid with a purity of 95% and a yield of 82%. Ms (ESI): m/z 370.1 [M+23] ⁺ .

¹ H-NMR(400MHz, CDCl ₃ )δ 10.06(s,1H),7.94(d,2H),7.70(d,2H),7.53(s,1H),7.47(d,2H),7.35(t, 1H),7.15(d,1H),6.55(m,1H).

8. Synthesis of 781F00A

In a 100mL three-necked flask, add 16-α-hydroxyprenisolone (1.2g, 3.0mmol, 1.0eq), 781F08 (1.1g, 3.17mmol, 1.05eq), anhydrous magnesium sulfate (1.8g, 15.0mmol, 5.0eq), acetonitrile (25mL) and stir. Under nitrogen protection, cool to below 0 degrees, add trifluoromethanesulfonic acid (2.3g, 15.0mmol, 5.0eq), keep stirring at about 0 degrees for 1 hour. Sample LCMS detection, the raw material reaction is complete, stop the reaction, filter, and the filtrate is directly prepared to obtain about 1.5g of white solid, purity 95%, yield 70%. Ms (ESI): m/z 606.3 [M + 1] ⁺ .

¹ H-NMR(400MHz,MeOD)δ 7.53(m,4H),7.44(m,2H),7.28(m,3H),6.23(dd,1H),5.99(t,1H),5.52(s,1H ),5.08(d,1H),4.63(d,1H),4.37(m,2H),2.65(td,1H),2.36(d,1H),2.25(m,1H),2.13(m,1H) ,1.95(dd,1H),1.80(m,4H),1.49(s,3H),1.11(dt,1H),1.10(m,4H).

9. Synthesis of 781F10

In a 25 mL three-necked flask, add raw material 781F00A (0.329 g, 0.544 mmol, 1.05 eq), raw material 78101-11 (0.25 g, 0.52 mmol, 1.0 eq), triethylamine (0.25 g, 1.56 mmol, 3.0 eq), and DMF (2 mL). After the addition, cool in an ice bath for 5-10 min to make the internal temperature reach -5°C, then slowly add T3P (50% DMF) (0.9 mL, 1.82 mmol, 3.5 eq). After the addition, stir for 16 hours under natural heating conditions. Sampling LCMS detection showed that the raw material reaction was complete, the target product was the main peak, and the byproduct of the target product dehydration was generated in a ratio of 2.5/1. The reaction was stopped and the reaction solution was directly purified by pre-HPLC to obtain a white solid product 781F10 (223 mg, yield 40%, purity 96%. Ms (ESI): m/z 1092.4 [M+Na] ⁺ .

10. Synthesis of 781F11

In a 50 mL three-necked flask, raw material 781F10 (0.22 g, 0.206 mmol, 1.0 eq), raw material tetrazole (0.20 g, 2.87 mmol, 14.0 eq), N,N-diethylphosphamide dibutyl tert-butyl ester (0.616 g, 2.47 mmol, 12.0 eq), and DMF (2.6 mL) were added. After the addition was complete, the mixture was reacted at room temperature for 2 hours. Samples were taken for LCMS detection. The raw materials were reacted completely and an intermediate was generated. The intermediate was two peaks. The temperature was lowered in an ice bath for 5-10 min to make the internal temperature reach 0°C. Then _H2O2 (30%) (0.13 g, 0.57 mmol, 5.5 eq) was slowly added. _After the addition was complete, the mixture was stirred at room temperature for 1 hour. Sampling LCMS detection showed that the raw material was completely reacted and the target product was the main peak. The reaction was stopped and the reaction solution was directly purified by pre-HPLC to obtain a white solid product 781F11 (184.1 mg, yield 70.8%, purity 98%. Ms (ESI): m/z 1284.6 [M+Na] ⁺ .

11. Synthesis of 781F12

Add raw material 781F11 (0.285 g, 0.233 mmol, 1.0 eq) and raw material piperidine (0.17 g, 1.96 mmol, 8.5 eq) and acetonitrile (5 mL) into a 25 mL single-mouth bottle, and stir at room temperature for 0.5 h. Sample LCMS detection shows that the raw material reacts completely and the target product is the main peak. Post-treatment is to reduce the pressure and spin dry the solvent, add 5 mL of petroleum ether for slurrying, stir at room temperature for 2 hours, and then filter. The filter cake is washed twice with 2 mL of petroleum ether to obtain a white solid product 781F12 (209 mg, yield 91%, purity 90%. Ms (ESI): m/z 1004.4 [M+H] ⁺ .

12. Synthesis of 781F13

Add raw material 2-bromoacetic acid (0.074g, 0.523mmol, 2.6eq) to a 25mL single-mouth bottle, add raw material EEDQ (0.13g, 0.523mmol, 2.6eq), DMF (1mL), stir at room temperature for 1 hour after addition, the reaction solution changes from colorless to yellow-green, then add 781F12 (0.21g, 0.201mmol, 1.0eq) DMF (0.5mL) solution, stir at room temperature for 1.6 hours after addition. Sample LCMS detection, the raw material reaction is complete, and the target product is the main peak. The reaction was stopped, and the mixture was diluted with dichloromethane (40 mL), then washed with 1 M HBr (10 mL x 2), then washed with saturated sodium bicarbonate (20 mL x 2), and finally washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and spun down with an oil pump to obtain a brown solid crude product 781F13 (230 mg, crude, purity 80%). Ms (ESI): m/z 1182.4 [M+Na] ⁺ .

13. Synthesis of compound 1

Add the raw material 781F13 (0.240 g, 0.201 mmol, 1.0 eq) and DCM (2 mL) to a 25 mL single-mouth bottle. After the addition, cool down in an ice bath for 5 min to make the internal temperature reach 0°C. Then slowly add trifluoroacetic acid (1 mL). After the addition, stir at room temperature for 40 minutes. Sample LCMS detection shows that the raw material has reacted completely and the target product is the main peak. The reaction solution is decompressed and dried under ice bath and then purified by pre-HPLC. Freeze-drying to obtain a white solid product compound 1 (78 mg, yield 39.2%, purity 99%. Ms (ESI): m/z 1014.2 [M + Na] ⁺ .

14. Synthesis of ADC-1 (9E6-L4H2-compound 1)

At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (2.5mM, 540.5uL, 1.35mmol) to the buffer solution A of the antibody 9E6-L4H2 (0.05M buffer solution at pH=6.3; 40.0mg/ml, 4.0mL, 0.27mmol), place in a water bath oscillator, oscillate at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C with a water bath. Add 1.0M Tris buffer pH=8.30 (560uL).

Compound 1 (2.68 mg, 2.70 mmol) was dissolved in 200 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (extraction phase: buffer A), and concentrated using an ultrafiltration tube to obtain buffer A buffer (1.97 mg/mL, 19.7 mL, yield 97%) of the title product ADC-1 (9E6-L4H2-Compound 1), which was stored frozen at 4°C. The theoretical DAR value is 4, and the measured DAR value is 4.

Example 19: Preparation of Antibody Drug Conjugate ADC-2 (9E6-L4H2-Compound 2)

1. Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)glycine glycine-L-phenylalanine tertiary butyl ester (Compound 1b)

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

MS (ESI): m/z 580.3[M+Na] ⁺ .

¹ H NMR(400MHz, DMSO-d6)δ 8.21-8.16(m,1H),8.12-8.05(m,1H),7.91-7.87(m,2H),7.72-7.68(m,2H),7.64-7.59 (m,1H),7.44-7.38(m,2H),7.36-7.25(m,4H),7.23-7.18(m,3H),4.41-4.18(m,4H),3.76-3.72(m,2H) ,3.68-3.61(m,2H),2.98-2.89(m,2H),2.69(s,2H),1.30(s,9H).

2. Preparation of Glycine Glycine-L-phenylalanine tertiary butyl ester (Compound 1c)

Place the raw material 1b (5.57 g, 10.0 mmol, 1.0 eq) in a 250 mL three-necked flask, add anhydrous DCM (60 mL) to dissolve, and cool in an ice bath. Slowly add piperidine (8.5 g, 100.0 mmol, 10.0 eq), and stir the reaction at room temperature for about 2 hours after the addition. Directly concentrate the reaction solution to dryness, and purify the crude product by column chromatography to obtain product 1c as a light yellow slurry (2.70 g, yield 81%). MS (ESI): m/z 358.3 [M+H] ⁺ .

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

Add raw materials 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; from Tonglai Biochemical, batch number: P110114) and 1c (0.69 g, 2.05 mmol, 1.0 eq) to a 100 mL three-necked flask, add anhydrous DMF (25 mL) to dissolve, 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 keep the reaction in an ice bath for 1 hour. Water was added to the reaction solution, extracted with ethyl acetate, washed with saturated saline, dried and concentrated under reduced pressure to obtain 1d as a light yellow oil (1.60 g, yield 97%).

MS(ESI): m/z 805.3[M+H] ⁺ .

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

Add raw material 1d (1.60g, 1.99mmol, 1.0eq) to a 100mL single-mouth bottle, and add anhydrous dichloromethane (16mL) to dissolve. Add trifluoroacetic acid (8mL) at room temperature and stir the reaction for 2 hours. Concentrate the reaction solution directly to dryness to obtain a yellow solid, add ethyl acetate to dissolve, wash with saturated salt water, dry, and reduce the pressure to concentrate to obtain 1e as a yellow oil (1.23g, yield 83%);

MS(ESI): m/z 749.3[M+H] ⁺ .

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

Add raw materials budesonide (1.29 g, 3.0 mmol, 1.0 eq) and (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (1.16 g, 3.15 mmol, 1.05 eq; prepared by reference to "Tetrahedron , 2018, 74 (15), 1951-1956 " ) and p-toluenesulfonic acid pyridinium salt (75 mg, 0.30 mmol, 0.1 eq) into a 100 mL three-necked flask. Add anhydrous tetrahydrofuran (20 mL) at room temperature and reflux for 4 hours. Directly concentrate the reaction solution to dryness, and purify the crude product by column chromatography to obtain product 1f as a white solid (0.54 g, yield 24%).

MS(ESI): m/z 739.4[M+H] ⁺ .

¹ H NMR(400MHz, DMSO-d6)δ 8.76-8.69(m,1H),7.91-7.84(m,2H),7.73-7.67(m,2H),7.63-7.57(m,1H),7.43-7.58 (m,2H),7.35-7.23(m,3H),6.13(d, J =7.5Hz,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).

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]dioxacyclopentan-8b-yl)-2-oxoethoxy)methyl)acetamide (Compound 1g)

Place the raw material 1f (540 mg, 0.73 mmol, 1.0 eq) in a 25 mL single-mouth bottle, and add anhydrous dichloromethane (10 mL) to dissolve. 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 liquid, extract the aqueous phase twice with dichloromethane, wash with saturated salt water, and reduce the pressure to concentrate to obtain 1 g of light yellow semisolid (450 mg of crude product containing fluorene, yield 100%), the crude product can be directly used in the next reaction.

MS(ESI): m/z 517.4[M+H] ⁺ .

7. (9H-fluoren-9-yl)methyl ((10S)-10-benzyl)-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b- Preparation of dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentan-8b-yl)-1,6,9,12,15,18-hexahydro-3,21,24,27,30-pentahydro-5,8,11,14,17-pentaaminetriacontan-32-yl)carbamate (Compound 1h)

Place 1g (0.45g crude product, equivalent to 0.73mmol, 1.0eq) and 1e (0.55g, 0.73mmol, 1.0eq) of raw materials in a 100mL three-necked flask, add anhydrous DMF (9mL) to dissolve. Cool in an ice bath, then add HATU (361mg, 0.95mmol, 1.3eq) and DIEA (189mg, 1.46mmol, 2.0eq) in sequence, and stir for 1 hour. Add water to the reaction solution, extract twice with dichloromethane, combine the organic phases, wash once with saturated brine, dry and concentrate under reduced pressure to obtain a yellow oily solid crude product. The crude solid was purified by slurrying in a mixed solvent of petroleum ether and ethyl acetate to obtain product 1h as a light yellow solid (710 mg, yield 78%).

MS (ESI): m/z 1269.7[M+Na] ⁺ .

8. 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- Preparation of dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentan-8b-yl)-1,6,9,12,15-pentaoxy-3-oxo-5,8,11,14-polytetraethylene glycol-16-yl-3,6,9,12-tetraoxypentadecan-15-amide (Compound 1i)

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

MS(ESI): m/z 1025.6[M+H] ⁺ .

9. 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- Preparation of naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxacyclopentan-8b-yl)-1,6,9,12,15-pentahydro-3-oxo-5,8,11,14-tetraethylene glycol-16-yl)-1-(2-bromoacetamido)-3,6,9,12-tetrahydropentadecan-15-amide (Compound 2)

Place bromoacetic acid solid (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 add anhydrous DMF (5 mL) to dissolve. Stir and react at room temperature for 1 hour. Dissolve the raw material 1i (130 mg, 0.127 mmol, 1.0 eq) in DMF (2 mL), and add this solution dropwise to the active ester solution prepared above. After the addition, maintain the reaction at room temperature for another 2 hours.

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

MS(ESI): m/z 1145.5/1147.6[M+1] ⁺ .

¹ H NMR (400MHz, CDCl ₃ )δ 8.62-8.57(m,1H),8.34-8.28(m,2H),8.19-8.11(m,2H),8.03-7.99(m,1H),7.30-7.17( m,6H),6.15(d, J =9.6Hz,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),3.06-2.84(m,2H),2.41-2.28(m,3H),2.06-1.73(m,2H),1.60-1.24(m,10H),1.01-0.82(m ,8H).

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

At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) (2.5mM, 170.3uL, 0.42mmol) to the buffer solution A of the antibody 9E6-L4H2 (0.05M buffer solution at pH=6.3; 15.0mg/ml, 1.5mL, 0.10mmol), place in a water bath oscillator, oscillate at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C with a water bath. Add 1.0M Tris buffer solution pH=8.30 (210uL).

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

Example 20. Affinity determination of anti-CD40-ADC to human CD40 and cynomolgus monkey CD40

The antibody to be tested and anti-CD40-ADC were affinity captured on the anti-human Fc chip, and then a series of concentration gradients of human or cynomolgus monkey CD40 antigens with His tags were passed through the chip surface. The reaction signal was detected in real time using the Biacore instrument to obtain the binding and dissociation curves. The buffer used in the experiment was HBS-EP+ 10× buffer solution (Cat.# BR-1006-69, GE), which was diluted to 1× (pH 7.4) with D.I. Water. The data obtained in the experiment were fitted with the (1:1) Binding model to obtain the affinity values, see Table 24.

Table 24. SPR affinity of anti-CD40-ADC to CD40 of different germ lines

Example 21. Activity of anti-CD40-ADC in CD40-mediated GRE reporter gene system

A reporter gene cell line stably transfected with MMTV-Luc was established in Hela cells using lentivirus. MMTV can respond to the activation of glucocorticoid receptors. This cell line is subsequently referred to as the GRE reporter gene cell line. The stably transfected Hela-MMTV-Luc cell line was plated in a 96-well plate (30,000 cells/well), and each well was transfected with empty plasmid or human CD40 plasmid (hCD40, 50 ng/well) using lipo3000. After overnight incubation with different concentrations of anti-CD40-ADC, the expression of luciferase in the reporter gene system was detected with Bright-Glo (promega) detection reagent 22 hours later.

The results are shown in Table 25 and Figures 11A (Experiment 1) and 11B (Experiment 2). ADC-1 and ADC-2 specifically activated the GRE reporter gene cell line expressing CD40, but were inactive in the GRE reporter gene cell line not expressing CD40. The control molecule ref-ADC (the control molecule is CN111465399A, the ADC molecule shown in Example 13-Hydrolysis in Table 6B; the antibody corresponds to Ab102, which contains the heavy chain shown in Sequence 3 and the light chain shown in Sequence 4; CN111465399A is incorporated into this disclosure by reference in its entirety) is more active in the CD40-GRE cell line, but exhibits non-specific activation activity in the GRE reporter gene cell line not expressing CD40.

Table 25. Activity of anti-CD40-ADC in CD40-mediated GRE reporter gene system

Example 22. Inhibitory activity of anti-CD40-ADC in B cell activation experimental system

CD40 is highly expressed on B cells. CD40L can induce B cell activation after binding to CD40, upregulating the expression of a series of activation markers. CD40 antagonist antibodies block the binding of CD40L to CD40, thereby eliminating the immune activation process of B cells.

Human PBMCs were plated at 2E5/well, 50 μL per well in a 96-well cell culture plate (medium: RPMI-1640, 10% FBS, 1% penicillin-streptomycin). 50 μL of gradient dilutions of the antibody or ADC to be tested were added to each well and incubated for 0.5 hours at 37°C, 5% CO _2. 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, the cells were washed twice with FACS buffer, and 100 μL of 1:1000 diluted Fixable viability dye EF780 (Invitrogen, 65086514) was added for staining at room temperature for 15 minutes. After washing the cells twice, 100 μL of 1:200 diluted human Fc blocker (BD, 564220) was added and blocked at room temperature for 10 minutes. After centrifugation, 100 μL of flow cytometry antibodies (PerCP/Cyanine5.5 anti-human CD19 (Biolegend, 302230, 1:100 dilution), APC anti-human CD69 (Biolegend, 310910, 1:200 dilution)) were added and incubated at 4°C for 0.5 hours. After centrifugation, the supernatant was removed, the cells were washed twice with FACS buffer, and the cells were resuspended with 200 μL FACS buffer. The fluorescence intensity on the cell surface was detected by flow cytometer (BD FACS Celesta).

The results are shown in Table 26 and Figures 12A and 12B. ADC-1 and ADC-2 have similar B cell inhibitory activity to monoclonal antibody 9E6-L4H2.

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

After adding antibodies or ADC, the CD19+CD69+ signal is lower than the background value, which may be because the background activation of B cells is inhibited to a certain extent (in addition to inhibiting the CD40L-induced signal).

Example 23. Mouse skin transplant rejection model

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

Female human CD40 transgenic mice, 6-7 weeks old, purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. Housing environment: SPF; Production license: SCXK (Su)-2016-0004; Human CD40 transgenic mouse certificate number: 320726200100179778.

The animals were adaptively raised for 7 days after arrival 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, donor Balb/c mice and C57BL6/J mice were anesthetized with 4% chloral hydrate, the tails of donor mice were removed, and the skin of the tail with a total 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, the skin of the same area was removed. The donor skin was placed on the incision of the recipient mouse, the edge skin was sutured with glue, and the mouse was placed in a cage to recover. The specific experimental process is shown in Figure 13A.

The mice were given the drug according to the dosing regimen in Table 27. After the mice recovered for 7 days, the survival of the mouse skin was observed every day, and the rejection score was recorded according to the rejection score. The scoring system is as follows: 3: The skin is not red and smooth; 2: Part of the skin is red, loses its luster, and is dry; 1: Most of the skin turns red, has no stripes, and shrinks; 0: Transplant rejection causes 80% skin necrosis.

Table 27. Drug administration scheme 1 for mouse skin transplant rejection model

The results are shown in Tables 28-29, Figure 13B and Figure 13C. Both 9E6-L4H2 and ADC-1 significantly improved the mouse skin graft scores and prolonged the survival time of skin grafts. Compared with the CD40 antagonist monoclonal antibody 9E6-L4H2, ADC-1 has better anti-graft rejection activity.

Table 28. Skin graft survival rate of anti-CD40-ADC (%)

Table 29. Skin graft scoring of anti-CD40-ADC

In addition, as shown in Figures 13D and 13E, ADC-1 has slightly better anti-transplant rejection activity than high-dose dexamethasone and better anti-transplant rejection activity than CD40 antagonist monoclonal antibody.

Table 30. Skin graft survival rate (%) of anti-CD40-ADC and high-dose dexamethasone

Table 31. Skin graft scores of anti-CD40-ADC and high-dose dexamethasone

In addition, mice were given the drug according to the dosing regimen in Table 32. After the mice recovered for 7 days, the skin survival of the mice was observed daily using the same method as above, and the rejection score was recorded according to the rejection score.

Table 32. Drug administration scheme 2 for mouse skin transplant rejection model

The results are shown in Figures 14A and 14B. ADC-1 and ADC-2 have similar anti-transplant rejection activities, and are significantly superior to CD40 antagonist monoclonal antibody 9E6-L4H2, monoclonal antibody combined with glucocorticoids, and slightly superior to high-dose dexamethasone.

Table 33. Skin graft survival rate of anti-CD40-ADC (%)

Table 34. Skin graft scoring of anti-CD40-ADC

Example 24. Evaluation of side effects of anti-CD40-ADC

On the last day of the in vivo efficacy experiment described in Example 23, 1 mpk ACTH was intraperitoneally injected into mice, and 30 minutes later, peripheral blood and plasma of mice were collected for detection of blood routine and hormone side effect related indicators. Endogenous corticosterone was quantified by LC-MS, and P1NP was detected by anti-mouse P1NP ELISA kit (Novusbio, NBP2-76466).

The results are shown in Table 35. High-dose dexamethasone exhibits obvious toxic side effects: reduced body weight, reduced immune cell count, and inhibition of endogenous HPA axis. ADC-1 dose-dependently reduces the number of peripheral blood immune cells in mice (possibly because anti-CD40-ADC enriches hormones in CD40-positive cells), but has no effect on endogenous corticosterone release and bone formation indicator P1NP, and its safety is better than the same-dose control molecule ref-ADC.

Table 35. Side effects of anti-CD40-ADC

In addition, as shown in Table 36, 10mg/kg ADC-1 and ADC-2 have good safety and no hormone-related side effects compared to high-dose dexamethasone.

Table 36. Side effects of anti-CD40-ADC

Although the above describes the specific implementation schemes of the present disclosure, those with ordinary knowledge in the relevant technical field should understand that these are only examples, and various changes or modifications can be made to these implementation schemes without departing from the principles and essence of the present disclosure.

TW202430226A_112151408_SEQL.xml

Claims (22)

An antibody-drug conjugate comprising an anti-CD40 antibody or an antigen-binding fragment thereof, wherein the anti-CD40 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein, a-1) the VH comprises HCDR1, HCDR2 and HCDR3 in any of the VHs shown in SEQ ID NOs: 13-14, and the VL comprises LCDR1, LCDR2 and LCDR3 in any of the VLs shown in SEQ ID NOs: 9-12; or a-2) 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; Where the CDR is defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. The antibody-drug conjugate as described in claim 1, wherein the anti-CD40 antibody or its antigen-binding fragment comprises: Heavy chain HCDR1, HCDR2 and HCDR3, which respectively comprise the sequences shown in SEQ ID NO: 3, 4 and 5; and light chain LCDR1, LCDR2 and LCDR3, which respectively comprise the sequences shown in SEQ ID NO: 6, 7 and 19; Preferably, the anti-CD40 antibody or its antigen-binding fragment comprises: Heavy chain HCDR1, HCDR2 and HCDR3, which respectively comprise the sequences shown in SEQ ID NO: 3, 4 and 5; light chain LCDR1, which comprises the sequence shown in SEQ ID NO: 6; light chain LCDR2, which comprises the sequence shown in SEQ ID NO: 7; and light chain LCDR3, which comprises the sequence shown in any one of SEQ ID NO: 8 and 15-18. The antibody-drug conjugate as described in claim 1 or 2, wherein the anti-CD40 antibody is selected from recombinant antibodies, rabbit antibodies, chimeric antibodies or humanized antibodies. The antibody-drug conjugate as described in claim 3, wherein, The heavy chain framework region of the humanized antibody or its antigen-binding fragment is derived from IGHV2-26*01, IGHV4-30-4*02, IGHV4-4*08, and IGHJ1*01; and/or, the light chain framework region is derived from IGkV1-13*02, IGkV1-9*01, IGkV1-6*01, and IGKJ4*01; Preferably, FR1-FR3 of the heavy chain framework region is derived from IGHV2-26*01, IGHV4-30-4*02, and IGHV4-4*08, and FR4 of the heavy chain framework region is derived from IGHJ1*01; FR1-FR3 of the light chain framework region is derived from IGkV1-13*02, IGkV1-9*01, and IGkV1-6*01, and FR4 of the light chain framework region is derived from IGKJ4*01. An antibody-drug conjugate as described in any one of claims 1 to 4, wherein the anti-CD40 antibody or its antigen-binding fragment comprises VH and VL, wherein, A-1) The VH comprises an amino acid sequence as shown in SEQ ID NO: 13 or having at least 90% identity thereto, and the VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 9-12 or having at least 90% identity thereto; A-2) the VH comprises an amino acid sequence as shown in SEQ ID NO: 14 or having at least 90% identity thereto, and the VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 9-12 or having at least 90% identity thereto; or A-3) The VH comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least 90% identity thereto, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 2 or having at least 90% identity thereto. The antibody-drug conjugate as described in any one of claims 1 to 5, wherein the anti-CD40 antibody or antigen-binding fragment thereof further contains the Fc region of an IgG antibody; preferably, the IgG antibody is an IgG1, IgG2 or IgG4 antibody; more preferably, the Fc region is the Fc region of IgG1, which contains any one or more mutations selected from 297A, 234A, 235A, 252Y, 254T, and 256E, and the mutations are numbered according to the EU numbering rules. The antibody-drug conjugate as described in any one of claims 1 to 6, wherein the antigen-binding fragment of the anti-CD40 antibody is a scFv, Fv, Fab or Fab' fragment. An antibody-drug conjugate as described in any one of claims 1 to 7, wherein the anti-CD40 antibody comprises a heavy chain and a light chain, wherein, The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 20 or 22 or having at least 90% identity thereto; the light chain comprises an amino acid sequence as shown in SEQ ID NO: 21 or having at least 90% identity thereto. The antibody-drug conjugate as described in any one of claims 1 to 8 is an antibody-drug conjugate having a structure shown in formula (I): Ab-(LD) _k (I); Wherein, Ab is an anti-CD40 antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 8; D is an effector molecule with immunosuppressive activity, preferably a glucocorticoid receptor agonist, a calcitonin phosphatase inhibitor, or a mTOR kinase inhibitor; L is a linker that covalently connects Ab to D, and k is an integer or decimal between 1 and 20. The antibody-drug conjugate as described in claim 9, wherein D is as shown in the following formula:

or

in, R _1a is independently selected from hydrogen, alkyl and alkoxy, and the alkyl and alkoxy are optionally substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl and hydroxyalkyl. Preferably, R _1a is independently selected from hydrogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy; Ring A is selected from aryl or heteroaryl groups which are optionally substituted by one or more substituents _Q1 ; Ring B is selected from aryl or heteroaryl groups which are optionally substituted by one or more substituents _Q1 ; X ₁ is selected from -(CR _5a R _5b ) m -, an aryl group or a heteroaryl group which is optionally substituted by one or more substituents Q ₁ ; R _5a , R _5b are each independently selected from hydrogen, halogen, hydroxyl, alkyl, deuterium, oxo, thio, nitro, cyano or the following groups which are optionally substituted with one or more substituents Q ₁ : alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k , -S(O)R _k , -S(O)OR _k , -S(O)(O)R _k , -S(O)(O)OR _k , -C(S)R _k , alkoxy, alkylthio, alkenyl and alkynyl; Ring C and Ring D are each independently selected from an aryl group, a heteroaryl group, a fused aryl group or a fused heteroaryl group which may be substituted with one or more substituents Q ₁ , and at least one of Ring C and Ring D is selected from a fused aryl group or a fused heteroaryl group which may be substituted with one or more substituents Q ₁ ; _X2 is selected _from -( _CR6aR6b )n-, aryl or heteroaryl optionally substituted by one or more substituents _Q1 , -O-, -S-, -S(O)-, -S(O)(O)-, -NR6c- _{, -CH2S- , -CH2O-} , _-NHCR6dR6e- , _-CR6f = _CR6g- , -C≡C-, or X is absent; R _6a , R _6b are each independently selected from hydrogen, halogen, hydroxyl, alkyl, deuterium, oxo, thio, nitro, cyano or the following groups which are optionally substituted with one or more substituents Q ₁ : alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k , -S(O)R _k , -S(O)OR _k , -S(O)(O)R _k , -S(O)(O)OR _k , -C(S)R _k , alkoxy, alkylthio, alkenyl and alkynyl, or R _6a , R _6b together with the adjacent carbon atoms form a 3- to 10-membered cycloalkyl group; R _6c , R _6d , R _6e , R _6f , and R _6g are each independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ halogenalkyl, or C ₁ -C ₆ alkoxy; _R1 is independently selected from hydrogen, alkyl or alkoxy, wherein the alkyl and alkoxy are independently substituted with one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl and hydroxyalkyl, preferably _R1 is independently selected from hydrogen, _C1 - _C6 alkyl or _C1 - _C6 alkoxy, more preferably hydrogen; R ₂ is each independently selected from -CH ₂ OH, -CH ₂ SH, -CH ₂ Cl, -SCH ₂ Cl, -SCH ₂ F, -SCH ₂ CF ₃ , -OH, -OCH ₂ CN, -OCH ₂ Cl. , -OCH ₂ F , -OCH ₃ , -OCH ₂ CH ₃ , -SCH ₂ CN,

R _2a are each independently selected from hydrogen or C ₁ -C ₆ alkyl; R _2b are each independently selected from C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy; R _2c are each independently selected from hydrogen, C ₁ -C ₆ alkyl, -CH ₂ OH or C ₁ -C ₆ alkoxy; R _2d and R _2e are each independently selected from hydrogen or C ₁ -C ₆ alkyl; _R3 are each independently selected from hydrogen or halogen; R ₄ are each independently selected from hydrogen, halogen or hydroxyl, preferably R ₄ are each independently selected from hydrogen; m and n are independently selected from integers between 1 and 6; The substituent groups _Q1 are each independently selected from _C1 - _C6 alkyl, halogen, deuterium, hydroxyl, _alkyl , _-NRiRj , pendoxirane, thio, -C(O) _Rk , -C(O)ORk, -S(O) _Rk , -S(O)ORk, -S(O)(O) _Rk , _-S (O)(O)ORk, -C(S)Rk, nitro, cyano, C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, C2-C6 alkynyl, _3-10 membered cycloalkyl, 3-10 membered heterocyclic group, 6-10 membered aryl, 5-10 membered heteroaryl, _8-12 membered fused cycloaryl and _5-12 membered fused heteroaryl. Preferably _Q1 is selected from _C1 - _C6 alkyl, halogen, deuterium, hydroxyl _{, alkyl} , -NRiRj, pendoxirane, _thio , -C(O)Rk, -C(O)ORk, -S(O)Rk, -S(O)(O) _Rk , _-S (O)(O)ORk, -C(S)Rk, nitro, cyano, C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, C2-C6 alkynyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclic group, 6-10 membered aryl, 5-10 membered heteroaryl, 8-12 membered fused cycloaryl and 5-12 membered fused heteroaryl. ₁ are each independently selected from halogen, hydroxyl, alkyl, deuterium, oxo, sulfhydryl, cyano, amine, carboxyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy or halogen C ₁ -C ₆ alkoxy; R _i and R _j are each independently selected from a hydrogen atom, a hydroxyl group, a C ₁ -C ₆ alkyl group, and a C ₁ -C ₆ alkoxy group; R _k is each independently selected from a hydrogen atom, a C ₁ -C ₆ alkyl group, a C ₁ -C ₆ halogenalkyl group, a C ₁ -C ₆ alkoxy group, a hydroxyl group, and -NR _i R _j , wherein the alkyl group, the alkoxy group, and the halogenalkyl group are optionally substituted with one or more substituents selected from a C ₁ -C ₆ alkyl group, a halogen group, a hydroxyl group, a hydroxyl group, -NR _i R _j , a pendoxy group, a thio group, a carboxyl group, a nitro group, a cyano group, a C ₁ -C ₆ alkoxy group, a C ₁ -C ₆ alkylthio group, a C ₂ -C ₆ alkenyl group, a C ₂ -C ₆ alkynyl group, a 3- to 10-membered cycloalkyl group, a 3- to 10-membered heterocyclo group, a 6- to 10-membered aryl group, and a 5- to 10-membered heteroaryl group. Preferably, R _k is each independently selected from a hydrogen atom, a C ₁ -C ₆ alkyl group, a C ₁ -C 6 ₆ halogenalkyl, C ₁ -C ₆ alkoxy, hydroxyl, -NR _i R _j ; Provided that when R _5a is hydrogen or alkyl, R _5b is not hydrogen or alkyl; Preferably, D is as shown in the following structure:

The antibody-drug conjugate of claim 10, wherein the linker comprises an amino acid member, and the amino acid member preferably comprises 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamate, aspartic acid, homolysine, n-methyl-valine,

The peptide residue is composed of amino acids, q is an integer of 1-6, more preferably valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe); phenylalanine-lysine (Phe-Lys), phenylalanine-homolysine (Phe-Homolys), n-methyl-valine-citrulline (Me-Val-Cit), alanine-alanine (Ala-Ala), glycine-glutamate (Gly-Glu), glutamate-alanine-alanine (Glu-Ala-Ala) and glycine-lysine (Gly-Lys), glycine-valine-citrulline (Glv-Val-Cit) and glycine-glycine-glycine (Gly-Gly-Gly),

, optimal glycine-glutamate (Gly-Glu); Preferably, the antibody-drug conjugate, wherein the linker comprises a stretching unit, preferably the stretching unit is selected from

,

or

, wherein p is independently selected from 1, 2, 3, 4, 5 or 6; Preferably, the connector is

. The antibody-drug conjugate as described in claim 10 or 11 has the following structure:

Where p is an integer from 1 to 6, and k is an integer or decimal from 1 to 10; Preferably, the antibody-drug conjugate has the following structure:

Where k is an integer or decimal between 1 and 10, preferably 4±0.4. The antibody-drug conjugate as described in claim 9, wherein D is a glucocorticoid, preferably comprising the following structure:

The antibody-drug conjugate as described in claim 13, wherein L-D is a chemical moiety represented by the following formula: -Str-Pep-Sp-D, Among them, Str is the stretching unit covalently linked to Ab; Sp is a spacer unit, preferably _{-NHCH2- ,} -NHCH2CH2- _, -NHCH2CH2CH2-, -NHCH2-O- _{CH2- , -NHCH2CH2} -O- _CH2- , -NH( _CH2 ) ₃ -C(O) _- , _{-NHCH2 -} O- _CH2 -C(O)-, -NH(CH2) _{2 -O - CH2 -} C(O)-,

,

,

,

,

or

; Pep is selected from an amino acid unit, a disulfide moiety, a sulfonamide moiety, or the following non-peptide chemical moieties:

,

or

, wherein W is -NH-heterocycloalkylene- or heterocycloalkylene; Y is heteroarylene, arylene, -C(O)C 1-6 alkylene, C 2-6 alkenylene, C _1-6 alkylene or -C _1-6 alkylene-NH-; each R ¹⁶ is independently selected from C 1-6 alkyl, C _2-6 alkenyl, -(C _1-6 alkylene)NHC(NH)NH 2 or -(C _1-6 alkylene)NHC(O)NH ₂ ; R 17 and R ₁₈ are each independently selected from hydrogen, C _{1-6 alkyl, C 2-6 alkenyl, aryl, heteroaryl, or R 17 and R 18 together can form a C 3-6 cycloalkyl} ^; _{R 19 and R 19 are each independently selected from hydrogen, C 1-6} ^alkyl _, C _2-6 alkenyl, aryl, heteroaryl, or R ¹⁹ and R ¹⁹ together can form a C _3-6 cycloalkyl; R ¹⁹ and R ^{R 20} are each independently selected from C _1-6 alkyl, C _2-6 alkenyl, aryl, heteroaryl, (C _1-6 alkyl)OCH ₂ —, or R ¹⁹ and R ²⁰ together can form a C _3-6 cycloalkyl ring. The antibody-drug conjugate as described in claim 14, wherein Str is selected from a chemical moiety represented by the following formula:

or

, wherein R ²¹ is selected from -W1-C(O)-, -C(O)-W1-C(O)-, (CH ₂ CH ₂ O) _p1 C(O)-, (CH ₂ CH ₂ O) _p1 CH ₂ C(O)-, (CH ₂ CH ₂ O) _p1 CH ₂ CH ₂ C(O)-, wherein W1 is selected from C _1-6 alkylene, C _1-6 alkylene-cycloalkyl or a straight-chain heteroalkyl group of 1 to 8 atoms, the heteroalkyl group containing 1 to 3 heteroatoms selected from N, O or S, wherein the alkyl, cycloalkyl and straight-chain heteroalkyl groups are each independently and optionally substituted by one or more groups selected from halogen, deuterium, hydroxyl, cyano, amino, C _1-6 alkyl, halogenated C _1-6 alkyl, deuterated C _1-6 alkyl, C substituted by C _1-6 alkoxy and C _3-6 cycloalkyl groups; ^L1 is selected from ^-NR22 _{( CH2CH2O} ) _p1CH2CH2C (O _{) -} , -NR22( _{CH2CH2O )} p1CH2C ₍ O)-, -S( _CH2 ) _p1C (O)-, -( _CH2 ) _p1C (O)- or a single bond, preferably a single bond; _p1 is an integer from 1 to ²⁰ , and ^R22 is selected from a hydrogen atom, a _C1-6 alkyl group, a halogenated _C1-6 alkyl group or a deuterated _C1-6 alkyl group; Preferably, R ²¹ is selected from C _1-6 alkylene C(O)-, -(CH ₂ -CH ₂ O) ₂ C(O)-, -(CH 2 -CH 2 O) ₂ CH ₂ C(O)-, -(CH ₂ -CH ₂ O) 2 CH 2 CH 2 C(O)-, -(CH ₂ -CH ₂ O) ₂ CH ₂ CH ₂ C(O)-, -(CH ₂ -CH ₂ O) ₃ C(O)- and -(CH ₂ -CH ₂ O) ₄ C(O)-. The antibody-drug conjugate as described in claim 14 or 15, wherein Pep is selected from valine-citrulline, alanine-alanine-asparagine, glycine-glycine-lysine, valine-lysine (Val-lys), valine-alanine, valine-phenylalanine or glycine-glycine-phenylalanine-glycine. The antibody-drug conjugate of any one of claims 13 to 16, wherein the linker L in the antibody-drug conjugate comprises: _-CH2C (O ₎ NH-(PEG) ₄ - _CH2CH2C (O)-Gly-Gly-Phe- _Gly , -CH2C(O)NH-(PEG) ₂ -Val-Cit, -CH2C(O)NH-(PEG) ₆ -Val-Cit, -CH2C(O)NH-(PEG) ₈ -Val-Cit _{, -CH2C} (O ₎ NH-(PEG _{) 4 - CH2CH2C} (O)-Val-lys, -CH2C(O)NH-( _CH2 ) ₅ -Val-Cit, -CH2C ₍ O)NH-( _CH2 ) ₅ -Val-lys, _-CH2C (O)NH-( _CH2 ) _5- -Gly-Gly-Phe-Gly, -CH2C(O)NH-(PEG) _{2 - CH2CH2C} (O)-Gly-Gly-Phe-Gly _, -CH2C(O ₎ NH-(PEG) ₂ -Ala-Ala-Asn, -CH2C(O)NH-(PEG _{) 6} -Ala-Ala-Asn, -CH2C ₍ O)NH-(PEG) ₈ -Ala-Ala-Asn, _-CH2C (O)NH-(PEG) ₄ -triazole-(PEG) ₃ -sulfonamide, -CH2C ₍ O)NH-(PEG) ₂ -CH2CH2C ₍ O)-Val-lys, _-CH2C (O)NH-(PEG) ₄ -triazole-(PEG) ₃ -sulfonamide or Mal- ₍ PEG) ₄ -triazole-(PEG) ₃ -disulfide. The antibody-drug conjugate as described in any one of claim 13 to 17 has the following structure:

Where k is an integer or decimal from 1 to 10, p1 is selected from 2, 4, 6 or 8, and p3 and p4 are independently selected from 0, 1 or 2; Preferably, the antibody-drug conjugate has the following structure:

Where k is an integer or decimal between 1 and 10, preferably 4±0.4. A pharmaceutical composition comprising an antibody-drug conjugate as described in any one of claims 1 to 18, and one or more pharmaceutically acceptable excipients. A method for preparing an antibody-drug conjugate as described in any one of claims 1 to 18, comprising the following steps: The anti-CD40 antibody or its antigen-binding fragment is reacted with a drug to obtain the antibody-drug conjugate; optionally, the method further comprises: purifying the antibody-drug conjugate. Use of an antibody-drug conjugate as described in any one of claims 1 to 18 or a pharmaceutical composition as described in claim 19 in the preparation of a drug, wherein the drug is used to treat or alleviate autoimmune diseases, graft-versus-host disease and/or transplant rejection reactions. A method for preventing or treating transplant rejection, graft-versus-host disease and/or autoimmune disease, comprising administering a preventive or therapeutically effective amount of an antibody-drug conjugate as described in any one of claims 1 to 18 or a pharmaceutical composition as described in claim 19 to a subject in need thereof; preferably, the subject is a subject who has undergone an organ or tissue transplant.