Anti-CD79b antibody-drug conjugate, and preparation method therefor and pharmaceutical use thereof

Abstract

Provided are an anti-CD79B antibody-drug conjugate, and a preparation method therefor and pharmaceutical use thereof. In particular, provided are an antibody-drug conjugate (ADC) which comprises an anti-CD79B antibody conjugated with an MMAE or a derivative thereof, exatecan or a derivative thereof, or Eribulin or a derivative thereof, a pharmaceutical composition containing the ADC, and use thereof in preparation of a drug for treatment of a CD79B-mediated disease or disorder, especially use thereof in preparation of an anti-cancer drug.

Description

Anti-CD79B antibody-drug conjugate, preparation method thereof and medical use thereof

[0001] This application claims priority to patent applications with application numbers CN202010730899.8 filed on July 27, 2020 and CN202010735910.X filed on July 28, 2020. Technical Field

[0002] The present application relates to an anti-CD79B antibody and antigen-binding fragment, a chimeric antibody and a humanized antibody comprising the CDR region of the anti-CD79B antibody, and a pharmaceutical composition comprising the human anti-CD79B antibody or its antigen-binding fragment, and its use as an anti-cancer drug. Background Art

[0003] Malignant tumors (cancer) are the second leading cause of death worldwide, second only to heart disease. Lymphoma is a malignant tumor originating from the lymphoid hematopoietic system and is the most common blood cancer in the world. Lymphoma is divided into two categories: non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL). Non-Hodgkin's lymphoma is a general term for a group of abnormal lymphocyte proliferation diseases with strong heterogeneity. Its incidence is much higher than that of Hodgkin's lymphoma, accounting for more than 80% of lymphomas. Among them, diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma in adults, accounting for approximately 32.5% of all non-Hodgkin's lymphomas; in Asian populations, this proportion is even higher, close to 40%. It is more common in elderly patients, with a median age of onset of 60-64 years old, and there are slightly more male patients than female patients.

[0004] The current standard first-line treatment for diffuse large B-cell lymphoma (DLBCL) is rituximab combined with chemotherapy (R-CHOP). Before the launch of rituximab, the anthracycline-based CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and prednisone) was the standard first-line treatment for DLBCL. R-CHOP has significantly improved the long-term survival of DLBCL patients. Clinical trial results show that, compared with the traditional CHOP regimen, R-CHOP significantly prolongs the median overall survival (OS) of patients in DLBCL by 4.9 years, the median disease-free survival (DFS) by over 6.6 years, and the 5-year progression-free survival (PFS) rate from 30% to 54%. However, 10% to 15% of refractory patients still fail to respond, and 20% to 30% experience relapse. Furthermore, not all DLBCL patients are suitable for R-CHOP, such as those over 80 years old, whose physical condition precludes standard R-CHOP treatment. R-CHOP may not be effective for more aggressive lymphoma types or for relapsed lymphoma.

[0005] Based on the lymphocyte origin, DLBCL is classified as a B-cell lymphoma. The B cell antigen receptor (BCR) complex is the most important molecule on the surface of B cells. The BCR complex is composed of membrane immunoglobulin (mIg), which recognizes and binds antigens, and heterodimers of Igα (CD79a) and Igβ (CD79B), which transmit antigen-stimulating signals. Igα and Igβ are glycoproteins of approximately 47 kDa and 37 kDa, respectively, and belong to the immunoglobulin superfamily. The genes encoding Igα and Igβ are called mb-1 and B29, respectively. Both Igα and Igβ have an Ig-like domain at the amino terminus of their extracellular membrane regions. Both Igα and Igβ serve as substrates for protein tyrosine kinases and participate in BCR signal transduction.

[0006] BCR is widely expressed on B cell lymphomas and normal B cells. In view of the clinical success and reliable safety of rituximab targeting CD20, the development of treatment methods targeting BCR should also have good efficacy and safety. Therapeutic antibodies targeting the CD79B antigen are beneficial, which produce minimal or no antigenicity when administered to patients (especially for long-term treatment). There is an urgent need in the art to develop effective CD79B antibodies and antibody-drug conjugates thereof for cancer treatment or delaying cancer progression. WO2020156439A discloses an anti-CD79B antibody and its use in treating tumors, and the present disclosure incorporates its content in its entirety.

[0007] Summary of the Invention

[0008] The present disclosure relates to anti-CD69B antibodies or antigen-binding fragments thereof and conjugates thereof with drugs (ADCs) and their medical uses, wherein an ADC drug is provided, which is conjugated with an anti-CD69B antibody or antigen-binding fragment and a cytotoxic substance (MMAE or its derivatives, exitecan or its derivatives, eribulin or its derivatives).

[0009] Anti-CD79B antibodies and antigen-binding fragments thereof

[0010] WO2020156439A discloses an anti-CD79B antibody and its use in treating tumors, and the present disclosure incorporates its content in its entirety.

[0011] The present disclosure provides an anti-CD79B antibody and an antigen-binding fragment thereof, comprising:

[0012] A heavy chain HCDR1 comprising the sequence GX1X2FX3X4Y (SEQ ID NO: 24), wherein X1 is S or Y, X2 is S or T, X3 is T or S, and X4 is S or T;

[0013] A heavy chain HCDR2 comprising the sequence shown as X5PRSGN (SEQ ID NO: 25), wherein X5 is F or Y;

[0014] Heavy chain HCDR3 comprises X6X7X8X9X 10 GDFX 11 Y (SEQ ID NO: 26), wherein X6 is absent or G, X7 is absent or S, X8 is G or D, X9 is D or Y, X 10 L or D, X 11 D or A;

[0015] Light chain LCDR1, which contains RSSQSIVHX 12 GNTYX 13 E (SEQ ID NO: 27), wherein X 12 S or H, X 13 is F or L;

[0016] A light chain LCDR2 comprising the sequence shown in SEQ ID NO: 11 or 17; and

[0017] A light chain LCDR3 comprising the sequence shown in SEQ ID NO: 12 or 18.

[0018] In some embodiments, the anti-CD79B antibody or antigen-binding fragment thereof comprises:

[0019] An antibody heavy chain variable region, the antibody heavy chain variable region comprising at least one HCDR selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15; and / or

[0020] An antibody light chain variable region, comprising at least one LCDR selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0021] In some embodiments, an anti-CD79B antibody or antigen-binding fragment thereof is provided, comprising:

[0022] (a) HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 23, 8, and 9, respectively, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 10, 11, and 12, respectively;

[0023] (b) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 7, 8, 9, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11, 12, respectively; or

[0024] (c) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 13, 14 and 15, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 16, 17 and 18, respectively.

[0025] In some specific embodiments, the aforementioned scheme (a) (i.e., an anti-CD79B antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 23, 8, and 9, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11, and 12, respectively) does not comprise an anti-CD79B antibody or an antigen-binding fragment thereof whose VH is as shown in SEQ ID NO: 19 and whose VL is as shown in SEQ ID NO: 20, or does not comprise an anti-CD79B antibody or an antigen-binding fragment thereof whose full-length heavy chain is as shown in SEQ ID NO: 28 and whose full-length light chain is as shown in SEQ ID NO: 29.

[0026] In some embodiments, the anti-CD79B antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody or a fragment thereof; for example, a humanized antibody or a fragment thereof.

[0027] In some embodiments, an anti-CD79B antibody or antigen-binding fragment thereof is provided, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0028] The heavy chain variable region (VH) comprises:

[0029] a sequence as set forth in SEQ ID NO: 3, 5, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 5;

[0030] and / or

[0031] The light chain variable region (VL) comprises:

[0032] A sequence as set forth in SEQ ID NO: 4, 6, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4, 6.

[0033] In some specific embodiments, the VH of the anti-CD79B antibody or antigen-binding fragment is as shown in SEQ ID NO: 3, and the VL is as shown in SEQ ID NO: 4; or the VH is as shown in SEQ ID NO: 5, and the VL is as shown in SEQ ID NO: 6.

[0034] In some other embodiments, an anti-CD79B antibody or antigen-binding fragment thereof is provided, wherein:

[0035] VH contains:

[0036] a sequence as set forth in SEQ ID NO: 19, 21, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 19, 21;

[0037] and / or VL, including:

[0038] A sequence as set forth in SEQ ID NO: 20, 22, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0039] In some specific embodiments, the VH of the anti-CD79B antibody or antigen-binding fragment is as shown in SEQ ID NO: 19, and the VL is as shown in SEQ ID NO: 20; or the VH is as shown in SEQ ID NO: 21, and the VL is as shown in SEQ ID NO: 22.

[0040] In some embodiments, the above-mentioned anti-CD79B antibody or its antigen-binding fragment comprises and light chain constant region; preferably, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, and the light chain constant region is selected from human antibody κ and λ chain constant regions and conventional variants thereof.

[0041] In some specific embodiments, the heavy chain constant region is human IgG1 or IgG2.

[0042] In some embodiments, an anti-CD79B antibody or fragment thereof is provided, wherein:

[0043] The heavy chain is represented by SEQ ID NO: 28 or a variant thereof; wherein the variant comprises 0 to 10 amino acid changes in the heavy chain;

[0044] The light chain is represented by SEQ ID NO: 29 or a variant thereof; the variant sequence comprises 0 to 10 amino acid changes in the light chain.

[0045] In some embodiments, an anti-CD79B antibody or fragment thereof is provided, wherein:

[0046] The heavy chain is represented by SEQ ID NO: 30 or a variant thereof; wherein the variant comprises 0 to 10 amino acid changes in the heavy chain;

[0047] The light chain is represented by SEQ ID NO: 31 or a variant thereof; the variant sequence comprises 0 to 10 amino acid changes in the light chain.

[0048] In some specific embodiments, the above-mentioned anti-CD79B antibodies or fragments thereof may be variants, wherein the variants have 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in VL and / or 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in VH.

[0049] In some specific embodiments, the variants have the same or similar biological functions or effects as the original anti-CD79B antibody or its fragment.

[0050] In some embodiments, the antigen-binding fragments of the above-mentioned anti-CD79B antibodies include Fab, Fv, sFv, F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, peptibodies, domain antibodies and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv).

[0051] In some embodiments, a polynucleotide, such as DNA or RNA, encoding an anti-CD79B antibody or an antigen-binding fragment thereof as described above is provided.

[0052] In some embodiments, an expression vector comprising the above-mentioned polynucleotide is provided, such as a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.

[0053] In some embodiments, host cells transformed with the above expression vectors are provided, such as eukaryotic cells or prokaryotic cells. In some specific embodiments, the host cells are bacteria (e.g., Escherichia coli), yeast (e.g., Pichia pastoris), or mammalian cells (e.g., Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells).

[0054] In some embodiments, a method for preparing the above-mentioned anti-CD79B antibody or antigen-binding fragment thereof is provided, comprising the steps of expressing the antibody or antigen-binding fragment thereof in a host cell as described above, and isolating the antibody or antigen-binding fragment thereof from the host cell.

[0055] In some embodiments, a method of treating or preventing a proliferative disease or slowing the progression of a proliferative disease is provided, comprising:

[0056] The subject is administered an effective amount of the above-mentioned anti-human CD79B antibody or its antigen-binding fragment or its encoded polynucleotide, or pharmaceutical composition for treating or delaying a disease, wherein the proliferative disease is cancer or tumor.

[0057] In some embodiments, the cancer or tumor is lymphoma or leukemia;

[0058] The lymphoma is selected from the group consisting of diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, small lymphocytic lymphoma, and mantle cell lymphoma;

[0059] The non-Hodgkin's lymphoma is selected from the group consisting of: aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, and refractory indolent NHL;

[0060] The leukemia is selected from the group consisting of chronic lymphocytic leukemia, hairy cell leukemia, and acute lymphocytic leukemia.

[0061] The present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0062] The drug is selected from MMAE or its derivatives, exitecan or its derivatives, eribulin or its derivatives;

[0063] The ligand is an anti-CD79B antibody or an antigen-binding fragment thereof, and the anti-CD79B antibody or an antigen-binding fragment thereof is any of the anti-CD79B antibodies or antigen-binding fragments disclosed above.

[0064] Ligand-drug (exitecan or its derivatives) conjugates

[0065] The present disclosure provides a ligand-exitecan or a derivative thereof conjugate or a pharmaceutically acceptable salt or solvate thereof, which is a ligand-exitecan or a derivative thereof conjugate or a pharmaceutically acceptable salt or solvate thereof represented by formula (I):

[0066]

[0067] in:

[0068] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0069] L 2 Selected from-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 is an integer from 1 to 20;

[0070] L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0071] R 1 is selected from hydrogen, halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0072] R 2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0073] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0074] R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0075] R 6 and R 7 are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

[0076] m is an integer from 0 to 4;

[0077] n is 1 to 10 and can be an integer or a decimal;

[0078] Pc is an anti-CD79B antibody or an antigen-binding fragment thereof provided by the present disclosure.

[0079] In some embodiments, the present disclosure provides a ligand-exitecan or a derivative thereof conjugate or a pharmaceutically acceptable salt or solvate thereof, which is a ligand-exitecan or a derivative thereof conjugate or a pharmaceutically acceptable salt or solvate thereof represented by formula (II):

[0080]

[0081] in:

[0082] s 1 is an integer from 2 to 8; preferably 5;

[0083] Pc, R 1 、R 2 、R 5 ~R 7 , m and n are as defined in formula (I).

[0084] In some embodiments, the linker unit -LY- of the ligand-exitecan or derivative conjugates disclosed herein includes, but is not limited to:

[0085]

[0086] In some embodiments, the present disclosure provides ligand-exitecan or its derivative conjugates or pharmaceutically acceptable salts or solvates thereof, including but not limited to:

[0087]

[0088] in:

[0089] n is 1 to 10 and can be an integer or a decimal;

[0090] Pc is the aforementioned anti-CD79B antibody or antigen-binding fragment thereof disclosed herein.

[0091] In some embodiments, the preparation is as shown in the general formula (Pc-L a -YD)-ixitecan or a derivative thereof or a pharmaceutically acceptable salt or solvate thereof, comprising the following steps:

[0092]

[0093] After Pc reduction, with the general formula (L a -YD) coupling reaction to obtain the general formula (Pc-L a -YD) shown in the compound;

[0094] Wherein, Pc is the anti-CD79B antibody or antigen-binding fragment thereof disclosed herein; W, L 2 , L 3 、R1 、R 2 、R 5 ~R 7 , m and n are as defined in formula (I).

[0095] In the above embodiments, Pc is any anti-CD79B antibody or antigen-binding fragment thereof disclosed herein; preferably, it is an antibody comprising a heavy chain as shown in SEQ ID NO: 28 and a light chain as shown in SEQ ID NO: 29, or an antibody comprising a heavy chain as shown in SEQ ID NO: 30 and a light chain as shown in SEQ ID NO: 31.

[0096] In some embodiments, the conjugates of the ligand-exitecan or its derivatives disclosed herein include tautomers, mesomers, racemates, enantiomers, diastereomers, deuterated forms, or mixtures thereof.

[0097] The compounds and preparation methods of WO2020063673 are incorporated herein in their entirety.

[0098] Ligand-drug (eribulin or its derivatives) conjugates

[0099] The present disclosure provides a ligand-eribulin or derivative conjugate or a pharmaceutically acceptable salt or solvate thereof, which is as shown in the formula Pc-(LD) k As shown:

[0100] Wherein, the ligand is Pc, and the Pc is any anti-CD79B antibody or antigen-binding fragment thereof disclosed herein,

[0101] L is a linker that covalently attaches Pc to D, and k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any number between any two numbers),

[0102] D is represented by the following formula (III):

[0103]

[0104] where R 1a Selected from hydrogen, alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 Cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, wherein the alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, one or more substituents are substituted; preferably R 1a It is a methyl group;

[0105] R 1b Selected from hydrogen, alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy, cycloalkyl (such as C 3-8 Cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, wherein the alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, one or more substituents are substituted; preferably R 1b is hydrogen; or

[0106] R 1a With R 1b Together with the atoms it is connected to form C 5-8 Heterocycloalkyl, the heteroalkyl is optionally replaced by an alkyl group (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), heterocyclyl, aryl and heteroaryl, and R 1a and R 1b Not hydrogen at the same time.

[0107] In some embodiments, in the ligand-eribulin or derivative thereof conjugate, R1a in D is methyl.

[0108] In some embodiments, D in the ligand-eribulin or its derivative conjugate is represented by the following formula:

[0109]

[0110] In some embodiments, Pc-(LD) k In the ligand-eribulin or derivative thereof conjugate shown, k is selected from 1 to 10 and can be an integer or a decimal.

[0111] In some embodiments, the linker is stable outside the cell, such that the ligand-eribulin or derivative thereof conjugate remains intact in the extracellular environment but is cleaved upon, for example, internalization into cancer cells.

[0112] In some embodiments, when the ligand drug conjugate enters a cell expressing the antigen, the drug portion of the conjugate is cleaved from the ligand portion, and the cleavage releases the drug (eg, eribulin or a derivative thereof).

[0113] In some embodiments, the linker comprises a cleavable moiety; wherein the cleavable moiety is positioned such that after cleavage, no linker and Pc remain in the drug (eg, eribulin derivative).

[0114] In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety.

[0115] In some embodiments, ligand drug conjugates comprising a cleavable peptide moiety exhibit lower aggregation levels, improved ligand to drug ratios, increased on-target killing of cancer cells, reduced off-target killing of non-cancerous cells, and / or higher drug loading (p).

[0116] In some embodiments, the addition of a cleavable portion increases cytotoxicity and / or efficacy relative to a non-cleavable linker. In some embodiments, efficacy and / or cytotoxicity are increased in cancers expressing moderate levels of antigen (e.g., CD79B). In some embodiments, the cleavable peptide portion is capable of being cleaved by an enzyme, and the linker is a linker that the enzyme is capable of cleaving. In some embodiments, the linker is a linker that cathepsin is capable of cleaving. In certain embodiments, an enzyme-cleavable linker (e.g., a cathepsin-cleavable linker) shows one or more of the above-mentioned improved properties.

[0117] In some embodiments, the linker comprises an amino acid unit (i.e., a peptide residue consisting of 2 to 7 amino acids), preferably the amino acids are selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, more preferably 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).

[0118] In some embodiments, the linker in the conjugate of the present disclosure is selected from:

[0119]

[0120]

[0121] In some embodiments, the Amino Acid unit comprises Valine-Citrulline (Val-Cit).

[0122] In some embodiments, ADCs comprising Val-Cit exhibit increased stability, reduced off-target cell killing, increased on-target cell killing, lower aggregation levels, and / or higher drug loading.

[0123] In another aspect, some embodiments provide linkers comprising a cleavable sulfonamide moiety, wherein the linker is cleavable under reducing conditions.

[0124] In some embodiments, the linker comprises a cleavable disulfide moiety, and the linker is capable of being cleaved under reducing conditions.

[0125] In another aspect, the linker of the present disclosure comprises at least one spacer unit that attaches D (eg, an eribulin derivative) to the cleavable moiety.

[0126] In some embodiments, the spacer unit comprises p-aminobenzyloxycarbonyl (PAB),

[0127]

[0128] In another aspect, some embodiments provide a conjugate represented by the formula:

[0129] k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8;

[0130] k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2;

[0131] k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8;

[0132] k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2;

[0133] k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8;

[0134] k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8;

[0135] k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8;

[0136] k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8;

[0137] k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8;

[0138] k is selected from 1 to 10 and can be an integer or a decimal; P1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2;

[0139] k is selected from 1 to 10 and can be an integer or a decimal; P1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2.

[0140] In some of the embodiments, the conjugate is represented by the following formula:

[0141]

[0142]

[0143] wherein k is selected from 1 to 10 and can be an integer or a decimal; further, R in D 1a Preferably, R 1b Preferred is hydrogen.

[0144] The present disclosure also provides a compound represented by formula D (Eribulin),

[0145] or a pharmaceutically acceptable salt thereof,

[0146] in,

[0147] R 1a Selected from hydrogen, alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 Cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, wherein the alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), , deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl, preferably R 1a It is a methyl group;

[0148] R 1b Selected from hydrogen, alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy, cycloalkyl (such as C 3-8 Cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, wherein the alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl, preferably R 1b is hydrogen, methyl; or

[0149] R 1a With R 1b Together with the carbon atom to which it is attached, it forms C 5-8 Heterocycloalkyl, the heteroalkyl is optionally replaced by an alkyl group (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), heterocyclyl, aryl and heteroaryl, and R 1a and R 1b Not hydrogen at the same time.

[0150] In some embodiments, R 1a With R 1b Each independently selected from C 1-6 Alkyl groups include, but are not limited to, methyl, ethyl, and isopropyl.

[0151] In some embodiments, R 1a Selected from C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl; R 1b Selected from hydrogen.

[0152] In some embodiments, R 1a With R 1b Together with the carbon atom to which it is attached, it forms C 6-8 Heterocycloalkyl.

[0153] In some embodiments, the compound represented by Formula D (Eribulin) is:

[0154]

[0155] In some embodiments, the compound represented by Formula D (Eribulin) is:

[0156]

[0157] In some embodiments, the compound represented by Formula D (Eribulin) is:

[0158]

[0159] The present disclosure also provides a compound represented by formula DZ (Eribulin),

[0160] or a pharmaceutically acceptable salt thereof,

[0161] Among them, R 1a Selected from hydrogen, alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 Cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, wherein the alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), , deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl, preferably R 1a It is a methyl group;

[0162] R 1b Selected from hydrogen, alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 Cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), alkoxy, aryl and heteroaryl, wherein the alkyl, cycloalkyl, aryl and heteroaryl are each independently selected from alkyl (such as C 1-6Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl, preferably R 1b It is hydrogen;

[0163] or R 1a With R 1b Together with the carbon atom to which it is attached, it forms C 5-8 Heterocycloalkyl, the heteroalkyl is optionally replaced by an alkyl group (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), heterocyclyl, aryl and heteroaryl; and R 1a and R 1b Not simultaneously hydrogen;

[0164] Y is selected from -O(CR a R b ) m2 -CR 8 R 9 -C(O)-、-NH-(CR a R b ) m2 -CR 8 R 9 -C(O)-, -O-CR 8 R 9 (CR a R b ) m2 -、-OCR 8 R 9 -C(O)-、-O(CR a R b ) m2 C(O)- or -S-(CR a R b ) m2 -CR 8 R 9 -C(O)-, where R a and R b are the same or different and are each independently selected from hydrogen, a deuterium atom, a halogen or an alkyl group; R 8 Selected from hydrogen, C 3-6 Cycloalkylalkyl or C3-6 Cycloalkyl; R 9 Selected from hydrogen, haloalkyl or C 3-6 Cycloalkyl, preferably hydrogen; or, R 8 and R 9 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; m2 is selected from 0, 1, 2 or 3.

[0165] In some embodiments, R 1a With R 1b Each independently selected from C 1-6 Alkyl groups include, but are not limited to, methyl, ethyl, and isopropyl.

[0166] In some embodiments, R 1a Selected from C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl; R 1b Selected from hydrogen.

[0167] In some embodiments, R 1a With R 1b Together with the carbon atom to which it is attached, it forms C 6-8 Heterocycloalkyl.

[0168] In some embodiments, the compound represented by DZ (Eribulin) is:

[0169] or a pharmaceutically acceptable salt thereof,

[0170] Where: R 8 Selected from hydrogen, C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl; R 9 Selected from hydrogen, haloalkyl or C 3-6 Cycloalkyl, preferably hydrogen; or, R 8 and R 9 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; m2 is selected from 0, 1, 2 or 3.

[0171] In some embodiments, the compound represented by DZ (Eribulin) is selected from:

[0172]

[0173]

[0174] On the other hand, some embodiments provide compounds represented by DZ (Eribulin) that may contain one or more asymmetric centers, such as Can be

[0175]

[0176] In the above embodiments, Pc is any anti-CD79B antibody or antigen-binding fragment thereof disclosed herein; preferably, it is an antibody comprising a heavy chain as shown in SEQ ID NO: 28 and a light chain as shown in SEQ ID NO: 29, or an antibody comprising a heavy chain as shown in SEQ ID NO: 30 and a light chain as shown in SEQ ID NO: 31.

[0177] In some embodiments, the conjugates of the present disclosure include tautomers, mesomers, racemates, enantiomers, diastereomers, deuterated forms, or mixtures thereof.

[0178] The compounds and preparation methods in CN202010073671.6 are introduced here in full.

[0179] Ligand-drug (MMAE or its derivatives) conjugates

[0180] The present disclosure provides an MMAE analogue / derivative, which is a compound represented by the general formula (D(MMAE)):

[0181]

[0182] , or a pharmaceutically acceptable salt thereof, wherein:

[0183] R 1 -R 6 is selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy and cycloalkyl;

[0184] R 7 is selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group, and a cycloalkyl group;

[0185] R 8 -R 11 Any two of them form a cycloalkyl group, and the remaining two groups are selected from hydrogen atoms, alkyl groups and cycloalkyl groups;

[0186] R 12 is selected from a hydrogen atom or an alkyl group;

[0187] R 13 -R 15 is selected from a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group or a halogen;

[0188] R 16is selected from an aryl group or a heteroaryl group, wherein the aryl group or the heteroaryl group is optionally further substituted by a substituent selected from a hydrogen atom, a halogen, a hydroxyl group, an alkyl group, an alkoxy group and a cycloalkyl group.

[0189] In some embodiments of the present disclosure, the compound represented by the general formula (D(MMAE)) is a compound represented by the general formula (D(MMAE)1):

[0190] or a pharmaceutically acceptable salt thereof,

[0191] R 9 With R 10 Formation of cycloalkyl groups;

[0192] R 2 -R 8 , R 11 -R 16 As defined in formula (D).

[0193] In some embodiments of the present disclosure, the compound represented by the general formula (D(MMAE)) is:

[0194]

[0195] Another aspect of the present disclosure relates to a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (-D(MMAE)):

[0196] or a pharmaceutically acceptable salt thereof,

[0197] in:

[0198] R 2 -R 6 is selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy and cycloalkyl;

[0199] R 7 is selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group, and a cycloalkyl group;

[0200] R 8 -R 11 Any two of them form a cycloalkyl group, and the remaining two groups are selected from hydrogen atoms, alkyl groups and cycloalkyl groups;

[0201] R 12 is selected from a hydrogen atom or an alkyl group;

[0202] R 13 -R 15 is selected from a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group or a halogen;

[0203] R16 is selected from aryl or heteroaryl, wherein the aryl or heteroaryl is optionally further substituted by a substituent selected from hydrogen, halogen, hydroxy, alkyl, alkoxy and cycloalkyl;

[0204] Wavy lines represent hydrogen atoms, either covalently attached to the linker or to the antibody.

[0205] In some embodiments of the present disclosure, the ligand-MMAE or derivative conjugate or a pharmaceutically acceptable salt or solvate thereof comprises a structure represented by formula (-D(MMAE)1):

[0206]

[0207] in:

[0208] R 9 With R 10 Formation of cycloalkyl groups;

[0209] Wavy line, R 2 -R 8 , R 11 -R 16 As defined in the general formula (D(MMAE)).

[0210] In some embodiments of the present disclosure, the ligand-MMAE or its derivative conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-MMAE or its derivative conjugate comprises a structure as shown below:

[0211]

[0212] Wavy lines represent hydrogen atoms, either covalently attached to the linker or to the antibody.

[0213] In some embodiments of the present disclosure, the ligand-MMAE or derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is represented by the general formula (IV):

[0214]

[0215] Among them, R 2 is a C1-C8 alkyl group;

[0216] R 3 is a C1-C8 alkyl group;

[0217] R 4 is a C1-C8 alkyl group;

[0218] R 5 It is H;

[0219] R 6is a C1-C8 alkyl group;

[0220] R 7 is a C1-C8 alkyl group;

[0221] R 8 Independently of each other, each is O-(C1-C8 alkyl);

[0222] R 9 It is H;

[0223] R 10 It is phenyl;

[0224] Z is O or NH;

[0225] R 11 Selected from H, C1-C 20 Alkyl or -(R 13 O)m-R14;

[0226] m is 3;

[0227] R 13 is a C2-C8 alkyl group;

[0228] R 14 is a C1-C8 alkyl group;

[0229] Pc is an anti-CD79B antibody or an antigen-binding fragment thereof disclosed herein; L is a linker; and n is 1 to 10, which can be an integer or a decimal.

[0230] In some specific embodiments, the ligand-MMAE or derivative conjugate or a pharmaceutically acceptable salt or solvate thereof comprises the structure shown below:

[0231]

[0232] In some embodiments of the present disclosure, the ligand-MMAE or derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is represented by the general formula (Pc-LD(MMAE)):

[0233]

[0234] in:

[0235] R 2 -R 16 As defined in the general formula (D(MMAE));

[0236] n is 1 to 10 and can be an integer or a decimal;

[0237] Pc is an anti-CD79B antibody or an antigen-binding fragment thereof disclosed herein; L is a linker.

[0238] In some embodiments of the present disclosure, the ligand-MMAE or derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, is a ligand-MMAE or derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, represented by the general formula (Pc-L-D1):

[0239]

[0240] in:

[0241] R 2 -R 16 As defined in the general formula (-D(MMAE));

[0242] Pc, L, and n are as defined in the general formula (Pc-LD(MMAE)).

[0243] In some embodiments of the present disclosure, the ligand-MMAE or derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is represented by the following general formula:

[0244]

[0245] Pc, L, and n are as defined in the general formula (Pc-LD(MMAE)).

[0246] In some embodiments of the present disclosure, the ligand-MMAE or derivative conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein n is 1 to 8, which may be an integer or a decimal; preferably 1 to 6, which may be an integer or a decimal.

[0247] In some embodiments of the present disclosure, the ligand-MMAE or its derivative conjugate or its pharmaceutically acceptable salt or solvate, wherein the linker is -YL 1 -L 2 -L 3 -L 4 ,

[0248] Y is a stretcher unit, selected from or chemical bond, X1 is selected from hydrogen, alkyl, alkoxy, aryl or halogen, X2 is selected from alkylene, said alkylene optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0249] L 1 is a stretching unit selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 17-WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0250] L 2 Selected from-NR 18 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 18 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20; preferably a chemical bond;

[0251] L 3 A peptide residue consisting of 2 to 7 amino acids, preferably valine, citrulline, and methylvaline; wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

[0252] R 17 and R 18 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0253] L 4 is an extension unit, preferably PAB.

[0254] In some embodiments of the present disclosure, the ligand-MMAE or its derivative conjugate or its pharmaceutically acceptable salt or solvate, wherein Y is selected from

[0255] In some embodiments of the present disclosure, the ligand-MMAE or its derivative conjugate or its pharmaceutically acceptable salt or solvate, wherein L 1 Selected from -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)-, where s 1 is an integer from 2 to 8; preferably

[0256] In some embodiments of the present disclosure, the ligand-MMAE or its derivative conjugate or its pharmaceutically acceptable salt or solvate, wherein L 3 is a dipeptide amino acid unit, preferably selected from valine-citrulline.

[0257] In some embodiments of the present disclosure, the ligand-MMAE or derivative conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the linker is selected from:

[0258]

[0259] The a-end is connected to the ligand and the b-end is connected to the drug.

[0260] In some embodiments of the present disclosure, the ligand-MMAE or derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is selected from the following structural formula:

[0261]

[0262] in:

[0263] n is 1 to 10 and can be an integer or a decimal;

[0264] Pc is an anti-CD79B antibody or an antigen-binding fragment thereof of the present disclosure; preferably an antibody comprising a heavy chain as shown in SEQ ID NO: 28 and a light chain as shown in SEQ ID NO: 29, or an antibody comprising a heavy chain as shown in SEQ ID NO: 30 and a light chain as shown in SEQ ID NO: 31.

[0265] Another aspect of the present disclosure relates to a method for preparing a compound of formula (D(MMAE)), or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0266]

[0267] The deprotection reaction of the general formula (-DA(MMAE)) yields a compound represented by the general formula (-D(MMAE)).

[0268] Where: R 2 -R 16 As defined in formula (D).

[0269] Another aspect of the present disclosure relates to a compound as shown below:

[0270]

[0271] , or a pharmaceutically acceptable salt thereof, can be used as an intermediate for preparing the ligand-drug conjugate of the present disclosure.

[0272] Another aspect of the present disclosure relates to a method for preparing Compound 2 (MMAE), or a pharmaceutically acceptable salt thereof, comprising the steps of:

[0273]

[0274] Compound 1 (MMAE) and compound 2a (MMAE) undergo condensation reaction to obtain compound 2 (MMAE).

[0275] Another aspect of the present disclosure relates to a method for preparing a ligand-drug conjugate of the general formula (Pc-LD(MMAE)) or a pharmaceutically acceptable salt or solvate thereof, comprising the following steps:

[0276]

[0277] After reducing Pc, coupling reaction with compound 2 (MMAE) was carried out to obtain a compound represented by the general formula (ADC (MMAE)-1);

[0278] wherein Pc and n are as defined in the general formula (Pc-LD(MMAE)).

[0279] In order to achieve the purpose of synthesizing MMAE and its diffractors disclosed herein, the present disclosure adopts the following synthesis technology scheme:

[0280] Option 1:

[0281] The present invention discloses a method for producing a compound represented by the general formula (D(MMAE)), or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0282]

[0283] The general formula (DA(MMAE)) is subjected to a deprotection reaction under alkaline conditions to obtain a compound represented by the general formula (D(MMAE)).

[0284] Where: R 2 -R 16 As defined in the general formula (D(MMAE)).

[0285] Reagents providing alkaline conditions include organic bases and inorganic bases, wherein the organic bases include but are not limited to triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide, and the inorganic bases include but are not limited to sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide; preferably diethylamine.

[0286] Option 2:

[0287] A method for preparing compound 2 (MMAE) or a pharmaceutically acceptable salt or solvate thereof disclosed herein, the method comprising:

[0288]

[0289] Compound (1(MMAE)) and compound (2a(MMAE)) are subjected to a condensation reaction under alkaline conditions with the addition of a condensing agent to obtain compound 2.

[0290] Reagents providing alkaline conditions include organic bases and inorganic bases, wherein the organic bases include but are not limited to triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide, and the inorganic bases include but are not limited to sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide, preferably N,N-diisopropylethylamine.

[0291] The condensing agent is selected from 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N,N',N'- Tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride or 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, preferably 1-hydroxybenzotriazole.

[0292] Option 3:

[0293] The present invention discloses a method for preparing a ligand-drug conjugate represented by the general formula (Pc-LD) or a pharmaceutically acceptable salt or solvate thereof, comprising the following steps:

[0294]

[0295] After reducing Pc, it is coupled with compound 2 (MMAE) to obtain a compound represented by the general formula (ADC (MMAE) -1); the reducing agent is preferably TCEP, and in particular, it is preferred to reduce the disulfide bonds on the antibody;

[0296] wherein Pc and n are as defined in the general formula (Pc-LD(MMAE)).

[0297] Example compounds

[0298] The present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, selected from:

[0299]

[0300] Wherein, Pc is any anti-CD79B antibody or antigen-binding fragment thereof disclosed herein, and n is 1 to 10, which can be an integer or a decimal.

[0301] In some specific embodiments, Pc is an anti-CD79B antibody or an antigen-binding fragment thereof in the embodiments of the present disclosure, for example, an antibody comprising a heavy chain as shown in SEQ ID NO: 28 and a light chain as shown in SEQ ID NO: 29, or an antibody comprising a heavy chain as shown in SEQ ID NO: 30 and a light chain as shown in SEQ ID NO: 31, and n is an integer or decimal between 1 and 6.

[0302] In some specific embodiments, the average DAR value of the antibody drug conjugates of the present disclosure can be any value between 1-10, such as 2-8, or 2-6, or 1-6, or 4-6.

[0303] Although all of the above formulae are presented as specific isomeric forms for simplicity, the present disclosure may include all isomers, such as tautomers, rotamers, geometric isomers, diastereomers, racemates, and enantiomers.

[0304] Tautomers are structural isomers that readily interconvert through a chemical reaction known as tautomerization. This reaction often results in the migration of a hydrogen atom or proton, accompanied by a switch in the alignment of a single bond and an adjacent double bond. Some common tautomeric pairs are keto-enol and lactam-lactim. An example of a lactam-lactim equilibrium is between A and B, shown below.

[0305]

[0306] All compounds in the present disclosure can be represented as either Form A or Form B. All tautomeric forms are within the scope of the present disclosure. The naming of the compounds does not exclude any tautomers.

[0307] Pharmaceutical composition

[0308] The present disclosure further provides a pharmaceutical composition comprising the conjugate as described above and a pharmaceutically acceptable excipient, diluent or carrier.

[0309] use

[0310] The present disclosure further provides the use of any one or combination of the following in preparing a medicament: an anti-CD79B antibody or antigen-binding fragment thereof according to the present disclosure, a conjugate according to the present disclosure, or a pharmaceutical composition according to the present disclosure; wherein the antibody or antigen-binding fragment thereof or drug conjugate thereof is used to treat a proliferative disease or delay the progression of a proliferative disease; the proliferative disorder may be cancer or a tumor; the cancer or tumor is selected from lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and / or mantle cell lymphoma.

[0311] Treatment

[0312] The present disclosure provides a method for treating or preventing a proliferative disease or delaying the progression of a proliferative disease, comprising administering to a subject an anti-CD79B antibody or antigen-binding fragment thereof according to the present disclosure, or a pharmaceutical composition according to the present disclosure, or an antibody-drug conjugate according to the present disclosure, effective for treating or delaying the disease; wherein the proliferative disorder is cancer or a tumor.

[0313] The present disclosure provides methods of treating or delaying progression of a B cell proliferative disorder or autoimmune disorder in a subject in need thereof. In some embodiments, the B cell proliferative disorder is cancer or a tumor.

[0314] The present disclosure provides methods for enhancing immune function in a subject having a cell proliferative disorder or an autoimmune disorder. In some embodiments, the cell proliferative disorder is cancer or a tumor.

[0315] The cancer or tumor in the above scheme can be selected from lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and / or mantle cell lymphoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0316] Figure 1: ELISA test results of serum titer of Balb / c mice immunized with human CD79B ECD-hFc protein.

[0317] Figure 2: FACS detection results of serum titer of Balb / c mice immunized with human CD79B ECD-hFc protein.

[0318] Figure 3: ELISA test results of serum titer of SJL mice immunized with human CD79B ECD-hFc protein.

[0319] Figure 4: FACS analysis of serum titers from SJL mice immunized with human CD79B ECD-hFc protein.

[0320] Figure 5: ELISA test results of the serum titer of SJL mice immunized with human CD79B ECD-his protein.

[0321] Figure 6: FACS detection results of serum titer of SJL mice immunized with human CD79B ECD-his protein.

[0322] Figure 7: ELISA test results of the serum titer of SJL mice immunized with monkey CD79B ECD-his protein.

[0323] Figure 8: Anti-human CD79B mouse monoclonal antibody ELISA test results.

[0324] FIG9A shows the FACS detection results of anti-human CD79B mouse monoclonal antibody.

[0325] FIG9B shows the detection results of anti-human CD79B mouse monoclonal antibody FACS, wherein hIgG1 is a negative control antibody and SN8 is a positive control antibody.

[0326] FIG10 : FACS detection results of cross-reactivity of anti-human CD79B mouse monoclonal antibody.

[0327] Figure 11: ELISA test results of anti-monkey CD79B mouse monoclonal antibody.

[0328] Figures 12A to 12G: FACS assay of binding of anti-monkey CD79B mouse monoclonal antibodies to monkey peripheral blood mononuclear cells. Figure 12A shows the results of a blank control FACS assay of monkey peripheral blood mononuclear cells; Figure 12B shows the results of a negative control antibody FACS assay of monkey peripheral blood mononuclear cells; Figure 12C shows the results of a positive control antibody FACS assay of monkey peripheral blood mononuclear cells; Figure 12D shows the results of a positive control antibody FACS assay of monkey peripheral blood mononuclear cells; Figure 12E shows the results of a positive control antibody FACS assay of monkey peripheral blood mononuclear cells; Figure 12F shows the results of a positive control antibody FACS assay of monkey peripheral blood mononuclear cells; Figure 12G shows the results of a positive control antibody FACS assay of monkey peripheral blood mononuclear cells. The positive control used was Genentech's SN8.

[0329] Figures 13A to 13C show the therapeutic effects of different ADCs on subcutaneous xenografts of human diffuse large B-cell lymphoma WSU-DLCL2 in nude mice.

[0330] Figure 14: Effects of different ADCs on the body weight of tumor-bearing nude mice.

[0331] Figure 15: Efficacy of different ADCs on subcutaneous xenografts of human diffuse large B-cell lymphoma WSU-DLCL2 in nude mice.

[0332] Figure 16: Effects of different ADCs on the body weight of WSU-DLCL2 tumor-bearing nude mice.

[0333] Figure 17: Tumor photos showing the efficacy of different ADCs on subcutaneous xenografts of human diffuse large B-cell lymphoma WSU-DLCL2 in nude mice.

[0334] Figure 18: Efficacy of different ADCs on subcutaneous xenografts of human follicular lymphoma DOHH-2 in nude mice.

[0335] Figure 19: Effects of different ADCs on the body weight of DOHH-2 tumor-bearing nude mice. DETAILED DESCRIPTION

[0336] the term

[0337] In order to make the present application easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this application belongs.

[0338] The three-letter and one-letter codes for amino acids used herein are as described in J. Biol. Chem, 243, p3558 (1968).

[0339] "Antibody" is used in the broadest sense to encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. An antibody may refer to an immunoglobulin, a tetrapeptide chain structure composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant regions of immunoglobulins' heavy chains vary. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Igs of the same class can be further divided into different subclasses based on the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each class of Ig can have either kappa or lambda chains.

[0340] The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (V region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region includes three hypervariable regions (CDRs) and four framework regions (FRs) whose sequences are relatively conserved. The three hypervariable regions determine the specificity of the antibody and are also known as the complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0341] In the present application, the antibody light chain variable region described herein may further comprise a light chain constant region, wherein the light chain constant region comprises a human or murine κ, λ chain or a variant thereof.

[0342] In the present application, the antibody heavy chain variable region described herein may further comprise a heavy chain constant region, wherein the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0343] The term "murine antibody" as used herein refers to a monoclonal antibody against human CD79B or an epitope thereof, prepared according to the knowledge and skill in the art. During preparation, a test subject is injected with the CD79B antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. In a specific embodiment of the present application, the murine CD79B antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa or lambda chain, or a variant thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or IgG4, or a variant thereof.

[0344] The term "fully human antibody" includes antibodies with variable and constant regions of human germline immunoglobulin sequences. The fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "fully human antibody" does not include "humanized antibodies."

[0345] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by transplanting non-human CDR sequences into the human antibody variable region framework. This overcomes the strong immune response induced by chimeric antibodies due to the large number of non-human protein components they carry. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the fully human antibody variable region to maintain activity.

[0346] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of an antibody of a first species with the constant region of an antibody of a second species, which can reduce the immune response induced by the antibody of the first species. As an example, to establish a chimeric antibody, it is necessary to first establish a rabbit that secretes rabbit-derived specific monoclonal antibodies, isolate the antibodies, clone the constant region genes of the fully human antibodies as needed, connect the rabbit variable region genes with the human constant region genes into a chimeric gene, and insert it into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of the fully human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgG1 or IgG4 heavy chain constant region, or using an IgG1 that is free of ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

[0347] The term "antigen-binding fragment" includes: single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).

[0348] Methods for producing and preparing these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009 / 040562, and its disulfide-stabilized form, Fab-dsFv, was first disclosed in WO2010 / 035012. Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. Multivalent antibodies may comprise multispecifics, such as bispecifics, or may be monospecifics (see, for example, WO92 / 22583 and WO05 / 113605), an example of which is Tri-Fab (or TFM) described in WO 92 / 22583.

[0349] The term "single-chain antibody", "single-chain Fv" or "scFv" refers to 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 using 1-4 repeats (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.

[0350] The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contributes to antigen binding. 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. One of the most commonly used definitions of the six CDRs is provided by Kabat EA 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 of the light chain variable domain, and CDR2 and CDR3 of the heavy chain variable domain.

[0351] 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) in "Sequences of Proteins of Immunological Interest", 5th ed., 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 et al. (1997) JMB 273:927-948). MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003) etc. 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 (LCDR 1), 50-52 (LCDR2) and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, CDR is composed 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 rule, the CDR amino acid residues in VH are numbered approximately as 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately as 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3). Following the IMGT rule, the CDR regions of antibodies can be determined using the program IMGT / DomainGap Align.

[0352] The term "antibody framework" refers to a portion of a 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.

[0353] The term "binding to CD79B" in the present application refers to being able to interact with CD79B or its epitope, and the CD79B or its epitope may be of human origin.

[0354] The term "antigen" refers to a vertebrate-derived molecule with immunological activity used for immunization to produce antibodies that recognize the antigen, or for screening expression libraries (e.g., phage, yeast, or ribosome display libraries). In the present disclosure, antigens are defined more broadly to include target molecules specifically recognized by antibodies, as well as a portion or mimetic of a molecule used in an immunization process for producing antibodies or in a library screening for selecting antibodies. Human CD79B, as well as truncated variants and other variants of human CD79B of the present disclosure, are referred to as antigens.

[0355] The term "epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope generally comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitope is bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0356] The terms "specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when human CD79B or its epitope is used as an analyte and an antibody is used as a ligand, the antibody binds to the epitope at a specific binding rate of less than 10 -7 M or even less equilibrium dissociation constant (KD) to a predetermined antigen or its epitope, and its affinity for binding to the predetermined antigen or its epitope is at least twice that of its affinity for binding to a non-specific antigen (such as BSA, etc.) other than the predetermined antigen (or its epitope) or a closely related antigen. The term "antibody that recognizes an antigen" can be used interchangeably with the term "specifically binding antibody" in this disclosure.

[0357] 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.

[0358] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. In one embodiment, a vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. In another embodiment, a vector is a viral vector. The vector disclosed herein can replicate autonomously in a host cell (e.g., a bacterial vector and an additional mammalian vector with a bacterial origin of replication) or can be integrated into the genome of the host cell, thereby replicating with the host genome (e.g., a non-additional mammalian vector).

[0359] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacteria, microorganisms, plants, or animal cells. Easily transformed bacteria 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. Suitable animal host cell lines include CHO (Chinese Hamster Ovary) and NSO cells.

[0360] 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 be stably transfected into CHO cells. As a more recommended existing technology, mammalian expression systems will lead to glycosylation of antibodies. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified using conventional techniques. For example, purification can be performed using A or G Sepharose FF columns. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected using SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product must be immediately frozen, such as at -70°C, or freeze-dried.

[0361] Amino acid sequence "identity" refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence, after aligning the amino acid sequences and, if necessary, introducing gaps to achieve the maximum sequence identity percentage (and not considering conservative substitutions as part of the sequence identity). To determine the percentage of amino acid sequence identity, alignment can be achieved in a variety of ways within the scope of the art, for example, using computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0362] The term "cross-reaction" refers to the ability of the antibody of the present application to bind to CD79B from different species. For example, the antibody of the present application that binds to human CD79B may also bind to CD79B of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (such as SPR and ELISA), or by binding or functional interactions with cells expressing CD79B. The method for determining cross-reactivity includes standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.

[0363] The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. Inhibition / blocking of CD79B preferably reduces or alters the normal level or type of activity that occurs when CD79B binding occurs in the absence of inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in CD79B binding affinity when in contact with an anti-CD79B antibody compared to CD79B not in contact with the anti-CD79B antibody.

[0364] The term "inhibit growth" (eg, with respect to cells) is intended to include any measurable decrease in cell growth.

[0365] The terms "induce an immune response" and "enhance an immune response" are used interchangeably and refer to an immune response (ie, passive or adaptive) to a specific antigenic stimulus.

[0366] "ADCC," as used herein, stands for antibody-dependent cell-mediated cytotoxicity, and refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the antibody's Fc region. Modification of the Fc region of IgG can reduce or eliminate the antibody's ADCC effector function. Such modifications include mutations in the heavy chain constant region of the antibody, such as N297A, L234A, or L235A in IgG1; or F235E or L234A / E235A mutations in IgG2 / 4 chimeras and IgG4.

[0367] Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the Cold Spring Harbor Laboratory Manual of Antibody Experimental Techniques (Chapters 5-8 and 15). For example, animals can be immunized with human CD79B or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments are genetically engineered to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained from ImMunoGeneTics (IMGT) or from the Journal of Immunoglobulins, 2001 ISBN 012441351.

[0368] The term "drug" refers to cytotoxic drugs or immunomodulators. Cytotoxic drugs are chemical molecules that have a strong ability to destroy the normal growth of cells. In principle, cytotoxic drugs can kill cells at sufficient concentrations, but due to the lack of specificity, while killing tumor cells, they can also cause apoptosis of normal cells, leading to serious side effects. The term includes toxins (such as small molecule toxins or enzyme-active toxins from bacteria, fungi, plants or animals), radioactive isotopes (such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 and radioisotopes of Lu), chemotherapeutic drugs, antibiotics, and nucleolytic enzymes. Immunomodulators are inhibitors of immune checkpoint molecules.

[0369] The term linker refers to a fragment or bond that is connected to a ligand at one end and to a drug at the other end. Other linkers can also be connected to the ligand or drug.

[0370] The joint can include one or more joint components. Exemplary joint components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), and those derived from coupling with joint reagents: N-succinimidyl 4-(2-pyridylthio) valerate ("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 joint can include a stretching unit, a spacer unit, an amino acid unit, and an extension unit, which can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The joint can be a "cleavable joint" for facilitating drug release in cells. For example, an acid-labile linker (eg, a hydrazone), a protease-sensitive (eg, a peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0371] The term "amino acid unit" refers to the amino acid unit of the following structure Y, if a stretcher unit is present. R The carbonyl group in is connected to the extension unit. If there is no extension unit, Y R The amino acid directly attached to the drug, in the embodiment of the present disclosure, is represented by -K k -:

[0372]

[0373] -K k - is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide, and the -K- units each independently have the following structural formula K a or K b , k is an integer between 0 and 10:

[0374]

[0375] in:

[0376] R in the above amino acid unit 23 is -H or methyl;

[0377] R 24is H, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

[0378]

[0379] R 25 It is -aryl-, -alkyl-aryl-, -cycloalkyl-, -alkyl-cycloalkyl-, -cycloalkyl-alkyl-, -alkyl-cycloalkyl-alkyl-, -heterocyclyl-, -alkyl-heterocyclyl-, -heterocyclyl-alkyl-, -alkyl-heterocyclyl-alkyl-, -aryl-, -alkyl-aryl-, -aryl-alkyl-, -alkyl-aryl-alkyl-, -heteroaryl-, -alkyl-heteroaryl-, -heteroaryl-alkyl-, -alkyl-heteroaryl-alkyl-.

[0380] In one embodiment, -K k - is a dipeptide, preferably -valine-citrulline-, -phenylalanine-lysine- or -N-methylvaline-citrulline-, more preferably -valine-citrulline-.

[0381] The term "stretcher" refers to a chemical structure fragment that is covalently linked to a ligand via a carbon atom at one end and to a drug (directly or indirectly) via a sulfur atom at the other end.

[0382] The term "spacer unit" is a bifunctional chemical structural fragment that can be used to couple a linker and a drug to ultimately form a ligand-drug conjugate. This coupling method can selectively connect the drug to the linker.

[0383] The term "amino acid" refers to organic compounds whose molecular structures contain an amino group and a carboxyl group, both of which are directly attached to a -CH- structure. The general formula is H2NCHRCOOH. Depending on the position of the amino group attached to the carbon atom in the carboxylic acid, amino acids are classified as α-, β-, γ-, δ-, ε-, and so on. In the biological world, the amino acids that make up natural proteins have a unique structural characteristic: their amino group is directly attached to the α-carbon atom, thus forming α-amino acids.

[0384] The term "stretcher unit" refers to a chemical structure that can couple the amino acid unit to the drug when the amino acid unit is present, or can couple the drug through the carbonyl group on YR when the amino acid unit is absent.

[0385] The extension unit in the present disclosure is PAB, which has a structure such as a 4-iminobenzylcarbamoyl fragment, and is connected to D as shown in the following formula:

[0386]

[0387] abbreviation

[0388] Connector components include but are not limited to:

[0389] MC=6-maleimidocaproyl, the structure is as follows:

[0390]

[0391] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease-cleavable linker);

[0392] Citrulline = 2-amino-5-ureidopentanic acid;

[0393] PAB = p-aminobenzyloxycarbonyl (exemplary of a "self-immolative" linker component);

[0394] Me-Val-Cit = N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to protect it from cleavage by cathepsin B);

[0395] MC(PEG)6-OH = maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines);

[0396] SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate;

[0397] SPDP = N-succinimidyl 3-(2-pyridyldithio) propionate;

[0398] SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate;

[0399] IT = iminothiolane;

[0400] PBS=phosphate-buffered saline;

[0401] The term "antibody drug conjugate" (ADC) refers to a ligand linked to a drug via a linker (or linker unit). In the present disclosure, "antibody drug conjugate" refers to a monoclonal antibody (or antigen-binding fragment) linked to a toxic drug via a linker unit.

[0402] The term "drug loading" (Drug-to-Antibody Ratio, DAR) refers to the average number of drugs coupled to each antibody in an antibody-drug conjugate population, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading range can be 1-20, preferably 1-10 drugs (D) connected to each antibody (Ab). In embodiments of the present disclosure, the drug loading is expressed as k or n, and exemplary can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean of any two values. Preferably 1-10, more preferably 1-8, or 2-8, or 2-7, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or 4-5 mean. Drug loading can be determined by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, mAb size variant determination (CE-SDS) and HPLC characterization.

[0403] The disclosed monoclonal antibody size variant determination method (CE-SDS) can employ capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) ultraviolet detection to quantitatively determine the purity of recombinant monoclonal antibody products based on molecular weight under reducing and non-reducing conditions according to capillary electrophoresis (Chinese Pharmacopoeia 2015 edition, 0542).

[0404] In one embodiment of the present disclosure, the drug is coupled to the N-terminal amino group and / or the ε-amino group of the lysine residue of the ligand via a linker. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.

[0405] The loading capacity of the ligand drug conjugate can be controlled by the following non-limiting methods, including:

[0406] (1) Control the molar ratio of the linker and the monoclonal antibody,

[0407] (2) Control reaction time and temperature,

[0408] (3) Select different reaction reagents.

[0409] Although the drug to antibody ratio has an exact value (e.g., n in Formula (I)) for a particular conjugate molecule, it will be understood that when used to describe a sample containing many molecules, the value will often be an average value, due to a certain degree of heterogeneity typically associated with the conjugation step. The average loading of the conjugate is referred to herein as the drug to antibody ratio or "DAR." In some embodiments, the DAR is between about 1 and about 6, and is typically about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0. In some embodiments, at least 50% of the sample by weight is a compound with an average DAR ± 2, and preferably at least 50% of the sample is a conjugate containing an average DAR ± 1. Examples include wherein the DAR is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 3.5, 4.5, 5.5, 6.5, 7.0, 7.5, 8.0. , 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 10.1, 10.2, 10.3, 11.4, 12.5, 13.9, 14.0, 15.1, 16.2, 17.3, 18.4, 19.5, 20.6, 21.2, 22.5, 23.6, 24.7, 25.8, 26.9, 27.0, 28.1, 29.

[0410] The detection method of DAR, for example, is to extrapolate the DAR value from the LC-MS data of reduced and deglycosylated samples. LC / MS allows the quantification of the average number of payload (drug moiety) molecules connected to the antibody in the ADC. HPLC separates the antibody into light and heavy chains, and also separates the heavy chain (HC) and light chain (LC) according to the number of linker-payload groups on each chain. Mass spectrometry data can identify the types of components in the mixture, such as LC, LC+1, LC+2, HC, HC+1, HC+2, etc. Based on the average loading amount of the LC and HC chains, the average DAR of the ADC can be calculated. The DAR of a given immunoconjugate sample represents the average number of drug (payload) molecules connected to a tetrameric antibody containing two light chains and two heavy chains. For example, the DAR detection method in WO2018142322.

[0411] 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 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-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-chain isomers thereof. More preferably, the alkyl group is a lower alkyl group containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-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, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituents may be substituted at any available point of attachment and are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.

[0412] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.

[0413] The term "alkylene" refers to a saturated straight or branched chain aliphatic hydrocarbon radical having a residue derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane, which is a straight or branched chain radical containing 1 to 20 carbon atoms, preferably containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, more preferably an alkylene radical containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-), and the like. The alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0414] The term "alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl), wherein alkyl or cycloalkyl are as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0415] 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, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, or bridged cycloalkyls.

[0416] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, excluding the ring portion of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. Preferably, the ring contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused ring or bridged ring heterocyclic groups.

[0417] The term "spiroheterocyclyl" refers to a polycyclic heterocyclic group with 5 to 20 ring members sharing one atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, 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. For example, it is 6 to 14 members, and for example, it is 7 to 10 members. According to the number of shared spiro atoms between the rings, the spiro heterocyclic group is divided into a monospiro heterocyclic group, a dispiro heterocyclic group or a polyspiro heterocyclic group, preferably a monospiro heterocyclic group or a dispiro heterocyclic group. For example, it is 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6 yuan monospiro heterocyclic group. Non-limiting examples of spiro heterocyclic groups include:

[0418]

[0419] The term "fused heterocyclyl" refers to a polycyclic heterocyclic group of 5 to 20 members, wherein 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, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. For example, it is 6 to 14 members, and another example is 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, for example, bicyclic or tricyclic, and another example is a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic group. Non-limiting examples of fused heterocyclic groups include:

[0420]

[0421] The term "bridged heterocyclyl" refers to a 5- to 14-membered polycyclic heterocyclic group 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 selected from nitrogen, oxygen or S(O) m(wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. For example, it is 6 to 14 members, and another example is 7 to 10 members. Depending on the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups, for example, bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0422]

[0423] The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:

[0424] wait.

[0425] The heterocyclic group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0426] 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 having a conjugated π electron system, for example, 6- to 10-membered, such as phenyl and naphthyl, with phenyl being a specific example. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring, non-limiting examples of which include:

[0427]

[0428] The aryl group may be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0429] 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 group is preferably 5 to 10-membered, more preferably 5-membered or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring, non-limiting examples of which include:

[0430]

[0431] The heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0432] The term "cycloalkylalkyl" refers to a hydrogen atom on an alkyl group replaced by one or more cycloalkyl groups, preferably one cycloalkyl group, wherein alkyl is as defined above and cycloalkyl is as defined above.

[0433] The term "haloalkyl" refers to an alkyl group where a hydrogen atom is replaced by one or more halogen atoms, wherein alkyl is as defined above.

[0434] The term "deuterated alkyl" refers to an alkyl group in which hydrogen atoms are replaced by one or more deuterium atoms, wherein alkyl is as defined above.

[0435] The term "hydroxy" refers to an -OH group.

[0436] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0437] The term "amino" refers to -NH2.

[0438] The term "nitro" refers to -NO2.

[0439] The abbreviation "Me" in the chemical formula is methyl.

[0440] "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 and instances where it does not. For example, "C1-C6 alkyl optionally substituted with halogen or cyano" means that halogen or cyano may but need not be present, and the description includes instances where the alkyl group is substituted with halogen or cyano and instances where the alkyl group is not substituted with halogen or cyano.

[0441] The compounds disclosed herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined by absolute stereochemistry as (R)- or (S)- or, for amino acids, (D)- or (L)-. The disclosure includes all possible isomers and their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or may be prepared using conventional methods such as chromatography and fractional crystallization. Conventional methods for preparing / isolating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemates (or racemates of salts or derivatives) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise indicated, it is intended that the compounds include both E and Z geometric isomers. Furthermore, all tautomeric forms are also meant to be included.

[0442] In the chemical structures of the compounds disclosed herein, when the configuration is not specified, the bond Can be or or include both and In the chemical structures of the compounds disclosed herein, when the configuration is not specified, the bond It can be a Z configuration or an E configuration, or contain both configurations. For example, Can be

[0443]

[0444] "Stereoisomers" refer to compounds composed of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. Various stereoisomers and mixtures thereof are contemplated in this disclosure, and include "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0445] Any isotopically labeled derivatives of the compounds of the present disclosure or their pharmaceutically acceptable salts, or their isomers are covered by the present disclosure. Atoms that can be isotopically labeled include, but are not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, iodine, and the like. They can be replaced by isotopes such as 2H (D), 3H, 11C, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 36Cl, and 125I, respectively. Unless otherwise indicated, when a position is specifically designated as deuterium (D), the position should be understood to have an abundance of deuterium (i.e., at least 45% deuterium incorporation) that is greater than the natural abundance of deuterium (which is 0.015%).

[0446] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms, in a group are replaced independently of one another by substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (e.g., olefinic) bonds.

[0447] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0448] The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to salts of the ligand-drug conjugates of the present disclosure, or salts of the compounds described herein, which are safe and effective when administered to a subject and possess the desired biological activity. The antibody-antibody drug conjugate compounds of the present disclosure contain at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0449] The term "solvate" refers to a pharmaceutically acceptable solvate formed between the ligand-drug conjugate compound of the present disclosure and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0450] The present disclosure relates to a class of cleavable linkers with specific structures and active substances with specific structures, as well as antibody-drug conjugates (ADCs) composed of the linkers, active substances, and antibodies. These ADCs are complexes formed by linking a toxic substance to an antibody via a spacer. The ADCs degrade in vivo to release the active molecule, thereby exerting anti-tumor effects.

[0451] The term "carrier," when used in the pharmaceutical compositions of the present disclosure, refers to a system that can change the way a drug enters a subject and its distribution within the body, control the rate of drug release, and deliver the drug to a target site. 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, with preferred examples being micelles, microemulsions, gels, liquid crystals, vesicles, and the like. These aggregates have the ability to encapsulate drug molecules while having good permeability to membranes, and can serve as excellent drug carriers.

[0452] The term "excipient" refers to any additive in a pharmaceutical preparation other than the active ingredient, also known as an adjuvant. Examples include binders, fillers, disintegrants, and lubricants in tablets; the base component of semisolid ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0453] The term "diluent," also known as filler, primarily increases the weight and volume of a tablet. The addition of a diluent not only maintains a certain volume but also reduces dosage variations of the main ingredient and improves the drug's compressibility. When the tablet contains an oily component, an absorbent is added to absorb the oil and maintain a "dry" state, facilitating tablet production. Examples include starch, lactose, inorganic calcium salts, and microcrystalline cellulose.

[0454] "Administer," "apply," and "treat" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administer," "apply," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0455] "Treatment" means administering to a subject an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof of the present application, or a conjugate thereof, wherein the subject has, is suspected of having, or is prone to having one or more diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms in the treated subject or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. The amount of a therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. Although the embodiments of the present application (e.g., treatment methods or articles of manufacture) may not be effective in alleviating the symptoms of the target disease in a subject, they alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0456] Example

[0457] The following embodiments are used for further description, but these embodiments are not intended to limit the scope of the present invention.

[0458] Experimental procedures in the Examples or Test Examples where specific conditions are not specified were generally performed under conventional conditions or according to the conditions recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents where the source is not specified were commercially available.

[0459] 1. Antibody Preparation:

[0460] Example 1-1. Cloning and expression of protein antigens

[0461] The antibody (including light and heavy chains) and antigen were constructed using overlap extension PCR methods known in the art. The DNA fragments obtained by overlap extension PCR were inserted into the expression vector pEE6.4 (Lonza Biologics) using the two restriction sites HindIII / BstBI and expressed in 293F cells (Invitrogen, Cat#R790-07). The resulting recombinant protein was used for immunization or screening. The amino acid sequence of human CD79B was obtained from NCBI (NP_000617.1), and its extracellular region (ECD) contains 159 amino acids (Met1-Asp159).

[0462] The amino acid sequence of the human CD79B extracellular domain (ECD) and human Fc domain fusion protein (human CD79B ECD-hFc) is shown in SEQ ID NO: 1:

[0463]

[0464] The amino acid sequence of the fusion protein of the human CD79B extracellular domain (ECD) and His tag (human CD79B ECD-His) is shown in SEQ ID NO: 2:

[0465]

[0466] Example 1-2. Preparation of mouse monoclonal antibodies

[0467] 1. Mouse immunization and serum titer detection

[0468] Balb / c and SJL mice were immunized with a fusion protein of the human CD79B extracellular domain (ECD) and human Fc domain (human CD79B ECD-hFc) and a fusion protein of the human CD79B extracellular domain (ECD) and His tag (human CD79B ECD-His), respectively, as immunogens via intraperitoneal injection to stimulate the production of antibodies against the human CD79B extracellular domain (ECD). Simultaneously, SJL mice were immunized with a fusion protein of the monkey CD79B extracellular domain (ECD) and His tag (cyno CD79B ECD-His) as an immunogen via intraperitoneal injection to stimulate the production of antibodies against the monkey CD79B extracellular domain (ECD).

[0469] The experimental steps are as follows:

[0470] 1) Intraperitoneal injection immunization. Calculate the amount of antigen required for each immunization according to the immunization schedule. Dilute the protein antigen to the appropriate concentration with PBS as required, then emulsify the antigen. Transfer the emulsified antigen and adjuvant mixture to a 2.0 ml sterile syringe and inject into the right abdominal area of ​​the mouse.

[0471] 2) Mouse serum collection. Label the corresponding serum tube for each mouse; collect approximately 100 μL of whole blood from the mouse's submandibular vein. Let the collected whole blood sample stand at room temperature for approximately 2 hours, then centrifuge to collect serum. Store the serum at 4°C for antibody titer and other testing.

[0472] 3) ELISA titer determination of serum from immunized mice. Coat 96-well plates with 50 μL of antigen per well at a concentration of 1 μg / mL in a 4°C refrigerator overnight. The next day, wash the coated plates once (washing solution: 1× PBST). After washing, block with 1% BSA blocking solution in 1× PBST at 37°C for 1 hour. Wash the plates three times with 1× PBST, then add serum to be tested at various dilutions and incubate at 37°C for 1 hour. Wash the plates three times with 1× PBST, then add 100 μL of a 1:5000 dilution of goat anti-mouse secondary antibody and incubate at 37°C for 0.5 hour. After washing, develop the color with a mixture of TMB colorimetric solutions A and B in a 1:1 ratio. After 15 minutes, terminate the color reaction with 1N hydrochloric acid. Fluorescence was measured at 450 nm on a Spectra Max M5 plate reader.

[0473] 4) FACS titer determination of serum from immunized mice. DoHH2 cells or monkey peripheral blood mononuclear cell suspensions were centrifuged and resuspended in PBS containing 0.1% BSA. The cells were counted and then added with the test serum from each group. After incubation at room temperature for 60 minutes, the cells were washed three times. Anti-mouse IgG (Fc-specific)-FITC secondary antibody was then added. After incubation at room temperature for 30 minutes in the dark, the cells were washed three times and gently resuspended in PBS containing 0.1% BSA for analysis.

[0474] The results of ELISA and FACS detection of serum titers of mice in each group are shown in Figures 1 to 7.

[0475] Five Balb / c mice, numbered 5491, 5492, 5493, 5494, and 5495, were immunized with human CD79B ECD-hFc protein. Serum titer ELISA results are shown in Figure 1. The results demonstrate that the titer of mouse immune sera reached over 1:100K. FACS analysis of mouse sera is shown in Figure 2, demonstrating that antibodies produced in mouse sera specifically recognize CD79B protein on the surface of DoHH2 cells.

[0476] Five SJL mice, numbered 5496, 5497, 5498, 5499, and 5500, were immunized with human CD79B ECD-hFc protein. Serum titer ELISA results are shown in Figure 3. The results demonstrate that the titer of mouse immune sera reached over 1:100K. FACS analysis of mouse sera is shown in Figure 4, demonstrating that antibodies produced in mouse sera specifically recognize CD79B protein on the surface of DoHH2 cells.

[0477] Five SJL mice, numbered 5726, 5727, 5728, 5729, and 5730, were immunized with human CD79B ECD-his protein. Serum titer ELISA results are shown in Figure 5 . The results demonstrate that the titer of mouse immune sera reached over 1:10K. FACS analysis of mouse sera is shown in Figure 6 , demonstrating that antibodies produced in mouse sera specifically recognize CD79B protein on the surface of DoHH2 cells.

[0478] Five SJL mice were immunized with monkey CD79B ECD-his protein, numbered 5501, 5502, 5503, 5504, and 5505. Serum titer ELISA test results are shown in Figure 7. The results showed that the titer of mouse immune sera reached above 1:10K.

[0479] The above results indicate that specific antibodies against CD79B are produced in the immunized mice. The above mice can be used for cell fusion to generate hybridoma cell lines capable of secreting specific antibodies against CD79B.

[0480] 2. Hybridoma preparation and antibody screening

[0481] The lymphocytes and myeloma cells SP2 / 0 (ATCC, CCL-121) of the immunized mice were electrofused. TM ) for fusion and subsequent antibody screening.

[0482] 1) Electrofusion experiment. One week before fusion, SP2 / 0 cells were expanded and cultured in 10% DMEM medium. The spleen and lymph nodes of the mice were removed, rinsed, and ground to collect lymphocytes. SP2 / 0 and lymphocytes were mixed in appropriate proportions and fused using an electrofusion instrument. After fusion, the cells were plated in a 96-well plate and cultured in a 37°C, 5% CO2 incubator. The cell status was observed daily. Five days after fusion, the cell fusion rate was calculated. Fused hybridoma cells were screened 9-14 days after fusion, and cells from positive wells were selected for expansion and culture in 24-well plates.

[0483] 2) Subcloning by limiting dilution. Resuspend the cell lines to be subcloned in 24-well culture wells and count them. Dilute each cell line to a concentration of 5-10 cells / ml. Add 0.2 ml of the diluted cell suspension to each well of a 96-well culture plate, containing 1-2 cells per well. Incubate the 96-well plate in a 37°C, 5% CO2 incubator. After 7-10 days, select positive clones and transfer them to 24-well wells for further confirmation.

[0484] 3) ELISA screening. Coat a 96-well plate with 50 μL of antigen per well at 1 μg / ml in a 4°C refrigerator overnight. The next day, wash the coated plate once (washing solution: 1× PBST). After washing, block with 1% BSA blocking solution in 1× PBST at 37°C for 1 hour. Wash the plate three times with 1× PBST, add 50 μL of the supernatant of the cells to be tested, and incubate at 37°C for 1 hour. Wash the plate three times with 1× PBST, add 100 μL of a 1:5000 diluted goat anti-mouse secondary antibody, and incubate at 37°C for 0.5 hour. After washing, mix TMB colorimetric solution A and solution B in a 1:1 ratio and develop color. After 15 minutes, terminate the color reaction with 1N hydrochloric acid. Detect fluorescence at 450 nm on a Spectra Max M5 plate reader.

[0485] 4) FACS screening. After centrifugation of the DoHH2 cell suspension, resuspend the cells in PBS containing 0.1% BSA and count them. Add the supernatant to be tested and incubate at room temperature for 60 minutes. After washing the cells, add anti-mouse IgG (Fc-specific)-FITC secondary antibody and incubate at room temperature for 30 minutes in the dark. Wash the cells three times, gently resuspend the cells in PBS containing 0.1% BSA, and analyze them.

[0486] 5) Identification of hybridoma positive clones. Multiple specific antibodies against the human CD79B antigen were obtained; the 17 hybridomas with the best binding ability in ELISA and FACS tests were used to produce and purify antibodies. The ELISA test results of anti-human CD79B hybridoma positive clones are shown in Table 1. The FACS test results of anti-human CD79B hybridoma positive clones are shown in Table 2. Specific antibodies against monkey CD79B antigen were also obtained, and the 4 hybridomas with the best binding ability in ELISA and FACS tests were used to produce and purify antibodies. The ELISA test results of anti-monkey CD79B hybridoma positive clone cells are shown in Table 3. The FACS test results of anti-monkey CD79B hybridoma positive clone cells are shown in Table 4. mIgG was used as a negative control in all cases.

[0487] Table 1. ELISA test results of anti-human CD79B hybridoma positive clones

[0488] Antibody number Clone number Test result (OD450) Negative control mIgG0.05mAb00112A11-1G13.26mAb00219F10-1D73.69mAb00351E5G63.02mAb00467B10C13.41mAb00578A9F43.73mAb00648F11D63.34mAb00761A11F13.40mAb00 863G2A23.56mAb00975F1E23.57mAb01066G3E73.83mAb01166E12H33.41mAb01273A8F33.45mA b01374C4F33.31mAb01470B8B33.10mAb01583B2G23.41mAb01683C2D43.46mAb01786F11F63.80

[0489] Table 2. FACS detection results of anti-human CD79B hybridoma positive clones

[0490] Antibody number Clone number Average fluorescence value Negative control mIgG58mAb00112A11-1G113032mAb00219F10-1D75943mAb00351E5G633918mAb00467B10C126000mAb00578A9F424454mAb00648F11D620120mAb00761A11F118039mAb00863G2A216453

[0491] mAb00975F1E216001mAb01066G3E715897mAb01166E12H314688mAb01273A8F314073mAb0137 4C4F312894mAb01470B8B38776mAb01583B2G210036mAb01683C2D49990mAb01786F11F68132

[0492] Table 3. ELISA test results of anti-monkey CD79B hybridoma positive clones

[0493]

[0494] Table 4. FACS detection results of anti-monkey CD79B hybridoma positive clones

[0495] Antibody number Clone number Average fluorescence value Negative control mIgG35mAb018121H1E91973mAb019152E5F61708mAb020159E3E51488mAb021134H2F51225

[0496] 3. Production, purification and identification of mouse monoclonal antibodies

[0497] 1) Mouse monoclonal antibody production and purification. Observe the hybridoma cells for antibody production under a microscope. If the cells have grown to ≥70% and are in good condition, collect the cells and count them using a Countstar IC1000 cell counter. Adjust the cell concentration to 1×10 5 to 5×10 5 Transfer the cells to a roller bottle at a concentration of 100 μg / ml. Place the roller bottle in an incubator at 37°C for 10-15 days, observing cell growth daily. When the culture medium turns orange and transparent, remove it for purification. Purify the antibody using a Protein A column as standard.

[0498] 2) Anti-human CD79B mouse monoclonal antibody ELISA. Coat a 96-well plate with 50 μL of antigen per well at 1 μg / mL in a refrigerator overnight at 4°C. The next day, wash the coated plate once (washing solution: 1× PBST). Block with 1% BSA blocking solution in 1× PBST at 37°C for 1 hour. Wash the plate three times with 1× PBST, then add 50 μL of 100 nM antibody diluted 1:10 and incubate at 37°C for 1 hour. Wash the plate three times with 1× PBST, then add 100 μL of a 1:5000 dilution of goat anti-mouse secondary antibody and incubate at 37°C for 0.5 hour. After washing, develop the plate with a 1:1 mixture of TMB colorimetric buffers A and B. After 15 minutes, stop the color reaction with 1N hydrochloric acid. Fluorescence was measured at 450 nm on a Spectra Max M5 plate reader. Among them, four anti-human CD79B mouse monoclonal antibodies had the strongest ELISA binding ability (mAb008, mAb015, mAb016 and mAb017). The specific data are shown in Figure 8.

[0499] 3) FACS detection of anti-human CD79B mouse monoclonal antibodies. After centrifugation of the DoHH2 cell suspension, the cells were resuspended in PBS containing 0.1% BSA and counted. 100 μL of 100 nM, 1:10 diluted antibody was added and incubated at room temperature for 1 hour. After washing the cells three times, anti-mouse IgG (Fc specific)-FITC secondary antibody was added. After incubation at room temperature for 30 minutes in the dark, the cells were washed three times, and the cells were gently resuspended in PBS containing 0.1% BSA and tested on the machine. Among them, 4 anti-human CD79B mouse monoclonal antibodies had the strongest FACS binding ability (mAb008, mAb015, mAb016 and mAb017). The specific data are shown in Figures 9A and 9B. Among them, hIgG1 is a negative control antibody and SN8 is a positive control antibody. SN8 is the antibody used in Roche's antibody-drug conjugate polatuzumab vedotin (sequence reference source: US20170362318A). Polatuzumab vedotin has been approved for marketing by the FDA. The results show that in FACS experiments, the three anti-human CD79B mouse monoclonal antibodies mAb015, mAb016, and mAb017 selected in this disclosure all showed superior binding to SN8.

[0500] 4) FACS detection of cross-reactivity of anti-human CD79B mouse monoclonal antibody. 293F-cynoCD79B cells were obtained by transient transfection. After centrifugation of the cell suspension, the cells were resuspended in PBS containing 0.1% BSA and counted. 100 μL of antibody was added at concentrations of 10 μg / ml and 1 μg / ml, respectively. Incubate at room temperature for 1 hour. After washing the cells three times, anti-mouse IgG (Fc specific)-FITC secondary antibody was added. After incubation at room temperature for 30 minutes in the dark, the cells were washed three times, and the cells were gently resuspended in PBS containing 0.1% BSA and detected on the machine. The results of FACS detection of cross-reactivity of anti-human CD79B mouse monoclonal antibody are shown in Figure 10.

[0501] 5) Anti-monkey CD79B mouse monoclonal antibody ELISA assay. Coat the plate with 50 μL of antigen per well at a μg / mL concentration overnight at 4°C. The next day, wash the coated plate once (washing solution: 1× PBST). After washing, block with 1% BSA blocking solution in 1× PBST at 37°C for 1 hour. Wash the plate three times with 1× PBST, then add 50 μL of 100 nM antibody diluted 1:10 and incubate at 37°C for 1 hour. Wash the plate three times with 1× PBST, then add 100 μL of a 1:5000 dilution of goat anti-mouse secondary antibody and incubate at 37°C for 0.5 hour. After washing, develop the plate with a 1:1 mixture of TMB colorimetric buffers A and B. After 15 minutes, terminate the color reaction with 1N hydrochloric acid. Fluorescence was measured at 450 nm on a Spectra Max M5 plate reader. The results are shown in Figure 11.

[0502] 6) FACS detection of the binding of anti-monkey CD79B mouse monoclonal antibodies to monkey peripheral blood mononuclear cells. Monkey peripheral blood mononuclear cells were extracted from fresh monkey blood. After the cell suspension was centrifuged, the cells were resuspended in PBS containing 0.1% BSA and counted. Anti-CD19 and anti-cynoCD79B antibodies were added respectively. Incubate at room temperature for 1 hour. After washing the cells three times, anti-mouse IgG (Fc specific)-FITC secondary antibody was added. After incubation at room temperature for 30 minutes in the dark, the cells were washed three times, and the cells were gently resuspended in PBS containing 0.1% BSA and detected on the machine. The FACS detection results of the binding of anti-monkey CD79B mouse monoclonal antibodies to monkey peripheral blood mononuclear cells are shown in Figures 12A to 12G. CD19 is a marker for B cells. As can be seen from the results, the four anti-monkey CD79B mouse monoclonal antibodies can all bind to monkey B cells.

[0503] 7) Anti-human CD79B mouse monoclonal antibody SPR detection. Surface plasmon resonance (SPR) was used to detect the affinity between the anti-human CD79B antibody and its antigen human CD79B-His. The antigen human CD79B-His protein was immobilized on a CM5 chip. The coupling level was set at 100RU. The running buffer was HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). The diluted antibody was flowed through the experimental channel and the control channel at a flow rate of 30μl / min for 3 minutes and dissociated for 5 minutes. Then, the regeneration buffer (10mM glycine, pH1.5) was run at a flow rate of 30μl / min for 30 seconds. The data were analyzed using Biacore 8K software.

[0504] Example 1-3. Determination of amino acid sequence of mouse monoclonal antibody variable region

[0505] The high-affinity hybridoma monoclonal cell line obtained in Example 1-2 was subjected to variable region amino acid sequence determination, and then recombinantly expressed as a human-mouse chimeric antibody (cAb), and further antibody identification was performed. The genes encoding the heavy chain variable region and the light chain variable region were amplified by reverse transcription PCR and connected to the vector; the light and heavy chain sequences were obtained by sequencing. First, the total RNA of the single cell line with good activity in Example 1-2 was extracted using an RNA purification kit (Qiagen, product number 74134). Then, a cDNA single strand was prepared using Invitrogen's product number 18080-051 cDNA synthesis kit. Using this as a template, the sequence of the light and heavy chain variable regions was synthesized by PCR, and the PCR product was cloned into the TA vector pMD-18T and then sent for sequencing. The obtained light and heavy chain sequences were cloned into expression vectors respectively to express recombinant monoclonal antibodies. After verifying the activity, humanization work was performed.

[0506] The amino acid residues of the VH / VL CDRs of the anti-human CD79B antibody were identified and annotated using the Chothia numbering system.

[0507] Sequence of mouse hybridoma monoclonal antibody mAb015:

[0508] Heavy chain variable region:

[0509]

[0510] Light chain variable region:

[0511]

[0512] Sequence of mouse hybridoma monoclonal antibody mAb017:

[0513] Heavy chain variable region:

[0514]

[0515] Light chain variable region:

[0516]

[0517] The mouse CDR sequences are shown in Table 5:

[0518] Table 5. CDR sequences of mouse anti-human CD79B antibodies

[0519]

[0520] Example 1-4. Humanization of anti-human CD79B antibody

[0521] The light and heavy chain sequences of the mouse anti-CD79B monoclonal antibodies obtained in Examples 1-3 were compared for homology in the antibody database to establish a humanized antibody model. Back mutations were selected based on the model to screen the optimal humanized anti-CD79B monoclonal antibody. Crystal structures similar to the obtained mouse candidate molecules were searched from the mouse Fab crystal structure model database (such as the PDB database) and high-resolution (such as ) Fab crystal structure, and establish a mouse Fab model. Align the light and heavy chain sequences of the mouse antibody with the sequences in the model, retain the sequences consistent with the mouse antibody sequence, and obtain the structural model of the mouse antibody. Inconsistent amino acids are possible back mutation sites. Use Swiss-pdb viewer software to run the mouse antibody structural model and optimize (minimize) the energy. Back mutations are performed on different amino acid sites in the model except CDR. Compare the activity of the resulting mutant antibody (humanized) and the antibody before humanization. Retain the humanized antibody with good activity. Optimize the CDR region, including avoiding glycosylation, deamidation, oxidation sites, etc.

[0522] The above antibodies were cloned, expressed, and purified. The most active humanized antibodies, hAb015-10 and hAb017-10, were selected through ELISA, FACS, and SPR assays. The data are shown in Table 6. The humanized antibodies hAb015-10 and hAb017-10 maintained similar affinity and related functions to those of mouse monoclonal antibodies.

[0523] Table 6. Identification results of humanized anti-CD79B antibodies

[0524]

[0525]

[0526] The sequences of humanized antibodies hAb015-10 and hAb017-10 are shown below.

[0527] hAb015-10 humanized antibody heavy chain variable region (VH):

[0528]

[0529] hAb015-10 humanized antibody light chain variable region (VL):

[0530]

[0531] hAb017-10 humanized antibody heavy chain variable region (VH):

[0532]

[0533] hAb017-10 humanized antibody light chain variable region (VL):

[0534]

[0535] hAb015-10 humanized antibody heavy chain:

[0536]

[0537] hAb015-10 humanized antibody light chain:

[0538]

[0539] hAb017-10 humanized antibody heavy chain:

[0540]

[0541] hAb017-10 humanized antibody light chain:

[0542]

[0543] The humanized mAb015 has a T30S mutation in its HCDR1 sequence, and the mutated HCDR1 is GSSFSSY (SEQ ID NO: 23). The anti-CD79B antibody disclosed herein has the general formula shown in Table 7:

[0544] Table 7. CDR general structure

[0545]

[0546] Example 1-5. Endocytosis of anti-CD79B antibodies

[0547] In order to detect whether the CD79B antibody in the present disclosure can be internalized into cells together with human CD79B after binding to human CD79B, a cell endocytosis experiment was performed using DOHH-2 cells (DSMZ, ACC 47) that highly express human CD79B protein to evaluate the endocytic ability of the antibody.

[0548] DOHH-2 cells were cultured according to conventional suspension cell methods. The complete medium consisted of RPMI 1640 medium (GIBCO, Cat No.: 11835-030) supplemented with 10% (v / v) fetal bovine serum (FBS) (GIBCO, Cat No.: 10099-141) and penicillin / streptomycin (GIBCO, Cat No.: 15070-063).

[0549] During the experiment, cells were collected by cold centrifugation at 1000 rpm for 5 minutes at 4°C. The cells were resuspended in 10-15 ml of ice-cold FACS buffer. The composition of FACS buffer is: phosphate-buffered saline (PBS), pH 7.4, plus 2% fetal bovine serum (FBS). The FACS buffer was kept cold on ice throughout the experiment. After counting and centrifugation, 300,000 cells were plated into a 96-well plate. After centrifugation and discarding the supernatant, 100 μl / well of Fc blocking buffer (12.5 μg / ml (BD, Cat No.: 564220)) was added. Blocking was performed at room temperature for 10 minutes. Then, 20 μg / ml of the CD79B antibody to be tested was added to the corresponding wells and incubated in the dark at 4°C for 1 hour. The cells were washed twice with cold PBS buffer to remove unbound antibody. Complete cell culture medium (RPMI 1640 medium with 10% fetal bovine serum) was added and incubated at 37°C, 5% CO₂ for 0, 1, 2, and 4 hours. After centrifugation and discarding the supernatant, 100 μl / well of 2% PFA buffer was added, the cells were resuspended, and allowed to stand for 10 minutes. The cells were then washed three times with FACS buffer, followed by the addition of 100 μl of secondary antibody solution (fluorescently labeled goat anti-human secondary antibody: 1:250 dilution, concentration 2 μg / ml, Biolegend, Cat#409304) and incubated at 4°C in the dark for half an hour. Pre-chilled PBS buffer was added, and the supernatant was discarded at 4°C. This was repeated three times. The cells were resuspended in FACS buffer at 200 μl / well and analyzed using a flow cytometer (BD FACS Calibur).

[0550] The results showed that when incubated at 4°C, none of the three antibodies, SN8, hAb015, and hAb017, were internalized by DOHH-2 cells. However, when incubated at 37°C, most of the antibodies were internalized by DOHH-2 cells after one hour, and antibody internalization reached its maximum after four hours. All three antibodies had good internalization.

[0551] 2. Preparation of Compounds

[0552] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). -6 The units are given in ppm.

[0553] NMR measurements were performed using a Bruker AVANCE-400 nuclear magnetic spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), and the internal standard was tetramethylsilane (TMS).

[0554] MS was measured using an Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS liquid spectrometer-mass spectrometer (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS).

[0555] Waters ACQuity UPLC-QD / SQD (Manufacturer: Waters, MS Model: Waters ACQuity Qda Detector / Waters SQ Detector) THERMO Ultimate 3000-Q Exactive (Manufacturer: THERMO, MS Model: THERMO Q Exactive).

[0556] High performance liquid chromatography (HPLC) analysis was performed using Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC e2695-2489 high pressure liquid chromatographs.

[0557] Chiral HPLC analysis was performed using an Agilent 1260 DAD high performance liquid chromatograph.

[0558] High performance liquid chromatography (HPLC) was performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson GX-281 preparative chromatographs.

[0559] Chiral preparations were performed using a Shimadzu LC-20AP preparative chromatograph.

[0560] The CombiFlash rapid preparation instrument is Combiflash Rf200 (TELEDYNE ISCO).

[0561] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate; the specification of the silica gel plate used in thin layer chromatography (TLC) is 0.15mm to 0.2mm; when separating and purifying products by thin layer chromatography, the specification used is 0.4mm to 0.5mm.

[0562] Silica gel column chromatography generally uses Yantai Huanghai Silica Gel 200 to 300 mesh silica gel as the carrier.

[0563] The known starting materials disclosed herein can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui Chemicals, and other companies.

[0564] Unless otherwise specified in the examples, the reactions can be carried out under an argon atmosphere or a nitrogen atmosphere. A hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1 L.

[0565] The pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator. The hydrogenation reaction is usually carried out by evacuating the vacuum and filling with hydrogen, and the operation is repeated three times.

[0566] A CEM Discover-S 908860 microwave reactor was used for the microwave reaction.

[0567] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0568] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0569] The eluent system for column chromatography and the developing solvent system for thin-layer chromatography used for purification include: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system, and C: petroleum ether / ethyl acetate system; the volume ratio of the solvent is adjusted according to the polarity of the compound, and can also be adjusted by adding alkaline or acidic reagents (such as triethylamine and acetic acid, etc.).

[0570] The drug components of the ADC disclosed herein are referenced in WO2020063676A, US7098308, US6884869, CN202010073671.6, and CN201911390425.7. The synthesis and testing of the relevant compounds are incorporated herein in their entirety. Non-limiting examples thereof are as follows:

[0571] Example 2-1. Compound A

[0572]

[0573] 1 (10 mg, 0.014 mmol, 1.0 eq) from this example, 2 (21 mg, 0.021 mmol, 1.5 eq) from this example, and a catalytic amount of HOBt (0.5 mg) were dissolved in anhydrous DMF (2 mL) under argon and stirred. 2.71 mg of DIEA and 0.08 mL of pyridine were added, and the reaction was heated to 40°C and stirred for 2 hours. Purification by preparative HPLC gave compound A (12.4 mg) in a 65.4% yield. LC / MS (ESI): m / z 1363.4 [M+1]. + .

[0574] HNMR(CDCl3,400MHz): δ0.63-0.95(m,32H),1.05-1.36(m,27H),1.60-1.98(m,4H) ,2.1-2.55(m,4H),2.78~3.02(m,3H),3.17-3.79(m,13H),3.93-4.20(m,2H),4.60 ~4.96(m,3H),5.14-5.36(m,2H),5.67(bs,1H),6.31-6.50(m,2H),6.64(s,2H),6. 91(d,J=14.8Hz,1H),7.19-7.30(m,7H),7.55(bs,2H),8.96(bs,1H),9.13(bs,1H).

[0575] Example 2-2-1. Compound B

[0576]

[0577] Under an ice-water bath, 1 (50 mg, 0.08 mmol) in this example was dissolved in 1.5 mL of N,N-dimethylformamide, and then DIPEA (18 mg, 0.14 mmol) was added, followed by di(p-nitrobenzene) carbonate (49 mg, 0.16 mmol). The mixture was stirred at room temperature for about 2-4 hours. After the reaction was complete as monitored by HPLC, 20 mL of methyl tert-butyl ether was added, stirred, and the solid was collected by filtration. After drying, the solid obtained 36 mg of crude product, which was directly used for the next step. LC / MS (ESI): m / z 784.1 [M+H] + .

[0578]

[0579] Under ice-water bath, compound 3 (72.91 mg, 0.1 mmol) of this example was dissolved in 10 mL of tetrahydrofuran, and Fmoc-OSu (41 mg, 0.12 mmol) was added. The mixture was stirred at room temperature for 3-5 hours. After completion of the reaction monitored by HPLC, the mixture was concentrated under reduced pressure to obtain a crude product, which was directly used in the next reaction.

[0580]

[0581] The crude product of Example 4 was dissolved in 10 mL of anhydrous ether, and silver oxide (34.8 mg, 0.15 mmol) was added, followed by iodomethane (28.4 mg, 0.2 mmol). The mixture was reacted at room temperature for about 10-16 hours. After monitoring the reaction to be substantially complete, the solid was removed by filtration, and the crude product was concentrated under reduced pressure to obtain the crude product, which was directly subjected to the next step.

[0582]

[0583] The crude product of Example 5 was dissolved in 10 mL of tetrahydrofuran, and 2 mL of diethylamine was added, followed by stirring at room temperature for about 2-4 hours. After completion of the reaction monitored by HPLC, the crude product was directly concentrated under reduced pressure to obtain the crude product, which was purified by silica gel column to obtain 40 mg of the product. LC / MS (ESI): m / z 744.2 [M+H] + .

[0584]

[0585] Compound 6 (13.5 mg, 0.018 mmol) from this example was dissolved in 1.5 mL of DMF, and DIPEA (7 mg, 0.054 mmol) was added. Compound 2 (18 mg, 1.3 mmol) from this example was then added in portions. The mixture was stirred for about 24-36 hours and concentrated under reduced pressure to obtain a crude product. HPLC separation was performed to obtain 12.5 mg of compound B with a purity of 96.95%. LC / MS (ESI): m / z 1388.3 [M+H] + .

[0586] HNMR (CDCl3, 400MHz): δ0.85~0.90(m,3H), 0.93~1.00(m,3H), 1.08~1.10(m,3H), 1.20~1.50 (m,15H),1.75~2.04(m,6H),2.13~2.55(m,16H),2.70~2.77(m,1H),2.80~2.96(m,2H),3.16 ~3.97(m,20H),3.99~4.39(m,8H),4.60~4.80(m,6H),4.88~5.10(m,5H),5.24~5.37(m,4H), 6.71(s,2H),7.03(d,J=6.8Hz,1H),7.18~7.30(m,3H),7.63(d,J=8.0Hz,2H),8.92(bs,1H).

[0587] Test Example 1: In vitro cytotoxic activity screening:

[0588] 1.1. Experimental principles and methods

[0589] This experiment uses CTG to detect ATP content to reflect the survival of tumor cells. First, cells of different densities were planted and cultured for 3 and 5 days. 50 The final culture conditions were determined based on the maximum inhibition rate and the killing effect of the toxin molecules was then tested under these conditions.

[0590] 1.2. Cell line selection

[0591] According to the purpose of the experiment, we referred to the literature reports and initially selected two disease models, breast cancer and NSCLC, and selected three cell lines, SKBR3 (HER2+), MDA-MB-468 (HER2-), and A549, for screening experiments.

[0592] 1.3. Determination of cell culture conditions

[0593] 1) Cell Plating: After trypsinizing A549 cells, terminate with culture medium. Count and transfer 4.3×105, 7.2×105, and 11.5×105 cells to a final volume of 26 ml. Add 180 μL of cell suspension to each well of columns 2 through 11 of a 96-well plate (3903) to achieve cell densities of 3K, 5K, and 8K per well, respectively. Add 200 μL of culture medium to column 12, and add PBS to the remaining wells. Repeat the above procedure for SKBR3 and MDA-MB-468 cells. Perform the procedure in duplicate.

[0594] 2) Drug Preparation: Prepare an eribulin positive control in a round-bottom 96-well plate (3788). Prepare 2 mM eribulin in the first column of plate 1 (the stock solution was diluted 10-fold with DMSO). Then, dilute the solution 10-fold in DMSO in a series of 10-fold dilutions through the tenth column, leaving the 11th column with DMSO. Add 95 μL of the corresponding culture medium to each well in columns 2 through 11 of plate 2. Pipette 5 μL of the solution from columns 2 through 11 of plate 1 into plate 2, mix thoroughly, and then pipette 20 μL of the solution onto the plated cells. Continue culturing for 3 and 5 days.

[0595] 3) CTG assay: Remove the plate on the 3rd and 5th day and equilibrate to room temperature. Add 90 μL CTG to each well and react at room temperature for 10 minutes in the dark. Read the luminescence value with a microplate reader and calculate the IC 50 .

[0596] 1.4. Drug efficacy testing

[0597] 1) Cell plating: A549, SKBR3, and MDA-MB-468 cells were trypsinized and resuspended in culture medium. 6.33*10^5 cells were added to 38 ml of culture medium and plated into 96-well plates (3903) with 180 μL per well to obtain 3K cells per well. The cells were cultured at 37°C for 24 h.

[0598] 2) Drug Preparation: Eribulin and compound D-1 (Compound 6 in Example 2-2-1) were prepared in a round-bottom 96-well plate (3788). The first column of plate 1 was diluted 10-fold with DMSO. From then on, column 10 was diluted 10-fold in DMSO, with column 11 containing DMSO. In plate 2, 95 μL of the corresponding culture medium was added to each well of columns 2 through 11. 5 μL of the solution from columns 2 through 11 of plate 1 was pipetted into plate 2. After mixing, 20 μL was pipetted into the plated cells, repeating in duplicate. Culture was continued for 5 days.

[0599] 3) CTG assay: Remove the plate and equilibrate to room temperature. Add 90 μL CTG to each well and react at room temperature for 10 minutes in the dark. Read the luminescence value with a microplate reader and calculate the IC 50 .

[0600] 1.5. Data Results

[0601] Table 8

[0602]

[0603] Conclusion: Compound D-1 has a good killing effect in all three tumor cell lines and is significantly better than the positive drug eribulin.

[0604] Example 2-2-2.

[0605]

[0606] At room temperature, 0.3 mL of 1,4-dioxane and 0.3 mL of water-soluble compound E-305 (31 mg, 0.042 mmol, synthesized according to the literature Bioorg. Med. Chem. Lett. 14 (2004) 5551–5554.) were measured, and then fluorenylmethyloxycarbonyl succinimide (17 mg, 0.050 mmol) and solid sodium carbonate (18 mg, 0.168 mmol) were added in sequence. Stir at room temperature overnight, and the raw material was detected to be almost completely converted. Water was added to the reaction mixture to quench the reaction, and the mixture was extracted with ethyl acetate and concentrated under reduced pressure. After purification on a silica gel column, 15 mg of the product was obtained. LC / MS (ESI): m / z 965.64 [M+H] + .

[0607]

[0608] At room temperature, the product from the previous step (7 mg, 0.007 mmol) was dissolved in dichloromethane. 4A molecular sieves (10 mg), trimethyloxonium tetrafluoroborate (11 mg, 0.07 mmol), and proton sponge (16 mg, 0.07 mmol) were then added sequentially and stirred at room temperature for 1 h. Conversion of the starting material was confirmed to be nearly complete. The reaction was quenched with water, extracted with methyl tert-butyl ether, and washed with 1N dilute hydrochloric acid. The mixture was concentrated under reduced pressure and purified on a silica gel column to obtain 7 mg of the product. LC / MS (ESI): m / z 979.68 [M+H] + .

[0609] Under an ice-water bath, dissolve the product from the previous step (10 mg, 0.01 mmol) in 1 mL of tetrahydrofuran, then add DBU (6 μL, 0.04 mmol) dropwise and stir until the reaction is complete. Quench the reaction with water, extract with dichloromethane, and concentrate under reduced pressure. HPLC separation yields 5 mg of product D-2. LC / MS (ESI): m / z 757.85 [M+H] + .

[0610] Example 2-2-3.

[0611]

[0612] In an ice-water bath, 2 mL of tetrahydrofuran was taken to dissolve compound 3 (6 mg, 0.008 mmol, synthesized according to the literature Bioorg. Med. Chem. Lett. 21 (2011) 1639–1643) in this example, and then lithium aluminum tetrahydride solution (80 μL, 1 M in THF, 0.08 mmol) was added dropwise. The mixture was stirred and the reaction temperature was slowly raised to 40°C. LCMS showed that the raw material was almost completely converted. The reaction was quenched with sodium sulfate decahydrate, stirred in an ice-water bath for half an hour, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was directly used in the next reaction. LC / MS (ESI): m / z 758.4 [M+H] + .

[0613]

[0614] At room temperature, the product from the previous step (7 mg, 0.007 mmol) was dissolved in dichloromethane. 4A molecular sieves (10 mg), trimethyloxonium tetrafluoroborate (11 mg, 0.07 mmol), and proton sponge (16 mg, 0.07 mmol) were then added sequentially and stirred at room temperature for 1 h. Conversion of the starting material was confirmed to be nearly complete. The reaction was quenched with water, extracted with methyl tert-butyl ether, and washed with 1N dilute hydrochloric acid. The mixture was concentrated under reduced pressure and purified on a silica gel column to obtain 7 mg of the product. LC / MS (ESI): m / z 979.68 [M+H] + .

[0615]

[0616] At room temperature, 0.5 mL of 1,4-dioxane and 0.5 mL of water were added to dissolve the product from the previous step. Fluorenylmethyloxycarbonyl succinimide (6.5 mg, 0.019 mmol) and sodium carbonate (6.8 mg, 0.064 mmol) were then added sequentially. Stirring was continued overnight at room temperature until the conversion of the starting material was nearly complete. The reaction was quenched with water, extracted with ethyl acetate, and concentrated under reduced pressure. The crude product was purified on a silica gel column to obtain 14 mg of the product. LC / MS (ESI): m / z 980.4 [M+H] + .

[0617]

[0618] Under an ice-water bath, dissolve the product from the previous step (14 mg, 0.014 mmol) in 1 mL of dichloromethane. Add Dess-Martin periodinane (18.2 mg, 0.042 mmol), stir, and allow the reaction to slowly warm to room temperature. Stir until the starting material is essentially converted, as determined by LCMS. Add aqueous sodium bicarbonate to quench the reaction, extract with dichloromethane, and concentrate. The crude product is purified on a silica gel column to obtain 8 mg of the product. LC / MS (ESI): m / z 978.4 [M+H]+ .

[0619]

[0620] Under an ice-water bath, 1 mL of tetrahydrofuran was added to dissolve the product obtained in the previous step (8 mg, 0.008 mmol), and DBU (6 μL, 0.032 mmol) was added dropwise. The mixture was stirred for 1 hour. The conversion of the raw material was basically complete after central control detection. Water was added to quench the reaction, and the mixture was extracted with dichloromethane and concentrated. The crude product was separated by HPLC to obtain the target product D-3 (1.3 mg). LC / MS (ESI): m / z 755.93 [M+H] + .

[0621] Test Example 2: In vitro cytotoxic activity screening

[0622] Experimental principles and methods

[0623] In this experiment, CTG was used to detect the ATP content, which reflects the survival of tumor cells.

[0624] 2.2. Determination of cell culture conditions

[0625] 1) Cell plating:

[0626] After trypsinization, A549, SKBR3, and MDA-MB-468 cells were resuspended in their respective culture medium and counted. The cell density was adjusted to 2.2 × 104 cells / ml. 135 μl of the cell suspension was added to each well of columns 2 to 11 of a 96-well plate, with column 12 serving as a blank control. The cells were cultured in a 37°C incubator with 5% CO2 for 24 h.

[0627] 2) Drug preparation:

[0628] a) Stock solution preparation: Dissolve the drug in DMSO to a stock solution concentration of 5 mM.

[0629] b) Plate 1: Dilute the stock solution 40-fold starting in column 1, and then serially dilute it 3-fold in columns 2 to 11. Column 12 is DMSO.

[0630] c) Well plate 2: Add 196 μL of the corresponding culture medium to columns 2 to 11, and pipette 4 μL from columns 3 to 12 of well plate 1 to columns 2 to 11 of well plate 2. Mix well.

[0631] 2.3. Cell Treatment

[0632] 15 μL was pipetted from well 2 and added to the cells, and culture was continued in a 37°C incubator with 5% CO 2 for 5 days.

[0633] 2.4. CTG Assay: Remove the plate and equilibrate to room temperature. Add 75 μL of CTG to each well and incubate at room temperature for 10 min in the dark. Read the luminescence value with a microplate reader and calculate the IC50.

[0634] 2.5. Data Results

[0635] Table 9

[0636]

[0637]

[0638] Example 2-2-4.

[0639]

[0640] Under ice-water bath, eribulin (9 mg, 0.012 mmol) was dissolved in 0.3 mL of DMF, and DIPEA (3.5 mg, 0.028 mmol) was added. Then, compound 2 (7.8 mg, 0.011 mmol) from this example was added portionwise. The mixture was stirred until the reaction was almost complete, and concentrated under reduced pressure to obtain a crude product. HPLC preparative separation yielded 4.95 mg of compound L-2 with a purity of 97%. LC / MS (ESI): m / z 1374.3 [M+H] + .

[0641] Example 2-2-5.

[0642]

[0643] Compound 4 (13.4 mg, 0.0316 mmol, 1.7 eq) and eribulin mesylate (15 mg, 0.0182 mmol, 1 eq) from this example were weighed and dissolved in DMF (0.5 ml). Triethylamine (10 mg, 0.0988 mmol, 5.4 eq) and DMTMM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 9.8 mg, 0.0332 mmol, 1.8 eq) were added under ice-cooling. The reaction mixture was naturally warmed to room temperature and stirred until nearly complete. Water (2 ml) and ethyl acetate (3 ml) were added to dilute the mixture. The aqueous phase was extracted with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate and concentrated. The crude product was purified on a preparative plate to obtain 16 mg of the product, with a yield of 86.7%. LC / MS (ESI): m / z 1136.3 [M+H]. + .

[0644]

[0645] Under ice-cooling, the product from the previous step (16 mg, 0.0141 mmol, 1 eq) was weighed and dissolved in THF (0.4 ml). Triethylamine (4.2 mg, 0.057 mmol, 4 eq) was added and stirred under ice-cooling until the reaction was nearly complete. Dichloromethane (5 ml) was added to dilute the mixture and the mixture was washed with water (2 ml x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude product, which was used directly in the next step. LC / MS (ESI): m / z 914.3 [M+H]. + .

[0646]

[0647] The product obtained in the previous step (16 mg, 0.0175 mmol, 1 eq) and compound 6 of this example (11.6 mg, 0.0246 mmol, 1.4 eq) were weighed and dissolved in DMF (0.5 ml). 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (9.9 mg, 0.026 mmol, 1.5 eq) and N,N-diisopropylethylamine (5.5 mg, 0.0426 mmol, 2.4 eq) were added and stirred in an ice bath until the reaction was almost complete. Water (2 ml) and ethyl acetate (3 ml) were added to dilute the solution. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative HPLC to give 10 mg of product L-3. LC / MS (ESI): m / z 1368.3 [M+H] + .

[0648] Example 2-2-6.

[0649]

[0650] Compound 4 (11.6 mg, 0.0273 mmol, 1.5 eq) from this example and compound D-1 (an eribulin derivative, 13.5 mg, 0.0181 mmol, 1 eq) from Example 2-2-1 were weighed and dissolved in N,N-dimethylformamide (0.5 ml). DMTMM (10.1 mg, 0.0343 mmol, 1.3 eq) was added under ice-cooling. The reaction mixture was allowed to react until the reaction was substantially complete. Water (2 ml) and ethyl acetate (3 ml) were added to terminate and dilute the reaction. The layers were separated, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated. The crude product was purified on a preparative plate to obtain 10 mg of the product, with a yield of 47.9%. LC / MS (ESI): m / z 1150.2 [M+H]. + .

[0651]

[0652] The product from the previous step (10 mg, 0.0087 mmol, 1 eq) was weighed and dissolved in THF (1 ml). 1,8-diazabicycloundec-7-ene (5.2 mg, 0.034 mmol, 4 eq) was added and stirred in an ice bath until the reaction was substantially complete. Dichloromethane (5 ml) was added for dilution and the mixture was washed with water (2 ml x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain the crude product, which was used directly in the next step. LC / MS (ESI): m / z 928.2 [M+H] + .

[0653]

[0654] The product from the previous step (16 mg, 0.0087 mmol, 1 eq) and compound 6 (7.8 mg, 0.0165 mmol, 1.9 eq) were weighed and dissolved in DMF (0.5 ml). 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (6.2 mg, 0.0163 mmol, 1.9 eq) and DIEA (5.7 mg, 0.0441 mmol, 5 eq) were added and stirred in an ice bath until the reaction was essentially complete. Water (2 ml) and ethyl acetate (3 ml) were added to dilute the solution. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated. The crude product was purified by preparative HPLC to give 3.5 mg of product L-4, with a two-step yield of 29.1%. LC / MS (ESI): m / z 1382.2 [M+H] + .

[0655] Example 2-3.

[0656] (S)-N-((3R,4S,5S)-1-((1S,3S,5S)-3-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)-2-azabicyclo[3.1.0]hexan-2-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide Compound 1 of this Example

[0657]

[0658] first step

[0659] (9H-fluoren-9-yl)methyl (1S,3S,5S)-3-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)-2-azabicyclo[3.1.0]hexane-2-carboxylate. Compound 1c of this example is (2R,3R)-3-((1S,3S,5S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)-2 -azabicyclo[3.1.0]hexan-3-yl)-3-methoxy-2-methylpropanoic acid Example compound 1a (1.05 g, 2.49 mmol, prepared by the method disclosed in "Step 7 on page 20 of the specification of patent application US2019 / 55223") and (1S,2R)-2-amino-1-phenylpropan-1-ol Example compound 1b (0.42 g, 2.78 mmol, prepared by the known method "Journal of of Organic Chemistry, 2012, vol. 77, # 12, p. 5454-5460) was added to a reaction flask, 10 mL of dichloromethane and 2 mL of N, N-dimethylformamide were added, and the atmosphere was replaced with argon three times. 2-(7-benzotriazole oxide)-N, N, N', N'-tetramethyluronium hexafluorophosphate (1.14 g, 3.00 mmol) and N, N-diisopropylethylamine (0.97 g, 7.47 mmol) were added with stirring, and the mixture was stirred at room temperature for 1 hour. 30 mL of water was added and the mixture was extracted with dichloromethane (15 mL × 4). The organic phase was washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with developing solvent System A to obtain the title product, Compound 1c of this example (1.38 g, yield: 99.8%).

[0660] MS m / z(ESI):555.2[M+1]

[0661] Step 2

[0662] (2R,3R)-3-((1S,3S,5S)-2-azabicyclo[3.1.0]hex-3-yl)-N-((1S,2R)-1-hydroxy-1-phenylprop-2-yl)-3-methoxy-2-methylpropionamide Compound 1d

[0663] Compound 1c (1.38 g, 2.49 mmol) of this example was dissolved in 10 mL of dichloromethane. 20 mL of diethylamine was added, and the atmosphere was replaced with argon three times. The reaction mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated, and the resulting residue was purified by silica gel column chromatography using developing solvent System A to obtain the title product, Compound 1d (805 mg, 97.3% yield).

[0664] MS m / z(ESI):333.2[M+1]

[0665] Step 3

[0666] (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((1S,3S,5S)-3-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)-2-azabicyclo[3.1.0]hex-2-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0667] (5S,8S,11S,12R)-11-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradec-14-oic acid, compound 1e of this embodiment (1.54 g, 2.41 mmol, supplier: Hao Yuan) was added to the reaction bottle, 30 mL of acetonitrile was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.10 g, 2.89 mmol) and N,N-diisopropylethylamine (0.94 g, 7.27 mmol) were added, and the mixture was stirred in an ice bath for 10 minutes. A suspension of Compound 1d (805 mg, 2.42 mmol) in 10 mL of acetonitrile was added and the mixture was stirred in an ice bath for 40 minutes. 60 mL of water was added and the mixture was extracted with ethyl acetate (20 mL x 4). The organic phase was washed with saturated sodium chloride solution (60 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using solvent system A to obtain the crude product, Compound 1f (2.9 g).

[0668] MS m / z(ESI):952.3[M+1]

[0669] Step 4

[0670] (S)-N-((3R,4S,5S)-1-((1S,3S,5S)-3-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)-2-azabicyclo[3.1.0]hexan-2-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide

[0671] Object 1

[0672] Crude Example Compound 1f (510 mg, 0.53 mmol) was dissolved in 2 mL of dichloromethane, and 4 mL of diethylamine was added. The atmosphere was replaced with argon three times, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated, and the resulting residue was purified by silica gel column chromatography using developing solvent System A to obtain the title product, Example Compound 1 (266 mg, 68.0% yield).

[0673] MS m / z(ESI):730.4[M+1]

[0674] 1 H NMR (400MHz, CD3OD): δ7.36-7.40(m,2H),7.31(t,2H),7.24(d,1H),4.69(d,1H),4.56(d,1H),4.17-4.28(m, 2H),4.06-4.14(m,1H),3.91(d,1H),3.78(t,1H),3.27-3.44(m,7H),3.15(s,3H),2.84-2.93(m,1H),2.60-2 .67(m,2H),2.30-2.37(m,3H),2.02-2.10(m,2H),1.79-1.95(m,4H),1.38-1.53(m,2H),1.25-1.36(m,2H),1 .21(d,1H),1.13-1.17(m,2H),1.07-1.11(m,2H),0.93-1.05(m,15H),0.83-0.89(m,4H),0.70-0.79(m,1H).

[0675] Examples 2-4.

[0676] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-3-methylbutyrylamino)-5-ureidopentanylamino)benzyl

[0677] ((S)-1-(((S)-1-(((3R,4S,5S)-1-((1S,3S,5S)-3-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)-2-azabicyclo[3.1.0]hexan-2-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate Compound 2 of this Example

[0678]

[0679] The compound 1 (30 mg, 0.041 mmol) of the present embodiment was added to 1 mL of N,N-dimethylformamide, and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate compound 2a (45 mg, 0.061 mmol, supplier: Ark) of the present embodiment was added, and 0.25 mL of pyridine was added. The atmosphere was replaced with argon three times, and then 1-hydroxybenzotriazole (12 mg, 0.089 mmol) and N,N-diisopropylethylamine (16 mg, 0.123 mmol) were added. After stirring at room temperature for 4 hours, 2a (45 mg, 0.061 mmol) was added and stirring was continued for 16 hours. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding fractions were collected and concentrated under reduced pressure to obtain the title product, compound 2 of this example (18 mg, yield: 33.0%).

[0680] MS m / z(ESI):1329.3[M+1]

[0681] 1H NMR (400MHz, CD3OD): δ7.58(d,2H),7.29-7.42(m,6H),7.20-7.26(m,1H),6.79(s,2H),5.04-5.20(m,4H),4.47-4.61(m,3H),4. 13-4.28(m,3H),4.06-4.12(m,1H),3.91(d,1H),3.75-3.82(m,1H),3.48(t,3H),3.27-3.41(m,7H),3.16-3.25(m,2H),3.18(s, 3H),2.91-2.97(m,2H),2.60-2.65(m,2H),2.27(t,2H),2.20(t,1H),2.01-2.10(m,3H),1.69-1.94(m,6H),1.63-1.68(m,6H),1 .46-1.51(m,1H),1.27-1.37(m,5H),1.12-1.21(m,3H),1.09(d,2H),0.93-1.04(m,11H),0.80-0.92(m,11H),0.70-0.77(m,2H).

[0682] 3. Preparation of anti-CD79B antibody-drug conjugates:

[0683] Experimental purpose and principle of ADC drug loading analysis:

[0684] ADC loading was determined using ultraviolet spectrophotometry (UV-Vis) (Thermo nanodrop 2000 UV spectrophotometer). The principle is that the total absorbance of the ADC at a certain wavelength is equal to the sum of the absorbances of the drug and antibody at that wavelength.

[0685] Experimental methods

[0686] Place the cuvette filled with sodium succinate buffer in the reference absorption cell and the sample measurement absorption cell respectively, deduct the solvent blank, and then place the cuvette filled with the test solution in the sample measurement absorption cell to measure the absorbance at 280nm and 370nm.

[0687] Result calculation:

[0688] A 280nm =ε mab-280 oeLh mab +ε Drug-280 oeLh Drug Formula (1)

[0689] ε Drug-280 : The average molar extinction coefficient of the drug at 280nm is 5100;

[0690] C Drug : drug concentration;

[0691] ε mab-280 : The average molar extinction coefficient of the monoclonal antibody at 280 nm is 214600;

[0692] C mab : mAb concentration;

[0693] b: The optical path length is 1 cm.

[0694] Similarly, the total absorbance equation of the sample at 370nm can be obtained:

[0695] A 370nm =ε mab-370 oeLh mab +ε Drug-370 oeLh Drug Formula (2)

[0696] ε Drug-370 : The average molar extinction coefficient of the drug at 370nm is 19000;

[0697] C Drug : drug concentration;

[0698] ε mab-370 : The extinction coefficient of monoclonal antibody at 370nm is 0;

[0699] C mab : mAb concentration;

[0700] b: The optical path length is 1 cm.

[0701] The drug loading capacity of ADC can be calculated by combining the extinction coefficient and concentration data of monoclonal antibody and drug at two detection wavelengths using equations (1) and (2): drug loading capacity = C Drug / C mab .

[0702] Example 3-1. ADC-1

[0703]

[0704] To a solution of hAb015-10 in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 1.5 mL, 0.101 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.3 μL, 0.253 μmol) at 37°C. The mixture was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0705] Compound B (1.41 mg, 1.015 μmol) from Example 2-2-1 was dissolved in 50 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product ADC-1 (i.e., hAb015-10-cys-B) in PBS buffer (0.84 mg / mL, 13.5 mL) of this example and stored frozen at 4°C.

[0706] HIC calculated mean: n=3.06.

[0707] Example 3-2. ADC-2

[0708]

[0709] To a solution of hAb015-10 antibody in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 3.5 mL, 0.236 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 59.1 μL, 0.591 μmol) at 37°C. The mixture was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0710] Compound A (3.2 mg, 2.348 μmol) was dissolved in 150 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product of this example, ADC-2 (i.e., hAb015-10-cys-Malei-PEG2-vc-PAB-MMAE), in PBS buffer (2.17 mg / mL, 16.4 mL), which was stored frozen at 4°C.

[0711] RP-HPLC calculated average value: n=3.68.

[0712] Example 3-3. ADC-3

[0713]

[0714] To a solution of hAb015-10 in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 5.0 mL, 0.338 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 85.0 μL, 0.850 μmol) at 37°C. The mixture was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0715] The compound MC-vc-PAB-MMAE (4.45 mg, 3.380 μmol) was dissolved in 250 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product of this example, ADC-3 (i.e., hAb015-10-cys-MC-vc-PAB-MMAE), in PBS buffer (2.79 mg / mL, 17.4 mL), which was stored frozen at 4°C.

[0716] Average value calculated by CE-SDS: n=3.09.

[0717] Example 3-4. ADC-4

[0718]

[0719] To a solution of hAb015-10 in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 1.8 mL, 0.122 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.4 μL, 0.304 μmol) at 37°C. The mixture was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0720]

[0721] Compound C (1.62 mg, 1.220 μmol) of this example was dissolved in 90 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product of this example, ADC-4 (i.e., hAb015-10-cys-C), in PBS buffer (1.37 mg / mL, 12.0 mL), which was stored frozen at 4°C.

[0722] RP-HPLC calculated average value: n=4.52.

[0723] Example 3-5. ADC-5

[0724]

[0725] To a solution of hAb015-10 antibody in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 1.5 mL, 0.101 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 16.2 μL, 0.162 μmol) at 37°C. The mixture was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0726]

[0727] Compound D (0.87 mg, 0.810 μmol) of this example was dissolved in 37 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain the title product of this example, ADC-5 (i.e., HAB015-10-cys-D, DAR value approximately 2), in PBS buffer (0.90 mg / mL, 14.0 mL), which was stored frozen at 4°C.

[0728] The preparation method of compound D is cited from WO2020063676A, for example, Example 9 thereof.

[0729] RP-HPLC calculated average value: n=1.81.

[0730] Example 3-6. ADC-6

[0731]

[0732] To a solution of hAb015-10 antibody in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 1.5 mL, 0.101 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.3 μL, 0.253 μmol) at 37°C. The mixture was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0733] Compound D (1.09 mg, 1.015 μmol) from Example 3-5 was dissolved in 45 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product of this example, ADC-6 (i.e., hAb015-10-cys-D, with a target DAR value of approximately 4), in PBS buffer (0.71 mg / mL, 14.0 mL), which was stored frozen at 4°C.

[0734] RP-HPLC calculated average value: n=3.46.

[0735] Example 3-7. ADC-7

[0736]

[0737] To a solution of hAb015-10 antibody in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 1.5 mL, 0.101 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 50.7 μL, 0.507 μmol) at 37°C. The mixture was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0738] Compound D (1.63 mg, 1.518 μmol) from Example 3-5 was dissolved in 68 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product of this example, ADC-7 (i.e., hAb015-10-cys-D, DAR value of approximately 6), in PBS buffer (0.81 mg / mL, 13.5 mL), which was stored frozen at 4°C.

[0739] RP-HPLC calculated average value: n=5.84.

[0740] Example 3-8. ADC-8

[0741]

[0742] At 37°C, a solution of antibody SN8 in PBS buffer (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 79 mL, 5.338 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 1.388 mL, 13.88 μmol). The mixture was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0743] The compound MC-VC-PAB-MMAE (70.3 mg, 53.40 μmol) was dissolved in 3.5 mL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the title product of this example, ADC-8 (i.e., SN8-cys-MC-PAB-MMAE, DAR value of approximately 4), in PBS buffer (5.83 mg / mL, 132 mL), which was stored frozen at 4°C.

[0744] CE-SDS calculated average value: n=3.59.

[0745] 4. Biological evaluation:

[0746] Example 4-1. Biacore affinity experiment

[0747] In this example, Biacore was used to determine the affinity of CD79B antibodies (hAb015-10 and SN8) and ADC to CD79B protein.

[0748] Experimental instruments, materials, and reagents: Biacore T200 (GE); biosensor chip CM5 (Cat.#BR-1005-30, GE); amino coupling kit (Cat.#BR-1000-50, GE); human anti-capture kit (Cat.#BR-1008-39, GE); human CD79B-His protein (Cat.#29750-H08H, Sino Biological); HBS-EP + 10X buffer solution (Cat.#BR-1006-69, GE) diluted to 1X (pH 7.4) with DI Water;

[0749] Experimental method: According to the method in the instructions of the human anti-capture kit, human anti-capture antibodies were covalently coupled to the CM5 biochip to affinity capture a certain amount of CD79B antibodies.

[0750] Data statistics and analysis: BIAevaluation version 4.1, GE software was used to fit the data with a (1:1) Langmuir model to obtain affinity values.

[0751] Experimental results and conclusions: The affinity test results of CD79B antibodies and ADCs binding to CD79B protein are shown in Table 10. Naked antibodies and different ADCs have similar binding affinity to human CD79B protein, and both are higher than the positive drug Polivy.

[0752] Among them, the sequence of SN8 (i.e. the antibody in Polivy) is:

[0753] >SN8 heavy chain amino acid sequence

[0754]

[0755] >SN8 light chain amino acid sequence

[0756]

[0757] Table 10. Binding affinity of antibodies and different ADCs to human CD79B protein

[0758] ADC / Antibody Human CD79B EC 50 (nM)hAb015-100.38ADC-10.51ADC-20.48ADC-30.40ADC-40.32ADC-50.80ADC-60.43ADC-70.53ADC-85.98

[0759] Example 4-2. In vitro endocytosis experiment

[0760] Cell endocytosis assay was performed using DOHH-2 cells (DSMZ, ACC 47) that highly express human CD79B protein to evaluate the endocytic ability of ADC.

[0761] DOHH-2 cells were cultured using standard suspension cell methods in complete medium consisting of RPMI 1640 medium (GIBCO, Cat No. 11835-030) supplemented with 10% (v / v) fetal bovine serum (FBS) (GIBCO, Cat No. 10099-141) and penicillin / streptomycin (GIBCO, Cat No. 15070-063). Cells were harvested by centrifugation at 1000 rpm for 5 minutes at 4°C. Cells were resuspended in 10-15 ml of ice-cold FACS buffer. FACS buffer consisted of phosphate-buffered saline (PBS), pH 7.4, supplemented with 2% fetal bovine serum (FBS). FACS buffer was kept on ice throughout the experiment. After counting and centrifugation, 300,000 cells / well were added to a 96-well plate. After centrifugation and discarding the supernatant, Fc blocking solution, 12.5μg / ml (BD, Cat No.: 564220), 100μl / well was added. Block for 10 minutes at room temperature. Then, 20μg / ml of the ADC to be tested was added to the corresponding wells and incubated in the dark at 4°C for 1 hour. Wash twice with pre-cooled PBS buffer to remove unbound ADC. Complete cell culture medium (RPMI 1640 medium with 10% fetal bovine serum) was added and incubated at 37°C, 5% CO2 for 0 hours and 4 hours. After centrifugation and discarding the supernatant, 100μl / well of 2% PFA buffer was added, the cells were resuspended and allowed to stand for 10 minutes. The cells were then washed three times with FACS buffer, followed by the addition of 100 μL of secondary antibody solution (fluorescently labeled goat anti-human secondary antibody, diluted 1:250, 2 μg / mL, Biolegend, Cat#409304). The cells were incubated at 4°C in the dark for half an hour. Pre-chilled PBS buffer was added, and the supernatant was discarded at 4°C. This was repeated three times. The cells were resuspended in FACS buffer (200 μL / well) and analyzed using a flow cytometer (BD FACS Calibur).

[0762] The results are shown in Table 11. The endocytosis rates of the different ADCs after incubation with DoHH2 cells for 4 hours were all greater than 65%, demonstrating good endocytosis capacity. The endocytosis rates of each ADC were comparable to those of the positive drug Polivy.

[0763] Table 11. Endocytosis of different ADCs in DoHH2 cells

[0764] EC 50 Endocytosis rate (4h) ADC-173% ADC-266% ADC-370% ADC-481% ADC-576% ADC-675% ADC-768% ADC-876%

[0765] Example 4-3. Cell proliferation experiment

[0766] This example evaluates the effects of different ADCs on the proliferation of DoHH2, WSU-DLCL2, and Raji cells cultured in vitro. According to literature reports (Leukemia. 2015 Jul; 29(7): 1578-86; Blood. 2007 Jul 15; 110(2): 616-23), DoHH2 is a cell that highly expresses CD79B, WSU-DLCL2 is a cell that lowly expresses CD79B, and Raji is a cell that negatively expresses CD79B.

[0767] Experimental Materials:

[0768] ADC-1: colorless, clear liquid, concentration 0.84 mg / mL, purity 99.17%;

[0769] ADC-2: colorless, clear liquid, concentration 2.17 mg / mL, purity 97.93%;

[0770] ADC-3: colorless, clear liquid, concentration 2.79 mg / mL, purity 98.28%;

[0771] ADC-4: colorless, clear liquid, concentration 1.36 mg / mL, purity 98.48%;

[0772] ADC-5: colorless, clear liquid, concentration 0.9 mg / mL, purity 98.49%;

[0773] ADC-6: colorless, clear liquid, concentration 0.71 mg / mL, purity 98.71%;

[0774] ADC-7: colorless, clear liquid, concentration 0.81 mg / mL, purity 98.27%;

[0775] ADC-8: colorless, clear liquid, concentration 5.83 mg / mL, purity 97.07%.

[0776] After actual testing, the DAR of ADC-1 is 3.06; the DAR of ADC-2 is 3.68; the DAR of ADC-3 is 3.09; the DAR of ADC-4 is 4.52; the DAR of ADC-5 is 1.81; the DAR of ADC-6 is 3.46; the DAR of ADC-7 is 5.84; and the DAR of ADC-8 is 3.59.

[0777] All the above drugs were kept in a light-proof and sealed container at 4°C.

[0778] Cell lines: DoHH2 cells were purchased from DSMZ, WSU-DLCL-2 cells were purchased from American Type Culture Collection (ATCC), and Raji cells were purchased from American Type Culture Collection (ATCC).

[0779] The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS).

[0780] Reagents and instruments: RPMI 1640 and FBS were purchased from Gibco; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Shanghai Bioengineering. A microplate reader was purchased from BioTek.

[0781] Experimental methods:

[0782] A certain number of cells in logarithmic growth phase were seeded into 96-well culture plates and treated with various concentrations of drugs for 72 hours. After the drug treatment period, MTT working solution was added for 4 hours, followed by the addition of a triple solution to dissolve the blue-purple formazan crystals. OD values ​​were measured at 570 nm and 690 nm using a microplate reader, and the cell growth inhibition rate was calculated using the following formula:

[0783] Inhibition rate = (OD570nm-OD690nm of control well - OD570nm-OD690nm of drug-treated well) /

[0784] Control well OD570nm-OD690nm×100%

[0785] Based on the inhibition rate at each concentration, the half-maximal inhibitory concentration (IC50) was calculated using PrismGraph 8.

[0786] The results are shown in Table 12.

[0787] Table 12. In vitro proliferation inhibition activity of different ADCs

[0788]

[0789] Example 4-4. Efficacy of ADC on subcutaneous transplanted tumors of human diffuse large B-cell lymphoma WSU-DLCL2 in nude mice

[0790] This example evaluates and compares the efficacy of various ADC drugs on subcutaneous transplanted tumors in nude mice.

[0791] hIgG1 (HRP00252): colorless, clear liquid, concentration 22.77 mg / mL, purity 99.03%, production date 2018.7.24, valid until 2020.01.24 (tentative 18 months); stored at -70°C.

[0792] 1. Drugs: ADC-1, ADC-2, ADC-3, ADC-4, ADC-6, and ADC-8 are the same as those in Example 4-3.

[0793] 2. Cells and Mice: Human diffuse large B-cell lymphoma WSU-DLCL2 cells were purchased from the American Type Culture Collection. WSU-DLCL2 cells were cultured in 10-cm dishes in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in an incubator containing 5% CO2. Cells were passaged 2-3 times per week. When cells reached the exponential growth phase, they were harvested, counted, and plated.

[0794] Nude mice (BALB / c-nu, 35 days old, ♀) were purchased from Beijing Huafukang Biotechnology Co., Ltd. Production license number: SCXK(Beijing)2019-0008, animal qualification certificate number: 1103222011004014. Housing environment: SPF grade.

[0795] 3. Experimental steps: Each nude mouse was subcutaneously inoculated with 2.1×10 7 WSU-DLCL2 cells, wait until the tumor grows to 100-150 mm 3 Afterwards, the animals were divided into groups based on tumor volume (D0). Mice were administered intravenously (IV) at a volume of 10 mL / kg; the specific dosage and dosing schedule are shown in Table X. Tumor volume was measured twice weekly, and the mice were weighed and the data were recorded.

[0796] 4. Experimental indicators and statistical analysis:

[0797] The experimental indicators are to examine the effect of drugs on tumor growth, and the specific indicators are T / C% or tumor inhibition rate TGI (%).

[0798] Tumor diameter was measured with a vernier caliper twice a week, and tumor volume (V) was calculated as follows:

[0799] V=1 / 2×a×b 2 , where a and b represent length and width respectively.

[0800] T / C(%) = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the start of the experiment. Here, T is the tumor volume of the group administered with the ADC, and C is the tumor volume of the group administered with IgG1 as the control group.

[0801] Tumor growth inhibition rate % (TGI%) = 100 - T / C(%);

[0802] When tumor regression occurs, tumor growth inhibition rate % (TGI%) = 100 - (T - T0) / T0 × 100;

[0803] If the tumor shrinks compared to the initial volume, i.e., T < T0 or C < C0, it is defined as partial tumor regression (PR); if the tumor completely disappears, it is defined as complete tumor regression (CR).

[0804] At the end of the experiment, when the experimental endpoint is reached, or when the average tumor volume of the solvent group reaches 1500 mm 3 , the animals are sacrificed by CO2 anesthesia, and then the tumors are dissected and photographed.

[0805] Unless otherwise specified, two-way ANOVA test is used for comparison between the two groups of tumor volumes, and P < 0.05 is defined as having statistically significant differences.

[0806] Result:

[0807] ADC-1 (3 mg / kg, 10 mg / kg, IV, D0) dose-dependently inhibits the growth of subcutaneous transplanted tumors of WSU-DLCL2 nude mice, and the tumor inhibition rates are 75% and 137% respectively. All tumors in the 10 mg / kg dose group show partial regression;

[0808] The tumor inhibition rates of ADC-2 (3 mg / kg, 10 mg / kg, IV, D0) against WSU-DLCL2 are 76% and 123% respectively. All tumors in the 10 mg / kg dose group show partial regression;

[0809] The tumor inhibition rate of ADC-3 (3 mg / kg, IV, D0) against WSU-DLCL2 is 76%, and 1 / 6 of the tumors show partial regression;

[0810] The tumor inhibition rates of ADC-4 (3 mg / kg, 10 mg / kg, IV, D0) against WSU-DLCL2 are 95% and 129% respectively. 1 / 6 and 6 / 6 of the tumors in the 3 mg / kg and 10 mg / kg dose groups show partial regression respectively;

[0811] The tumor inhibition rate of ADC-6 (3 mg / kg, IV, D0) against WSU-DLCL2 is 86%, and 2 / 6 of the tumors show partial regression;

[0812] The tumor inhibition rates of ADC-8 (3 mg / kg, 10 mg / kg, IV, D0) against WSU-DLCL2 were 39% and 93%, respectively. In the 10 mg / kg dose group, 4 / 6 tumors partially regressed.

[0813] Tumor-bearing mice tolerated all of the above drugs well, with no significant weight loss or other symptoms. IgG1 was used as a negative control. See Table 13 and Figures 13A to 13C and 14 for the results.

[0814] Table 13. Efficacy of ADC on WSU-DLCL2 subcutaneous xenograft tumors in nude mice

[0815]

[0816] D0: time of first administration; P value refers to comparison with vehicle; IV: intravenous injection; partial regression: tumor volume on D21 was smaller than that on D0.

[0817] 6. Conclusion

[0818] A single intravenous injection of 3 mg / kg or 10 mg / kg of ADC-1, ADC-2, ADC-3, ADC-4, ADC-6, and ADC-8 demonstrated significant efficacy against subcutaneous WSU-DLCL2 xenografts in nude mice, causing partial tumor regression. The drugs exhibited a clear dose-dependent effect, and at equivalent doses, each ADC exhibited superior efficacy to the active ingredient ADC-8 (i.e., Polivy). All of these drugs were well tolerated by tumor-bearing mice.

[0819] Example 4-5. Efficacy of ADC on subcutaneous transplanted tumors of human diffuse large B-cell lymphoma WSU-DLCL2 in nude mice

[0820] This example further evaluates and compares the efficacy of ADC drugs on WSU-DLCL2 subcutaneous xenograft tumors in nude mice.

[0821] 1. Drugs: Dilute ADC-5, ADC-6, ADC-7, ADC-1, and ADC-8 drugs with normal saline to the concentrations in Example 4-3.

[0822] 2. Cells and mice: Same as Example 4-4.

[0823] 3. Experimental steps: Each nude mouse was subcutaneously inoculated with 2×10 7 WSU-DLCL2 cells, wait until the tumor grows to 100-150 mm 3 Afterwards, the animals were divided into groups based on tumor volume (D0). Mice were administered intravenously (IV) at a volume of 10 mL / kg; the specific dosage and dosing schedule are shown in Table 12. Tumor volume was measured twice weekly, and the mice were weighed and the data were recorded.

[0824] 4. The experimental parameters are the same as those in Example 4-4, and the statistical analysis is as follows:

[0825] Unless otherwise specified, the tumor volumes between the two groups were compared using the two-tailed Student's t test, and P < 0.05 was defined as a statistically significant difference.

[0826] 5. Results

[0827] The tumor inhibition rates of ADC-5 (3 mg / kg, 6 mg / kg, 12 mg / kg, IV, D0) on subcutaneous transplanted tumors in WSU-DLCL2 nude mice were 69%, 86%, and 88%, respectively. Among them, 1 / 6 and 1 / 6 tumors in the 6 mg / kg and 12 mg / kg dose groups, respectively, partially regressed.

[0828] ADC-6 (1.5 mg / kg, 3 mg / kg, 6 mg / kg, IV, D0) showed inhibition rates of 66%, 108%, and 125% on WSU-DLCL2 subcutaneous xenografts, respectively. Partial regression was observed in 5 / 6 and 6 / 6 tumors in the 3 mg / kg and 6 mg / kg dose groups, respectively.

[0829] ADC-7 (1 mg / kg, IV, D0) had an inhibition rate of 91% on WSU-DLCL2 subcutaneous xenografts, with 1 / 6 of the tumors partially regressing.

[0830] The tumor inhibition rate of ADC-1 (3 mg / kg, IV, D0) on WSU-DLCL2 subcutaneous xenografts was 44%;

[0831] The tumor inhibition rate of ADC-8 (3 mg / kg, IV, D0) on WSU-DLCL2 subcutaneous transplanted tumors was 10%.

[0832] Tumor-bearing mice tolerated all the above drugs well and did not experience symptoms such as weight loss.

[0833] Please see Table 14 and Figures 15 to 17 for specific results.

[0834] Table 14. Dosage regimen and experimental results

[0835]

[0836] Wherein, D0: time of first administration; P value refers to comparison with vehicle; IV: intravenous injection.

[0837] Example 4-6. Efficacy of ADC on subcutaneous transplanted tumors of human B cell lymphoma DoHH2 in nude mice

[0838] This example further evaluates and compares the efficacy of ADC drugs on DoHH2 subcutaneous xenograft tumors in nude mice.

[0839] 1. Drugs: Dilute ADC-1, ADC-6, and ADC-8 drugs with physiological saline to the concentrations in Example 4-3.

[0840] 2. Cells and Mice: DOHH-2 cells were purchased from DSMZ, Germany. DOHH-2 cells were cultured in 10-cm dishes in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in an incubator containing 5% CO₂. Cells were passaged 2-3 times per week. When cells reached the exponential growth phase, they were harvested, counted, and plated.

[0841] Nude mice (BALB / c-nu, 4–5 weeks old, ♀) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. Production license number: SCXK(Shanghai)2018-0003, Animal Certification Certificate number: 20180003010222. Housing environment: SPF grade.

[0842] 3. Experimental steps: Each nude mouse was subcutaneously inoculated with 3×10 7 DOHH-2 cells, wait until the tumor grows to 100-150 mm 3 Afterwards, the animals were divided into groups based on tumor volume (D0). Mice were administered intravenously (IV) at a volume of 10 mL / kg; the specific dosage and dosing schedule are shown in Table 15. Tumor volume was measured twice weekly, and the mice were weighed and the data were recorded.

[0843] 4. Experimental indicators and statistical analysis:

[0844] The experimental indicators are to examine the effect of drugs on tumor growth, and the specific indicators are T / C% or tumor inhibition rate TGI (%).

[0845] Tumor diameter was measured with a vernier caliper twice a week, and tumor volume (V) was calculated as follows:

[0846] V=1 / 2×a×b 2 Where a and b represent length and width respectively.

[0847] T / C (%) = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. T is the tumor volume in the ADC-treated group, and C is the tumor volume in the IgG1-treated control group.

[0848] Tumor growth inhibition rate % (TGI%) = 100-T / C ​​(%);

[0849] When tumor regression occurs, tumor growth inhibition rate % (TGI%) = 100-(T-T0) / T0×100;

[0850] If the tumor shrinks compared to the initial volume, i.e., when T < T0 or C < C0, it is defined as partial tumor regression (PR); if the tumor completely disappears, it is defined as complete tumor regression (CR).

[0851] At the end of the experiment, when the experimental endpoint is reached, or when the average tumor volume in the solvent group reaches 1500 mm 3 , the animals were sacrificed by CO2 anesthesia, and then the tumors were dissected and photographed.

[0852] Unless otherwise specified, two-way ANOVA was used to compare the tumor volumes between the two groups, and P < 0.05 was defined as having a statistically significant difference.

[0853] 5. Results:

[0854] The tumor inhibition rates of ADC-1, ADC-6, and ADC-8 (1 mg / kg, IV, D0) on the subcutaneous transplanted tumors of DOHH-2 nude mice were 82% (1 / 6 PR), 127% (5 / 6 PR), and 41% respectively; the tumor-bearing mice could well tolerate the above drugs, and no obvious symptoms such as weight loss occurred.

[0855] For the results, please refer to Table 15 and Figures 18 and 19.

[0856] Table 15. Efficacy of different ADCs on subcutaneous transplanted tumors of human follicular lymphoma DOHH-2 nude mice

[0857]

[0858] D0: The time of the first drug administration; P value refers to compared with the solvent; IV: intravenous injection; partial regression: the tumor volume at D21 is less than that at D0.

[0859] 6. Conclusions

[0860] Single intravenous injection of 1 mg / kg of ADC-1 and ADC-6 both had obvious efficacy on the subcutaneous transplanted tumors of human follicular lymphoma DoHH2 nude mice, causing partial tumor regression; the drug effects were better than those of the positive drug ADC-8 (i.e., Polivy). The tumor-bearing mice could well tolerate the above drugs.

[0861] The use and welfare of experimental animals in this disclosure were carried out in accordance with the regulations of the "Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)". The health status and death of animals were monitored daily, and routine inspections included observing the effects of the test substances and drugs on the daily behavior of animals such as behavioral activities, weight changes, and appearance signs.

Claims

1. A ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, in: The drug is selected from: microtubule aggregation inhibitors, Topo I inhibitors, MMAE or its derivatives; preferably, the drug is selected from MMAE or its derivatives, exitecan or its derivatives, eribulin or its derivatives; The ligand is an anti-CD79B antibody or an antigen-binding fragment thereof, which comprises: A heavy chain HCDR1 comprising the sequence shown in SEQ ID NO: 24; A heavy chain HCDR2 comprising the sequence shown in SEQ ID NO: 25; A heavy chain HCDR3 comprising the sequence shown in SEQ ID NO: 26; A light chain LCDR1 comprising the sequence shown in SEQ ID NO: 27; A light chain LCDR2 comprising the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 17; and A light chain LCDR3 comprising the sequence shown in SEQ ID NO: 12 or SEQ ID NO:

18.

2. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the anti-CD79B antibody or antigen-binding fragment thereof comprises: a) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 23, 8 and 9, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11 and 12, respectively; b) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 7, 8, 9, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11, 12, respectively; or c) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 13, 14, 15, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 16, 17, 18, respectively.

3. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 2, wherein the anti-CD79B antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody or a fragment thereof; Preferably, it is a humanized antibody or a fragment thereof.

4. The ligand-drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-CD79B antibody or antigen-binding fragment thereof comprises: a heavy chain variable region as set forth in SEQ ID NO: 3, or at least 90% identical thereto, and a light chain variable region as set forth in SEQ ID NO:4 or at least 90% identical thereto; or a heavy chain variable region as set forth in SEQ ID NO: 5, or at least 90% identical thereto, and a light chain variable region as set forth in SEQ ID NO:6 or at least 90% identical thereto; or a heavy chain variable region as set forth in SEQ ID NO: 19, or at least 90% identical thereto, and a light chain variable region as set forth in SEQ ID NO: 20 or at least 90% identical thereto; or a heavy chain variable region as set forth in SEQ ID NO: 21, or at least 90% identical thereto, and A light chain variable region as set forth in SEQ ID NO: 22, or at least 90% identical thereto.

5. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof as claimed in any one of the preceding claims, wherein the anti-CD79B antibody or antigen-binding fragment thereof comprises a constant region Fc, Preferably, the Fc is IgG1, IgG2 or IgG4; More preferably, the Fc is IgG1 or IgG2.

6. The ligand-drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein the antigen-binding fragment of the anti-CD79B antibody is a scFv, Fv, Fab or Fab' fragment.

7. The ligand-drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-CD79B antibody comprises: A heavy chain as represented by SEQ ID NO: 28, or at least 90% identical thereto, and a light chain as set forth in SEQ ID NO:29 or at least 90% identical thereto; or A heavy chain as set forth in SEQ ID NO: 30, or at least 90% identical thereto, and A light chain as set forth in SEQ ID NO: 31 or at least 90% identical thereto.

8. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 7, which is a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof represented by formula (I): in: W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-、-NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-、-S(CH 2 ) 1 C(O)- or chemical bond, p 1 is an integer from 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally substituted by one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; R 1 is selected from a hydrogen atom, a halogen, a cycloalkylalkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; m is an integer from 0 to 4; n is 1 to 10 and can be an integer or a decimal; Pc is an anti-CD79B antibody or an antigen-binding fragment thereof as defined in any one of claims 1-7.

9. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 8, which is a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof represented by formula (II): in: s 1 is an integer from 2 to 8; preferably 5; Pc, R 1 , R 2 , R 5 , R 6 , R 7 , m and n are as defined in claim 8.

10. The ligand-drug conjugate according to claim 8 or 9, or a pharmaceutically acceptable salt or solvate thereof, wherein -L a -Y- or -L b -Y-Selected from:

11. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 10, which is selected from the following structural formulas: in: n is 1 to 10, which can be an integer or a decimal, preferably n is an integer or a decimal from 1 to 6; Pc is an anti-CD79B antibody or an antigen-binding fragment thereof as defined in any one of claims 1-7.

12. A method for preparing the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 11, wherein The following steps are involved: After Pc reduction, it reacts with the general formula L a -YD to obtain the compound of the general formula Pc-L a -Ligand-drug conjugate shown in YD; in: Pc is an anti-CD79B antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 7; W.L. 2 , L 3 , R 1 , R 2 , R 5 , R 6 , R 7 , m and n are as defined in claim 8.

13. The ligand-drug conjugate according to any one of claims 1 to 7 or a pharmaceutically acceptable salt or solvate thereof, which is of the general formula Pc-(LD) k As shown, in: Pc is an anti-CD79B antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 7, L is the connector, k is an integer or decimal from 1 to 20, D is represented by formula (III): in, R 1a is selected from hydrogen, alkyl, cycloalkyl, aryl and heteroaryl, Optionally, the alkyl, cycloalkyl, aryl and heteroaryl groups are each independently substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups, preferably R 1a It is methyl; R 1b is selected from hydrogen, alkyl, alkoxy, cycloalkyl, aryl and heteroaryl, Optionally, the alkyl, cycloalkyl, aryl and heteroaryl groups are each independently substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups, preferably R 1b is hydrogen; or R 1a With R 1b Together with the atoms it is attached to, it forms C 5-8 Heterocycloalkyl; optionally, the heteroalkyl is substituted with one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, and R 1a and R 1b Not hydrogen at the same time. 14 . The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 13 , wherein k is selected from 1 to 10 and can be an integer or a decimal.

15. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 13, wherein the linker comprises a cleavable peptide portion.

16. The ligand-drug conjugate according to claim 15 or a pharmaceutically acceptable salt or solvate thereof, wherein the cleavable peptide portion can be cleaved by an enzyme, preferably the enzyme is cathepsin, more preferably cathepsin B.

17. The ligand-drug conjugate according to claim 15 or 16, or a pharmaceutically acceptable salt or solvate thereof, in: The linker comprises a peptide residue consisting of 2 to 7 amino acids, The amino acids are selected from the group consisting of: phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid; More preferably, the peptide residues are selected from the group consisting of: valine-citrulline, alanine-alanine-asparagine, glycine-glycine-lysine, valine-lysine, valine-alanine, valine-phenylalanine, glycine-glycine-phenylalanine-glycine.

18. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 13, wherein the linker comprises a cleavable sulfonamide moiety or a cleavable disulfide moiety. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 18 , wherein the linker is cleavable under reducing conditions.

20. The ligand-drug conjugate of any one of claims 13 to 19, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker comprises a spacer unit attached to D. 21 . The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 20 , wherein the spacer unit comprises p-aminobenzyloxycarbonyl (PAB).

22. The ligand-drug conjugate according to any one of claims 13 to 21, or a pharmaceutically acceptable salt or solvate thereof, which is represented by any one of the following structures: k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2; k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2; k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2; k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2.

23. The ligand-drug conjugate according to claim 22 or a pharmaceutically acceptable salt or solvate thereof, which is represented by any one of the following structures: Wherein k is selected from 1 to 10 and can be an integer or a decimal; Preferably, R in D 1a is methyl, R 1b It's hydrogen.

24. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 7, which is represented by formula (IV): in, R 2 It is C 1 -C 8 alkyl; R 3 It is C 1 -C 8 alkyl; R 4 It is C 1 -C 8 alkyl; R 5 It is H; R 6 It is C 1 -C 8 alkyl; R 7 It is C 1 -C 8 alkyl; R 8 Independently of each other are O-(C 1 -C 8 alkyl); R 9 It is H; R 10 It is phenyl; Z is O or NH; R 11 Selected from H, C 1 -C 20 Alkyl or -(R 13 O) 3 -R14; R 13 It is C 2 -C 8 alkyl; R 14 It is C 1 -C 8 alkyl; Pc is an anti-CD79B antibody or an antigen-binding fragment thereof as defined in claims 1-7; L is the connector; n is 1 to 10 and can be an integer or a decimal.

25. The ligand-drug conjugate of claim 24 or a pharmaceutically acceptable salt or solvate thereof, comprising the structure shown below:

26. The ligand-drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt or solvate thereof, comprising a structure as shown in formula (V): in, R 2 -R 6 is selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy and cycloalkyl; R 7 is selected from the group consisting of a hydrogen atom, an alkyl group, an alkoxy group and a cycloalkyl group; R 8 -R 11 Any two of them form a cycloalkyl group, and the remaining two can be selected from a hydrogen atom, an alkyl group and a cycloalkyl group; R 12 is selected from a hydrogen atom or an alkyl group; R 13 -R 15 is selected from a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group or a halogen; R 16 is selected from aryl or heteroaryl, optionally substituted by a substituent selected from hydrogen, halogen, hydroxy, alkyl, alkoxy and cycloalkyl; n is 1 to 10 and can be an integer or a decimal; Pc is an anti-CD79B antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 7; L is a connector.

27. The ligand-drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt or solvate thereof, comprising a structure as shown in formula (VI): in, n、R 2 -R 16 As defined in claim 26; Pc is the anti-CD79B antibody or antigen-binding fragment thereof according to any one of claims 1 to 7; L is a linker.

28. The ligand-drug conjugate according to any one of claims 1 to 7 or a pharmaceutically acceptable salt or solvate thereof, which is represented by formula (VII): n is 1 to 10 and can be an integer or a decimal; Pc is an anti-CD79B antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 7; L is a connector.

29. The ligand-drug conjugate according to any one of claims 24 to 28, or a pharmaceutically acceptable salt or solvate thereof, in: n is 1 to 8 and can be an integer or a decimal; Preferably, n is 1 to 6, and can be an integer or a decimal.

30. The ligand-drug conjugate of any one of claims 24-29 or a pharmaceutically acceptable salt or solvate thereof, wherein the linker is -YL 1 -L 2 -L 3 -L 4 , Y is selected from or chemical bond, X 1 is selected from hydrogen, alkyl, alkoxy, aryl or halogen, X 2 is selected from alkylene, optionally substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 1 Selected from -(succinimidyl-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 17 -WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the optional C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 18 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-、-NR 18 (CH 2 CH 2 O)p 1 CH 2 C(O)-、-S(CH 2 ) 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20; preferably L 2 is a chemical bond; L 3 A peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are preferably selected from valine, citrulline, and methylvaline; wherein the amino acids are optionally substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl; R 17 and R 18 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; L 4 is an extension unit, preferably L 4 For PAB.

31. The ligand-drug conjugate of claim 30 or a pharmaceutically acceptable salt or solvate thereof, wherein Y is 32. The ligand-drug conjugate of claim 30 or a pharmaceutically acceptable salt or solvate thereof, wherein L 1 Selected from -(succinimidyl-3-yl-N)-(CH 2 )s 1 -C(O)-, where s 1 is an integer from 2 to 8; Preferred L 1 for 33. The ligand-drug conjugate of any one of claims 30 to 32, or a pharmaceutically acceptable salt or solvate thereof, wherein L 3 is a dipeptide amino acid unit, preferably L 3 It's valine-citrulline.

34. The ligand-drug conjugate of any one of claims 24 to 33, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker is selected from: The a end is connected to the Pc, and the b end is connected to the drug.

35. The ligand-drug conjugate according to any one of claims 24 to 34, or a pharmaceutically acceptable salt or solvate thereof, which is selected from the following structural formula: in: n is 1 to 10 and can be an integer or a decimal; Pc is an anti-CD79B antibody or an antigen-binding fragment thereof as defined in any one of claims 1-7.

36. A method for preparing the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 35, wherein The following steps are involved: wherein Pc,n is as defined in claim 35.

37. A ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, which is selected from any one of the following: in, Pc is an anti-CD79B antibody or an antigen-binding fragment thereof as defined in claims 1-7, and n is 1 to 10, which can be an integer or a decimal; Preferably, n is an integer or decimal between 1 and 6.

38. A ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, which is a deuterated product of the ligand-drug conjugate according to any one of claims 1-11, 13-35, and 37, or a mixture thereof.

39. A pharmaceutical composition comprising: a pharmaceutically acceptable carrier, diluent or excipient; and A therapeutically effective amount of the ligand-drug conjugate according to any one of claims 1-11, 13-35, 37-38, or a pharmaceutically acceptable salt or solvate thereof.

40. Use of the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-11, 13-35, 37-38, or the pharmaceutical composition according to claim 39 in the preparation of a drug, in: The drug is used to treat a proliferative disease or delay the progression of a proliferative disease. Preferably, the proliferative disorder is cancer or a tumor; More preferably, the cancer or tumor is selected from lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and / or mantle cell lymphoma.

41. A method for treating or preventing a proliferative disease or delaying the progression of a proliferative disease, the method include: Administering to a subject a therapeutically effective amount or a disease-delaying effective amount of the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-11, 13-35, 37-38, or a pharmaceutical composition according to claims 39-40, Preferably, the proliferative disorder is cancer or a tumor; More preferably, the cancer or tumor is selected from lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and / or mantle cell lymphoma.

42. A method of enhancing immune function in a subject having a B cell proliferative disorder or an autoimmune disorder, include: Administering to a subject an effective amount of the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-11, 13-35, 37-38, or the pharmaceutical composition according to claims 39-40 for treating or delaying a disease, Preferably, the B cell proliferative disorder is cancer or a tumor; More preferably, the B cell proliferative disorder is lymphoma, B cell lymphoma, diffuse large B cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and / or mantle cell lymphoma.

43. An anti-CD79B antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 23, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; and The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

44. The anti-CD79B antibody or antigen-binding fragment thereof of claim 43, comprising: a heavy chain variable region as set forth in SEQ ID NO: 19, or at least 90% identical thereto, and A light chain variable region as set forth in SEQ ID NO: 20, or at least 90% identical thereto.

45. The anti-CD79B antibody or antigen-binding fragment thereof according to claim 43 or 44, comprising a constant region Fc, Preferably, the Fc is IgG1, IgG2 or IgG4.

46. ​​An anti-CD79B antibody or antigen-binding fragment thereof as claimed in any one of claims 43 to 45, comprising a heavy chain as shown in SEQ ID NO: 28 or having at least 90% identity thereto, and A light chain as set forth in SEQ ID NO: 29 or at least 90% identical thereto.

47. A polynucleotide encoding the anti-CD79B antibody or antigen-binding fragment thereof according to any one of claims 43 to 46.

48. A host cell comprising the polynucleotide of claim 47, Preferably, the host cell is a bacterial, yeast, or mammalian cell; More preferably, the host cell is Escherichia coli, Pichia pastoris, Chinese hamster ovary cells or human embryonic kidney 293 cells.

49. A method for preparing an anti-CD79B antibody or an antigen-binding fragment thereof, include: Expressing the anti-human CD76B antibody or its antigen-binding fragment in the host cell of claim 48, and isolating the anti-CD79B antibody or its antigen-binding fragment from the culture.

50. A pharmaceutical composition comprising any one or any combination thereof selected from the following: The anti-CD79B antibody or antigen-binding fragment thereof as described in any one of claims 43 to 46 or the polynucleotide as described in claim 47; and Optionally, a pharmaceutically acceptable excipient, diluent or carrier.

51. A method for treating or preventing a proliferative disease or delaying the progression of a proliferative disease, include: Administering to a subject an effective amount of the anti-CD79B antibody or antigen-binding fragment thereof according to any one of claims 43 to 46, or the polynucleotide according to claim 47, or the pharmaceutical composition according to claim 50 for treating or delaying a disease, Preferably, the B cell proliferative disorder is cancer or a tumor; More preferably, the B cell proliferative disorder is lymphoma, B cell lymphoma, diffuse large B cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and / or mantle cell lymphoma.