Antibody conjugate, preparation method therefor, and application thereof

Abstract

The present application relates to the field of biomedicine, and pertains to an antibody conjugate and an application thereof. The antibody conjugate comprises a heterodimeric antibody, a linker, and an active agent. Also provided is a method for preparing the antibody conjugate. The provided antibody conjugate exhibits excellent anti-tumor activity and / or a relatively good safety profile. The provided antibody conjugate can be used in treatment of tumors or autoimmune diseases.

Description

Antibody-conjugates, their preparation methods and applications Technical Field

[0001] This disclosure pertains to the field of biomedicine and relates to antibody-drug conjugates and their applications, wherein the antibody-drug conjugates comprise linked heterodimeric antibodies, linkers, and active agents. This disclosure also relates to methods for preparing the antibody-drug conjugates and their use in the preparation of medicaments for treating tumors or autoimmune diseases. Background Technology

[0002] Antibody-conjugates, in which a target molecule is conjugated to an antibody via a linker, are known in the field. Among them, antibody-conjugates in which the target molecule is a drug (e.g., a cytotoxic drug) have been extensively studied.

[0003] Antibody-drug conjugates (ADCs) are a class of drugs that combine the specificity of an antibody with the activity of a drug, with the antibody and drug moieties linked by an intermediate linker. Compared to traditional chemotherapy drugs, ADCs can bind precisely to tumor cells and reduce the impact on normal cells.

[0004] Since the first antibody-drug conjugate, Mylotarg, was launched in the United States in 2000, at least ten ADCs have been launched globally. At the same time, there are also reports in the literature on the research of various novel antibody-drug conjugates in the field of anti-tumor therapy. However, the availability of these conjugates is still limited to meet clinical needs, and more options are still needed. Summary of the Invention

[0005] This disclosure relates to novel antibody-conjugate structures and assembly methods, which exhibit superior performance in several aspects. For example, bispecific antibody-conjugates (including bispecific antibody-conjugates targeting different epitopes of the same antigen) can be assembled in vitro based on two monospecific antibodies as raw materials. These bispecific antibody-conjugates can be linked to different active agents (e.g., toxins). Alternatively, monospecific antibody-conjugates linked to different active agents (e.g., toxins) can also be assembled in vitro based on monospecific antibodies.

[0006] Antibody-conjugate

[0007] This disclosure provides an antibody-conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-conjugate comprises a heterodimeric antibody comprising a first hapten and a second hapten, the first hapten comprising a first CH3 region and the second hapten comprising a second CH3 region, wherein, according to EU designations, the first and / or second CH3 regions have at least one amino acid substitution at positions 366, 394, 405, and / or 407 to promote heterodimer formation, and the amino acid substitution positions of the first and second CH3 regions are different. In some embodiments, the first and second haptens may be conjugated to the same or different active agents or therapeutic agents. In some embodiments, the first and second haptens may be conjugated to the same or different active agents or therapeutic agents via the same or different linkers. In some embodiments, this disclosure relates to the use of a first half-antibody comprising a first CH3 region and a second half-antibody comprising a second CH3 region in the preparation of an antibody-conjugate comprising a heterodimeric antibody, wherein, according to EU designation, the first and / or second CH3 regions have at least one amino acid substitution at positions 366, 394, 405 and / or 407 to promote the formation of the heterodimer, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0008] This disclosure provides an antibody-conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent. The heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region, and the second hapten comprising a second CH3 region. According to EU designations, the first and / or second CH3 regions have at least one amino acid substitution at positions 366, 394, 405, and / or 407, and the amino acid substitution positions of the first and second CH3 regions are different. The first hapten is linked to an active agent via a linker, and the second hapten is linked to an active agent via a linker. The active agent linked to the first hapten may be the same as or different from the active agent linked to the second hapten. In some embodiments, the heterodimeric antibody comprises an amino acid substitution that promotes the formation of the heterodimer, wherein the amino acid substitution promoting the formation of the heterodimer is such that the first and / or second CH3 regions have at least one amino acid substitution at positions 366, 394, 405, and / or 407, and the amino acid substitution positions of the first and second CH3 regions are different.

[0009] In some embodiments, the heavy and light chains of the first hapten are derived from the same antibody. In some embodiments, the heavy and light chains of the second hapten are derived from the same antibody. In some embodiments, the heavy chains of the first and second haptens are derived from different antibodies, but the light chains of the first and second haptens are derived from the same antibody.

[0010] In some embodiments, the heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region and the second hapten comprising a second CH3 region, wherein, according to EU designations, the amino acid substitution combinations of the first and / or second CH3 regions include:

[0011] (1) The first CH3 region has an amino acid substitution at position 366, and the second CH3 region has an amino acid substitution at position 407;

[0012] (2) The first CH3 region has an amino acid substitution at position 405, and the second CH3 region has an amino acid substitution at position 394; or

[0013] (3) The first CH3 region has an amino acid substitution at position 405, and the second CH3 region has an amino acid substitution at position 407.

[0014] In some embodiments, the antibody-conjugate comprises a heterodimeric antibody, which comprises a first hapten and a second hapten, the first hapten containing a first CH3 region and the second hapten containing a second CH3 region, wherein, according to EU designation,

[0015] (1) The first CH3 region has an amino acid substitution at position 366, and the second CH3 region has an amino acid substitution at position 407; and / or

[0016] (2) The first CH3 region has an amino acid substitution at position 405, and the second CH3 region has an amino acid substitution at position 394.

[0017] In some embodiments, the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent, wherein the heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region, and the second hapten comprising a second CH3 region, wherein, according to EU designations,

[0018] (1) The first CH3 region has an amino acid substitution at position 366, and the second CH3 region has an amino acid substitution at position 407; and / or

[0019] (2) The first CH3 region has an amino acid substitution at position 405, and the second CH3 region has an amino acid substitution at position 394;

[0020] The first half-antibody is attached to an active agent via a linker, and the second half-antibody is attached to an active agent via a linker. The active agent attached to the first half-antibody may be the same as or different from the active agent attached to the second half-antibody. In some embodiments, the heterodimeric antibody contains an amino acid substitution that promotes the formation of the heterodimer, wherein the amino acid substitution that promotes the formation of the heterodimer is selected from amino acid substitutions selected from (1) and / or (2).

[0021] In some embodiments, the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent. The heterodimeric antibody comprises a first hapten and a second hapten. The first hapten contains a first CH3 region, and the second hapten contains a second CH3 region. According to EU designations, the first and / or second CH3 regions have the amino acid Met, Ile, or Leu at position 366, the amino acid Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394, the amino acid Ala, Thr, Ser, Asp, Glu, or Gly at position 405, and / or the amino acid Thr, Ala, or Val at position 407. The amino acid substitution positions of the first and second CH3 regions are different. The first hapten is linked to an active agent via a linker, and the second hapten is linked to an active agent via a linker. The active agent linked to the first hapten may be the same as or different from the active agent linked to the second hapten. In some embodiments, the heterodimeric antibody comprises amino acid substitutions that promote the formation of the heterodimer. These substitutions, according to EU designations, include amino acids Met, Ile, or Leu at position 366, Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394, Ala, Thr, Ser, Asp, Glu, or Gly at position 405, and / or Thr, Ala, or Val at position 407, with the amino acid substitution positions of the first and second CH3 regions being different.

[0022] In some embodiments, the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent. The heterodimeric antibody comprises a first hapten and a second hapten. The first hapten contains a first CH3 region, and the second hapten contains a second CH3 region. According to EU designations, the first and / or second CH3 regions have the amino acid Met, Ile, or Leu at position 366, the amino acid Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394, the amino acid Ala, Thr, Ser, Asp, or Glu at position 405, and / or the amino acid Thr, Ala, or Val at position 407. The amino acid substitution positions of the first and second CH3 regions are different. The first hapten is linked to an active agent via a linker, and the second hapten is linked to an active agent via a linker. The active agent linked to the first hapten may be the same as or different from the active agent linked to the second hapten. In some embodiments, the heterodimeric antibody comprises amino acid substitutions that promote the formation of the heterodimer, wherein the amino acid substitutions that promote the formation of the heterodimer are: the first and / or the second CH3 region has amino acid Met, Ile, or Leu at position 366, amino acid Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394, amino acid Ala, Thr, Ser, Asp, or Glu at position 405, and / or amino acid Thr, Ala, or Val at position 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0023] In some embodiments, the heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region and the second hapten comprising a second CH3 region, wherein, according to EU designations, the amino acid substitution combinations of the first and / or second CH3 regions include:

[0024] (1) The amino acid at position 366 of the first CH3 region is replaced with Met, Ile or Leu, and the amino acid at position 407 of the second CH3 region is replaced with Thr, Ala or Val;

[0025] (2) The amino acid at position 405 of the first CH3 region is substituted with Ala, Thr, Ser, Asp, Glu, or Gly, and the amino acid at position 394 of the second CH3 region is substituted with Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg; or

[0026] (3) The amino acid at position 405 of the first CH3 region is replaced with Ala, Thr, Ser, Asp, Glu or Gly, and the amino acid at position 407 of the second CH3 region is replaced with Thr, Ala or Val.

[0027] In some embodiments, the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent, wherein the heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region, and the second hapten comprising a second CH3 region, wherein, according to EU designations,

[0028] (1) The first CH3 region has Met, Ile, or Leu at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407; or

[0029] (2) The first CH3 region has Ala, Thr, Ser, Asp, Glu or Gly at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys or Arg at bit 394.

[0030] The first half-antibody is attached to an active agent via a linker, and the second half-antibody is attached to an active agent via a linker. The active agent attached to the first half-antibody may be the same as or different from the active agent attached to the second half-antibody. In some embodiments, the heterodimeric antibody contains an amino acid substitution that promotes the formation of the heterodimer, wherein the amino acid substitution that promotes the formation of the heterodimer is selected from amino acid substitutions selected from (1) and / or (2).

[0031] In some embodiments, the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent, wherein the heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region, and the second hapten comprising a second CH3 region, wherein, according to EU designations,

[0032] (1) The first CH3 region has Met, Ile, or Leu at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407; or

[0033] (2) The first CH3 region has Ala, Thr, Ser, Asp or Glu at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys or Arg at bit 394;

[0034] The first half-antibody is attached to an active agent via a linker, and the second half-antibody is attached to an active agent via a linker. The active agent attached to the first half-antibody may be the same as or different from the active agent attached to the second half-antibody. In some embodiments, the heterodimeric antibody contains an amino acid substitution that promotes the formation of the heterodimer, wherein the amino acid substitution that promotes the formation of the heterodimer is selected from amino acid substitutions selected from (1) and / or (2).

[0035] In some embodiments, the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent. The heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region, and the second hapten comprising a second CH3 region. According to EU designations, the first CH3 region has Met, Ile, or Leu at position 366, and the second CH3 region has Thr, Ala, or Val at position 407. The first hapten is linked to an active agent via a linker, and the second hapten is linked to an active agent via a linker. The active agent linked to the first hapten may be the same as or different from the active agent linked to the second hapten. In some embodiments, the heterodimeric antibody contains an amino acid substitution that promotes the formation of the heterodimer. This amino acid substitution is: the first CH3 region has Met, Ile, or Leu at position 366, and the second CH3 region has Thr, Ala, or Val at position 407. In some embodiments, the first CH3 region has Met at position 366, and the second CH3 region has Thr, Ala, or Val at position 407. In some embodiments, the first CH3 region has Ile at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407. In some embodiments, the first CH3 region has Leu at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407. In some embodiments, the first CH3 region has Met or Leu at bit 366, and the second CH3 region has Thr or Ala at bit 407. In some embodiments, the first CH3 region has Met at bit 366, and the second CH3 region has Thr or Ala at bit 407. In some specific embodiments, the first CH3 region has Met at bit 366, and the second CH3 region has Thr at bit 407. In some specific embodiments, the first CH3 region has Met at bit 366, and the second CH3 region has Ala at bit 407. In some specific embodiments, the first CH3 region has Met at position 366, the second CH3 region has Ala at position 407, and the heterodimeric antibody does not contain amino acid substitutions at one or more of positions 409, 349, 370, 357, 364, and 356 in either the first or second CH3 region. In some specific embodiments, the first CH3 region has Met at position 366, the second CH3 region has Ala at position 407, and the heterodimeric antibody does not contain any of the amino acid substitutions from K409F, Y349S, K370Y, K409V, E357D, S364Q, E356G, and S364R. In some specific embodiments, the first CH3 region has Leu at position 366, and the second CH3 region has Thr at position 407.In some specific embodiments, the first CH3 region has Leu at position 366, and the second CH3 region has Ala at position 407. In some specific embodiments, the first CH3 region has Met at position 366, and the second CH3 region has Val at position 407. In some specific embodiments, the first CH3 region has Met at position 366, and the second CH3 region has Val at position 407, and the heterodimeric antibody does not contain amino acid substitutions at one or more of positions 351, 368, 397, 405, and 394 in either the first or second CH3 region. In some specific embodiments, the first CH3 region has Met at position 366, and the second CH3 region has Val at position 407, and the heterodimeric antibody does not contain one or more amino acid substitutions from L351Y, L368M, V397T, F405M, and T394W. In some specific embodiments, the first CH3 region has Ile at position 366, and the second CH3 region has Ala at position 407. In some specific embodiments, the first CH3 region has Ile at position 366, and the second CH3 region has Thr at position 407. In some specific embodiments, the first CH3 region has Ile at position 366, and the second CH3 region has Val at position 407. In some specific embodiments, the first CH3 region has Ile at position 366, and the second CH3 region has Val at position 407, and the heterodimeric antibody does not contain amino acid substitutions at one or more of positions 351, 405, and 392 in either the first or second CH3 region. In some specific embodiments, the first CH3 region has Ile at position 366, and the second CH3 region has Val at position 407, and the heterodimeric antibody does not contain one or more amino acid substitutions from L351Y, F405A, and K392M. In some specific embodiments, the first CH3 region has Leu at position 366, and the second CH3 region has Val at position 407. In some specific embodiments, the first CH3 region has a Leu at position 366, the second CH3 region has a Val at position 407, and the heterodimer does not contain amino acid substitutions at one or more of the following positions: 405, 394, 351, 392, 350, 400, and 390. In some specific embodiments, the first CH3 region has a Leu at position 366, the second CH3 region has a Val at position 407, and the heterodimer does not contain one or more amino acid substitutions from the following: F405A, T394W, L351Y, K392M, T350V, K392L, F405T, S400E, T350V, N390R, K392M, and F405S.In some specific embodiments, the first CH3 region has a Leu at position 366, and the second CH3 region has an Ala at position 407. In some specific embodiments, the first CH3 region has a Leu at position 366, and the second CH3 region has an Ala at position 407, and the heterodimeric antibody does not contain amino acid substitutions at one or more of positions 409, 351, 399, and 409 in either the first or second CH3 region. In some specific embodiments, the first CH3 region has a Leu at position 366, and the second CH3 region has an Ala at position 407, and the heterodimeric antibody does not contain one or more amino acid substitutions from K409F, L351Y, D399C, and K409P.

[0036] In some embodiments, the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent. The heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region, and the second hapten comprising a second CH3 region. According to EU designations, the first CH3 region has Ala, Thr, Ser, Asp, Glu, or Gly at position 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394. The first hapten is linked to an active agent via a linker, and the second hapten is linked to an active agent via a linker. The active agent linked to the first hapten may be the same as or different from the active agent linked to the second hapten. In some embodiments, the heterodimeric antibody comprises amino acid substitutions that promote the formation of the heterodimer, wherein the amino acid substitutions promoting the formation of the heterodimer are: the first CH3 region has Ala, Thr, Ser, Asp, Glu, or Gly at position 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394. In some embodiments, the first CH3 region has Ala or Asp at position 405, and the second CH3 region has Trp, Phe, Tyr, Ile, Lys, or Arg at position 394. In some embodiments, the first CH3 region has Ala at position 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394. In some embodiments, the first CH3 region has Thr at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Ser at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Asp at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Glu at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Gly at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Asp at bit 405, and the second CH3 region has Trp, Phe, Tyr, Ile, Lys, or Arg at bit 394.In some implementations, the first CH3 region has Ala, Thr, Ser, or Asp at bit 405, and the second CH3 region has Trp, Phe, Tyr, or Lys at bit 394. In some specific implementations, the first CH3 region has Ala at bit 405, and the second CH3 region has Trp at bit 394. In some specific implementations, the first CH3 region has Ala at bit 405, and the second CH3 region has Phe at bit 394. In some specific implementations, the first CH3 region has Ala at bit 405, and the second CH3 region has Tyr at bit 394. In some specific implementations, the first CH3 region has Ala at bit 405, and the second CH3 region has Lys at bit 394. In some specific implementations, the first CH3 region has Thr at bit 405, and the second CH3 region has Trp at bit 394. In some specific implementations, the first CH3 region has Thr at bit 405, and the second CH3 region has Phe at bit 394. In some specific implementations, the first CH3 region has Thr at bit 405, and the second CH3 region has Tyr at bit 394. In some specific implementations, the first CH3 region has Thr at bit 405, and the second CH3 region has Lys at bit 394. In some specific implementations, the first CH3 region has Ser at bit 405, and the second CH3 region has Trp at bit 394. In some specific implementations, the first CH3 region has Ser at bit 405, and the second CH3 region has Phe at bit 394. In some specific implementations, the first CH3 region has Ser at bit 405, and the second CH3 region has Tyr at bit 394. In some specific implementations, the first CH3 region has Ser at bit 405, and the second CH3 region has Lys at bit 394. In some specific implementations, the first CH3 region has Asp at bit 405, and the second CH3 region has Trp at bit 394. In some specific implementations, the first CH3 region has Asp at bit 405, and the second CH3 region has Phe at bit 394. In some specific implementations, the first CH3 region has Asp at bit 405, and the second CH3 region has Tyr at bit 394. In some specific implementations, the first CH3 region has Asp at bit 405, and the second CH3 region has Lys at bit 394.

[0037] In some specific implementations, the first CH3 region and the second CH3 region have any of the exemplary mutation combinations in Table A.

[0038] Table A: Exemplary Mutation Combinations

[0039] In some embodiments, the sequences of the first CH3 region and the second CH3 region are different, such that the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction between the first CH3 region and the second CH3 region respectively. In some embodiments, the first CH3 region and the second CH3 region are stably associated.

[0040] In some embodiments, the first CH3 region is the CH3 region of IgG. In some embodiments, the first CH3 region is the CH3 region of human IgG. In some embodiments, the first CH3 region is a CH3 region selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the first CH3 region is a CH3 region selected from IgG1 and IgG4.

[0041] In some embodiments, the second CH3 region is the CH3 region of IgG. In some embodiments, the second CH3 region is the CH3 region of human IgG. In some embodiments, the second CH3 region is a CH3 region selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the second CH3 region is a CH3 region selected from IgG1 and IgG4.

[0042] In some embodiments, both the first CH3 region and the second CH3 region are CH3 regions of IgG1. In some embodiments, both the first CH3 region and the second CH3 region are CH3 regions of human IgG1. In some embodiments, both the first CH3 region and the second CH3 region are CH3 regions of IgG4. In some embodiments, both the first CH3 region and the second CH3 region are CH3 regions of human IgG4. In some embodiments, one CH3 region of the first CH3 region and the second CH3 region is a CH3 region of IgG1, and the other CH3 region is a CH3 region of IgG4. In some embodiments, one CH3 region of the first CH3 region and the second CH3 region is a CH3 region of human IgG1, and the other CH3 region is a CH3 region of human IgG4.

[0043] In some implementations, the first and second haptens are IgG1 type haptens.

[0044] IgG antibodies can exist in various allotypes. In some embodiments, the first CH3 region is a CH3 region selected from IgG1 having allotypes G1m1, nG1m1, G1m3, G1m17, G1m17,1, G1m17,1,2, and G1m3,1. In some embodiments, the first CH3 region is a CH3 region selected from human IgG1 having allotypes G1m1, nG1m1, G1m3, G1m17, G1m17,1, G1m17,1,2, and G1m3,1. In some embodiments, the second CH3 region is a CH3 region selected from IgG1 having allotypes G1m1, nG1m1, G1m3, G1m17, G1m17,1, G1m17,1,2, and G1m3,1. In some embodiments, the second CH3 region is a CH3 region selected from human IgG1 having allotypes G1m1, nG1m1, G1m3, G1m17, G1m17,1, G1m17,1,2 and G1m3,1.

[0045] In some embodiments, the first half-antibody comprises a first Fc polypeptide containing a first CH3 region, and the second half-antibody comprises a second Fc polypeptide containing a second CH3 region. The first and second Fc polypeptides can stably associate with each other to form an Fc domain. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, or an IgG4 Fc domain. In some embodiments, the Fc domain is an IgG4 Fc domain. In some specific embodiments, the Fc domain is a human IgG4 Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain. In some specific embodiments, the Fc domain is a human IgG1 Fc domain.

[0046] In some embodiments, the first and second CH3 regions, in addition to the specified amino acid substitutions, comprise the sequence illustrated in SEQ ID NO: 3. In some embodiments, compared to the sequence shown in SEQ ID NO: 3, the first and second CH3 regions comprise at least one of the following amino acid substitutions, and the positions of the amino acid substitutions in the first and second CH3 regions differ: 366M, 366L, 405A, 405T, 405D, 405E, 405S, 405G, 407A, 407T, 394W, 394F, 394M, 394Y, 394I, 394L, 394K, and 394R. In some embodiments, compared to the sequence shown in SEQ ID NO: 3, the first CH3 region and the second CH3 region contain at least one of the following amino acid substitutions, and the positions of the amino acid substitutions in the first CH3 region and the second CH3 region are different: 366M, 366L, 405A, 405T, 405D, 405S, 407A, 407T, 394W, 394F, 394M, 394Y, 394I, 394L, 394K, and 394R.

[0047] In some embodiments, the first and second Fc polypeptides comprise, in addition to the specified amino acid substitutions, the sequence described in SEQ ID NO: 1. In some embodiments, the first and second Fc polypeptides comprise at least one of the following amino acid substitutions, and the positions of the amino acid substitutions in the first and second Fc polypeptides differ: 366M, 366L, 405A, 405T, 405D, 405E, 405S, 405G, 407A, 407T, 394W, 394F, 394M, 394Y, 394I, 394L, 394K, and 394R. In some embodiments, the first Fc polypeptide and the second Fc polypeptide contain at least one of the following amino acid substitutions, and the amino acid substitution positions of the first Fc polypeptide and the second Fc polypeptide are different: 366M, 366L, 405A, 405T, 405D, 405S, 407A, 407T, 394W, 394F, 394M, 394Y, 394I, 394L, 394K and 394R.

[0048] In some embodiments, the first CH3 region and the second CH3 region comprise sequences shown as any one of SEQ ID NO:58 to SEQ ID NO:75, and the sequences of the first CH3 region and the second CH3 region are different. In some embodiments, the first CH3 region and the second CH3 region comprise sequences shown as any one of SEQ ID NO:58 to SEQ ID NO:62, SEQ ID NO:64, and SEQ ID NO:66 to SEQ ID NO:75, and the sequences of the first CH3 region and the second CH3 region are different.

[0049] In some embodiments, the heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region and the second hapten comprising a second CH3 region, wherein the first CH3 region and the second CH3 region comprise any of the following sequence combinations:

[0050] (1) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0051] (2) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0052] (3) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0053] (4) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0054] (5) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0055] (6) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0056] (7) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0057] (8) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0058] (9) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0059] (10) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0060] (11) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0061] (12) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0062] (13) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0063] (14) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0064] (15) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0065] (16) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0066] (17) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0067] (18) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0068] (19) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0069] (20) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0070] (21) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0071] (22) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0072] (23) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0073] (24) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0074] (25) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0075] (26) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0076] (27) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0077] (28) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0078] (29) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0079] (30) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0080] (31) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0081] (32) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0082] (33) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0083] (34) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0084] (35) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0085] (36) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0086] (37) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0087] (38) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0088] (39) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0089] (40) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0090] (41) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0091] (42) The first CH3 region contains the sequence shown in SEQ ID NO:65, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0092] (43) The first CH3 region contains the sequence shown in SEQ ID NO:65, and the second CH3 region contains the sequence shown in SEQ ID NO:69; or

[0093] (44) The first CH3 region contains the sequence shown in SEQ ID NO:65, and the second CH3 region contains the sequence shown in SEQ ID NO:71.

[0094] In some embodiments, the heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region and the second hapten comprising a second CH3 region, wherein the first CH3 region and the second CH3 region comprise any of the following sequence combinations:

[0095] (1) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0096] (2) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0097] (3) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0098] (4) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0099] (5) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0100] (6) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0101] (7) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0102] (8) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0103] (9) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0104] (10) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0105] (11) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0106] (12) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0107] (13) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0108] (14) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0109] (15) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0110] (16) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0111] (17) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0112] (18) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0113] (19) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0114] (20) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0115] (21) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0116] (22) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0117] (23) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0118] (24) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0119] (25) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0120] (26) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0121] (27) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0122] (28) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0123] (29) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0124] (30) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0125] (31) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0126] (32) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0127] (33) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0128] (34) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0129] (35) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:75; or

[0130] (36) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:75.

[0131] In some embodiments, the heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region and the second hapten comprising a second CH3 region, wherein the first CH3 region and the second CH3 region comprise any of the following sequence combinations:

[0132] (1) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0133] (2) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0134] (3) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0135] (4) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0136] (5) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:68; or

[0137] (6) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:71.

[0138] In some embodiments, the first half-antibody and / or the second half-antibody, in addition to containing the CH3 region or the Fc region, contain one or more or all other regions of the antibody, such as the CH1 region, VH region, CL region and / or VL region.

[0139] In some embodiments, the first hapten includes a first antigen-binding portion, and the second hapten includes a second antigen-binding portion. In some embodiments, the first hapten includes a first antigen-binding portion and a first Fc polypeptide, and the second hapten includes a second antigen-binding portion and a second Fc polypeptide.

[0140] In some embodiments, the first antigen-binding portion and the second antigen-binding portion bind to the same antigen. In some embodiments, the first antigen-binding portion and the second antigen-binding portion bind to different antigens. In some embodiments, the first antigen-binding portion and the second antigen-binding portion bind to the same epitope. In some embodiments, the first antigen-binding portion and the second antigen-binding portion bind to different epitopes. The epitopes may be located on different antigens or the same antigen.

[0141] In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety are each independently in the form of Fab, ScFv, VHH, or ScFab (single-chain Fab). In some embodiments, both the first and second antigen-binding moiety are Fab. In vitro assembly methods can overcome the light-heavy chain mismatch problem, especially for antibodies with at least two antigen-binding moieties that are Fab.

[0142] In some embodiments, the first and / or second hapten includes at least one additional antigen-binding moiety. In some embodiments, the first hapten further includes at least one additional antigen-binding moiety linked to the first antigen-binding moiety and / or the first Fc peptide. In some embodiments, the second hapten includes at least one additional antigen-binding moiety linked to the second antigen-binding moiety and / or the second Fc peptide. In some other embodiments, the first hapten includes at least one additional antigen-binding moiety linked to the first antigen-binding moiety and / or the first Fc peptide, and the second hapten includes at least one additional antigen-binding moiety linked to the second antigen-binding moiety and / or the second Fc peptide. The additional antigen-binding moiety may bind the same or different antigenic epitopes to the first and / or second antigen-binding moiety, thereby increasing the antibody's valence and / or specificity. The additional antigen-binding moiety may be in the form of Fab, ScFv, VHH, or ScFab, etc.

[0143] In some implementations, the heterodimeric antibody is divalent or multivalent (e.g., divalent, trivalent, tetravalent, etc.).

[0144] In some embodiments, the heterodimeric antibody is monospecific or multispecific (e.g., bispecific, trispecific, tetraspecific, etc.). In some specific embodiments, the heterodimeric antibody is a bivalent monospecific antibody. In some specific embodiments, the heterodimeric antibody is a bivalent bispecific antibody. In some specific embodiments, the heterodimeric antibody is a trivalent bispecific antibody. In some specific embodiments, the heterodimeric antibody is a tetravalent bispecific antibody. In some specific embodiments, the heterodimeric antibody is a bivalent bispecific antibody. In some specific embodiments, the heterodimeric antibody is a trivalent bispecific antibody. In some specific embodiments, the heterodimeric antibody is a tetravalent bispecific antibody. In some specific embodiments, the heterodimeric antibody is a trivalent trispecific antibody.

[0145] In some embodiments, the first and / or second hapten contains a hinge region. In some embodiments, the first hapten does not contain a Cys-Pro-Pro-Cys sequence in its hinge region. In some embodiments, the second hapten does not contain a Cys-Pro-Pro-Cys sequence in its hinge region. In some embodiments, neither the first nor the second hapten contains a Cys-Pro-Pro-Cys sequence in its hinge region. In some embodiments, the first hapten contains a Cys-Pro-Pro-Cys sequence in its hinge region. In some embodiments, the second hapten contains a Cys-Pro-Pro-Cys sequence in its hinge region. In some embodiments, both the first and second hapten contain a Cys-Pro-Pro-Cys sequence in their hinge regions. In some embodiments, the heterodimeric antibody does not contain a Cys-Pro-Pro-Cys sequence in its hinge region. In some embodiments, the heterodimeric antibody contains a Cys-Pro-Pro-Cys sequence in its hinge region. In some embodiments, the first and / or second half-antibody contains a hinge region comprising the sequence shown in SEQ ID NO:2.

[0146] The adapter disclosed herein can be linked to a heterodimeric antibody (or, a first hemiantibody and / or a second hemiantibody in a heterodimeric antibody) by any method known in the art. The adapter can be any structure adapted to link the active agent and the heterodimeric antibody. The linking can be site-directed or random, for example, site-directed linking mediated by sorting enzymes, glutaminases, etc., glycan-based site-directed linking, site-directed linking by introducing non-natural amino acids, cysteine ​​(e.g., thiomab) site-directed linking, etc., to achieve the linker portion to the antibody. In some embodiments, the adapter is a adapter containing a maleimide group (e.g., maleimide hexanoyl (MC)), such as adapters containing maleimide-PEG, MC-GGFG (MC-GlyGlyPheGly), or MC-VC-PAB.

[0147] In some embodiments, the linker is connected to the heterodimeric antibody via a thiol group and / or an amino group. In some more preferred embodiments, the linker is connected to the heterodimeric antibody via a thiol group. In some embodiments, the chemical bond between the heterodimeric antibody and the linker is a thioether bond formed at a disulfide bond site on the heterodimeric antibody (e.g., the hinge region of the heterodimeric antibody).

[0148] In the antibody-conjugates or pharmaceutically acceptable salts thereof disclosed herein, the active agents linked to the first and second hemiantibodies can each be independently selected from microtubule inhibitors, DNA damaging agents, immunomodulators, topoisomerase inhibitors, or other target molecules with the desired efficacy. These active agents include, but are not limited to, taxanes (including, but not limited to, paclitaxel, paclitaxel liposomes, albumin-bound paclitaxel, cabazitaxel, and docetaxel), platinum-based drugs (including, but not limited to, oxaliplatin, cisplatin, carboplatin, nedaplatin, bicycloplatin, miplatin, lobaplatin, picoplatin, lobaplatin, triplatinum tetranitrate, phenanthreneplatin, and saxaplatin), Bcl-xL inhibitors, RNA splicing inhibitors, transcription inhibitors, protease inhibitors, and photosensitizers (e.g., IRDye). 700DX), eribulin or its derivatives, olistatin drugs (including but not limited to olistatin, MMAF and MMAE), maytansine drugs (including but not limited to maytansine, DM1 and DM4), camptothecin drugs (including but not limited to camptothecin, hydroxycamptothecin, aminocamptothecin, irinotecan, topotecan, esanotecan, rubitecan, lurtotecan, gemmatine, karenitecin, 7-ethylcamptothecin, SN-38 and their derivatives, such as esanotecan derivative DXd), chachiomycin drugs (e.g. chachiomycin, N-acetyl-γ-calicheamicin), Pyrrolobenzodiazepine (PBD) drugs (trametesin, DSB-120 and SJG-136), TLR agonists, STING agonists, etc.

[0149] In some embodiments, the active agent linked to the first half-antibody and the active agent linked to the second half-antibody are the same, specifically selected from eribulin or its derivatives, or esanotecan or its derivatives (e.g., DXd, deuterated esanotecan, deuterated DXd, such as DDDXd), or olprestatin drugs (e.g., MMAE or MMAF).

[0150] In some embodiments, the active agent linked to the first half-antibody and the active agent linked to the second half-antibody are different, specifically selected from eribulin or a derivative thereof, or esanotecan or a derivative thereof (e.g., DXd, deuterated esanotecan, deuterated DXd, such as DDDXd), or olistatin drugs (e.g., MMAE or MMAF). In some embodiments, one of the active agents linked to the first half-antibody and the second half-antibody is eribulin or a derivative thereof, and the other is esanotecan or a derivative thereof. In some embodiments, the active agent linked to the first half-antibody is eribulin or a derivative thereof, and the active agent linked to the second half-antibody is esanotecan or a derivative thereof. In some embodiments, the active agent linked to the first half-antibody is esanotecan or a derivative thereof, and the active agent linked to the second half-antibody is eribulin or a derivative thereof. In some embodiments, the active agent linked to the first half-antibody is MMAE, and the active agent linked to the second half-antibody is MMAF. In some embodiments, the active agent linked to the first half-antibody is MMAF, and the active agent linked to the second half-antibody is MMAE. In some embodiments, the first hapten is conjugated to MC-GGFG-DDDXd, and the second hapten is conjugated to MC-GGFG-Eribulin. In some embodiments, the first hapten is conjugated to MC-GGFG-Eribulin, and the second hapten is conjugated to MC-GGFG-DDDXd. In some embodiments, the first hapten is conjugated to MC-VC-PAB-MMAE, and the second hapten is conjugated to MC-VC-PAB-MMAF. In some embodiments, the first hapten is conjugated to MC-VC-PAB-MMAF, and the second hapten is conjugated to MC-VC-PAB-MMAE.

[0151] In some embodiments, one of the first and second half-antibody-linked active agents has the structure shown in Formula I, and the other active agent has the structure shown in Formula II.

[0152] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0153] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0154] In some embodiments, the active agent linked to the first half-antibody has the structure shown in Formula I, and the active agent linked to the second half-antibody has the structure shown in Formula II.

[0155] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0156] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0157] In some embodiments, the active agent linked to the first half-antibody has the structure shown in Formula II, and the active agent linked to the second half-antibody has the structure shown in Formula I.

[0158] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0159] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R...1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0160] In some embodiments, the first hapten is transmitted via a linker with the structure shown in Formula IIIa.

[0161] The structure shown in formula Ia is connected.

[0162] The second half-antibody is connected to the structure shown in Formula IIa via a linker of the structure shown in Formula IIIa.

[0163] The hapten is attached to the position shown * in the adapter, and the structures shown in Formula Ia and Formula IIa are attached to the positions shown # in the adapter.

[0164] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0165] R 3 R 4 R 5 and R 6Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0166] In some embodiments, the first hapten is transmitted via a linker with the structure shown in Formula IIIa.

[0167] The structure shown in Formula IIa is connected.

[0168] The second half-antibody is connected to the structure shown in Formula Ia via a linker of the structure shown in Formula IIIa.

[0169] The hapten is attached to the position shown * in the adapter, and the structures shown in Formula Ia and Formula IIa are attached to the positions shown # in the adapter.

[0170] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0171] R 3 R 4 R 5 and R6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0172] In some of the above embodiments, the R 1 and R 2 Each is independently selected from hydrogen atoms or C atoms. 1-5 Alkyl (preferably C) 1-4 Alkyl, such as C 1-3 Alkyl group). In some of the above embodiments, the R 1 and R 2 Each is independently selected from hydrogen atoms, methyl, ethyl, propyl, or isopropyl. In some of the above embodiments, the R... 1 and R 2 It is a hydrogen atom. In some of the above embodiments, the R... 3 For deuterium atoms, R 4 It is a deuterium atom.

[0173] In some specific embodiments, this disclosure provides antibody-conjugates or pharmaceutically acceptable salts thereof, wherein the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent, the heterodimeric antibody comprising a first hapten and a second hapten, the first hapten comprising a first CH3 region, and the second hapten comprising a second CH3 region, wherein, according to EU designation, the first CH3 region has Met, Ile, or Leu at position 366, and the second CH3 region has Thr, Ala, or Val at position 407.

[0174] The first hapten via a linker with the structure shown in Formula IIIa

[0175] The structure shown in formula Ia is connected.

[0176] The second half-antibody is connected to the structure shown in Formula IIa via a linker of the structure shown in Formula IIIa.

[0177] The hapten is attached to the position shown * in the adapter, and the structures shown in Formula Ia and Formula IIa are attached to the positions shown # in the adapter.

[0178] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form an optionally substituted 5- to 8-membered heterocyclic group (e.g., containing 1-3 heteroatoms selected from N, O, or S); preferably, the R 1 and R 2 It is a hydrogen atom;

[0179] R 3 R 4 R 5 and R 6 Each is independently selected from hydrogen atoms or deuterium atoms; preferably, the R 3 For deuterium atoms, R 4 It is a deuterium atom. Further preferably, R 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0180] In some specific embodiments, this disclosure provides antibody-conjugates or pharmaceutically acceptable salts thereof, wherein the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent, the heterodimeric antibody comprising a first hapten and a second hapten, the first hapten comprising a first CH3 region, and the second hapten comprising a second CH3 region, wherein, according to EU designation,

[0181] The first half-antibody contains a first CH3 region, and the second half-antibody contains a second CH3 region. According to the EU designation, the first CH3 region has Ala, Thr, Ser, Asp, Glu, or Gly at position 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394.

[0182] The first hapten via a linker with the structure shown in Formula IIIa

[0183] The structure shown in formula Ia is connected.

[0184] The second half-antibody is connected to the structure shown in Formula IIa via a linker of the structure shown in Formula IIIa.

[0185] The hapten is attached to the position shown * in the adapter, and the structures shown in Formula Ia and Formula IIa are attached to the positions shown # in the adapter.

[0186] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form an optionally substituted 5- to 8-membered heterocyclic group (e.g., containing 1-3 heteroatoms selected from N, O, or S); preferably, the R 1 and R 2 It is a hydrogen atom;

[0187] R 3 R 4 R 5 and R 6 Each is independently selected from hydrogen atoms or deuterium atoms; preferably, the R 3 For deuterium atoms, R4 It is a deuterium atom. Further preferably, R 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0188] In some of the above embodiments, the R 1 and R 2 Each is independently selected from hydrogen atoms or C atoms. 1-5 Alkyl (preferably C) 1-4 Alkyl, such as C 1-3 Alkyl group). In some of the above embodiments, the R 1 and R 2 Each is independently selected from hydrogen atoms, methyl, ethyl, propyl, or isopropyl. In some of the above embodiments, the R... 1 and R 2 It is a hydrogen atom. In some of the above embodiments, the R... 3 For deuterium atoms, R 4 It is a deuterium atom.

[0189] In other specific embodiments, this disclosure provides antibody-conjugates or pharmaceutically acceptable salts thereof, wherein the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent, the heterodimeric antibody comprising a first hapten and a second hapten, the first hapten comprising a first CH3 region, and the second hapten comprising a second CH3 region, wherein, according to EU designations, the first CH3 region has Met, Ile, or Leu at position 366, and the second CH3 region has Thr, Ala, or Val at position 407, and the first hapten is coupled via a linker of the structure shown in Formula IIIa.

[0190] The structure shown in Formula IIa is connected.

[0191] The second half-antibody is connected to the structure shown in Formula Ia via a linker of the structure shown in Formula IIIa.

[0192] The hapten is attached to the position shown * in the adapter, and the structures shown in Formula Ia and Formula IIa are attached to the positions shown # in the adapter.

[0193] Among them, R1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form an optionally substituted 5- to 8-membered heterocyclic group (e.g., containing 1-3 heteroatoms selected from N, O, or S); preferably, the R 1 and R 2 It is a hydrogen atom;

[0194] R 3 and R 4 Each is independently selected from hydrogen atoms or deuterium atoms; preferably, the R 3 For deuterium atoms, R 4 It is a deuterium atom. Further preferably, R 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0195] In other specific embodiments, this disclosure provides antibody-conjugates or pharmaceutically acceptable salts thereof, wherein the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent, the heterodimeric antibody comprising a first hapten and a second hapten, the first hapten comprising a first CH3 region, and the second hapten comprising a second CH3 region, wherein, according to EU designation, the first CH3 region has Ala, Thr, Ser, Asp, Glu, or Gly at position 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394.

[0196] The first hapten via a linker with the structure shown in Formula IIIa

[0197] The structure shown in Formula IIa is connected.

[0198] The second half-antibody is connected to the structure shown in Formula Ia via a linker of the structure shown in Formula IIIa.

[0199] The hapten is attached to the position shown * in the adapter, and the structures shown in Formula Ia and Formula IIa are attached to the positions shown # in the adapter.

[0200] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form an optionally substituted 5- to 8-membered heterocyclic group (e.g., containing 1-3 heteroatoms selected from N, O, or S); preferably, the R 1 and R 2 It is a hydrogen atom;

[0201] R 3 and R 4Each is independently selected from hydrogen atoms or deuterium atoms; preferably, the R 3 For deuterium atoms, R 4 It is a deuterium atom. Further preferably, R 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0202] In some of the above embodiments, the linker of the structure shown in IIIa is attached to the disulfide bond site of the heterodimeric antibody at the position shown in *, preferably attached to the first half-antibody and / or the second half-antibody via a thioether bond.

[0203] This disclosure provides an antibody-conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent, the heterodimeric antibody comprising a first hapten and a second hapten, and one of the active agents linked to the first hapten and the second hapten having a structure shown in Formula I, and the other active agent having a structure shown in Formula II.

[0204] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0205] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0206] This disclosure provides an antibody-conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent, the heterodimeric antibody comprising a first hapten and a second hapten, the first hapten being linked to an active agent having a structure shown in Formula I, and the second hapten being linked to an active agent having a structure shown in Formula II.

[0207] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0208] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0209] This disclosure provides an antibody-conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent, the heterodimeric antibody comprising a first hapten and a second hapten, the first hapten being linked to an active agent having a structure shown in Formula II, and the second hapten being linked to an active agent having a structure shown in Formula I.

[0210] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0211] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0212] In some embodiments, the first hapten is transmitted via a linker with the structure shown in Formula IIIa.

[0213] The structure shown in formula Ia is connected.

[0214] The second half-antibody is connected to the structure shown in Formula IIa via a linker of the structure shown in Formula IIIa.

[0215] The hapten is attached to the position shown * in the adapter, and the structures shown in Formula Ia and Formula IIa are attached to the positions shown # in the adapter.

[0216] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0217] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0218] In some embodiments, the first hapten is transmitted via a linker with the structure shown in Formula IIIa.

[0219] The structure shown in Formula IIa is connected.

[0220] The second half-antibody is connected to the structure shown in Formula Ia via a linker of the structure shown in Formula IIIa.

[0221] The hapten is attached to the position shown * in the adapter, and the structures shown in Formula Ia and Formula IIa are attached to the positions shown # in the adapter.

[0222] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0223] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0224] In some implementations, the C 1-6 Alkyl, C 3-7 cycloalkyl, 3- to 7-membered heterocyclic groups, C 6-10 The aryl group or 5- to 12-membered heteroaryl group may be substituted by one or more groups selected from the following: deuterium, hydroxyl, halogen, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl groups.

[0225] In some of the above embodiments, the DAR (drug-antibody ratio) of the antibody-drug conjugate is 1-10, 2-8, 3.5-8, 4-8, 3.5-4.5, 6-8, 7-8, 7.5-8, 7.6-8, 7.7-8, 7.8-8, or 7.9-8. In some embodiments, the DAR is 2, 3, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 5, 6, 7, 7.5, 7.6, 7.7, 7.73, 7.79, 7.8, 7.86, 7.9, or 8.

[0226] The number of active agents linked to the heterodimeric antibody (or the first or second half-antibody) in the antibody-conjugate or its pharmaceutically acceptable salt provided in this disclosure can vary, such that the antibody-conjugate can be homogeneous or heterogeneous. The heterogeneous type is that the antibody-conjugate includes heterodimeric antibodies linked with different numbers of active agents. For example, one molecule of heterodimeric antibody may be linked with 0 (i.e., no active agent), 1, 2, 3, 4, 5, 6, 7, 8, or more molecular active agents. Another example is that one molecule of half-antibody (e.g., the first or second half-antibody) may be linked with 0 (i.e., no active agent), 1, 2, 3, 4, or more molecular active agents.

[0227] The antibody-conjugates or pharmaceutically acceptable salts thereof provided in this disclosure include, but are not limited to, monoclonal antibodies, monospecific antibodies, bispecific antibodies, multispecific antibodies, and nanobodies. The antibody-conjugates or pharmaceutically acceptable salts thereof provided in this disclosure can bind to any disease-associated antigen known in the art. When the disease is a tumor, the antigen can be selected from any tumor-associated antigen, including but not limited to HER2, HER3, EGFR, ROR1, CLDN18.2, B7-H3, B7-H4, TROP-2, CD19, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, PD-1, or PD-L1. In some embodiments, the heterodimeric antibody is a heterodimeric antibody targeting at least one (e.g., one, two, or three) targets selected from HER2, HER3, EGFR, ROR1, CLDN18.2, B7-H3, B7-H4, TROP-2, CD19, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, PD-1, and PD-L1. In some embodiments, the heterodimeric antibody is a heterodimeric antibody targeting different epitopes of the same target. In one specific embodiment, the heterodimeric antibody is a bispecific antibody targeting B7-H3 and B7-H4. In one specific embodiment, the heterodimeric antibody is a bispecific antibody targeting c-MET and MUC1. In one specific embodiment, the heterodimeric antibody is a bispecific antibody targeting c-MET and EGFR. In one specific embodiment, the heterodimeric antibody is a bispecific antibody targeting CD19 and CD20. In another specific embodiment, the heterodimeric antibody is a bispecific antibody targeting CD33 and CD70. In some embodiments, the heterodimeric antibody is a monospecific antibody targeting EGFR.

[0228] In some embodiments, the DAR of the antibody-drug conjugate or its pharmaceutically acceptable salt disclosed herein is 4-8, 7-8, 7.5-8, 7, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.

[0229] In some embodiments, the antibody-conjugates or pharmaceutically acceptable salts thereof provided in this disclosure exhibit one or more combinations of the following properties:

[0230] (1) Combined with human c-MET;

[0231] (2) Combined with human MUC1;

[0232] (3) It exhibits internalization in cells expressing c-MET;

[0233] (4) It exhibits internalization in cells expressing MUC1;

[0234] (5) It has killing activity against tumor cells expressing c-MET and / or MUC1;

[0235] (6) It has therapeutic effects on diseases related to c-MET and / or MUC1 expression; and

[0236] (7) It has the bystander effect.

[0237] In some embodiments, the antibody-conjugates or pharmaceutically acceptable salts thereof provided in this disclosure exhibit one or more combinations of the following properties:

[0238] (1) Combined with human B7-H3;

[0239] (2) Combined with human B7-H4;

[0240] (3) It exhibits internalization in cells expressing B7-H3;

[0241] (4) It exhibits internalization in cells expressing B7-H4;

[0242] (5) It has killing activity against tumor cells expressing B7-H3 and / or B7-H4;

[0243] (6) It has therapeutic effects on diseases related to B7-H3 and / or B7-H4 expression; and

[0244] (7) It has the bystander effect.

[0245] In some embodiments, the antibody-conjugates or pharmaceutically acceptable salts thereof provided in this disclosure exhibit one or more combinations of the following properties:

[0246] (1) Combined with human c-MET;

[0247] (2) Combined with human EGFR;

[0248] (3) It exhibits internalization in cells expressing c-MET;

[0249] (4) It exhibits internalization in cells expressing EGFR;

[0250] (5) It has killing activity against tumor cells expressing c-MET and / or EGFR;

[0251] (6) It has therapeutic effects on diseases related to c-MET and / or EGFR expression; and

[0252] (7) It has the bystander effect.

[0253] In some embodiments, the antibody-conjugates or pharmaceutically acceptable salts thereof provided in this disclosure exhibit one or more combinations of the following properties:

[0254] (1) Combined with human CD19;

[0255] (2) Combined with human CD20;

[0256] (3) It exhibits internalization in cells expressing CD19;

[0257] (4) It exhibits internalization in cells expressing CD20;

[0258] (5) It has killing activity against tumor cells expressing CD19 and / or CD20;

[0259] (6) It has therapeutic effects on diseases related to CD19 and / or CD20 expression; and

[0260] (7) It has the bystander effect.

[0261] In some embodiments, the antibody-conjugates or pharmaceutically acceptable salts thereof provided in this disclosure exhibit one or more combinations of the following properties:

[0262] (1) Combined with human CD33;

[0263] (2) Combined with human CD70;

[0264] (3) It exhibits internalization in cells expressing CD33;

[0265] (4) It exhibits internalization in cells expressing CD70;

[0266] (5) It has killing activity against tumor cells expressing CD33 and / or CD70;

[0267] (6) It has therapeutic effects on diseases related to CD33 and / or CD70 expression; and

[0268] (7) It has the bystander effect.

[0269] In some embodiments, the antibody-conjugates provided in this disclosure have low levels of aggregates and low molecular weight impurities, and the ADC products containing the first and second CH3 regions of the heterodimeric antibody described herein have high ADC monomer purity. The heterodimeric antibody therein can be assembled in vitro with high assembly efficiency (e.g., higher than 80%, higher than 85%, or even higher than 90%), exhibits excellent thermal stability, can effectively bind to and internalize target antigens at different expression levels in cells, and shows good killing effect on cells with different expression levels of target antigens.

[0270] The antibody-drug conjugates provided in this disclosure can be assembled in vitro. The antibodies and antibody-drug conjugates are of high purity and stable. The antibody-drug conjugates or pharmaceutically acceptable salts provided in this disclosure achieve excellent efficacy and / or safety. The antibody-drug conjugates or pharmaceutically acceptable salts provided in this disclosure achieve excellent antitumor efficacy and / or safety. In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts have good in vivo efficacy. In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts have good in vivo antitumor activity. In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts have excellent safety. In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts are not prone to aggregation. In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts have better solubility (e.g., water solubility).

[0271] Preparation method

[0272] This disclosure provides a method for preparing an antibody-conjugate or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0273] (1) Provides a first homodimeric antibody comprising a first CH3 region, wherein, according to the EU number, the first CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407;

[0274] (2) The first homodimer antibody was treated with a reducing agent to reduce the interchain disulfide bonds of the first homodimer antibody; the first homodimer antibody was linked to the active agent through a coupling reaction, and the first homodimer conjugate was obtained by purification.

[0275] (3) Provide a second homodimer antibody comprising a second CH3 region, wherein, according to the EU number, the second CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0276] (4) The second homodimer antibody is treated with a reducing agent to reduce the interchain disulfide bonds of the second homodimer antibody; the second homodimer antibody is coupled with an active agent through a coupling reaction, and the second homodimer conjugate is purified.

[0277] Wherein, the active agent linked to the first homodimeric antibody may be the same as or different from the active agent linked to the second homodimeric antibody;

[0278] (5) Incubate the first homodimeric conjugate and the second homodimeric conjugate together; and

[0279] (6) Obtain the antibody-conjugate or a pharmaceutically acceptable salt thereof.

[0280] This disclosure provides a method for preparing an antibody-conjugate or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0281] (1) Provides a first homodimeric antibody comprising a first CH3 region, wherein, according to the EU number, the first CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407;

[0282] (2) The first homodimer antibody was treated with a reducing agent to reduce the interchain disulfide bonds of the first homodimer antibody; the first homodimer antibody with thiol group was linked to the active agent through a coupling reaction and purified to obtain the first homodimer conjugate.

[0283] (3) Provide a second homodimer antibody comprising a second CH3 region, wherein, according to the EU number, the second CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0284] (4) The second homodimer antibody is treated with a reducing agent to reduce the interchain disulfide bonds of the second homodimer antibody; the second homodimer antibody with thiol group is coupled to the active agent through a coupling reaction, and the second homodimer conjugate is purified.

[0285] Wherein, the active agent linked to the first homodimeric antibody may be the same as or different from the active agent linked to the second homodimeric antibody;

[0286] (5) Incubate the first homodimeric conjugate and the second homodimeric conjugate together; and

[0287] (6) Obtain the antibody-conjugate or a pharmaceutically acceptable salt thereof.

[0288] This disclosure also provides a method for preparing an antibody-conjugate or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0289] (1) Provides a first homodimeric antibody comprising a first CH3 region, wherein, according to the EU number, the first CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407;

[0290] (2) The first homodimer antibody is treated with a reducing agent to reduce the interchain disulfide bonds of the first homodimer antibody; through a coupling reaction, the first homodimer antibody with thiol groups is linked to the active agent through a linker to obtain the first reaction solution;

[0291] (3) Provide a second homodimer antibody comprising a second CH3 region, wherein, according to the EU number, the second CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0292] (4) The second homodimer antibody is treated with a reducing agent to reduce the interchain disulfide bonds of the second homodimer antibody; through a coupling reaction, the second homodimer antibody with thiol groups is linked to the activator through a linker to obtain the second reaction solution;

[0293] Wherein, the active agent linked to the first homodimeric antibody may be the same as or different from the active agent linked to the second homodimeric antibody;

[0294] (5) Incubate the first reaction solution together with the second reaction solution; and

[0295] (6) Obtain the antibody-conjugate or a pharmaceutically acceptable salt thereof.

[0296] In some embodiments of the method, step (6) includes subjecting the reaction solution obtained in step (5) to oxidative conditions sufficient to allow cysteine ​​in the antibody-conjugate to be oxidized to interchain disulfide bonds. In other embodiments of the method, step (6) includes removing a reducing agent from the reaction system. Removing the reducing agent from the reaction system reduces the reaction system to a non-reducing or less reducing state. The step of removing the reducing agent may be, for example, ultrafiltration, etc.

[0297] This disclosure also provides a method for preparing an antibody-conjugate or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0298] (1) Provides a first homodimeric antibody comprising a first CH3 region, wherein, according to the EU number, the first CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407;

[0299] (2) The first homodimeric antibody is linked to the active agent through a linker to obtain the first homodimeric conjugate;

[0300] (3) Provide a second homodimer antibody comprising a second CH3 region, wherein, according to the EU number, the second CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0301] (4) The second homodimeric antibody is linked to the active agent through a linker to obtain the second homodimeric conjugate;

[0302] Wherein, the active agent linked to the first homodimeric antibody may be the same as or different from the active agent linked to the second homodimeric antibody;

[0303] (5) Incubate the first homodimeric coupling compound and the second homodimeric coupling compound together in the presence of a reducing agent, wherein the presence of the reducing agent is sufficient to allow the reduction of interchain disulfide bonds in the hinge regions of the first and second homodimeric coupling compounds, and

[0304] (6) Obtain the antibody-conjugate or a pharmaceutically acceptable salt thereof.

[0305] In some embodiments of the method, after step (5), the composition obtained from step (5) is subjected to oxidative conditions sufficient to allow cysteine ​​in the antibody-conjugate to be oxidized to interchain disulfide bonds. In other embodiments of the method, step (6) includes the removal of a reducing agent from the reaction system. Removing the reducing agent from the reaction system reduces the reaction system to a non-reducing or less reducing state. The step of removing the reducing agent may be, for example, ultrafiltration, etc.

[0306] In some implementations, rather than assembling to form homodimeric antibodies, the first half-antibody generated by the reduction of the first homodimeric antibody and the second half-antibody generated by the reduction of the second homodimeric antibody preferentially assemble to form heterodimeric antibodies.

[0307] In some implementations, the first hemidimeric antibody and the second hemidimeric antibody can be coupled to the same or different active agents via the same or different linkers.

[0308] Furthermore, in one embodiment of the above preparation methods, the reducing agent is selected from: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dierythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, β-mercaptoethanol, or salts thereof; preferably selected from: 2-MEA, DTT, and TCEP, or salts thereof. A salt of TCEP may, for example, be TCEP hydrochloride (TCEP·HCl). In some embodiments, the concentration of the reducing agent is 1-150mM, 50-150mM, 50-100mM, 1-50mM, 1-30mM, 10-20mM, 60-80mM, 60-75mM, 70-80mM, 75-80mM, etc., specifically such as 1mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, or a range consisting of any two of the aforementioned values.

[0309] The reduction temperature is 10-40℃, 10-30℃, 15-30℃, 20-30℃, 25-30℃, 15-40℃, 20-40℃, 25-40℃, etc., specifically 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.

[0310] The restoration time is 0.5-12 hours, 1-10 hours, 1-8 hours, 1-6 hours, 2-4 hours, 1-4 hours, etc., specifically for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, etc.

[0311] When the first homodimer conjugate and the second homodimer conjugate are incubated together, the molar ratio of the two is 1:1.

[0312] After removing the reducing agent from the reaction system, the reaction system can optionally be kept at 1-6°C for 1-30 hours. In some embodiments, the reaction system is kept at 1-6°C for 15-20 hours. In some specific examples, the reaction system is kept at 1°C, 2°C, 3°C, 4°C, 5°C, or 6°C for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, or 26 hours.

[0313] In some embodiments, according to the EU designation, the first and / or second CH3 regions have amino acids Met, Ile, or Leu at position 366, amino acids Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394, amino acids Ala, Thr, Ser, Asp, Glu, or Gly at position 405, and / or amino acids Thr, Ala, or Val at position 407, and the amino acid substitution positions of the first and second CH3 regions are different.

[0314] In some embodiments, according to the EU designation, the first and / or second CH3 regions have amino acids Met, Ile, or Leu at position 366, amino acids Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394, amino acids Ala, Thr, Ser, Asp, or Glu at position 405, and / or amino acids Thr, Ala, or Val at position 407, and the amino acid substitution positions of the first and second CH3 regions are different.

[0315] In some embodiments, according to the EU designation, the first and / or second CH3 regions have the amino acid Met, Ile, or Leu at position 366, the amino acid Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394, the amino acid Ala, Thr, Ser, Asp, Glu, or Gly at position 405, or the amino acid Thr, Ala, or Val at position 407, and the amino acid substitution positions of the first and second CH3 regions are different.

[0316] In some embodiments, according to the EU designation, the first and / or second CH3 regions have amino acids Met, Ile, or Leu at position 366, amino acids Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394, amino acids Ala, Thr, Ser, Asp, or Glu at position 405, or amino acids Thr, Ala, or Val at position 407, and the amino acid substitution positions of the first and second CH3 regions are different.

[0317] In some of the above embodiments, the first and second homodimer antibodies contain amino acid substitutions that promote heterodimer formation, wherein the amino acid substitutions that promote heterodimer formation are selected from the first and / or second CH3 regions described above.

[0318] In some implementation schemes, according to EU designation,

[0319] (1) The amino acid at position 366 of the first CH3 region is replaced with Met, Ile or Leu, and the amino acid at position 407 of the second CH3 region is replaced with Thr, Ala or Val;

[0320] (2) The amino acid at position 405 of the first CH3 region is substituted with Ala, Thr, Ser, Asp, Glu, or Gly, and the amino acid at position 394 of the second CH3 region is substituted with Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg; or

[0321] (3) The amino acid at position 405 of the first CH3 region is replaced with Ala, Thr, Ser, Asp, Glu or Gly, and the amino acid at position 407 of the second CH3 region is replaced with Thr, Ala or Val.

[0322] In some implementation schemes, according to EU designation,

[0323] (1) The first CH3 region has Met, Ile, or Leu at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407; or

[0324] (2) The first CH3 region has Ala, Thr, Ser, Asp, Glu, or Gly at position 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394. In some embodiments, the first and second homodimer antibodies contain amino acid substitutions that promote heterodimer formation, said amino acid substitutions being selected from (1) or (2) above.

[0325] In some implementations, depending on the EU number, the first CH3 region has Met or Leu at bit 366, and the second CH3 region has Thr or Ala at bit 407.

[0326] In some implementation schemes, according to EU designation,

[0327] (1) The first CH3 region has Met, Ile, or Leu at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407; or

[0328] (2) The first CH3 region has Ala, Thr, Ser, Asp, or Glu at position 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394. In some embodiments, the first and second homodimer antibodies contain amino acid substitutions that promote heterodimer formation, said amino acid substitutions being selected from (1) or (2) above.

[0329] In some implementations, according to the EU designation, the first CH3 region and the second CH3 region are stably associated by introducing at least one amino acid substitution at positions 366, 394, 405 and / or 407 of the first CH3 region and the second CH3 region.

[0330] In some embodiments, the first CH3 region has Met at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407. In some embodiments, the first CH3 region has Ile at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407. In some embodiments, the first CH3 region has Leu at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407. In some embodiments, the first CH3 region has Met or Leu at bit 366, and the second CH3 region has Thr or Ala at bit 407. In some embodiments, the first CH3 region has Met at bit 366, and the second CH3 region has Thr or Ala at bit 407. In some specific embodiments, the first CH3 region has Met at bit 366, and the second CH3 region has Thr at bit 407. In some specific embodiments, the first CH3 region has Met at bit 366, and the second CH3 region has Ala at bit 407. In some specific embodiments, the first CH3 region has Met at position 366, the second CH3 region has Ala at position 407, and the heterodimeric antibody does not contain any of the amino acid substitutions of K409F, Y349S, K370Y, K409V, E357D, S364Q, E356G, or S364R. In some specific embodiments, the first CH3 region has Leu at position 366, and the second CH3 region has Thr at position 407. In some specific embodiments, the first CH3 region has Leu at position 366, and the second CH3 region has Ala at position 407. In some specific embodiments, the first CH3 region has Met at position 366, and the second CH3 region has Val at position 407. In some specific embodiments, the first CH3 region has Met at position 366, the second CH3 region has Val at position 407, and the heterodimeric antibody does not contain one or more amino acid substitutions from L351Y, L368M, V397T, F405M, and T394W. In some specific embodiments, the first CH3 region has Ile at position 366, and the second CH3 region has Ala at position 407. In some specific embodiments, the first CH3 region has Ile at position 366, and the second CH3 region has Thr at position 407. In some specific embodiments, the first CH3 region has Ile at position 366, and the second CH3 region has Val at position 407. In some specific embodiments, the first CH3 region has Ile at position 366, the second CH3 region has Val at position 407, and the heterodimeric antibody does not contain one or more amino acid substitutions from L351Y, F405A, and K392M.In some specific embodiments, the first CH3 region has a Leu at position 366, and the second CH3 region has a Val at position 407. In some specific embodiments, the first CH3 region has a Leu at position 366, and the second CH3 region has a Val at position 407, and the heterodimer does not contain one or more amino acid substitutions from F405A, T394W, L351Y, K392M, T350V, K392L, F405T, S400E, T350V, N390R, K392M, and F405S. In some specific embodiments, the first CH3 region has a Leu at position 366, and the second CH3 region has an Ala at position 407. In some specific embodiments, the first CH3 region has a Leu at position 366, and the second CH3 region has an Ala at position 407, and the heterodimer antibody does not contain one or more amino acid substitutions from K409F, L351Y, D399C, and K409P.

[0331] In some embodiments, the first CH3 region has Ala at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Thr at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Ser at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Asp at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Glu at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Gly at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Asp at bit 405, and the second CH3 region has Trp, Phe, Tyr, Ile, Lys, or Arg at bit 394. In some embodiments, the first CH3 region has Ala, Thr, Ser, or Asp at bit 405, and the second CH3 region has Trp, Phe, Tyr, or Lys at bit 394. In some specific embodiments, the first CH3 region has Ala at bit 405, and the second CH3 region has Trp at bit 394. In some specific implementations, the first CH3 region has Ala at bit 405, and the second CH3 region has Phe at bit 394. In some specific implementations, the first CH3 region has Ala at bit 405, and the second CH3 region has Tyr at bit 394. In some specific implementations, the first CH3 region has Ala at bit 405, and the second CH3 region has Lys at bit 394. In some specific implementations, the first CH3 region has Thr at bit 405, and the second CH3 region has Trp at bit 394. In some specific implementations, the first CH3 region has Thr at bit 405, and the second CH3 region has Phe at bit 394. In some specific implementations, the first CH3 region has Thr at bit 405, and the second CH3 region has Tyr at bit 394.In some specific implementations, the first CH3 region has Thr at bit 405, and the second CH3 region has Lys at bit 394. In some specific implementations, the first CH3 region has Ser at bit 405, and the second CH3 region has Trp at bit 394. In some specific implementations, the first CH3 region has Ser at bit 405, and the second CH3 region has Phe at bit 394. In some specific implementations, the first CH3 region has Ser at bit 405, and the second CH3 region has Tyr at bit 394. In some specific implementations, the first CH3 region has Ser at bit 405, and the second CH3 region has Lys at bit 394. In some specific implementations, the first CH3 region has Asp at bit 405, and the second CH3 region has Trp at bit 394. In some specific implementations, the first CH3 region has Asp at bit 405, and the second CH3 region has Phe at bit 394. In some specific implementations, the first CH3 region has Asp at bit 405, and the second CH3 region has Tyr at bit 394. In some specific implementations, the first CH3 region has Asp at bit 405, and the second CH3 region has Lys at bit 394.

[0332] In some specific implementations, the first CH3 region and the second CH3 region have any of the exemplary mutation combinations in Table A.

[0333] In some implementations, depending on the EU number, the first CH3 region has Ala or Asp at bit 405, and the second CH3 region has Trp, Phe, Tyr, Ile, Lys, or Arg at bit 394.

[0334] In some implementations, the sequences of the first CH3 region and the second CH3 region are different, such that the heterodimer interaction between the first CH3 region and the second CH3 region is stronger than the homodimer interaction between the first CH3 region and the second CH3 region.

[0335] In some embodiments, the first CH3 region is the CH3 region of IgG. In some embodiments, the first CH3 region is the CH3 region of human IgG. In some embodiments, the first CH3 region is a CH3 region selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the first CH3 region is a CH3 region selected from IgG1 and IgG4.

[0336] In some embodiments, the second CH3 region is the CH3 region of IgG. In some embodiments, the second CH3 region is the CH3 region of human IgG. In some embodiments, the second CH3 region is a CH3 region selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the second CH3 region is a CH3 region selected from IgG1 and IgG4.

[0337] In some embodiments, both the first CH3 region and the second CH3 region are CH3 regions of IgG1. In some embodiments, both the first CH3 region and the second CH3 region are CH3 regions of human IgG1. In some embodiments, both the first CH3 region and the second CH3 region are CH3 regions of IgG4. In some embodiments, both the first CH3 region and the second CH3 region are CH3 regions of human IgG4. In some embodiments, one CH3 region of the first CH3 region and the second CH3 region is a CH3 region of IgG1, and the other CH3 region is a CH3 region of IgG4. In some embodiments, one CH3 region of the first CH3 region and the second CH3 region is a CH3 region of human IgG1, and the other CH3 region is a CH3 region of human IgG4.

[0338] IgG antibodies can exist in various allotypes. In some embodiments, the first CH3 region is a CH3 region selected from IgG1 having allotypes G1m1, nG1m1, G1m3, G1m17, G1m17,1, G1m17,1,2, and G1m3,1. In some embodiments, the first CH3 region is a CH3 region selected from human IgG1 having allotypes G1m1, nG1m1, G1m3, G1m17, G1m17,1, G1m17,1,2, and G1m3,1. In some embodiments, the second CH3 region is a CH3 region selected from IgG1 having allotypes G1m1, nG1m1, G1m3, G1m17, G1m17,1, G1m17,1,2, and G1m3,1. In some embodiments, the second CH3 region is a CH3 region selected from human IgG1 having allotypes G1m1, nG1m1, G1m3, G1m17, G1m17,1, G1m17,1,2 and G1m3,1.

[0339] In some embodiments, the first homodimeric antibody comprises a first Fc polypeptide containing a first CH3 region, and the second homodimeric antibody comprises a second Fc polypeptide containing a second CH3 region. Two first Fc polypeptides associate to form a first Fc domain. Two second Fc polypeptides associate to form a second Fc domain. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, or an IgG4 Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain. In some specific embodiments, the Fc domain is a human IgG1 Fc domain.

[0340] In some embodiments, the first homodimeric antibody includes a first antigen-binding portion, and the second homodimeric antibody includes a second antigen-binding portion. In some embodiments, the first homodimeric antibody includes a first antigen-binding portion and a first Fc domain, and the second homodimeric antibody includes a second antigen-binding portion and a second Fc domain.

[0341] In some embodiments, the first and / or second CH3 regions contain the sequence illustrated in SEQ ID NO: 3, in addition to the specified amino acid substitutions. In some embodiments, compared to the sequence shown in SEQ ID NO: 3, the first and second CH3 regions contain at least one of the following amino acid substitutions, and the positions of the amino acid substitutions in the first and second CH3 regions differ: 366M, 366L, 405A, 405T, 405D, 405E, 405S, 405G, 407A, 407T, 394W, 394F, 394M, 394Y, 394I, 394L, 394K, and 394R. In some embodiments, compared to the sequence shown in SEQ ID NO: 3, the first CH3 region and the second CH3 region contain at least one of the following amino acid substitutions, and the positions of the amino acid substitutions in the first CH3 region and the second CH3 region are different: 366M, 366L, 405A, 405T, 405D, 405S, 407A, 407T, 394W, 394F, 394M, 394Y, 394I, 394L, 394K, and 394R.

[0342] In some embodiments, the first and / or second Fc regions contain the sequence defined in SEQ ID NO: 1, in addition to the specified amino acid substitutions. In some embodiments, the first and second Fc polypeptides contain at least one of the following amino acid substitutions, and the positions of the amino acid substitutions in the first and second Fc polypeptides are different: 366M, 366L, 405A, 405T, 405D, 405E, 405S, 405G, 407A, 407T, 394W, 394F, 394M, 394Y, 394I, 394L, 394K, and 394R. In some embodiments, the first Fc polypeptide and the second Fc polypeptide contain at least one of the following amino acid substitutions, and the amino acid substitution positions of the first Fc polypeptide and the second Fc polypeptide are different: 366M, 366L, 405A, 405T, 405D, 405S, 407A, 407T, 394W, 394F, 394M, 394Y, 394I, 394L, 394K and 394R.

[0343] In some embodiments, the first CH3 region and the second CH3 region comprise sequences shown as any one of SEQ ID NO:58 to SEQ ID NO:75, and the sequences of the first CH3 region and the second CH3 region are different. In some embodiments, the first CH3 region and the second CH3 region comprise sequences shown as any one of SEQ ID NO:58 to SEQ ID NO:62, SEQ ID NO:64, and SEQ ID NO:66 to SEQ ID NO:75, and the sequences of the first CH3 region and the second CH3 region are different.

[0344] In some embodiments, the heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region and the second hapten comprising a second CH3 region, wherein the first CH3 region and the second CH3 region comprise any of the following sequence combinations:

[0345] (1) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0346] (2) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0347] (3) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0348] (4) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0349] (5) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0350] (6) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0351] (7) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0352] (8) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0353] (9) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0354] (10) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0355] (11) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0356] (12) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0357] (13) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0358] (14) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0359] (15) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0360] (16) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0361] (17) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0362] (18) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0363] (19) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0364] (20) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0365] (21) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0366] (22) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0367] (23) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0368] (24) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0369] (25) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0370] (26) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0371] (27) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0372] (28) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0373] (29) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0374] (30) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0375] (31) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0376] (32) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0377] (33) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0378] (34) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0379] (35) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0380] (36) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0381] (37) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0382] (38) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0383] (39) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0384] (40) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0385] (41) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0386] (42) The first CH3 region contains the sequence shown in SEQ ID NO:65, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0387] (43) The first CH3 region contains the sequence shown in SEQ ID NO:65, and the second CH3 region contains the sequence shown in SEQ ID NO:69; or

[0388] (44) The first CH3 region contains the sequence shown in SEQ ID NO:65, and the second CH3 region contains the sequence shown in SEQ ID NO:71.

[0389] In some embodiments, the heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region and the second hapten comprising a second CH3 region, wherein the first CH3 region and the second CH3 region comprise any of the following sequence combinations:

[0390] (1) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0391] (2) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0392] (3) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0393] (4) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0394] (5) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0395] (6) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0396] (7) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0397] (8) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:68;

[0398] (9) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0399] (10) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0400] (11) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0401] (12) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:69;

[0402] (13) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0403] (14) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0404] (15) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0405] (16) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:70;

[0406] (17) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0407] (18) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0408] (19) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0409] (20) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:71;

[0410] (21) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0411] (22) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0412] (23) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0413] (24) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:72;

[0414] (25) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0415] (26) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0416] (27) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0417] (28) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:73;

[0418] (29) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0419] (30) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0420] (31) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0421] (32) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:74;

[0422] (33) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0423] (34) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:75;

[0424] (35) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:75; or

[0425] (36) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:75.

[0426] In some embodiments, the heterodimeric antibody comprises a first hapten and a second hapten, the first hapten comprising a first CH3 region and the second hapten comprising a second CH3 region, wherein the first CH3 region and the second CH3 region comprise any of the following sequence combinations:

[0427] (1) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0428] (2) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0429] (3) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:66;

[0430] (4) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:67;

[0431] (5) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:68; or

[0432] (6) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:71.

[0433] In some embodiments, the first homodimeric antibody and / or the second homodimeric antibody, in addition to containing the CH3 region or the Fc region, also contain one or more or all other regions of the antibody, such as the CH1 region, VH region, CL region and / or VL region.

[0434] In some embodiments, the first antigen-binding portion and the second antigen-binding portion bind to the same antigen. In some embodiments, the first antigen-binding portion and the second antigen-binding portion bind to different antigens. In some embodiments, the first antigen-binding portion and the second antigen-binding portion bind to the same epitope. In some embodiments, the first antigen-binding portion and the second antigen-binding portion bind to different epitopes. The epitopes may be located on different antigens or the same antigen.

[0435] In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety are each independently in the form of Fab, ScFv, VHH, or ScFab. In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety are both Fab. In some embodiments, the first homodimeric antibody comprises two first antigen-binding moieties. In some embodiments, the second homodimeric antibody comprises two second antigen-binding moieties. In vitro assembly methods can overcome the light-heavy chain mismatch problem, especially for antibodies with at least two antigen-binding moieties being Fab.

[0436] In some embodiments, the first and / or second homodimeric antibody includes at least one additional antigen-binding moiety. In some embodiments, the first homodimeric antibody further includes at least one additional antigen-binding moiety linked to the first antigen-binding moiety and / or the first Fc domain. In some embodiments, the second homodimeric antibody includes at least one additional antigen-binding moiety linked to the second antigen-binding moiety and / or the second Fc domain. In some other embodiments, the first homodimeric antibody further includes at least one additional antigen-binding moiety linked to the first antigen-binding moiety and / or the first Fc domain, and the second homodimeric antibody includes at least one additional antigen-binding moiety linked to the second antigen-binding moiety and / or the second Fc domain. The additional antigen-binding moiety may bind the same or different antigenic epitopes to the first and / or second antigen-binding moiety, thereby increasing the antibody's valence and / or specificity. The additional antigen-binding moiety may be in the form of Fab, ScFv, VHH, or ScFab, etc.

[0437] In some implementations, the first and / or homodimeric antibody is divalent or multivalent (e.g., divalent, trivalent, tetravalent, etc.).

[0438] In some embodiments, the first and / or homodimeric antibody is monospecific or multispecific (e.g., bispecific, trispecific, tetraspecific, etc.). In some specific embodiments, the first and / or homodimeric antibody is a bivalent monospecific antibody. In some specific embodiments, the first and / or homodimeric antibody is a bivalent bispecific antibody.

[0439] In some embodiments, the first and / or second homodimeric antibody comprises a hinge region. In some embodiments, the first homodimeric antibody does not contain a Cys-Pro-Pro-Cys sequence in the hinge region. In some embodiments, the second homodimeric antibody does not contain a Cys-Pro-Pro-Cys sequence in the hinge region. In some embodiments, both the first and second homodimeric antibodies do not contain a Cys-Pro-Pro-Cys sequence in the hinge region. In some embodiments, the first homodimeric antibody contains a Cys-Pro-Pro-Cys sequence in the hinge region. In some embodiments, the second homodimeric antibody contains a Cys-Pro-Pro-Cys sequence in the hinge region. In some embodiments, both the first and second homodimeric antibodies contain a Cys-Pro-Pro-Cys sequence in the hinge region. In some embodiments, the first and / or second haploidentical antibody comprises a hinge region containing the sequence shown in SEQ ID NO:2.

[0440] In some embodiments, the first homodimeric antibody and / or the second homodimeric antibody are full-length IgG antibodies. In some embodiments, the first homodimeric antibody and / or the second homodimeric antibody comprise heavy and light chains.

[0441] The first and second isodimeric antibodies are linked by a linker to an active agent. The linker can be linked to the antibody using any method known in the art. The linker can be any structure adapted to link the active agent and the antibody. The linking can be site-directed or random, for example, site-directed linking mediated by sorting enzymes, glutaminases, etc., glycan-based site-directed linking, site-directed linking by introducing non-natural amino acids, cysteine ​​(e.g., thiomab) site-directed linking, etc., to achieve the linker portion to the antibody. In some embodiments, the linker is a linker containing a maleimide group (e.g., maleimide hexanoyl (MC)), such as linkers containing maleimide-PEG, MC-GGFG (MC-GlyGlyPheGly), or MC-VC-PAB.

[0442] In some embodiments, the linker is connected to the homodimeric antibody via a thiol group. In some embodiments, the chemical bond between the homodimeric antibody and the linker is a thioether bond formed at a disulfide bond site on the homodimeric antibody (e.g., the hinge region of the homodimeric antibody).

[0443] In the method provided in this disclosure, the active agents of the first and second homodimer conjugates can each be independently selected from tubulin inhibitors, DNA damaging agents, immunomodulators, topoisomerase inhibitors, or other target molecules with the desired efficacy. The active agents, including but not limited to taxanes (including but not limited to paclitaxel, paclitaxel liposomes, albumin-bound paclitaxel, cabazitaxel, and docetaxel), platinum-based drugs (including but not limited to oxaliplatin, cisplatin, carboplatin, nedaplatin, bicycloplatin, miplatin, lobaplatin, picoplatin, lobaplatin, triplatinum tetranitrate, phenanthreneplatin, and saxaplatin), Bcl-xL inhibitors, RNA splicing inhibitors, and transcription inhibitors... Pharmaceutical formulations, protease inhibitors, photosensitizers (e.g., IRDye700DX), eribulin or its derivatives, olistatin drugs (including but not limited to olistatin, MMAF, and MMAE), maytansine drugs (including but not limited to maytansine, DM1, and DM4), camptothecin drugs (including but not limited to camptothecin, hydroxycamptothecin, aminocamptothecin, irinotecan, topotecan, ethanotecan, rubitecan, lurtotecan, gematotecan, karenitecin, 7-ethylcamptothecin, SN-38, and their derivatives, such as ethanotecan derivative DXd), chachiomycin drugs (e.g., chachiomycin, N-acetyl-γ-calicheamicin), pyrrolobenzodiazepine (PBD) drugs (trametesin, DSB-120, and SJG-136), TLR agonists, STING agonists, etc.

[0444] In some embodiments, the active agent linked to the first homodimer antibody is the same as the active agent linked to the second homodimer antibody, specifically selected from eribulin or its derivatives, or esanotecan or its derivatives (e.g., DXd, deuterated esanotecan, deuterated DXd, such as DDDXd), or olistatin drugs.

[0445] In some embodiments, the active agent linked to the first homodimeric antibody is different from the active agent linked to the second homodimeric antibody, specifically selected from eribulin or its derivatives, or ethanotecan or its derivatives (e.g., DXd, deuterated ethanotecan, deuterated DXd, such as DDDXd), or olprestatin drugs (e.g., MMAE or MMAF). In some embodiments, the active agent of one of the first and second homodimeric conjugates is eribulin or its derivative, and the active agent of the other homodimeric conjugate is ethanotecan or its derivative. In some embodiments, the active agent of the first homodimeric conjugate is eribulin or its derivative, and the active agent of the second homodimeric conjugate is ethanotecan or its derivative. In some embodiments, the active agent of the first homodimeric conjugate is ethanotecan or its derivative, and the active agent of the second homodimeric conjugate is eribulin or its derivative. In some embodiments, the active agent linked to the first half-antibody is MMAE, and the active agent linked to the second half-antibody is MMAF. In some embodiments, the active agent linked to the first half-antibody is MMAF, and the active agent linked to the second half-antibody is MMAE.

[0446] In some embodiments, one of the activators in the first and second homodimer coupling agents has the structure shown in Formula I, and the other activator has the structure shown in Formula II.

[0447] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0448] R3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0449] In some embodiments, the activator of the first homodimeric coupling compound has the structure shown in Formula I, and the activator of the second homodimeric coupling compound has the structure shown in Formula II.

[0450] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0451] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0452] In some embodiments, the activator of the first homodimeric coupling compound has the structure shown in Formula II, and the activator of the second homodimeric coupling compound has the structure shown in Formula I.

[0453] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0454] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6 All are hydrogen atoms.

[0455] In some embodiments, the first homodimeric antibody is transmitted via a linker with the structure shown in Formula IIIa.

[0456] The structure shown in formula Ia is connected.

[0457] The second homodimeric antibody is connected to the structure shown in Formula IIa via a linker of the structure shown in Formula IIIa.

[0458] The homodimeric antibody is attached to the position shown * in the linker, and the structures shown in Formula Ia and Formula IIa are attached to the positions shown # in the linker.

[0459] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0460] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R 5 and R 6All are hydrogen atoms.

[0461] In some embodiments, the first homodimeric antibody is transmitted via a linker with the structure shown in Formula IIIa.

[0462] The structure shown in Formula IIa is connected.

[0463] The second homodimeric antibody is connected to the structure shown in Formula Ia via a linker of Formula IIIa.

[0464] The homodimeric antibody is attached to the position shown * in the linker, and the structures shown in Formula Ia and Formula IIa are attached to the positions shown # in the linker.

[0465] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5- to 12-membered heteroaryl groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 Cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S), optionally substituted C 6-10 Aryl, optionally substituted 5 to 12-membered heteroaryl groups (e.g., containing 1 to 3 heteroatoms selected from N, O, or S); or, R 1 and R 2 Together with the atoms connected thereto, they form optional substituted 5- to 8-membered heterocyclic groups (e.g., containing 1-3 heteroatoms selected from N, O, or S);

[0466] R 3 R 4 R 5 and R 6 Each atom is independently selected from either hydrogen or deuterium. More preferably, R... 1 R 2 R 5 and R 6 All are hydrogen atoms, R 3 R 4 All are deuterium atoms. Another preferred option is R. 1 R 2 R 3 R 4 R5 and R 6 All are hydrogen atoms.

[0467] In some of the above embodiments, the R 1 and R 2 Each is independently selected from hydrogen atoms or C atoms. 1-5 Alkyl (preferably C) 1-4 Alkyl, such as C 1-3 Alkyl group). In some of the above embodiments, the R 1 and R 2 Each is independently selected from hydrogen atoms, methyl, ethyl, propyl, or isopropyl. In some of the above embodiments, the R... 1 and R 2 It is a hydrogen atom. In some of the above embodiments, the R... 3 For deuterium atoms, R 4 It is a deuterium atom.

[0468] In some implementations, the C 1-6 Alkyl, C 3-7 cycloalkyl, 3- to 7-membered heterocyclic groups, C 6-10 The aryl group or 5- to 12-membered heteroaryl group may be substituted by one or more groups selected from the following: deuterium, hydroxyl, halogen, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl groups.

[0469] In some of the above embodiments, the linker of the structure shown in IIIa is attached to the disulfide bond site of the homodimeric antibody at the position shown in *, preferably attached to the first homodimeric antibody and / or the second homodimeric antibody via a thioether bond.

[0470] In some of the above embodiments, the DAR of the antibody-conjugate prepared by the method of this disclosure is 1-10, 2-8, 3-8, 4-8, 3.5-4.5, 6-8, 7-8, 7.5-8, 7.6-8, 7.7-8, 7.8-8, or 7.9-8. In some embodiments, the DAR of the antibody-conjugate prepared by the method of this disclosure is 2, 3, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 5, 6, 7, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.

[0471] In the methods provided in this disclosure, the homodimeric antibody includes, but is not limited to, monoclonal antibodies, monospecific antibodies, and nanobodies. In the methods provided in this disclosure, the homodimeric antibody can bind to any disease-related antigen known in the art. When the disease is a tumor, the antigen can be selected from any tumor-related antigen, including but not limited to HER2, HER3, EGFR, ROR1, CLDN18.2, B7-H3, B7-H4, TROP-2, CD19, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, PD-1, or PD-L1. In some embodiments, the homodimeric antibody targets HER2, HER3, EGFR, ROR1, CLDN18.2, B7-H3, B7-H4, TROP-2, CD19, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, PD-1, or PD-L1. In one specific embodiment, the first homodimer targets c-MET, and the second homodimer targets MUC1. In one specific embodiment, the first homodimer targets B7-H3, and the second homodimer targets B7-H4. In one specific embodiment, the first homodimer targets c-MET, and the second homodimer targets EGFR. In one specific embodiment, the first homodimer targets CD19, and the second homodimer targets CD20. In one specific embodiment, the first homodimer targets CD33, and the second homodimer targets CD70. In one specific implementation, the first homodimer targets EGFR, and the second homodimer also targets EGFR. In some implementations, the aforementioned targets of the first and second homodimers can be interchanged; for example, the first homodimer targets MUC1, and the second homodimer targets c-MET.

[0472] In some embodiments, the antibody-conjugates or pharmaceutically acceptable salts thereof provided in this disclosure exhibit one or more combinations of the following properties:

[0473] (1) Combined with human c-MET;

[0474] (2) Combined with human MUC1;

[0475] (3) It exhibits internalization in cells expressing c-MET;

[0476] (4) It exhibits internalization in cells expressing MUC1;

[0477] (5) It has killing activity against tumor cells expressing c-MET and / or MUC1;

[0478] (6) It has therapeutic effects on diseases related to c-MET and / or MUC1 expression; and

[0479] (7) It has the bystander effect.

[0480] In some embodiments, the antibody-conjugate or its pharmaceutically acceptable salt prepared by the methods of this disclosure exhibits one or more combinations of the following properties:

[0481] (1) Combined with human B7-H3;

[0482] (2) Combined with human B7-H4;

[0483] (3) It exhibits internalization in cells expressing B7-H3;

[0484] (4) It exhibits internalization in cells expressing B7-H4;

[0485] (5) It has killing activity against tumor cells expressing B7-H3 and / or B7-H4;

[0486] (6) It has therapeutic effects on diseases related to B7-H3 and / or B7-H4 expression; and

[0487] (7) It has the bystander effect.

[0488] In some embodiments, the antibody-conjugate or its pharmaceutically acceptable salt prepared by the methods of this disclosure exhibits one or more combinations of the following properties:

[0489] (1) Combined with human c-MET;

[0490] (2) Combined with human EGFR;

[0491] (3) It exhibits internalization in cells expressing c-MET;

[0492] (4) It exhibits internalization in cells expressing EGFR;

[0493] (5) It has killing activity against tumor cells expressing c-MET and / or EGFR;

[0494] (6) It has therapeutic effects on diseases related to c-MET and / or EGFR expression; and

[0495] (7) It has the bystander effect.

[0496] In some embodiments, the antibody-conjugate or its pharmaceutically acceptable salt prepared by the methods of this disclosure exhibits one or more combinations of the following properties:

[0497] (1) Combined with human CD19;

[0498] (2) Combined with human CD20;

[0499] (3) It exhibits internalization in cells expressing CD19;

[0500] (4) It exhibits internalization in cells expressing CD20;

[0501] (5) It has killing activity against tumor cells expressing CD19 and / or CD20;

[0502] (6) It has therapeutic effects on diseases related to CD19 and / or CD20 expression; and

[0503] (7) It has the bystander effect.

[0504] In some embodiments, the antibody-conjugate or its pharmaceutically acceptable salt prepared by the methods of this disclosure exhibits one or more combinations of the following properties:

[0505] (1) Combined with human CD33;

[0506] (2) Combined with human CD70;

[0507] (3) It exhibits internalization in cells expressing CD33;

[0508] (4) It exhibits internalization in cells expressing CD70;

[0509] (5) It has killing activity against tumor cells expressing CD33 and / or CD70;

[0510] (6) It has therapeutic effects on diseases related to CD33 and / or CD70 expression; and

[0511] (7) It has the bystander effect.

[0512] Antibody-conjugates or pharmaceutically acceptable salts thereof prepared by the methods of this disclosure:

[0513] (1) It has achieved excellent efficacy and / or safety;

[0514] (2) It achieved excellent anti-tumor effects and / or safety;

[0515] (3) It has good in vivo efficacy;

[0516] (4) It has good in vivo antitumor activity;

[0517] (5) It has excellent safety features;

[0518] (6) Not easily aggregated; and / or

[0519] (7) It has better solubility (e.g., water solubility).

[0520] In some embodiments, the method provided in this disclosure can assemble high-purity target ADCs in vitro with high assembly efficiency, resulting in products with low aggregate and low molecular weight impurity content, and excellent thermal stability.

[0521] Pharmaceutical Composition

[0522] In one aspect, this disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof. In some embodiments, this disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other reagents. The pharmaceutical composition can be formulated using conventional techniques. The actual dose level of the active ingredient in the pharmaceutical composition can be varied as needed, and the pharmaceutical composition can be administered via any suitable route and manner.

[0523] use

[0524] This disclosure provides for the use of the antibody-drug conjugates of this disclosure or pharmaceutically acceptable salts thereof. This disclosure also provides for the use of the pharmaceutical compositions of this disclosure. Uses may include the treatment of tumors or autoimmune diseases.

[0525] In one aspect, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases expressing antigens bound to the heterodimeric antibody. In some embodiments, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors or autoimmune diseases. In some embodiments, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases expressing c-MET and / or MUC1. In some embodiments, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases expressing B7-H3 and / or B7-H4. In some embodiments, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases expressing c-MET and / or EGFR. In some embodiments, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases expressing CD19 and / or CD20. In some embodiments, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases expressing CD33 and / or CD70.

[0526] In one aspect, this disclosure provides the use of the pharmaceutical compositions of this disclosure in the preparation of a medicament for treating diseases expressing antigens to which the heterodimeric antibody binds. In some embodiments, this disclosure provides the use of the pharmaceutical compositions of this disclosure in the preparation of a medicament for treating tumors or autoimmune diseases. In some embodiments, this disclosure provides the use of the pharmaceutical compositions of this disclosure in the preparation of a medicament for treating diseases expressing c-MET and / or MUC1. In some embodiments, this disclosure provides the use of the pharmaceutical compositions of this disclosure in the preparation of a medicament for treating diseases expressing B7-H3 and / or B7-H4. In some embodiments, this disclosure provides the use of the pharmaceutical compositions of this disclosure in the preparation of a medicament for treating diseases expressing c-MET and / or EGFR. In some embodiments, this disclosure provides the use of the pharmaceutical compositions of this disclosure in the preparation of a medicament for treating diseases expressing CD19 and / or CD20. In some embodiments, this disclosure provides the use of the pharmaceutical compositions of this disclosure in the preparation of a medicament for treating diseases expressing CD33 and / or CD70.

[0527] In one aspect, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents, in the preparation of a medicament for treating diseases expressing antigens bound to the heterodimeric antibody. In some embodiments, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents, in the preparation of a medicament for treating tumors or autoimmune diseases. In some embodiments, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents, in the preparation of a medicament for treating diseases expressing c-MET and / or MUC1. In some embodiments, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents, in the preparation of a medicament for treating diseases expressing B7-H3 and / or B7-H4, c-MET and / or EGFR, CD19 and / or CD20, CD33 and / or CD70.

[0528] In one aspect, this disclosure provides the use of the pharmaceutical compositions of this disclosure and one or more additional therapeutic agents in the preparation of a medicament for treating diseases expressing antigens bound to the heterodimeric antibodies. In some embodiments, this disclosure provides the use of the pharmaceutical compositions of this disclosure and one or more additional therapeutic agents in the preparation of a medicament for treating tumors or autoimmune diseases. In some embodiments, this disclosure provides the use of the pharmaceutical compositions of this disclosure and one or more additional therapeutic agents in the preparation of a medicament for treating diseases expressing c-MET and / or MUC1. In some embodiments, this disclosure provides the use of the pharmaceutical compositions of this disclosure and one or more additional therapeutic agents in the preparation of a medicament for treating diseases expressing B7-H3 and / or B7-H4, c-MET and / or EGFR, CD19 and / or CD20, CD33 and / or CD70.

[0529] In one aspect, this disclosure provides a method of treating a disease expressing an antigen bound to the heterodimeric antibody, the method comprising administering to a subject an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, this disclosure provides a method of treating a tumor or autoimmune disease, the method comprising administering to a subject an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, this disclosure provides a method of treating a disease expressing c-MET and / or MUC1, the method comprising administering to a subject an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, this disclosure provides a method of treating a disease expressing B7-H3 and / or B7-H4, c-MET and / or EGFR, CD19 and / or CD20, CD33 and / or CD70, the method comprising administering to a subject an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure. In some embodiments, the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of this disclosure, is administered to the subject in a therapeutically effective amount.

[0530] In one aspect, this disclosure provides a method of treating a disease expressing an antigen bound to the heterodimeric antibody, the method comprising administering to a subject an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents. In some embodiments, this disclosure provides a method of treating a tumor or autoimmune disease, the method comprising administering to a subject an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents. In some embodiments, this disclosure provides a method of treating a disease expressing c-MET and / or MUC1, the method comprising administering to a subject an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents. In some embodiments, this disclosure provides a method of treating a disease expressing B7-H3 and / or B7-H4, c-MET and / or EGFR, CD19 and / or CD20, CD33 and / or CD70, the method comprising administering to a subject an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents. In some embodiments, the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, along with one or more other therapeutic agents, is administered to the subject in a therapeutically effective amount.

[0531] In one aspect, this disclosure provides a method for treating a disease expressing an antigen bound to the heterodimeric antibody, the method comprising administering to a subject a pharmaceutical composition of the present disclosure and one or more additional therapeutic agents. In some embodiments, this disclosure provides a method for treating a tumor or autoimmune disease, the method comprising administering to a subject a pharmaceutical composition of the present disclosure and one or more additional therapeutic agents. In some embodiments, this disclosure provides a method for treating a disease expressing c-MET and / or MUC1, the method comprising administering to a subject a pharmaceutical composition of the present disclosure and one or more additional therapeutic agents. In some embodiments, this disclosure provides a method for treating a disease expressing B7-H3 and / or B7-H4, c-MET and / or EGFR, CD19 and / or CD20, CD33 and / or CD70, the method comprising administering to a subject a pharmaceutical composition of the present disclosure and one or more additional therapeutic agents. In some embodiments, the pharmaceutical composition of the present disclosure and one or more additional therapeutic agents are administered to a subject in a therapeutically effective amount.

[0532] In some embodiments, the tumor is a tumor expressing c-MET and / or MUC1, B7-H3 and / or B7-H4, c-MET and / or EGFR, CD19 and / or CD20, CD33 and / or CD70. In some embodiments, the disease expressing c-MET and / or MUC1, B7-H3 and / or B7-H4, c-MET and / or EGFR, CD19 and / or CD20, CD33 and / or CD70 is a tumor expressing c-MET and / or MUC1, B7-H3 and / or B7-H4, c-MET and / or EGFR, CD19 and / or CD20, CD33 and / or CD70. In some embodiments, the additional therapeutic agent may be a tumor therapeutic agent known in the art.

[0533] In some embodiments, the method includes contacting tumor cells with the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, thereby killing tumor cells or inhibiting tumor cell growth.

[0534] In some embodiments, the method includes contacting tumor cells with the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, and simultaneously or sequentially contacting the tumor cells with one or more additional therapeutic agents, thereby killing tumor cells or inhibiting tumor cell growth.

[0535] In some embodiments, administration of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of this disclosure, to a subject can kill tumor cells or inhibit tumor cell growth.

[0536] In some embodiments, administration of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, along with one or more other therapeutic agents, to a subject can kill tumor cells or inhibit tumor cell growth. In some embodiments, administration of the pharmaceutical composition of this disclosure, along with one or more other therapeutic agents, to a subject can kill tumor cells or inhibit tumor cell growth.

[0537] In one aspect, this disclosure provides an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure, for treating diseases expressing antigens to which the heterodimeric antibody binds. In some embodiments, this disclosure provides an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure, for treating tumors or autoimmune diseases.

[0538] In one aspect, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of this disclosure, for treating diseases expressing antigens to which the heterodimeric antibody binds. In some embodiments, this disclosure provides the use of the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of this disclosure, for treating tumors or autoimmune diseases.

[0539] In some implementations, the tumor is biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, melanoma, pharyngeal cancer, oral cancer, skin cancer, lung cancer, urethral cancer, urothelial carcinoma, bone cancer, soft tissue cancer, gallbladder cancer, testicular cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, liver cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, or myeloma.

[0540] Reagent test kit

[0541] This disclosure provides a kit comprising the antibody-drug conjugate of this disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of this disclosure.

[0542] This disclosure describes kits comprising the antibody-drug conjugates described herein or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions described herein. The kits can be used to implement the use of the antibody-drug conjugates described herein or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions described herein, or for other uses. In some embodiments, the kit may include the antibody-drug conjugates described herein or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions described herein; optionally, the kit may also include instructions for use. The kit may also include other materials required from a commercial and user perspective, such as other buffers, diluents, needles, syringes, etc. Attached Figure Description

[0543] Figure 1 shows the binding curves of anti-MUC1 / anti-c-MET bispecific antitoxin ADC to HCA-7 cells as detected by FACS.

[0544] Figures 2A and 2B show the binding curves of anti-B7-H3 / anti-B7-H4 bispecific antitoxin ADC to MX-1 or MDA-MB-468 cells as detected by FACS. Figure 2A shows the binding curves of MX-1 cells, and Figure 2B shows the binding curves of MDA-MB-468 cells.

[0545] Figure 3 shows the killing activity of the anti-B7-H3 / anti-B7-H4 dual antitoxin ADC against NIH:OVCAR3 tumor cells.

[0546] Figure 4 shows the binding activity of the dual-antibody dual-toxin ADC on NCI-H292 cells.

[0547] Figure 5 shows the binding activity of the dual-antibody dual-toxin ADC on HCA-7 cells.

[0548] Figure 6 shows the internalization activity of the dual-antibody dual-toxin ADC on NCI-H292 cells.

[0549] Figure 7 shows the internalization activity of dual-antibody dual-toxin ADC on HCA-7 cells.

[0550] Figure 8 shows the killing activity of the dual-antibody dual-toxin ADC on NCI-H292 cells.

[0551] Figure 9 shows the killing activity of dual-antibody dual-toxin ADC on HCA-7 cells.

[0552] Figures 10A-10G show the hydrophobic chromatographic detection results of some bispecific antibody-dual toxin ADC samples. Figure 10A shows E-1 / C-1-DDDXd+Eribulin-4+4, Figure 10B shows E-3 / C-3-DDDXd+Eribulin-4+4, Figure 10C shows E-3 / C-4-DDDXd+Eribulin-4+4, Figure 10D shows E-4 / C-3-DDDXd+Eribulin-4+4, Figure 10E shows E-4 / C-4-DDDXd+Eribulin-4+4, Figure 10F shows E-5 / C-5-DDDXd+Eribulin-4+4, and Figure 10G shows E-6 / C-6-DDDXd+Eribulin-4+4.

[0553] Figures 11A-11D show exemplary structures of antibody-conjugates of this disclosure. Figure 11A shows a bivalent monospecific antibody-dual toxin ADC, with the antigen-binding portion being Fab, and the monospecific antibody linking two different toxins. Figure 11B shows a bivalent bispecific antibody-dual toxin ADC, with the antigen-binding portion being Fab, and the bispecific antibody linking two different toxins. Figures 11C and 11D show half-antibody-linked toxins.

[0554] Explanation and definition

[0555] Unless otherwise stated, the following terms as used in this disclosure have the following meanings. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0556] The terms "first" or "second" used in this disclosure to refer to halves, antigen-binding moieties, Fc peptides, Fc domains, CH3 regions, homodimeric antibodies, reaction solutions, etc., are used for ease of distinction when more than one type of moieties is present. Unless explicitly stated otherwise, the use of these terms is not intended to assign a specific order, orientation, or priority.

[0557] The term "substituted" refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxoation does not occur on the aromatic group. "Optionally substituted" means that it may or may not be substituted, unless otherwise specified; the type and number of substituents can be arbitrary to the extent chemically feasible.

[0558] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Therefore, for example, if a group is substituted by two Rs, each R has an independent option.

[0559] The term "thiol" refers to the -SH group.

[0560] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration representing the center of a solid.

[0561] Unless otherwise specified, when a group has a connectable site, the connection position of that site with other groups can be indicated by an asterisk (*) or a hash (#).

[0562] As used in this article, compounds formed by replacing atoms or groups of atoms in a parent compound (e.g., ADC, linker-payload) molecule with other atoms or groups of atoms are called “derivatives” of the parent compound.

[0563] The compounds disclosed herein may exist in specific geometric or stereoisomer forms. This disclosure envisions all such compounds, including cis and trans isomers, levorotatory and dextrorotatory isomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this disclosure.

[0564] Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" arise because the single bonds of double bonds or cyclic carbon atoms cannot rotate freely.

[0565] Unless otherwise stated, the term "enantiomer" refers to stereoisomers that are mirror images of each other.

[0566] Unless otherwise stated, the term "diastereomer" refers to a stereoisomer of a molecule having two or more chiral centers and being in a non-mirror relationship with each other.

[0567] The compounds and intermediates disclosed herein may also exist in different tautomer forms, and all such forms are included within the scope of this disclosure. The terms "tautomer" or "tautomer form" refer to structural isomers of different energies that can interconvert via low energy barriers. For example, proton tautomers (also known as proton transfer tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerization. A specific example of a proton tautomer is the imidazole moiety, in which a proton can migrate between two ring nitrogens. Valence tautomers include interconversions via the recombination of some bonding electrons.

[0568] The term "treatment" means administering the compounds (e.g., ADCs) or pharmaceutical compositions described in this disclosure to improve or eliminate a disease or one or more symptoms associated with said disease, and includes, but is not limited to:

[0569] (i) Suppress the disease or disease state, that is, curb its development;

[0570] (ii) Relieve the disease or disease state, even if the disease or disease state subsides;

[0571] (iii) To reduce any direct or indirect pathological consequences of disease or disease state.

[0572] The term "therapeutic effective amount" means (i) the amount of the disclosed compound used to treat a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the disclosed compound or pharmaceutical composition constituting a "therapeutic effective amount" can vary depending on factors such as the compound or pharmaceutical composition and its ability to elicit a desired response in an individual, the disease state and its severity, the route of administration, and the age, sex, and weight of the mammal to be treated. Therapeutic effective amounts can also routinely be determined by those skilled in the art based on their own knowledge and the content of this disclosure.

[0573] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0574] The term "pharmaceutically acceptable salt" refers to a salt of a compound (such as the antibody-conjugate of this disclosure) that is safe and effective when used in mammals and has the intended biological activity. For example, it can be a metal salt, an ammonium salt, a salt formed with an organic base, a salt formed with an inorganic acid, a salt formed with an organic acid, a salt formed with a basic or acidic amino acid, etc.

[0575] The term "excipient" refers to any component other than the active ingredient (e.g., the antibody-conjugate of this disclosure). The selection of excipients will largely depend on factors such as the specific method of administration, the efficacy of the excipient in terms of solubility and stability, and the nature of the dosage form.

[0576] The term "solvent" refers to a substance formed by the association of a compound with solvent molecules.

[0577] The terms “Fc domain,” “Fc,” or “Fc region” are used herein to define the C-terminal region of the immunoglobulin heavy chain, which contains at least a portion of the constant region. This term includes both native sequence Fc and variant Fc. The C-terminal lysine (Lys447) of the Fc may or may not be present. Unless otherwise stated, the amino acid residues in the Fc or constant region are numbered according to the EU numbering system, also known as the EU index. As used herein, one “Fc polypeptide” of the Fc domain refers to one of the two polypeptides that form the dimer Fc domain. For example, the Fc polypeptide of the IgG Fc domain contains IgG CH2 and IgG CH3.

[0578] The term "antibody" is used in its broadest sense to encompass natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), as well as various antibody structures of single-chain antibodies, as long as they exhibit the desired antigen-binding activity.

[0579] The term "multispecific" refers to an antibody's ability to specifically bind to multiple different antigenic determinants, such as two or more different antigenic determinants. In this article, antigenic determinant is synonymous with antigenic epitope. Typically, a bispecific antibody contains two antigen-binding sites, each specific to a different antigenic determinant. Different antigenic determinants can be expressed on the same or different cells. Different antigenic determinants can differ depending on the antigen (e.g., binding to antigens EGFR and c-Met) or can be present on the same antigen. An antigenic determinant is a specific chemical group with a certain composition and structure on the surface or other sites of an antigen molecule, capable of specifically binding to its corresponding antibody or sensitized lymphocyte. An example of an antigenic determinant is c-Met, which has multiple antigenic determinants with known or unknown structures. In this article, any antibody that can bind to two different antigenic determinants on an antigen is called a bispecific antibody. A specific bispecific antibody, for example, can bind to EGFR and c-Met.

[0580] The term "N-valent antibody" indicates that the antibody has N antigen-binding sites. For example, "bivalent antibody" or "antibody is bivalent" means that the antibody has two antigen-binding sites, and "trivalent antibody" or "antibody is trivalent" means that the antibody has three antigen-binding sites. Natural human immunoglobulin molecules typically have two antigen-binding sites, Fab molecules typically have a single antigen-binding site, and monovariable domain and scFv molecules typically have a single antigen-binding site.

[0581] The term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. The specific antigen-binding moiety can be Fab, scFv, or a single variable domain.

[0582] The term "single variable domain (or VHH)" refers to a variable domain that can specifically bind to an antigenic epitope without pairing with other variable domains. A single variable domain typically has three CDRs (CDR1, CDR2, and CDR3) located on a single domain.

[0583] The term "Fab" refers to a protein composed of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of an immunoglobulin. In this text, Fab refers to the Fab molecule in its native or modified form, specifically the Fab heavy chain (VH-CH1, natively N-to-C-terminal) consisting of the VH variable region and CH1 constant region of the heavy chain, and the Fab light chain (VL-CL, natively N-to-C-terminal) consisting of the VH variable region and CL constant region of the light chain. A modified Fab can be, for example, a Fab with amino acid substitutions introduced into the CH1 / CL domain and / or the VH / VL domain. A specific example of a modified Fab is a Fab with amino acid substitutions introduced into the CL domain.

[0584] The term "scFv" encompasses the VH and VL domains of an immunoglobulin, wherein these domains are present within a single polypeptide chain. In some embodiments, the scFv also includes a peptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding.

[0585] The antibodies disclosed herein may be IgG1, IgG2, IgG3, or IgG4 isotypes. The term "isotype" refers to the type of antibody encoded by the heavy chain constant region gene. In some embodiments, the antibodies disclosed herein are IgG1 isotypes. The antibodies disclosed herein may be derived from any species, including but not limited to mice, rats, rabbits, non-human primates (such as chimpanzees, cynomolgus monkeys, spider monkeys, and rhesus monkeys), llamas, and humans. The antibodies disclosed herein may be murine antibodies, chimeric antibodies, humanized antibodies, or human antibodies.

[0586] The term "mouse antibody" or "mouse-derived antibody" refers to an antibody in which both the backbone region and the CDR region in the variable region are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from mouse germline immunoglobulin sequences. The murine antibodies of this disclosure may include amino acid residues not encoded by mouse germline immunoglobulin sequences, but "mouse antibody" or "mouse-derived antibody" does not include antibodies in which a CDR sequence derived from other mammalian germlines is inserted into the mouse backbone sequence.

[0587] Chimeric antibodies are antibodies formed by fusing the variable region of a murine antibody with the constant region of a human antibody. They can reduce the immune response induced by murine antibodies. To create chimeric antibodies, firstly, a hybridoma that secretes murine-specific monoclonal antibodies is established. Then, the variable region gene is cloned from the hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody is expressed in a eukaryotic or prokaryotic system.

[0588] "Humanized antibody" is an antibody that contains a complementarity-determining region (CDR) derived from a non-human antibody, a backbone region derived from a human antibody, and a constant region.

[0589] The term "CDR" (complementarity-determining region) is also known as the "hypervariant region." Natural four-chain antibodies typically contain six CDRs: three in the heavy chain variable region and three in the light chain variable region.

[0590] The term "variable region" refers to a domain of approximately 100 to 110 or more amino acids, defined by the N-terminal domain of the light or heavy chain of an antibody, that is primarily responsible for antigen recognition. The terms light chain variable region (VL) and heavy chain variable region (VH) refer to these light chain and heavy chain domains, respectively.

[0591] The term "half-antibody" or "half-body" refers to a portion of an antibody that forms an antibody structure with another half-antibody. In this disclosure, a half-antibody refers to an antibody structure that contains at least an antibody CH3 polypeptide. For example, in one specific example, a half-antibody contains an Fc polypeptide, and the two Fc polypeptides of two half-antibodies associate to form a dimer antibody. In another specific example, a half-antibody contains a linked Fab antigen-binding moiety and an Fc polypeptide. Alternatively, a half-antibody is a molecule that constitutes half of a complete monoclonal antibody (i.e., one heavy chain and one light chain). In this document, the half-antibody may further contain additional antigen-binding moieties in addition to the antigen-binding moiety it contains; that is, the half-antibody may contain multiple antigen-binding moieties (e.g., two Fab fragments linked together, two VHHs linked together, or VHHs and Fab linked together). In other embodiments, a half-antibody contains a linked VHH antigen-binding moiety and an Fc polypeptide.

[0592] As used in this article, the term "EC" 50 "Colony" refers to the half-maximal effective concentration, which is the antibody concentration at which the induced response is 50% of the maximum response, i.e., half the distance between the maximum response and the baseline. 50 It can be measured by ELISA or FACS analysis or any other method known in the art.

[0593] “K D "Refers to the equilibrium dissociation constant, which is the self-dissociation rate constant (k d ) relative binding rate constant (k a (that is, k) d / k a The ratio of K to α is expressed as molar concentration (M). The K of the antibody D The value can be determined using methods well-established in the art. The K value used for antibody determination... D The preferred method is to use surface plasmon resonance (SPR) technology, preferably using a biosensor system such as the Biacore surface plasmon resonance system for analysis.

[0594] The term "DAR" refers to the drug-antibody ratio, which means the average number of effective payloads conjugated to the antibody or antibody fragment in an antibody-drug conjugate.

[0595] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Preferably, the subject according to this disclosure is a human. Unless otherwise stated, the terms "patient" or "subject" may be used interchangeably.

[0596] As used herein, “about” means within the acceptable range of error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” may, in accordance with art practice, mean within one or more standard deviations. Alternatively, “about” may mean a range of up to ±5%, such as fluctuations within ±2%, ±1%, or ±0.5% of a given specific numerical range. When a particular value is given in the scope of this disclosure, unless otherwise stated, “about” shall be understood to mean within the acceptable range of error for that particular value. In this document, unless otherwise stated, the values ​​of step parameters or conditions are implicitly modified by “about”.

[0597] The terms “comprise,” “containing,” and “comprising” and their equivalents (e.g., contain, contains, containing, include, includes, and including) shall be understood as “including but not limited to,” meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be covered.

[0598] In this document, unless the context clearly indicates otherwise, singular terms encompass plural referents, and vice versa. Detailed Implementation

[0599] This disclosure also provides the following specific implementation schemes, but the scope of protection of this disclosure is not limited thereto:

[0600] Implementation Scheme 1. An antibody-conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent, the heterodimeric antibody comprising a first hapten and a second hapten, the first hapten comprising a first CH3 region, and the second hapten comprising a second CH3 region, wherein, according to EU designation,

[0601] The first and / or second CH3 region has an amino acid Met, Ile or Leu at position 366, an amino acid Trp, Phe, Met, Tyr, Ile, Leu, Lys or Arg at position 394, an amino acid Ala, Thr, Ser, Asp or Glu at position 405, and / or an amino acid Thr, Ala or Val at position 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different;

[0602] Furthermore, the first half-antibody is connected to an active agent via a connector, and the second half-antibody is connected to an active agent via a connector. The active agent connected to the first half-antibody may be the same as or different from the active agent connected to the second half-antibody.

[0603] Implementation Scheme 2. The antibody-conjugate or its pharmaceutically acceptable salt according to Implementation Scheme 1, wherein the heterodimeric antibody comprises an amino acid substitution that promotes the formation of the heterodimer, the amino acid substitution being as follows: the first and / or second CH3 region has an amino acid Met, Ile, or Leu at position 366, an amino acid Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394, an amino acid Ala, Thr, Ser, Asp, or Glu at position 405, and / or an amino acid Thr, Ala, or Val at position 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0604] Implementation Scheme 3. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in Implementation Scheme 1 or 2, wherein, according to the EU designation,

[0605] (1) The first CH3 region has Met, Ile, or Leu at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407; and / or

[0606] (2) The first CH3 region has Ala, Thr, Ser, Asp, Glu or Gly at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys or Arg at bit 394.

[0607] Implementation Scheme 4. The antibody-conjugate or its pharmaceutically acceptable salt according to Implementation Scheme 3, wherein the first CH3 region has Met or Leu at position 366, and the second CH3 region has Thr or Ala at position 407.

[0608] Implementation Scheme 5. The antibody-conjugate or its pharmaceutically acceptable salt according to Implementation Scheme 4, wherein the first CH3 region has Met at position 366 and the second CH3 region has Thr at position 407; the first CH3 region has Met at position 366 and the second CH3 region has Ala at position 407; the first CH3 region has Leu at position 366 and the second CH3 region has Thr at position 407; or the first CH3 region has Leu at position 366 and the second CH3 region has Ala at position 407.

[0609] Implementation Scheme 6. The antibody-conjugate or its pharmaceutically acceptable salt according to Implementation Scheme 3, wherein the first CH3 region has Ala, Thr, Ser or Asp at position 405, and the second CH3 region has Trp, Phe, Tyr or Lys at position 394.

[0610] Implementation Scheme 7. The antibody-conjugate or its pharmaceutically acceptable salt according to Implementation Scheme 6, wherein the first CH3 region has Ala at position 405 and the second CH3 region has Trp at position 394; the first CH3 region has Ala at position 405 and the second CH3 region has Phe at position 394; the first CH3 region has Ala at position 405 and the second CH3 region has Tyr at position 394; the first CH3 region has Ala at position 405 and the second CH3 region has Lys at position 394; the first CH3 region has Thr at position 405 and the second CH3 region has Trp at position 394; the first CH3 region has Thr at position 405 and the second CH3 region has Phe at position 394; the first CH3 region has Thr at position 405 and the second CH3 region has Tyr at position 394; the first CH3 region has Thr at position 405 and the... The second CH3 region has Lys at bit 394; the first CH3 region has Ser at bit 405, and the second CH3 region has Trp at bit 394; the first CH3 region has Ser at bit 405, and the second CH3 region has Phe at bit 394; the first CH3 region has Ser at bit 405, and the second CH3 region has Tyr at bit 394; the first CH3 region has Ser at bit 405, and the second CH3 region has Lys at bit 394; the first CH3 region has Asp at bit 405, and the second CH3 region has Trp at bit 394; the first CH3 region has Asp at bit 405, and the second CH3 region has Phe at bit 394; the first CH3 region has Asp at bit 405, and the second CH3 region has Tyr at bit 394; or, the first CH3 region has Asp at bit 405, and the second CH3 region has Lys at bit 394.

[0611] Implementation Scheme 8. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-7, wherein the first CH3

[0612] The region is the CH3 region of IgG, preferably the CH3 region of human IgG.

[0613] Implementation Scheme 9. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-8, wherein the first CH3

[0614] The region is a CH3 region selected from IgG1, IgG2, IgG3 and IgG4, preferably a CH3 region selected from IgG1 and IgG4.

[0615] Implementation Scheme 10. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-9, wherein the second CH3

[0616] The region is the CH3 region of IgG, preferably the CH3 region of human IgG.

[0617] Implementation Scheme 11. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-10, wherein the second CH3

[0618] The region is a CH3 region selected from IgG1, IgG2, IgG3 and IgG4, preferably a CH3 region selected from IgG1 and IgG4.

[0619] Implementation Scheme 12. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-11, wherein the first CH3

[0620] Both the first and second CH3 regions are CH3 regions of human IgG1 or CH3 regions of human IgG4.

[0621] Implementation Scheme 13. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-12, wherein the first CH3

[0622] The sequences of the first CH3 region and the second CH3 region are different, which makes the heterodimer interaction between the first CH3 region and the second CH3 region stronger than the homodimer interaction between the first CH3 region and the second CH3 region.

[0623] Implementation Scheme 14. An antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-13, wherein the first half-antibody comprises a first Fc polypeptide, the first Fc polypeptide comprises a first CH3 region, the second half-antibody comprises a second Fc polypeptide, the second Fc polypeptide comprises a second CH3 region, and the first Fc polypeptide and the second Fc polypeptide constitute an Fc domain.

[0624] Implementation Scheme 15. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to Implementation Scheme 14, wherein the Fc domain is human IgG Fc

[0625] The preferred domains are IgG1 Fc domain, IgG2 Fc domain, IgG3 Fc domain or IgG4 Fc domain.

[0626] Implementation Scheme 16. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to Implementation Scheme 14 or 15, wherein the Fc domain comprises an amino acid substitution that reduces or eliminates the binding of the CH3 region of an Fc polypeptide in the Fc domain to protein A.

[0627] Implementation Scheme 17. An antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-16, wherein the first half-antibody comprises a first antigen-binding portion and the second half-antibody comprises a second antigen-binding portion.

[0628] Implementation Scheme 18. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-17, wherein the first half

[0629] The antibody comprises a first antigen-binding moiety and a first Fc polypeptide, and the second half-antibody comprises a second antigen-binding moiety and a second Fc polypeptide.

[0630] Implementation Scheme 19. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 17-18, wherein the first antigen-binding portion and the second antigen-binding portion bind the same or different antigens, or bind the same or different epitopes.

[0631] Implementation Scheme 20. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 17-19, wherein the first antigen-binding portion and the second antigen-binding portion are each independently Fab, ScFv, VHH or ScFab, preferably, both the first antigen-binding portion and the second antigen-binding portion are Fab.

[0632] Implementation Scheme 21. An antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-20, wherein the heterodimeric antibody comprises a Cys-Pro-Pro-Cys sequence in the hinge region.

[0633] Implementation Scheme 22. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-21, wherein the active agent to which the first half-antibody and the second half-antibody are linked is independently selected from: microtubule inhibitors, DNA damaging agents, immunomodulators, topoisomerase inhibitors or other active agents.

[0634] Implementation Scheme 23. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-22, wherein the active agent to which the first and second half-antibody are linked is each independently selected from: eribulin or a derivative thereof, olipatin, MMAF, MMAE, maytansine, DM1, DM4, camptothecin, hydroxycamptothecin, aminocamptothecin, irinotecan, topotecan, esaxatecan or a derivative thereof, rubitecan, letopotecan, gimarotecan, kareniteci n, 7-ethylcamptothecin, SN-38, DXd, chachiin, N-acetyl-γ-calicheamicin, atrazomycin, DSB-120, SJG-136, TLR agonists, STING agonists, paclitaxel, paclitaxel liposomes, albumin-bound paclitaxel, cabazitaxel, docetaxel, oxaliplatin, cisplatin, carboplatin, nedaplatin, bicycloplatin, miplatin, lobaplatin, pyrplatin, levoplatin, triplatinum tetranitrate, phenanthreneplatin and saplatin and IRDye700DX.

[0635] Implementation Scheme 24. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-23, wherein,

[0636] (1) The active agent linked to the first half-antibody is eribulin or a derivative thereof, and the active agent linked to the second half-antibody is essanotecan or a derivative thereof; or

[0637] (2) The active agent linked to the first half-antibody is essanotecan or its derivative, and the active agent linked to the second half-antibody is eribulin or its derivative.

[0638] Implementation Scheme 25. The antibody-conjugate or a pharmaceutically acceptable salt thereof as described in Implementation Scheme 24, wherein,

[0639] (1) The active agent linked to the first half-antibody has the structure shown in Formula I, and the active agent linked to the second half-antibody has the structure shown in Formula II; or;

[0640] (2) The active agent linked to the first half-antibody has the structure shown in Formula II, and the active agent linked to the second half-antibody has the structure shown in Formula I.

[0641] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6-10 aryl, optionally substituted 5 to 12 heteroaryl groups, R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6- 10 aryl, optionally substituted 5 to 12 heteroaryl groups; or, R 1 and R 2 Together with the atoms connected to it, they form optional 5- to 8-membered heterocyclic groups;

[0642] R 3 R 4 R 5 and R 6 Each is independently selected from either hydrogen or deuterium atoms.

[0643] Implementation Scheme 26. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-25, wherein,

[0644] (1) The first hapten antibody is transmitted through a linker with the structure shown in Formula IIIa below.

[0645] The structure shown in formula Ia is connected.

[0646] The second half-antibody is connected to the structure shown in Formula IIa via a linker of the structure shown in Formula IIIa.

[0647] or

[0648] (2) The first hapten via a linker with the structure shown in Formula IIIa

[0649] The structure shown in Formula IIa is connected.

[0650] The second half-antibody is connected to the structure shown in Formula Ia via a linker of the structure shown in Formula IIIa.

[0651] The hapten is attached to the linker at the position shown *, and the structures shown in Formula Ia and Formula IIa are attached to the linker at the position shown #, wherein R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6-10 aryl, optionally substituted 5 to 12 heteroaryl groups, R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6- 10 aryl, optionally substituted 5 to 12 heteroaryl groups; or, R 1 and R 2 Together with the atoms connected to it, they form optional 5- to 8-membered heterocyclic groups;

[0652] R 3 R 4 R 5 and R 6 Each is independently selected from either hydrogen or deuterium atoms.

[0653] Implementation Scheme 27. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to Implementation Scheme 25 or 26, wherein the R 1 and R 2 respective

[0654] Independently selected from hydrogen atoms or C atoms 1-5 alkyl.

[0655] Implementation Scheme 28. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to Implementation Scheme 27, wherein the R 1 and R 2 Independent

[0656] The R is selected from hydrogen atom, methyl, ethyl, propyl or isopropyl; preferably, the R 1 and R 2 It is a hydrogen atom.

[0657] Implementation Scheme 29. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 25 or 26, wherein the R 3

[0658] For deuterium atoms, R 4 It is a deuterium atom; or R 3 For hydrogen atoms, R 4 It is a hydrogen atom.

[0659] Implementation Scheme 30. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-29, wherein the antibody-conjugate

[0660] The DAR values ​​of the objects are 1-10, 2-8, 3.5-8, 4-8, 3.5-4.5, 6-8, 7-8, 7.5-8, 7.6-8, 7.7-8, 7.8-8, or 7.9-8.

[0661] Implementation Scheme 31. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-30, wherein the DAR is 2, 3, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 5, 6, 7, 7.5, 7.6, 7.7, 7.73, 7.79, 7.8, 7.86, 7.9, or 8.

[0662] Implementation Scheme 32. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-31, wherein the heterodimeric antibody is a heterodimeric antibody targeting one or two targets selected from HER2, HER3, EGFR, ROR1, CLDN18.2, B7-H3, B7-H4, TROP-2, CD19, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, PD-1, and PD-L1.

[0663] Implementation Scheme 33. A method for preparing an antibody-conjugate or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0664] (1) Provides a first homodimeric antibody comprising a first CH3 region, wherein, according to the EU number, the first CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407;

[0665] (2) The first homodimer antibody was treated with a reducing agent to reduce the interchain disulfide bonds of the first homodimer antibody; the first homodimer antibody with thiol group was linked to the active agent through a coupling reaction and purified to obtain the first homodimer conjugate.

[0666] (3) Provide a second homodimer antibody comprising a second CH3 region, wherein, according to the EU number, the second CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0667] (4) The second homodimer antibody is treated with a reducing agent to reduce the interchain disulfide bonds of the second homodimer antibody; the second homodimer antibody with thiol group is coupled to the active agent through a coupling reaction, and the second homodimer conjugate is purified.

[0668] Wherein, the active agent linked to the first homodimeric antibody may be the same as or different from the active agent linked to the second homodimeric antibody;

[0669] (5) Incubate the first homodimeric conjugate and the second homodimeric conjugate together; and

[0670] (6) Obtain the antibody-conjugate or a pharmaceutically acceptable salt thereof.

[0671] Implementation Scheme 34. A method for preparing an antibody-conjugate or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0672] (1) Provides a first homodimeric antibody comprising a first CH3 region, wherein, according to the EU number, the first CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407;

[0673] (2) The first homodimer antibody is treated with a reducing agent to reduce the interchain disulfide bonds of the first homodimer antibody; through a coupling reaction, the first homodimer antibody with thiol groups is linked to the active agent through a linker to obtain the first reaction solution;

[0674] (3) Provide a second homodimer antibody comprising a second CH3 region, wherein, according to the EU number, the second CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0675] (4) The second homodimer antibody is treated with a reducing agent to reduce the interchain disulfide bonds of the second homodimer antibody; through a coupling reaction, the second homodimer antibody with thiol groups is linked to the activator through a linker to obtain the second reaction solution;

[0676] Wherein, the active agent linked to the first homodimeric antibody may be the same as or different from the active agent linked to the second homodimeric antibody;

[0677] (5) Incubate the first reaction solution together with the second reaction solution; and

[0678] (6) Obtain the antibody-conjugate or a pharmaceutically acceptable salt thereof.

[0679] Implementation Scheme 35. A method for preparing an antibody-conjugate or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0680] (1) Provides a first homodimeric antibody comprising a first CH3 region, wherein, according to the EU number, the first CH3 region has at least one amino acid substitution at positions 366, 368, 370, 399, 405, 407 and / or 409;

[0681] (2) The first homodimeric antibody is linked to the active agent through a linker to obtain the first homodimeric conjugate;

[0682] (3) Provide a second homodimer antibody comprising a second CH3 region, wherein, according to the EU number, the second CH3 region has at least one amino acid substitution at positions 366, 368, 370, 399, 405, 407 and / or 409, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0683] (4) The second homodimeric antibody is linked to the active agent through a linker to obtain the second homodimeric conjugate;

[0684] Wherein, the active agent linked to the first homodimeric antibody may be the same as or different from the active agent linked to the second homodimeric antibody;

[0685] (5) Incubate the first homodimeric coupling compound and the second homodimeric coupling compound together in the presence of a reducing agent, wherein the presence of the reducing agent is sufficient to allow the reduction of interchain disulfide bonds in the hinge regions of the first and second homodimeric coupling compounds, and

[0686] (6) Obtain the antibody-conjugate or a pharmaceutically acceptable salt thereof.

[0687] Implementation Scheme 36. The method according to any one of Implementation Schemes 33-35, wherein step (6) comprises subjecting the reaction solution obtained in step (5) to oxidative conditions sufficient to allow cysteine ​​in the antibody-conjugate to be oxidized to interchain disulfide bonds; or step (6) comprises removing a reducing agent from the reaction system.

[0688] Implementation Scheme 37. The method according to any one of Implementation Schemes 33-36, wherein the reducing agent is selected from: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dierythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, β-mercaptoethanol, or salts thereof; preferably, the reducing agent is selected from: 2-MEA, DTT and TCEP, or salts thereof.

[0689] Implementation Scheme 38. The method according to any one of Implementation Schemes 33-37, wherein the first homodimeric antibody and the second homodimeric antibody contain amino acid substitutions that promote heterodimer formation, wherein the amino acid substitutions that promote heterodimer formation are: the first CH3 region has at least one amino acid substitution at positions 366, 368, 370, 399, 405, 407 and / or 409; the second CH3 region has at least one amino acid substitution at positions 366, 368, 370, 399, 405, 407 and / or 409, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0690] Implementation Scheme 39. The method according to any one of Implementation Schemes 33-38, according to EU designation,

[0691] The first and / or second CH3 region has an amino acid Met, Ile, or Leu at position 366, an amino acid Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg at position 394, an amino acid Ala, Thr, Ser, Asp, or Glu at position 405, and / or an amino acid Thr, Ala, or Val at position 407, and the amino acid substitution positions of the first and second CH3 regions are different.

[0692] Implementation Scheme 40. The method according to any one of Implementation Schemes 33-39, wherein, according to the EU designation,

[0693] (1) The first CH3 region has Met, Ile, or Leu at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407; and / or

[0694] (2) The first CH3 region has Ala, Thr, Ser, Asp, Glu or Gly at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys or Arg at bit 394.

[0695] Implementation Scheme 41. The method according to any one of Implementation Schemes 33-40, according to the EU number, wherein the first CH3 region has Met or Leu at bit 366, and the second CH3 region has Thr or Ala at bit 407.

[0696] Implementation Scheme 42. The method according to Implementation Scheme 40, wherein the first CH3 region has Met at bit 366 and the second CH3 region has Thr at bit 407; the first CH3 region has Met at bit 366 and the second CH3 region has Ala at bit 407; the first CH3 region has Leu at bit 366 and the second CH3 region has Thr at bit 407; or the first CH3 region has Leu at bit 366 and the second CH3 region has Ala at bit 407.

[0697] Implementation Scheme 43. The method according to any one of Implementation Schemes 33-40, according to the EU number, wherein the first CH3 region has Ala, Thr, Ser or Asp at bit 405, and the second CH3 region has Trp, Phe, Tyr or Lys at bit 394.

[0698] Implementation Scheme 44. According to the method of Implementation Scheme 42, according to the EU number, wherein the first CH3 region has Ala at bit 405 and the second CH3 region has Trp at bit 394; the first CH3 region has Ala at bit 405 and the second CH3 region has Phe at bit 394; the first CH3 region has Ala at bit 405 and the second CH3 region has Tyr at bit 394; the first CH3 region has Ala at bit 405 and the second CH3 region has Lys at bit 394; the first CH3 region has Thr at bit 405 and the second CH3 region has Trp at bit 394; the first CH3 region has Thr at bit 405 and the second CH3 region has Phe at bit 394; the first CH3 region has Thr at bit 405 and the second CH3 region has Tyr at bit 394; the first CH3 region has Thr at bit 405 and the second CH3 region has Phe at bit 394; the first CH3 region has Thr at bit 405 and the second CH3 region has Tyr at bit 394; the first CH3 region has Thr at bit 405 and the second CH3 region has Tyr at bit 394. The CH3 region has Lys at bit 394; the first CH3 region has Ser at bit 405, and the second CH3 region has Trp at bit 394; the first CH3 region has Ser at bit 405, and the second CH3 region has Phe at bit 394; the first CH3 region has Ser at bit 405, and the second CH3 region has Tyr at bit 394; the first CH3 region has Ser at bit 405, and the second CH3 region has Lys at bit 394; the first CH3 region has Asp at bit 405, and the second CH3 region has Trp at bit 394; the first CH3 region has Asp at bit 405, and the second CH3 region has Phe at bit 394; the first CH3 region has Asp at bit 405, and the second CH3 region has Tyr at bit 394; or, the first CH3 region has Asp at bit 405, and the second CH3 region has Lys at bit 394.

[0699] Implementation Scheme 45. The method according to any one of Implementation Schemes 33-43, wherein the first CH3 region is the CH3 region of IgG, preferably the CH3 region of human IgG.

[0700] Implementation Scheme 46. The method according to any one of Implementation Schemes 33-45, wherein the first CH3 region is a CH3 region selected from IgG1, IgG2, IgG3 and IgG4, preferably a CH3 region selected from IgG1 and IgG4.

[0701] Implementation Scheme 47. The method according to any one of Implementation Schemes 33-46, wherein the second CH3 region is the CH3 region of IgG, preferably the CH3 region of human IgG.

[0702] Implementation Scheme 48. The method according to any one of Implementation Schemes 33-47, wherein the second CH3 region is a CH3 region selected from IgG1, IgG2, IgG3 and IgG4, preferably a CH3 region selected from IgG1 and IgG4.

[0703] Implementation Scheme 49. The method according to any one of Implementation Schemes 33-48, wherein the first CH3 region and the second CH3 region are both human IgG1.

[0704] The CH3 region is either the CH3 region of human IgG4 or it is the CH3 region of human IgG4.

[0705] Implementation Scheme 50. The method according to any one of Implementation Schemes 33-49, wherein the sequences of the first CH3 region and the second CH3 region are not...

[0706] This results in a stronger heterodimer interaction between the first CH3 region and the second CH3 region than the homodimer interaction between the first CH3 region and the second CH3 region.

[0707] Implementation Scheme 51. The method according to any one of Implementation Schemes 33-50, wherein the first homodimeric antibody comprises a first Fc polypeptide.

[0708] The first Fc polypeptide contains the first CH3 region, the second homodimeric antibody contains the second Fc polypeptide, the second Fc polypeptide contains the second CH3 region, the two first Fc polypeptides constitute the first Fc domain, and the two second Fc polypeptides constitute the second Fc domain.

[0709] Implementation Scheme 52. The method according to Implementation Scheme 51, wherein the Fc domain is a human IgG Fc domain, preferably an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, or an IgG4 Fc domain.

[0710] Implementation Scheme 53. The method according to any one of Implementation Schemes 33-52, wherein the first homodimeric antibody comprises a first antigen-binding portion, and the second homodimeric antibody comprises a second antigen-binding portion.

[0711] Implementation Scheme 54. The method according to any one of Implementation Schemes 33-53, wherein the first homodimeric antibody comprises a first antigen-binding portion and a first Fc domain, and the second homodimeric antibody comprises a second antigen-binding portion and a second Fc domain.

[0712] Implementation Scheme 55. The method according to Implementation Scheme 53 or 54, wherein the first antigen-binding portion and the second antigen-binding portion bind the same or different antigens, or bind the same or different epitopes.

[0713] Implementation Scheme 56. The method according to any one of Implementation Schemes 34-55, wherein the first antigen-binding portion and the second antigen-binding portion are each independently Fab, ScFv, VHH or ScFab, preferably, both the first antigen-binding portion and the second antigen-binding portion are Fab.

[0714] Implementation Scheme 57. The method according to any one of Implementation Schemes 34-56, wherein the first homodimeric antibody and the second homodimeric antibody both contain a Cys-Pro-Pro-Cys sequence in their hinge regions.

[0715] Implementation Scheme 58. The method according to any one of Implementation Schemes 34-57, wherein the active agent of the first homodimer conjugate and the active agent of the second homodimer conjugate are each independently selected from: microtubule inhibitors, DNA damage agents, immunomodulators, topoisomerase inhibitors or other active agents.

[0716] Implementation Scheme 59. The method according to any one of Implementation Schemes 34-58, wherein the active agent of the first homodimer conjugate and the active agent of the second homodimer conjugate are each independently selected from: eribulin or its derivatives, oliquistatin, MMAF, MMAE, maytansine, DM1, DM4, camptothecin, hydroxycamptothecin, aminocamptothecin, irinotecan, topotecan, ethanotecan or its derivatives, rubitecan, letopotecan, gimarettecan, karenitecin, 7-Ethylcamptothecin, SN-38, DXd, cazithromycin, N-acetyl-γ-calicheamicin, atrazomycin, DSB-120, SJG-136, TLR agonists, STING agonists, paclitaxel, paclitaxel liposomes, albumin-bound paclitaxel, cabazitaxel, docetaxel, oxaliplatin, cisplatin, carboplatin, nedaplatin, bicycloplatin, miplatin, lobaplatin, pyrplatin, levoplatin, triplatinum tetranitrate, phenanthreneplatin, saxaplatin, and IRDye700DX.

[0717] Implementation Scheme 60. The method according to any one of Implementation Schemes 34-59, wherein,

[0718] (1) The active agent of the first homodimeric conjugate is eribulin or a derivative thereof, and the active agent of the second homodimeric conjugate is ethatecan or a derivative thereof; or

[0719] (2) The active agent of the first homodimer conjugate is ethatecan or its derivative, and the active agent of the second homodimer conjugate is eribulin or its derivative.

[0720] Implementation Scheme 61. The method according to Implementation Scheme 60, wherein,

[0721] (1) The active agent of the first homodimer coupling compound has the structure shown in Formula I, and the active agent of the second homodimer coupling compound has the structure shown in Formula II; or;

[0722] (2) The activator of the first homodimer coupling compound has the structure shown in Formula II below, and the activator of the second homodimer coupling compound has the structure shown in Formula I below;

[0723] Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6-10 aryl, optionally substituted 5 to 12 heteroaryl groups, R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6- 10 aryl, optionally substituted 5 to 12 heteroaryl groups; or, R 1 and R 2 Together with the atoms connected to it, they form optional 5- to 8-membered heterocyclic groups;

[0724] R 3 R 4 R 5 and R 6 Each is independently selected from either hydrogen or deuterium atoms.

[0725] Implementation Scheme 62. The method according to any one of Implementation Schemes 34-61, wherein,

[0726] (1) The first homodimeric antibody is transmitted through a linker with the structure shown in Formula IIIa below.

[0727] The structure shown in formula Ia is connected.

[0728] The second homodimeric antibody is connected to the structure shown in Formula IIa via a linker of the structure shown in Formula IIIa.

[0729] or

[0730] (2) The first homodimeric antibody is connected via a linker with the structure shown in Formula IIIa below.

[0731] The structure shown in Formula IIa is connected.

[0732] The second homodimeric antibody is connected to the structure shown in Formula Ia via a linker of Formula IIIa.

[0733] The homodimeric antibody is attached to the linker at the position shown *, and the structures shown in Formula Ia and Formula IIa are attached to the linker at the position shown #, wherein R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6-10 aryl, optionally substituted 5 to 12 heteroaryl groups, R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6- 10 aryl, optionally substituted 5 to 12 heteroaryl groups; or, R 1 and R 2 Together with the atoms connected to it, they form optional 5- to 8-membered heterocyclic groups;

[0734] R 3 R 4 R 5 and R 6 Each is independently selected from either hydrogen or deuterium atoms.

[0735] Implementation Scheme 63. The method according to Implementation Scheme 61 or 62, wherein the R 1 and R 2 Each is independently selected from hydrogen atoms or C atoms. 1-5 alkyl.

[0736] Implementation Scheme 64. The method according to Implementation Scheme 63, wherein the R 1 and R 2 Each is independently selected from hydrogen atoms, methyl, ethyl, propyl, or isopropyl; preferably, the R 1 and R 2 It is a hydrogen atom.

[0737] Implementation Scheme 65. The method according to Implementation Scheme 61 or 62, wherein the R 3 For deuterium atoms, R 4 It is a deuterium atom; or R 3 and R 4 It is a hydrogen atom.

[0738] Implementation Scheme 66. The method according to any one of Implementation Schemes 33-65, wherein the DAR of the antibody-conjugate is 1-10, 2-8, 3-8, 4-8, 3.5-4.5, 6-8, 7-8, 7.5-8, 7.6-8, 7.7-8, 7.8-8, or 7.9-8.

[0739] Implementation Scheme 67. The method according to any one of Implementation Schemes 33-65, wherein the DAR is 2, 3, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 5, 6, 7, 7.5, 7.6, 7.7, 7.73, 7.79, 7.8, 7.9 or 8.

[0740] Implementation Scheme 68. The method according to any one of Implementation Schemes 33-67, wherein the homodimer antibody targets HER2, HER3, EGFR, ROR1, CLDN18.2, B7-H3, B7-H4, TROP-2, CD19, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, PD-1, or PD-L1.

[0741] Implementation Scheme 69. The method according to any one of Implementation Schemes 33-68, wherein the concentration of the reducing agent is 1-150mM, 50-150mM, 50-100mM, 1-50mM, 1-30mM, 10-20mM, 60-80mM, 60-75mM, 70-80mM or 75-80mM; and / or the reduction temperature is 10-40℃, 10-30℃, 15-30℃, 20-30℃, 25-30℃, 15-40℃, 20-40℃ or 25-40℃; and / or the reduction time is 0.5-12 hours, 1-10 hours, 1-8 hours, 1-6 hours, 2-4 hours or 1-4 hours.

[0742] Implementation Scheme 70. The method according to any one of Implementation Schemes 33-69, wherein when the first homodimeric conjugate and the second homodimeric conjugate are incubated together, the molar ratio of the two is 1:1.

[0743] Implementation Scheme 71. The antibody-conjugate or its pharmaceutically acceptable salt or method according to any of the preceding embodiments, wherein the first CH3 region and the second CH3 region have the amino acid substitutions described therein relative to the CH3 region sequence shown in SEQ ID NO: 3.

[0744] Implementation Scheme 72. A pharmaceutical composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-32.

[0745] Implementation Scheme 73. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-32, or the pharmaceutical composition of Implementation Scheme 72, in the preparation of a medicament for treating tumors.

[0746] Implementation Scheme 74. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of Implementation Schemes 1-32, or the pharmaceutical composition of Implementation Scheme 72, and one or more other therapeutic agents in the preparation of a medicament for treating tumors or autoimmune diseases.

[0747] Implementation Scheme 75. The use according to Implementation Scheme 73 or 74, wherein the tumor is biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, melanoma, pharyngeal cancer, oral cancer, skin cancer, lung cancer, urethral cancer, urothelial carcinoma, bone cancer, soft tissue cancer, gallbladder cancer, testicular cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, liver cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, or myeloma.

[0748] Implementation Scheme 76. A method for treating tumors or autoimmune diseases, comprising administering to a subject in need a therapeutically effective amount of any one of Implementation Schemes 1-32, an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in Implementation Scheme 72.

[0749] Implementation Scheme 77. The method according to Implementation Scheme 76, the method comprising contacting tumor cells with any one of Implementation Schemes 1-32, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of Implementation Scheme 72, thereby killing tumor cells or inhibiting tumor cell growth.

[0750] Implementation Scheme 78. The method according to Implementation Scheme 76 or 77, wherein the method further comprises administering one or more additional therapeutic agents; preferably, the additional therapeutic agents are tumor therapeutic agents.

[0751] Implementation Scheme 79. The method according to any one of Implementation Schemes 76-78, wherein the tumor is biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, melanoma, pharyngeal cancer, oral cancer, skin cancer, lung cancer, urethral cancer, urothelial carcinoma, bone cancer, soft tissue cancer, gallbladder cancer, testicular cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, liver cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, or myeloma.

[0752] Implementation Scheme 80. A kit comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of Implementation Schemes 1-32, or the pharmaceutical composition described in Implementation Scheme 72.

[0753] Example

[0754] For clarity, this disclosure is further illustrated by embodiments, but these embodiments are not intended to limit the scope of this disclosure. Those skilled in the art will readily identify various non-critical parameters that can be changed or modified to produce substantially similar results.

[0755] Example 1: Fc region mutation design

[0756] This embodiment details the design of Fc region mutations (or amino acid substitutions) of parental antibodies, as shown in Table 1-1 below.

[0757] Table 1-1. Mutation design of the Fc region of parental antibodies (based on EU designations)

[0758] First, taking the IgG1 subtype antibody (whose Fc region sequence is shown in SEQ ID NO:1 and hinge region sequence is shown in SEQ ID NO:2) as an example, various mutations were performed at the aforementioned sites of the corresponding parental antibodies, and GenScript was commissioned to express and purify all antibodies. Based on the antibody report issued by GenScript and the preferred mutation design based on Table 1-1, the SEC analysis purity of each mutated parental antibody was very high (data not shown).

[0759] Secondly, we assembled bispecific antibodies in vitro using the mutation combinations shown in Table 1-1, obtaining heterodimeric bispecific antibodies according to the preparation process described in Example 23. Based on the purity and molecular weight evaluation results of the obtained bispecific antibodies, three mutation combinations (T366M and Y407A; T366M and Y407T; T394W and F405A) were selected to prepare bispecific antibody-dextrin ADCs targeting different targets, as detailed in Examples 2-4 below. Table 1-2 below summarizes the codes, mutations, and target information of the parental antibodies used in the examples of this disclosure:

[0760] Table 1-2. Parental antibody codes, mutations, and target information used in the embodiments of this disclosure.

[0761] Example 2: Preparation of the joint-load capacity

[0762] Example 2-1: Preparation of MC-GGFG-deuterated DXd (MC-GGFG-DDDXd)

[0763] Step 1 Synthesis of Intermediate A

[0764] Under nitrogen protection, 80 g of ethyl diazonium acetate was added to a 3 L single-necked flask, along with 800 mL of dichloromethane and 800 mL of 1% deuterated acetic acid solution (8 g of deuterated acetic acid dissolved in 800 mL of deuterated water). The flask was then placed in the dark and stirred at room temperature for 75 hours. The organic phase was collected by separation, and the aqueous phase was extracted twice with dichloromethane (200 mL × 2). The organic phases were combined. The organic phase was washed with 200 mL of deuterated water, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and the filtrate was concentrated to dryness under reduced pressure at 20 °C to obtain 49.25 g of intermediate A; the CAS No. of intermediate A is 1356471-71-0.

[0765] Step 2 Synthesis of intermediate B

[0766] Weigh 50 g of N-fluorenemethoxycarbonyl-glycyl-glycine into a 2 L round-bottom flask, add 750 mL of tetrahydrofuran and 150 mL of glacial acetic acid, stir at 40 °C for 20 minutes, add 100 g of lead tetraacetate, raise the temperature to 80 °C and continue the reaction for 3 hours. Cool to room temperature, filter, and wash the filter cake with 250 mL of ethyl acetate. Concentrate the filtrate to dryness, add 330 mL of dichloromethane and 670 mL of ethyl acetate to dissolve and obtain the organic phase. Wash the organic phase three times with 30% potassium bicarbonate aqueous solution (500 mL × 3), then dry with anhydrous sodium sulfate and filter. Concentrate the filtrate to dryness under reduced pressure, add 100 mL of dichloromethane to dissolve, then add 100 mL of n-hexane, stir at room temperature until a solid precipitates, then add 300 mL of a mixed solution of n-hexane and dichloromethane (n-hexane:dichloromethane = 1:1), and stir overnight. The filter cake was filtered and dried in a vacuum oven at 40°C for 4 hours to obtain 37.3 g of intermediate B. LC-MS (ESI) m / z: 391.09 [M+Na] + The CAS No. of intermediate B is 1599440-06-8.

[0767] Step 3 Synthesis of intermediate C

[0768] Weigh 78g of intermediate B and add it to a 3000mL single-necked flask. Add 800mL of dichloromethane and 45g of intermediate A. Cool the 3000mL single-necked flask to 0℃ in an ice-water bath. Dissolve 16g of lithium tert-butoxide in 400mL of dichloromethane to prepare a lithium tert-butoxide solution. Add the lithium tert-butoxide solution to the 3000mL single-necked flask and react at 0℃ for 3 hours. Transfer to room temperature and add 800mL of water with stirring. Separate the liquid and collect the organic phase. Extract the aqueous phase with 400mL of dichloromethane and combine the organic phases. Wash the organic phase once with 800mL of saturated brine, dry to anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and perform silica gel column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain 61g of intermediate C. LC-MS (ESI) m / z: 437.34 [M+Na] + The CAS No. of intermediate C is 2760715-83-9.

[0769] Step 4: Synthesis of Compound D

[0770] 31.2 g of intermediate C was added to 270 mL of deuterated methanol and 70 mL of heavy water, stirred in an ice bath, followed by the addition of 5.5 g of NaOH, and stirred overnight at room temperature. The reaction mixture was extracted with 300 mL of ethyl acetate and 300 mL of water. The pH of the aqueous layer was adjusted to 2-3 with 20 mL of glacial acetic acid, resulting in the precipitation of a solid. 20.3 g of compound D was obtained by filtration. 1H-NMR (500MHz, DMSO-d6) δ8.70(t,J=6.6Hz,1H),7.89(d,J=7.5Hz,2H),7.72(d,J=7.5Hz,2H),7.57(t,J=6.0Hz,1H),7.42(t,J=7.5Hz,2H ),7.34(t,J=7.5Hz,2H),4.61(d,J=6.6Hz,2H),4.30(d,J=7.1Hz,2H),4.23(1H,m),3.64(d,J=6.0Hz,2H); LC-MS(ESI)m / z:409.08[M+Na] + .

[0771] Step 5: Synthesis of Compound E

[0772] Weigh 2.0 g of ethatecan mesylate dihydrate and 1.63 g of compound D into a 100 mL round-bottom flask. Add 40 mL of N,N-dimethylformamide and stir. Cool to 0 °C. Add 2.0 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and 1.82 g of N,N-diisopropylethylamine sequentially, and react at 0 °C for 3 h. Pour the reaction solution into 120 mL of ice water and stir for 1 h. Filter and dissolve the filter cake in dichloromethane. Perform silica gel column chromatography (100 g of 100-200 mesh silica gel, dichloromethane:methanol = 30:1, 2 L) to obtain 2.6 g of compound E. 1 H-NMR (500MHz, DMSO-d6), δ8.78(t,J=6.6Hz,1H),8.47(d,J=9.0Hz,1H),7.86(d,J=7.5Hz,2H),7.73(d,J=11.0 Hz,1H),7.68(d,J=7.5Hz,2H),7.53(t,J=6.0Hz,1H),7.39(t,J=7.5Hz,2H),7.30(m,2H),7.29(s,1H),6.50(br s,1H),5.56(m,1H),5.39(m,2H),5.14(m,2H),4.64(m,2H),4.25(d,J=6.8Hz,2H),4.19(m,1H),3.62(d,J=6.0H z,2H),3.15(m,2H),2.35(s,3H),2.17(m,2H),1.83(m,2H),0.85(t,J=7.3Hz,3H); LC-MS(ESI)m / z:804.84[M+H] + .

[0773] Step 6: Preparation of compound F

[0774] Weigh 0.38 g of 1,8-diazabicyclo[5.4.0]undec-7-ene and add it to a 100 mL round-bottom flask. Then add 20 mL of tetrahydrofuran to the round-bottom flask and stir. Cool to 0 °C. Weigh 2.0 g of compound E and prepare a solution with 20 mL of tetrahydrofuran. Slowly add the prepared solution of compound E to the 100 mL round-bottom flask and allow it to warm naturally to room temperature. React for 3 h. Filter under nitrogen protection to obtain 1.45 g of compound F. 1 H-NMR (500MHz, DMSO-d6) δ8.76(m,1H),7.72(m,1H),7.28(s,1H),5.48(m,3H),5.16(m,2H),4.61(m,2H),3.40( m,2H),3.20(m,2H),2.35(s,3H),2.17(m,2H),1.83(m,2H),0.86(t,J=7.1Hz,3H); LC-MS(ESI)m / z:582.39[M+H] + .

[0775] Step 7: Preparation of compound H

[0776] Weigh 1.00 g of compound F and 0.97 g of compound G into a 100 mL round-bottom flask, and add 10 mL of N,N-dimethylformamide. Cool to -20 °C, add 0.34 g of 1-hydroxybenzotriazole and 0.49 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and react at -20 °C for 3 h. Add 20 mL of dichloromethane and 20 mL of water to the reaction mixture, stir for 0.5 h, let stand, separate the layers, and collect the organic phase. Dry the organic phase with anhydrous sodium sulfate and filter. Concentrate the filtrate to dryness under reduced pressure. Analyze by silica gel column chromatography (dichloromethane:methanol = 15:1) to obtain 400 mg of compound H, MS m / z: 1037.08 [M+H]. + . 1H-NMR (500MHz, DMSO-d6) δ8.62(t,J=6.5Hz,1H),8.49(d,J=8.5Hz,1H),8.2 9(t,J=5.5Hz,1H),8.12(d,J=8.0Hz,1H),8.06(t,J=5.5Hz,1H),8.00(t,J=5 .5Hz,1H),7.74(d,J=10.5Hz,1H),7.30(s,1H),7.27-7.12(m,5H),6.98(s,2 H),6.51(brs,1H),5.61-5.58(m,1H),5.45-5.37(m,2H),5.22-5.13(m,2H), 4.64(d,J=6.5Hz,2H),4.49-4.45(m,1H),3.76-3.57(m,6H),3.37-3.32(m, 2H),3.24-3.09(m,2H),3.02(dd,J=4.5Hz,14.0Hz,1H),2.77(dd,J=9.5Hz,1 3.5Hz,1H),2.36(s,3H),2.23-2.14(m,2H),2.09(t,J=7.5Hz,2H),1.91-1.7 9(m,2H),1.49-1.42(m,4H),1.20-1.14(m,2H),0.87(t,J=7.5Hz,3H); HR-MS m / z: 1036.4194 [M+H] + .

[0777] Example 2-2: Preparation of MC-GGFG-Eribulin

[0778] Compound I (100 mg, 0.12 mmol) and compound J (75 mg, 0.145 mmol) were weighed and added to a 20 mL reaction tube (CAS No. 441045-17-6 for compound I and CAS No. 2413428-36-9 for compound J). 2 mL of N,N-dimethylformamide was added. The mixture was cooled to 0 °C. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (70 mg, 0.18 mmol) and N,N-diisopropylethylamine (49 mg, 0.36 mmol) were added, and the mixture was reacted at 0 °C for 1 h. Approximately 70 mg of compound MC-GGFG-Eribulin was obtained by liquid chromatography-HPLC.

[0779] ESI-MS analysis showed that MC-GGFG-Eribulin had an m / z ratio of 1241.72 [M+H]. + The proton spectrum is as follows:

[0780] 1 H NMR (500MHz, DMSO) δ8.22(t,J=5.3Hz,1H),8.12(d,J=8.0Hz,1H),8.06(t,J=5.4Hz,1H),7.9 9(t,J=5.3Hz,1H),7.65(d,J=5.2Hz,1H),7.30-7.21(m,4H),7.18(d,J=6.4Hz,1H),6.99(s, 2H),5.02(d,J=26.0Hz,2H),4.79(d,J=38.9Hz,2H),4.65-4.60(m,2H),4.56(d,J=3.7Hz,1H ),4.50-4.42(m,1H),4.26(d,J=10.1Hz,1H),4.21-4.14(m,1H),4.10(s,3H),4.05-3.98(m,1 H),3.86-3.64(m,8H),3.60-3.45(m,4H),3.37-3.30(m,2H),3.26(s,4H),3.17-3.09(m,1H) ,3.08-2.99(m,2H),2.84(d,J=10.4Hz,1H),2.86-2.66(m,3H),2.56-2.50(m,1H),2.37-2.1 8(m,5H),2.15-2.05(m,3H),2.05-1.96(m,2H),1.95-1.84(m,4H),1.75-1.57(m,6H),1.55- 1.38(m,6H),1.35-1.25(m,3H),1.23-1.12(m,3H),1.03(d,J=6.0Hz,3H),1.00-0.92(m,1H).

[0781] Example 3: Preparation of anti-MUC1 / anti-c-MET bispecific antitoxin ADC

[0782] Example 3-1: Preparation of anti-MUC1 / anti-c-MET bispecific antitoxin ADC DV13-DDDxd-4-DV15-Eribulin-4

[0783] Reagents:

[0784] The antibodies were DV13 (T366M) and DV15 (Y407T) as shown in Tables 1-2 of Example 1, and the Linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin synthesized in Example 2.

[0785] Experimental procedure:

[0786] 1. Reduction of DV13 antibody: Displace DV13 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DV13 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DV13 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 1.

[0787] 2. Coupling of DV13 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 2.

[0788] 3. Reduction of DV15 antibody: Displace DV15 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DV15 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DV15 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 3.

[0789] 4. Conjugation of DV15 antibody with MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to connect the antibody with the adapter-loaded substance, thus obtaining reaction solution 4.

[0790] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with pH 7.4 phosphate buffer, it is allowed to stand at 4℃ for 17 hours to obtain anti-MUC1 / anti-c-MET bispecific antibody-dual toxin ADC, namely DV13-DDDXd-4-DV15-Eribulin-4.

[0791] Mass spectrometry analysis confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0792] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0793] This bispecific antibody-dextrin ADC contains a bispecific antibody, a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the DV13 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the DV15 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0794] Example 3-2: Preparation of anti-MUC1 / anti-c-MET bispecific antitoxin ADC DV13-DDDxd-4-DV16-Eribulin-4

[0795] Reagents:

[0796] The antibodies were DV13 (T366M) and DV16 (Y407A) as shown in Tables 1-2 of Example 1, and the Linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin synthesized in Example 2.

[0797] Experimental procedure:

[0798] 1. Reduction of DV13 antibody: Displace DV13 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DV13 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DV13 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 1.

[0799] 2. Coupling of DV13 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 2.

[0800] 3. Reduction of DV16 antibody: Displace DV16 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DV16 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DV16 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 3.

[0801] 4. Conjugation of DV16 antibody with MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded substance, thus obtaining reaction solution 4.

[0802] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with pH 7.4 phosphate buffer, let it stand at 4℃ for 17 hours to obtain anti-MUC1 / anti-c-MET bispecific antibody-dual toxin ADC, namely DV13-DDDXd-4-DV16-Eribulin-4.

[0803] Mass spectrometry analysis confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0804] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0805] This bispecific antibody-dextrin ADC contains a bispecific antibody, a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the DV13 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the DV16 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0806] Example 3-3: Preparation of anti-MUC1 / anti-c-MET bispecific antitoxin ADC DV14-DDDxd-4-DV17-Eribulin-4

[0807] Reagents:

[0808] The antibodies were DV14 (F405A) and DV17 (T394W) as shown in Tables 1-2 of Example 1, and the Linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin synthesized in Example 2.

[0809] Experimental procedure:

[0810] 1. Reduction of DV14 antibody: Displace DV14 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DV14 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DV14 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 1.

[0811] 2. Conjugation of DV14 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to connect the antibody with the adapter-loaded vector, thus obtaining reaction solution 2.

[0812] 3. Reduction of DV17 antibody: Displace DV17 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DV17 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DV17 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 3.

[0813] 4. Conjugation of DV17 antibody with MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to connect the antibody with the adapter-loaded agent, thus obtaining reaction solution 4.

[0814] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with pH 7.4 phosphate buffer, it is allowed to stand at 4℃ for 17 hours to obtain anti-MUC1 / anti-c-MET bispecific antibody-dual toxin ADC, namely DV14-DDDXd-4-DV17-Eribulin-4.

[0815] Mass spectrometry analysis confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0816] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0817] This bispecific antibody-dextrin ADC contains a bispecific antibody, a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the DV14 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the DV17 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0818] Examples 3-4: Preparation of anti-MUC1 / anti-c-MET bispecific antitoxin ADC DM-DDDxd-4-DC-Eribulin-4

[0819] Reagents:

[0820] The antibodies were DM (F405L) and DC (K409R) as shown in Tables 1-2 of Example 1, and the Linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin synthesized in Example 2.

[0821] Experimental procedure:

[0822] 1. Reduction of DM antibody: Displace the DM antibody into phosphate buffer at pH 7.4, and adjust the concentration of DM antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DM antibody solution to a light-proof glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 1.

[0823] 2. Coupling of DM antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 2.

[0824] 3. Reduction of DC antibody: Displace the DC antibody into phosphate buffer at pH 7.4, and adjust the DC antibody concentration to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DC antibody solution to a light-proof glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the antibody to TCEP·HCl molar ratio 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between the antibody chains, to obtain reaction solution 3.

[0825] 4. Conjugation of DC antibody to MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to connect the antibody to the adapter-loaded substance, thus obtaining reaction solution 4.

[0826] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with pH 7.4 phosphate buffer, it is allowed to stand at 4℃ for 17 hours to obtain anti-MUC1 / anti-c-MET bispecific antibody-dual toxin ADC, namely DM-DDDXd-4-DC-Eribulin-4.

[0827] Mass spectrometry analysis confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0828] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0829] This bispecific antibody-dextrin-toxin ADC contains a bispecific antibody, a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the DM monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the DC monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0830] Example 4: Preparation of anti-B7-H3 / anti-B7-H4 bispecific antitoxin ADC

[0831] Example 4-1: Preparation of anti-B7-H3 / anti-B7-H4 bispecific antitoxin ADC DN1-17-DDDxd-4-DV10-Eribulin-4

[0832] Reagents:

[0833] The antibodies were DN1-17 (T366M) and DV10 (Y407T) as shown in Tables 1-2 of Example 1, and the Linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin synthesized in Example 2.

[0834] Experimental procedure:

[0835] 1. Reduction of DN1-17 antibody: Displace the DN1-17 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DN1-17 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DN1-17 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 1.

[0836] 2. Coupling of DN1-17 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded substance, thus obtaining reaction solution 2.

[0837] 3. Reduction of DV10 antibody: Displace DV10 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DV10 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DV10 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 3.

[0838] 4. Conjugation of DV10 antibody with MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 4.

[0839] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with pH 7.4 phosphate buffer, let it stand at 4℃ for 17 hours to obtain anti-B7-H3 / anti-B7-H4 bispecific antibody-dual toxin ADC, namely DN1-17-DDDxd-4-DV10-Eribulin-4.

[0840] Mass spectrometry analysis confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0841] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0842] This bispecific antibody-dextrin ADC contains a bispecific antibody, a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the DN1-17 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the DV10 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0843] Example 4-2: Preparation of anti-B7-H3 / anti-B7-H4 bispecific antitoxin ADC DN1-17-DDDxd-4-DV11-Eribulin-4

[0844] Reagents:

[0845] The antibodies were DN1-17 (T366M) and DV11 (Y407A) as shown in Tables 1-2 of Example 1, and the Linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin synthesized in Example 2.

[0846] Experimental procedure:

[0847] 1. Reduction of DN1-17 antibody: Displace the DN1-17 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DN1-17 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DN1-17 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 1.

[0848] 2. Coupling of DN1-17 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded substance, thus obtaining reaction solution 2.

[0849] 3. Reduction of DV11 antibody: Displace DV11 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DV11 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DV11 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 3.

[0850] 4. Conjugation of DV11 antibody with MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to connect the antibody with the adapter-loaded substance, thus obtaining reaction solution 4.

[0851] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with pH 7.4 phosphate buffer, let it stand at 4℃ for 17 hours to obtain anti-B7-H3 / anti-B7-H4 bispecific antibody-dual toxin ADC, namely DN1-17-DDDxd-4-DV11-Eribulin-4.

[0852] Mass spectrometry analysis confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0853] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0854] This bispecific antibody-dextrin-toxin ADC contains a bispecific antibody, a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the DN1-17 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the DV11 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0855] Example 4-3: Preparation of anti-B7-H3 / anti-B7-H4 bispecific antitoxin ADC DN1-19-DDDxd-4-DV12-Eribulin-4

[0856] Reagents:

[0857] The antibodies were DN1-19 (F405A) and DV12 (T394W) as shown in Tables 1-2 of Example 1, and the Linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin synthesized in Example 2.

[0858] Experimental procedure:

[0859] 1. Reduction of DN1-19 antibody: Displace the DN1-19 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DN1-19 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DN1-19 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 1.

[0860] 2. Coupling of DN1-19 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 2.

[0861] 3. Reduction of DV12 antibody: Displace DV12 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DV12 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DV12 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 3.

[0862] 4. Conjugation of DV12 antibody with MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 4.

[0863] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with pH 7.4 phosphate buffer, let it stand at 4℃ for 17 hours to obtain anti-B7-H3 / anti-B7-H4 bispecific antibody-dual toxin ADC, namely DN1-19-DDDxd-4-DV12-Eribulin-4.

[0864] Mass spectrometry analysis confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0865] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0866] This bispecific antibody-dextrin-toxin ADC contains a bispecific antibody, a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the DN1-19 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the DV12 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0867] Example 4-4: Preparation of anti-B7-H3 / anti-B7-H4 bispecific antitoxin ADC DV1-DDDxd-4-DV4-Eribulin-4

[0868] Reagents:

[0869] The antibodies were DV1 (F405L) and DV4 (K409R) as shown in Tables 1-2 of Example 1, and the Linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin synthesized in Example 2.

[0870] Experimental procedure:

[0871] 1. Reduction of DV1 antibody: Displace DV1 antibody into phosphate buffer at pH 7.4, and adjust the concentration of DV1 antibody to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DV1 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the molar ratio of antibody to TCEP·HCl 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 1.

[0872] 2. Coupling of DV1 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 2.

[0873] 3. Reduction of DV4 antibody: Displace DV4 antibody into phosphate buffer at pH 7.4, and adjust the DV4 antibody concentration to approximately 10 mg / mL with phosphate buffer (pH 7.4). Add the DV4 antibody solution to a light-protected glass bottle, and add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution to make the antibody to TCEP·HCl molar ratio 1:10. Incubate at 37°C in the dark for 1 hour, stirring gently to reduce the disulfide bonds between antibody chains, to obtain reaction solution 3.

[0874] 4. Conjugation of DV4 antibody to MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:12. Incubate at 22°C in the dark for 1 hour with gentle stirring to connect the antibody to the adapter-loaded substance, thus obtaining reaction solution 4.

[0875] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with pH 7.4 phosphate buffer, let it stand at 4℃ for 17 hours to obtain anti-B7-H3 / anti-B7-H4 bispecific antibody-dual toxin ADC, namely DV1-DDDxd-4-DV4-Eribulin-4.

[0876] Mass spectrometry analysis confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0877] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0878] This bispecific antibody-dextrin ADC contains a bispecific antibody, a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the DV1 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the DV4 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0879] Example 5: SEC detection of bispecific antibody-bispecific toxin ADC

[0880] The components of the prepared ADC sample were separated using gel permeation chromatography (GPC). A neutral pH buffer containing 10% isopropanol was used as the mobile phase for elution, and the components were eluted sequentially in descending order of molecular weight. The column used was an ACQUITY UPLC Protein BEH SEC Column. A 1.7 μm, 4.6 × 300 mm gel chromatographic column was used. The mobile phase was 50 mmol / L phosphate buffer-200 mmol / L sodium chloride-10% isopropanol, pH 7.0 (12.53 g of disodium hydrogen phosphate dodecahydrate, 2.33 g of sodium dihydrogen phosphate dihydrate, and 11.69 g of sodium chloride were weighed, added to approximately 800 mL of ultrapure water, stirred until fully dissolved, and then diluted to 1000 mL with ultrapure water. 100 mL of isopropanol was added to this solution to bring the total volume to 1000 mL, mixed well, and filtered through a 0.22 μm filter membrane). A precise 20 μg sample was injected into the HPLC system and detected at 280 nm. The flow rate was 0.3 mL / min, and isocratic elution was performed for 15 min. The data were processed, and the results were quantitatively analyzed using the area normalization method. The peak area percentages of aggregates, ADC monomers, and low molecular weight impurities were calculated separately, with the aggregates preceding the main peak, the ADC monomers at the main peak, and the low molecular weight impurities following the main peak. The analytical results showed that the purity of each bispecific antibody-dual toxin ADC sample in Examples 3 and 4 exceeded 90%.

[0881] Example 6: Cell-binding activity of anti-MUC-1 / anti-c-MET bispecific antitoxin ADC

[0882] The cell binding activity of the anti-MUC-1 / anti-c-MET bispecific antitoxin ADC prepared in Example 3 against human colon adenocarcinoma cells HCA-7 (low expression of c-MET and MUC1, source: Nanjing Kebai Biotechnology, catalog number: CBP60035) was detected by flow cytometry (FACS).

[0883] In short, HCA-7 tumor cells were diluted to 2×10⁻⁶. 6The concentration of the bispecific antibody-dextrin ADC was 50 μL / well, added to each well of a 96-well plate, and incubated together with 50 μL / well of the anti-MUC-1 / anti-c-MET bispecific antibody-dextrin ADC. A negative control of IgG-Eribulin-8 + IgG-DDDxd-8 toxin was included (IgG was IgG1 subtype, purchased from Sinocare, catalog number: HG1K; the control was prepared according to the method in Example 3). The initial concentration of the bispecific antibody-dextrin ADC was 666.7 nM, and it was serially diluted 5-fold with FACS buffer (Miltenyi Biotec, catalog number 130-091-221) to a total of 8 concentration gradients. The plates were incubated at 4°C for 60 minutes. Afterwards, the plates were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Wash three times with pre-chilled FACS buffer to remove excess ADC. Add 100 μL / well of goat anti-human IgG Fcγ-PE secondary antibody (Jackson immunoresearch, catalog number 109-116-170) diluted 1:200 (v / v) with FACS buffer. Incubate at 4°C for 20 min. After incubation, wash three times with pre-chilled FACS buffer to remove excess secondary antibody. Resuspend cells in 50 μL of FACS buffer and analyze fluorescence signals using flow cytometry (Sartorius, iQUE3). The binding activity of the anti-MUC-1 / anti-c-MET bispecific antibody-drug ADC to cell surface MUC1 and / or c-MET was measured by the mean fluorescence intensity (MFI) signal. Data analysis was performed using software, and the results are shown in Figure 1. The calculated EC50 values ​​were... 50 As shown in Table 2 below, the results indicate that DV13-DDDXd-4-DV15 Eribulin-4, DV13-DDDXd-4-DV16-Eribulin-4, and DV14-DDDXd-4-DV17-Eribulin-4 can effectively bind to HCA-7 cells with low c-MET and MUC1 expression levels, and are superior to or comparable to DM-DDDXd-4-DC-Eribulin-4.

[0884] Table 2. Binding of anti-MUC-1 / anti-c-MET dual antitoxin ADC to cells EC 50 And the maximum combined value (Top)

[0885] Example 7: Cell-binding activity of anti-B7-H3 / B7-H4 bispecific antitoxin ADC

[0886] The cell binding activity of the anti-B7-H3 / B7-H4 bispecific antitoxin ADC prepared in Example 4 on human breast cancer cells MX-1 (high expression of B7-H4, low expression of B7-H3, Nanjing Kebai Biotechnology, catalog number: CBP60640) and MDA-MB-468 (medium expression of B7-H4, low expression of B7-H3, source: Beina Biotechnology, catalog number: BNCC339862) was detected by flow cytometry.

[0887] In short, MX-1 and MDA-MB-468 cells were diluted to 2 × 10⁻⁶. 6 The concentration of the bispecific antibody-dextrin ADC was 50 μL / well added to each well of a 96-well plate and incubated with 50 μL / well of the anti-B7-H3 / B7-H4 bispecific antibody-dextrin ADC. A negative control of IgG-Eribulin-8 + IgG-DDDxd-8 toxin was included (IgG was IgG1 subtype, purchased from Sinocare, catalog number: HG1K). The initial concentration of the bispecific antibody-dextrin ADC was 100 nM, and it was serially diluted 5-fold with FACS buffer (Miltenyi Biotec, catalog number 130-091-221) to create 8 concentration gradients. The plates were incubated at 4°C for 60 minutes. Afterwards, the plates were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Wash three times with pre-chilled FACS buffer to remove excess supernatant. Add 100 μL / well of goat anti-human IgG Fcγ-PE secondary antibody (Jackson immunoresearch, catalog number 109-116-170) diluted 1:200 (v / v) with FACS buffer. Incubate at 4°C for 20 min. After incubation, wash three times with pre-chilled FACS buffer to remove excess secondary antibody. Resuspend in 50 μL of FACS buffer and analyze fluorescence signals using flow cytometry (Sartorius, iQUE3). The binding activity of the anti-B7-H3 / B7-H4 bispecific antibody-drug ADC to B7-H3 and / or B7-H4 on the cell surface was measured by the mean fluorescence intensity (MFI) signal. Data analysis was performed using software, and the results are shown in Figures 2A-2B. The calculated EC50 values ​​were... 50 As shown in Table 3 below, the results indicate that DN1-17-DDDXd-4-DV10-Eribulin-4, DN1-17-DDDXd-4-DV11-Eribulin-4, and DN1-19-DDDXd-4-DV12-Eribulin-4 can effectively bind to cells with different expression levels of B7-H3 and B7-H4, and their activity is comparable to that of DV1-DDDXd-4-DV4-Eribulin-4.

[0888] Table 3. Binding of bispecific anti-B7-H3 / anti-B7-H4 ADC to cells (EC)50 And the maximum combined value (Top)

[0889] Example 8: Killing of tumor cells by a dual anti-B7-H3 / B7-H4 toxin ADC

[0890] To detect the killing effect of the anti-B7-H3 / anti-B7-H4 bispecific antitoxin ADC prepared in Example 4 on B7-H3 and B7-H4 positive tumor cells, the killing activity was tested using natural human ovarian cancer cells NIH:OVCAR3 (high expression of B7-H3, medium to low expression of B7-H4, source: Cell Bank of Chinese Academy of Sciences, catalog number: THu228).

[0891] NIH:OVCAR3 cells in logarithmic growth phase were diluted to 3.5 × 10⁻⁶. 4 Cells / mL were added to 96-well plates at 100 μL / well, and the plates were incubated at 37℃ and 5% CO2 for 4–6 h. The anti-B7-H3 / anti-B7-H4 bispecific antitoxin ADCs were diluted to the desired concentrations using the vendor-recommended NIH:OVCAR3 complete medium: the initial concentrations of DN1-17-DDDXd-4-DV10 Eribulin-4, DN1-17-DDDXd-4-DV11 Eribulin-4, DN1-19-DDDXd-4-DV12 Eribulin-4, and DV1-DDDXd-4-DV4-Eribulin-4 were 95.8 nM, 94.1 nM, 94.1 nM, and 101.6 nM, respectively, in a 4-fold serial dilution, for a total of 10 concentration gradients. Cells were removed from the adherent culture. The experimental group was treated with 50 μL / well of diluted bispecific antibody and toxin ADC, while the blank control group was treated with 50 μL / well of the corresponding complete culture medium. After culturing for 144 hours, the cells were detected using the CellCounting-Lite 2.0 Luminescent Cell Viability Assay kit (Vazyme, catalog number: DD1101-03). The specific steps included removing the cells from the 96-well plate, adding 75 μL of CellCounting-Lite assay solution (Vazyme, catalog number: DD1101-03) to each well, vortexing to mix, incubating at room temperature in the dark for 10 minutes, then transferring 170 μL from each well to an opaque white plate, removing air bubbles, and reading the chemiluminescence value using a microplate reader (PE, Envision 2105) to calculate the cell killing rate.

[0892] The data was analyzed, and the results are shown in Figure 3. The calculated EC 50 As shown in Table 4 below, the results indicate that all samples had comparable cytotoxic effects on tumor cells.

[0893] Table 4. Killing effect of bispecific anti-and bitoxin ADCs targeting B7-H3 and B7-H4 on tumor cells

[0894] Example 9: Fc region mutation design

[0895] This embodiment discloses in detail the Fc region mutation design of the parental antibody, as shown in Table 5-1 below.

[0896] Table 5-1. Mutation design of the Fc region of parental antibodies (based on EU designations)

[0897] First, taking the IgG1 subtype antibody (whose Fc region sequence is shown in SEQ ID NO:1 and hinge region sequence is shown in SEQ ID NO:2) as an example, various mutations were performed at the aforementioned sites on the corresponding parental antibodies, and GenScript was commissioned to express and purify all antibodies. Based on the antibody report issued by GenScript and the preferred mutations in Table 5-1, the SEC analysis purity of each mutated parental antibody was very high (data not shown).

[0898] Secondly, we assembled naked antibodies in vitro according to the mutation combinations shown in Table 5-1, and obtained heterodimeric bispecific antibodies according to the preparation process described in Example 23. Based on the purity and molecular weight evaluation results of the obtained bispecific antibodies, several mutation combinations were selected (see Table 5-2) to prepare bispecific antibody-dextrin ADCs targeting different targets, as detailed in the examples below. Table 5-2 below summarizes the codes, mutations, and target information of the parental antibodies used in the examples of this disclosure:

[0899] Table 5-2. Parental antibody codes, mutations and target information used in the embodiments of this disclosure.

[0900] Example 10: Preparation of anti-EGFR / anti-cMet bispecific antitoxin ADC

[0901] Example 10-1: Preparation of anti-EGFR / anti-cMet bispecific antitoxin ADC E-1 / C-1-DDDXd+Eribulin-4+4

[0902] Reagents:

[0903] The antibodies were E-1 and C-1, and the linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin.

[0904] Experimental procedure:

[0905] 1. Displace the E-1 antibody into histidine buffer at pH 6.0, and adjust the antibody concentration to approximately 10 mg / mL using histidine buffer (pH 6.0). Adjust the pH to approximately 7.0 using 300 mM Na2HPO4 aqueous solution. Add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution to make the antibody to TCEP·HCl molar ratio 1:10. Place the solution on a rotary incubator and incubate at 120 rpm and 37°C in the dark for 1 hour to obtain reaction solution 1.

[0906] 2. Coupling of E-1 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:14. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 2.

[0907] 3. Displace the C-1 antibody into histidine buffer at pH 6.0, and adjust the antibody concentration to approximately 10 mg / mL using histidine buffer (pH 6.0). Adjust the pH to approximately 7.0 using 300 mM Na2HPO4 aqueous solution. Add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) aqueous solution to make the antibody to TCEP·HCl molar ratio 1:10. Place the solution on a rotary incubator and incubate at 120 rpm and 37°C in the dark for 1 hour to obtain reaction solution 3.

[0908] 4. Conjugation of C-1 antibody to MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:14. Incubate at 22°C in the dark for 1 hour with gentle stirring to connect the antibody to the adapter-loaded substance, thus obtaining reaction solution 4.

[0909] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with histidine buffer, anti-EGFR / anti-cMet bispecific antibody-dual toxin ADC is obtained, namely E-1 / C-1-DDDXd+Eribulin-4+4 (or named E-1+C-1-DDDXd+Eribulin-4+4, and other names are similar).

[0910] Hydrophobic chromatography confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0911] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0912] This bispecific antibody-dextrin ADC contains a bispecific antibody (E-1-C-1), a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the E-1 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the C-1 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0913] Example 10-2: Preparation of anti-EGFR / anti-cMet bitoxic ADC E-3 / C-3-DDDXd+Eribulin-4+4

[0914] Reagents:

[0915] The antibodies were E-3 and C-3, and the linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin.

[0916] Experimental procedure:

[0917] 1. Displace the E-3 antibody into histidine buffer at pH 6.0, and adjust the antibody concentration to approximately 10 mg / mL using histidine buffer (pH 6.0). Adjust the pH to approximately 7.0 using 300 mM Na2HPO4 aqueous solution. Add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) aqueous solution to make the antibody to TCEP·HCl molar ratio 1:10. Place the solution on a rotary incubator and incubate at 120 rpm and 37°C in the dark for 1 hour to obtain reaction solution 1.

[0918] 2. Coupling of E-3 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:14. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 2.

[0919] 3. Displace the C-3 antibody into histidine buffer at pH 6.0, and adjust the antibody concentration to approximately 10 mg / mL using histidine buffer (pH 6.0). Adjust the pH to approximately 7.0 using 300 mM Na2HPO4 aqueous solution. Add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) aqueous solution to make the antibody to TCEP·HCl molar ratio 1:10. Place the solution on a rotary incubator and incubate at 120 rpm and 37°C in the dark for 1 hour to obtain reaction solution 3.

[0920] 4. Conjugation of C-3 antibody with MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:14. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded substance, thus obtaining reaction solution 4.

[0921] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with histidine buffer, anti-EGFR / anti-cMet bispecific antibody-dual toxin ADC, namely E-3 / C-3-DDDXd+Eribulin-4+4, is obtained.

[0922] Hydrophobic chromatography confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0923] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0924] This bispecific antibody-dextrin-toxin ADC contains a bispecific antibody (E-3-C-3), a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the E-3 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the C-3 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0925] Example 10-3: Preparation of anti-EGFR / anti-cMet bitoxic ADC E-3 / C-4-DDDXd+Eribulin-4+4

[0926] Reagents:

[0927] The antibodies were E-3 and C-4, and the linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin.

[0928] Experimental procedure:

[0929] 1. Displace the E-3 antibody into histidine buffer at pH 6.0, and adjust the antibody concentration to approximately 10 mg / mL using histidine buffer (pH 6.0). Adjust the pH to approximately 7.0 using 300 mM Na2HPO4 aqueous solution. Add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) aqueous solution to make the antibody to TCEP·HCl molar ratio 1:10. Place the solution on a rotary incubator and incubate at 120 rpm and 37°C in the dark for 1 hour to obtain reaction solution 1.

[0930] 2. Coupling of E-3 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:14. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 2.

[0931] 3. Displace the C-4 antibody into histidine buffer at pH 6.0, and adjust the antibody concentration to approximately 10 mg / mL using histidine buffer (pH 6.0). Adjust the pH to approximately 7.0 using 300 mM Na2HPO4 aqueous solution. Add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution to make the antibody to TCEP·HCl molar ratio 1:10. Place the solution on a rotary incubator and incubate at 120 rpm and 37°C in the dark for 1 hour to obtain reaction solution 3.

[0932] 4. Conjugation of C-4 antibody with MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:14. Incubate at 22°C in the dark for 1 hour with gentle stirring to connect the antibody with the adapter-loaded substance, thus obtaining reaction solution 4.

[0933] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with histidine buffer, anti-EGFR / anti-cMet bispecific antibody-dual toxin ADC, namely E-3 / C-4-DDDXd+Eribulin-4+4, is obtained.

[0934] Hydrophobic chromatography confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0935] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0936] This bispecific antibody-dextrin ADC contains a bispecific antibody (E-3-C-4), a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the E-3 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the C-4 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0937] Example 10-4: Preparation of anti-EGFR / anti-cMet bispecific antitoxin ADC E-4 / C-3-DDDXd+Eribulin-4+4

[0938] Reagents:

[0939] The antibodies were E-4 and C-3, and the linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin.

[0940] Experimental procedure:

[0941] 1. Displace the E-4 antibody into histidine buffer at pH 6.0, and adjust the antibody concentration to approximately 10 mg / mL using histidine buffer (pH 6.0). Adjust the pH to approximately 7.0 using 300 mM Na2HPO4 aqueous solution. Add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) aqueous solution to make the antibody to TCEP·HCl molar ratio 1:10. Place the solution on a rotary incubator and incubate at 120 rpm and 37°C in the dark for 1 hour to obtain reaction solution 1.

[0942] 2. Coupling of E-4 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:14. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 2.

[0943] 3. Displace the C-3 antibody into histidine buffer at pH 6.0, and adjust the antibody concentration to approximately 10 mg / mL using histidine buffer (pH 6.0). Adjust the pH to approximately 7.0 using 300 mM Na2HPO4 aqueous solution. Add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) aqueous solution to make the antibody to TCEP·HCl molar ratio 1:10. Place the solution on a rotary incubator and incubate at 120 rpm and 37°C in the dark for 1 hour to obtain reaction solution 3.

[0944] 4. Conjugation of C-3 antibody with MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:14. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded substance, thus obtaining reaction solution 4.

[0945] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with histidine buffer, anti-EGFR / anti-cMet bispecific antibody-dual toxin ADC, namely E-4 / C-3-DDDXd+Eribulin-4+4.

[0946] Hydrophobic chromatography confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0947] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0948] This bispecific antibody-dextrin ADC contains a bispecific antibody (E-4-C-3), a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the E-4 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the C-3 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0949] Examples 10-5: Preparation of anti-EGFR / anti-cMet bispecific antitoxin ADC E-4 / C-4-DDDXd+Eribulin-4+4

[0950] Reagents:

[0951] The antibodies were E-4 and C-4, and the linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin.

[0952] Experimental procedure:

[0953] 1. Displace the E-4 antibody into histidine buffer at pH 6.0, and adjust the antibody concentration to approximately 10 mg / mL using histidine buffer (pH 6.0). Adjust the pH to approximately 7.0 using 300 mM Na2HPO4 aqueous solution. Add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) aqueous solution to make the antibody to TCEP·HCl molar ratio 1:10. Place the solution on a rotary incubator and incubate at 120 rpm and 37°C in the dark for 1 hour to obtain reaction solution 1.

[0954] 2. Coupling of E-4 antibody with MC-GGFG-DDDXd: Add reaction solution 1 to a 10 mg / mL MC-GGFG-DDDXd solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-DDDXd 1:14. Incubate at 22°C in the dark for 1 hour with gentle stirring to link the antibody with the adapter-loaded vector, thus obtaining reaction solution 2.

[0955] 3. Displace the C-4 antibody into histidine buffer at pH 6.0, and adjust the antibody concentration to approximately 10 mg / mL using histidine buffer (pH 6.0). Adjust the pH to approximately 7.0 using 300 mM Na2HPO4 aqueous solution. Add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution to make the antibody to TCEP·HCl molar ratio 1:10. Place the solution on a rotary incubator and incubate at 120 rpm and 37°C in the dark for 1 hour to obtain reaction solution 3.

[0956] 4. Conjugation of C-4 antibody with MC-GGFG-Eribulin: Add reaction solution 3 to a 10 mg / mL MC-GGFG-Eribulin solution dissolved in DMF (N,N-dimethylformamide) to make the molar ratio of antibody to MC-GGFG-Eribulin 1:14. Incubate at 22°C in the dark for 1 hour with gentle stirring to connect the antibody with the adapter-loaded substance, thus obtaining reaction solution 4.

[0957] 5. Synthesis and purification of bispecific antibody-dual toxin ADC: Mix reaction solution 2 and reaction solution 4 to make the molar ratio of the two antibodies 1:1 to obtain reaction solution 5. After ultrafiltration of reaction solution 5 with histidine buffer, anti-EGFR / anti-cMet bispecific antibody-dual toxin ADC, namely E-4 / C-4-DDDXd+Eribulin-4+4, is obtained.

[0958] Hydrophobic chromatography confirmed the availability of the target bispecific antibody-dual toxin ADC, and its main component structure is as follows:

[0959] Each Linker-payload portion is coupled to the corresponding antibody by forming a thioether bond with the thiol group in the cysteine ​​residue of the antibody hinge region.

[0960] This bispecific antibody-dextrin ADC contains a bispecific antibody (E-4-C-4), a linker, and a cytotoxic drug. The bispecific antibody comprises a first half-antibody and a second half-antibody. The first half-antibody consists of one heavy chain and one light chain of the E-4 monoclonal antibody, and the second half-antibody consists of one heavy chain and one light chain of the C-4 monoclonal antibody. The first half-antibody is conjugated to MC-GGFG-DDDXd, and the second half-antibody is conjugated to MC-GGFG-Eribulin.

[0961] Examples 10-6: Preparation of anti-EGFR / anti-cMet bispecific antitoxin ADC E-5 / C-5-DDDXd+Eribulin-4+4

[0962] Reagents:

[0963] The antibodies were E-5 and C-5, and the linker-payload was MC-GGFG-DDDXd and MC-GGFG-Eribulin.

[0964] Experimental procedure:

[0965] 1. Displace the E-5 antibody into histidine buffer at pH 6.0, and adjust the antibody concentration to approximately 10 mg / mL using histidine buffer (pH 6.0). Adjust the pH to approximately 7.0 using 300 mM Na2HPO4 aqueous solution. Add 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) aqueo...

Claims

An antibody-conjugate or a pharmaceutically acceptable salt thereof, wherein, The antibody-conjugate comprises a heterodimeric antibody, a linker, and an active agent. The heterodimeric antibody comprises a first half-antibody and a second half-antibody, the first half-antibody containing a first CH3 region, and the second half-antibody containing a second CH3 region. According to EU designations, The first and / or second CH3 region has an amino acid Met, Ile or Leu at position 366, an amino acid Trp, Phe, Met, Tyr, Ile, Leu, Lys or Arg at position 394, an amino acid Ala, Thr, Ser, Asp or Glu at position 405, and / or an amino acid Thr, Ala or Val at position 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different; Furthermore, the first half-antibody is connected to an active agent via a connector, and the second half-antibody is connected to an active agent via a connector. The active agent connected to the first half-antibody may be the same as or different from the active agent connected to the second half-antibody. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein, According to EU designation, (1) The amino acid at position 366 of the first CH3 region is replaced with Met, Ile or Leu, and the amino acid at position 407 of the second CH3 region is replaced with Thr, Ala or Val; (2) The amino acid at position 405 of the first CH3 region is substituted with Ala, Thr, Ser, Asp, Glu, or Gly, and the amino acid at position 394 of the second CH3 region is substituted with Trp, Phe, Met, Tyr, Ile, Leu, Lys, or Arg; or (3) The amino acid at position 405 of the first CH3 region is substituted with Ala, Thr, Ser, Asp, Glu, or Gly, and the amino acid at position 407 of the second CH3 region is substituted with Thr, Ala, or Val; preferably (1) The first CH3 region has Met, Ile, or Leu at bit 366, and the second CH3 region has Thr, Ala, or Val at bit 407; and / or (2) The first CH3 region has Ala, Thr, Ser, Asp, Glu or Gly at bit 405, and the second CH3 region has Trp, Phe, Met, Tyr, Ile, Leu, Lys or Arg at bit 394. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to claim 2, wherein, The first CH3 region has Met at bit 366, and the second CH3 region has Thr at bit 407; The first CH3 region has Met at bit 366, and the second CH3 region has Ala at bit 407; The first CH3 region has Leu at bit 366, and the second CH3 region has Thr at bit 407; The first CH3 region has Leu at bit 366, and the second CH3 region has Ala at bit 407; The first CH3 region has Ile at bit 366, and the second CH3 region has Val at bit 407; The first CH3 region has Ala at bit 405, and the second CH3 region has Trp at bit 394; The first CH3 region has Thr at bit 405, and the second CH3 region has Trp at bit 394; The first CH3 region has Asp at bit 405, and the second CH3 region has Trp at bit 394; The first CH3 region has Glu at bit 405, and the second CH3 region has Trp at bit 394; The first CH3 region has Ser at bit 405, and the second CH3 region has Trp at bit 394; The first CH3 region has Ala at bit 405, and the second CH3 region has Phe at bit 394; The first CH3 region has Thr at bit 405, and the second CH3 region has Phe at bit 394; The first CH3 region has Asp at bit 405, and the second CH3 region has Phe at bit 394; The first CH3 region has Glu at bit 405, and the second CH3 region has Phe at bit 394; The first CH3 region has Ser at bit 405, and the second CH3 region has Phe at bit 394; The first CH3 region has Ala at bit 405, and the second CH3 region has Met at bit 394; The first CH3 region has Thr at bit 405, and the second CH3 region has Met at bit 394; The first CH3 region has Asp at bit 405, and the second CH3 region has Met at bit 394; The first CH3 region has Ser at bit 405, and the second CH3 region has Met at bit 394; The first CH3 region has Ala at bit 405, and the second CH3 region has Tyr at bit 394; The first CH3 region has Thr at bit 405, and the second CH3 region has Tyr at bit 394; The first CH3 region has Asp at bit 405, and the second CH3 region has Tyr at bit 394; The first CH3 region has Glu at bit 405, and the second CH3 region has Tyr at bit 394; The first CH3 region has Ser at bit 405, and the second CH3 region has Tyr at bit 394; The first CH3 region has Ala at bit 405, and the second CH3 region has Ile at bit 394; The first CH3 region has Thr at bit 405, and the second CH3 region has Ile at bit 394; The first CH3 region has Asp at bit 405, and the second CH3 region has Ile at bit 394; The first CH3 region has Ser at bit 405, and the second CH3 region has Ile at bit 394; The first CH3 region has Ala at bit 405, and the second CH3 region has Leu at bit 394; The first CH3 region has Thr at bit 405, and the second CH3 region has Leu at bit 394; The first CH3 region has Asp at bit 405, and the second CH3 region has Leu at bit 394; The first CH3 region has Ser at bit 405, and the second CH3 region has Leu at bit 394; The first CH3 region has Ala at bit 405, and the second CH3 region has Lys at bit 394; The first CH3 region has Thr at bit 405, and the second CH3 region has Lys at bit 394; The first CH3 region has Asp at bit 405, and the second CH3 region has Lys at bit 394; The first CH3 region has Glu at bit 405, and the second CH3 region has Lys at bit 394; The first CH3 region has Ser at bit 405, and the second CH3 region has Lys at bit 394; The first CH3 region has Ala at bit 405, and the second CH3 region has Arg at bit 394; The first CH3 region has Thr at bit 405, and the second CH3 region has Arg at bit 394; The first CH3 region has Asp at bit 405, and the second CH3 region has Arg at bit 394; The first CH3 region has Glu at bit 405, and the second CH3 region has Arg at bit 394; The first CH3 region has Ser at bit 405, and the second CH3 region has Arg at bit 394; The first CH3 region has Gly at bit 405, and the second CH3 region has Trp at bit 394; The first CH3 region has Gly at bit 405, and the second CH3 region has Phe at bit 394; or The first CH3 region has Gly at bit 405, and the second CH3 region has Tyr at bit 394; Preferably, the first CH3 region has Met at bit 366, and the second CH3 region has Thr at bit 407; The first CH3 region has Met at bit 366, and the second CH3 region has Ala at bit 407; The first CH3 region has Leu at bit 366, and the second CH3 region has Thr at bit 407; or The first CH3 region has Leu at bit 366, and the second CH3 region has Ala at bit 407. The antibody-conjugate or its pharmaceutically acceptable salt according to any one of claims 1-3, wherein, The first CH3 region is the CH3 region of IgG, preferably the CH3 region of human IgG; and / or The second CH3 region is the CH3 region of IgG, preferably the CH3 region of human IgG; Preferably, the first CH3 region is a CH3 region selected from IgG1, IgG2, IgG3, and IgG4, and more preferably a CH3 region selected from IgG1 and IgG4; and / or The second CH3 region is a CH3 region selected from IgG1, IgG2, IgG3 and IgG4, preferably a CH3 region selected from IgG1 and IgG4. The antibody-conjugate or its pharmaceutically acceptable salt according to any one of claims 1-4, wherein, The first CH3 region and the second CH3 region are both CH3 regions of human IgG1 or both CH3 regions of human IgG4. The antibody-conjugate or its pharmaceutically acceptable salt according to any one of claims 1-5, wherein, The first CH3 region and the second CH3 region contain any of the following sequence combinations: (1) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:66; (2) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:67; (3) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:66; (4) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:67; (5) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:68; (6) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:68; (7) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:68; (8) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:68; (9) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:68; (10) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:69; (11) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:69; (12) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:69; (13) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:69; (14) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:69; (15) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:70; (16) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:70; (17) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:70; (18) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:70; (19) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:71; (20) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:71; (21) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:71; (22) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:71; (23) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:71; (24) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:72; (25) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:72; (26) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:72; (27) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:72; (28) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:73; (29) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:73; (30) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:73; (31) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:73; (32) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:74; (33) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:74; (34) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:74; (35) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:74; (36) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:74; (37) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:75; (38) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:75; (39) The first CH3 region contains the sequence shown in SEQ ID NO:62, and the second CH3 region contains the sequence shown in SEQ ID NO:75; (40) The first CH3 region contains the sequence shown in SEQ ID NO:63, and the second CH3 region contains the sequence shown in SEQ ID NO:75; (41) The first CH3 region contains the sequence shown in SEQ ID NO:64, and the second CH3 region contains the sequence shown in SEQ ID NO:75; (42) The first CH3 region contains the sequence shown in SEQ ID NO:65, and the second CH3 region contains the sequence shown in SEQ ID NO:68; (43) The first CH3 region contains the sequence shown in SEQ ID NO:65, and the second CH3 region contains the sequence shown in SEQ ID NO:69; or (44) The first CH3 region contains the sequence shown in SEQ ID NO:65, and the second CH3 region contains the sequence shown in SEQ ID NO:71; Preferably, the first CH3 region and the second CH3 region comprise any of the following sequence combinations: (1) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:66; (2) The first CH3 region contains the sequence shown in SEQ ID NO:58, and the second CH3 region contains the sequence shown in SEQ ID NO:67; (3) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:66; (4) The first CH3 region contains the sequence shown in SEQ ID NO:59, and the second CH3 region contains the sequence shown in SEQ ID NO:67; (5) The first CH3 region contains the sequence shown in SEQ ID NO:60, and the second CH3 region contains the sequence shown in SEQ ID NO:68; or (6) The first CH3 region contains the sequence shown in SEQ ID NO:61, and the second CH3 region contains the sequence shown in SEQ ID NO:

71. The antibody-conjugate or its pharmaceutically acceptable salt according to any one of claims 1-6, wherein, The first half-antibody contains a first Fc polypeptide, the first Fc polypeptide contains a first CH3 region, the second half-antibody contains a second Fc polypeptide, the second Fc polypeptide contains a second CH3 region, and the first Fc polypeptide and the second Fc polypeptide constitute an Fc domain. Preferably, the Fc domain is a human IgG Fc domain, and more preferably an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, or an IgG4 Fc domain; More preferably, the Fc domain comprises amino acid substitutions that reduce or eliminate the binding of the CH3 region of an Fc polypeptide in the Fc domain to protein A. The antibody-conjugate or its pharmaceutically acceptable salt according to any one of claims 1-7, wherein, The first half antibody includes a first antigen-binding portion, and the second half antibody includes a second antigen-binding portion. The first antigen-binding portion and the second antigen-binding portion bind to the same or different antigens, or bind to the same or different epitopes. Preferably, the first antigen-binding portion and the second antigen-binding portion are each independently Fab, ScFv, VHH or ScFab; more preferably, the first antigen-binding portion and the second antigen-binding portion are both Fab. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-8, wherein, The heterodimeric antibody contains a Cys-Pro-Pro-Cys sequence in its hinge region. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-9, wherein, The active agents linked to the first and second halves of the antibody are each independently selected from: microtubule inhibitors, DNA damage agents, immunomodulators, topoisomerase inhibitors, or other active agents; Preferably, the active agents linked to the first and second half-antibodies are each independently selected from: eribulin or its derivatives, olipatin, MMAF, MMAE, maytansine, DM1, DM4, camptothecin, hydroxycamptothecin, aminocamptothecin, irinotecan, topotecan, esanotecan or its derivatives, rubitecan, letopotecan, gimarotecan, karenitecin, 7-ethylcamptothecin, SN-38, DXd, chachiin, N-acetyl-γ-calicheamicin, atrazomycin, DSB-120, SJG-136, TLR agonists, STING agonists, paclitaxel, paclitaxel liposomes, albumin-bound paclitaxel, cabazitaxel, docetaxel, oxaliplatin, cisplatin, carboplatin, nedaplatin, bicycloplatin, miplatin, lobaplatin, pyrplatin, levoplatin, triplatinum tetranitrate, phenanthreneplatin, saxaplatin, and IRDye700DX; More preferably, (1) The active agent linked to the first half-antibody is eribulin or a derivative thereof, and the active agent linked to the second half-antibody is essanotecan or a derivative thereof; or (2) The active agent linked to the first half-antibody is esanotecan or its derivative, and the active agent linked to the second half-antibody is eribulin or its derivative; More preferably, (1) The active agent linked to the first half-antibody has the structure shown in Formula I, and the active agent linked to the second half-antibody has the structure shown in Formula II; or; (2) The active agent linked to the first half-antibody has the structure shown in Formula II, and the active agent linked to the second half-antibody has the structure shown in Formula I. Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6-10 aryl, optionally substituted 5 to 12 heteroaryl groups, R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6-10 aryl, optionally substituted 5 to 12 heteroaryl groups; or, R 1 and R 2 Together with the atoms attached thereto, they form optional 5- to 8-membered heterocyclic groups; R 3 R 4 R 5 and R 6 Each is independently selected from either hydrogen or deuterium atoms. The antibody-conjugate or its pharmaceutically acceptable salt according to any one of claims 1-10, wherein, (1) The first hapten antibody is transmitted through a linker with the structure shown in Formula IIIa below. The structure shown in formula Ia is connected. The second half-antibody is connected to the structure shown in Formula IIa via a linker of the structure shown in Formula IIIa. or (2) The first hapten via a linker with the structure shown in Formula IIIa The structure shown in Formula IIa is connected. The second half-antibody is connected to the structure shown in Formula Ia via a linker of the structure shown in Formula IIIa. The hapten is attached to the position shown * in the adapter, and the structures shown in Formula Ia and Formula IIa are attached to the positions shown # in the adapter. Among them, R 1 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6-10 aryl, optionally substituted 5 to 12 heteroaryl groups, R 2 Selected from hydrogen atoms, deuterium atoms, and optionally substituted C atoms 1-6 Alkyl groups, optionally substituted C 3-7 cycloalkyl, optionally substituted 3- to 7-membered heterocyclic groups, optionally substituted C 6-10 aryl, optionally substituted 5 to 12 heteroaryl groups; or, R 1 and R 2 Together with the atoms attached thereto, they form optional 5- to 8-membered heterocyclic groups; R 3 R 4 R 5 and R 6 Each is independently selected from either hydrogen or deuterium atoms; Preferably, the R 1 and R 2 Each is independently selected from hydrogen atoms or C atoms. 1-5 alkyl; The R 3 For deuterium atoms, R 4 It is a deuterium atom; or R 3 For hydrogen atoms, R 4 It is a hydrogen atom. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-11, wherein the DAR of the antibody-drug conjugate is 1-10, 2-8, 3.5-8, 4-8, 3.5-4.5, 6-8, 7-8, 7.5-8, 7.6-8, 7.7-8, 7.8-8, or 7.9-8, 2, 3, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 5, 6, 7, 7.5, 7.6, 7.7, 7.73, 7.79, 7.8, 7.86, 7.9, or 8. The antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-12, wherein, The heterodimeric antibody is a heterodimeric antibody targeting at least one of the following targets: HER2, HER3, EGFR, ROR1, CLDN18.2, B7-H3, B7-H4, TROP-2, CD19, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, PD-1, and PD-L1. A method for preparing the antibody-conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-13, comprising the following steps: (1) Provides a first homodimeric antibody comprising a first CH3 region, wherein, according to the EU number, the first CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407; (2) The first homodimer antibody was treated with a reducing agent to reduce the interchain disulfide bonds of the first homodimer antibody; the first homodimer antibody with thiol group was linked to the active agent through a coupling reaction and purified to obtain the first homodimer conjugate. (3) Provide a second homodimer antibody comprising a second CH3 region, wherein, according to the EU number, the second CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different. (4) The second homodimer antibody is treated with a reducing agent to reduce the interchain disulfide bonds of the second homodimer antibody; the second homodimer antibody with thiol group is coupled to the active agent through a coupling reaction, and the second homodimer conjugate is purified. Wherein, the active agent linked to the first homodimeric antibody may be the same as or different from the active agent linked to the second homodimeric antibody; (5) Incubate the first homodimeric conjugate and the second homodimeric conjugate together; and (6) Obtain the antibody-conjugate or a pharmaceutically acceptable salt thereof; Alternatively, it may include the following steps: (1) Provides a first homodimeric antibody comprising a first CH3 region, wherein, according to the EU number, the first CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407; (2) The first homodimer antibody is treated with a reducing agent to reduce the interchain disulfide bonds of the first homodimer antibody; through a coupling reaction, the first homodimer antibody with thiol groups is linked to the active agent through a linker to obtain the first reaction solution; (3) Provide a second homodimer antibody comprising a second CH3 region, wherein, according to the EU number, the second CH3 region has at least one amino acid substitution at positions 366, 394, 405 and / or 407, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different. (4) The second homodimer antibody is treated with a reducing agent to reduce the interchain disulfide bonds of the second homodimer antibody; through a coupling reaction, the second homodimer antibody with thiol groups is linked to the activator through a linker to obtain the second reaction solution; Wherein, the active agent linked to the first homodimeric antibody may be the same as or different from the active agent linked to the second homodimeric antibody; (5) Incubate the first reaction solution together with the second reaction solution; and (6) Obtain the antibody-conjugate or a pharmaceutically acceptable salt thereof; Alternatively, it may include the following steps: (1) Provides a first homodimeric antibody comprising a first CH3 region, wherein, according to the EU number, the first CH3 region has at least one amino acid substitution at positions 366, 368, 370, 399, 405, 407 and / or 409; (2) The first homodimeric antibody is linked to the active agent through a linker to obtain the first homodimeric conjugate; (3) Provide a second homodimer antibody comprising a second CH3 region, wherein, according to the EU number, the second CH3 region has at least one amino acid substitution at positions 366, 368, 370, 399, 405, 407 and / or 409, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different. (4) The second homodimeric antibody is linked to the active agent through a linker to obtain the second homodimeric conjugate; Wherein, the active agent linked to the first homodimeric antibody may be the same as or different from the active agent linked to the second homodimeric antibody; (5) Incubate the first homodimeric coupling compound and the second homodimeric coupling compound together in the presence of a reducing agent, wherein the presence of the reducing agent is sufficient to allow the reduction of interchain disulfide bonds in the hinge regions of the first and second homodimeric coupling compounds, and (6) Obtain the antibody-conjugate or a pharmaceutically acceptable salt thereof. The method according to claim 14, wherein, Step (6) includes subjecting the reaction solution obtained in step (5) to oxidative conditions sufficient to allow cysteine ​​in the antibody-conjugate to be oxidized to interchain disulfide bonds; or step (6) includes removing a reducing agent from the reaction system. According to the method of claim 14 or 15, the reducing agent is selected from: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dierythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, β-mercaptoethanol, or salts thereof; preferably, the reducing agent is selected from: 2-MEA, DTT and TCEP, or salts thereof. A pharmaceutical composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-13. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-13, or the pharmaceutical composition according to claim 17, and optionally one or more other therapeutic agents, in the preparation of a medicament for treating tumors. The use according to claim 18, wherein, The tumors mentioned are biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, melanoma, pharyngeal cancer, oral cancer, skin cancer, lung cancer, urethral cancer, urothelial carcinoma, bone cancer, soft tissue cancer, gallbladder cancer, testicular cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, liver cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, or myeloma. A kit comprising the antibody-drug conjugate of any one of claims 1-13 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 17.

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