Heterodimer antibody, preparation method therefor, and use thereof

Abstract

Provided are a heterodimeric antibody, a method for preparing the heterodimeric antibody, and use of the antibody in the preparation of a medicament for treating tumors and autoimmune diseases.

Description

Heterodimeric antibodies, their preparation methods and applications Technical Field

[0001] This disclosure pertains to the field of biomedicine and relates to heterodimeric antibodies, methods for preparing such heterodimeric antibodies, and the use of such antibodies in the preparation of medicaments for treating tumors or autoimmune diseases. Background Technology

[0002] Bispecific antibodies, with their dual-targeting advantage, have become a hot research area in the treatment of tumors, autoimmune diseases, and infectious diseases. Since catuxomab (EpCAM×CD3) was approved by the FDA in 2009, 14 bispecific antibody drugs have been approved for marketing worldwide. The construction and preparation technologies of bispecific antibodies have also undergone a series of innovations, one of the goals of which is to solve the mismatch problem and improve production efficiency. In recent years, scientists have developed many strategies and technology platforms that can design and express bispecific antibodies with different structures. The two parental antibodies are often co-expressed as half-molecules or expressed separately, and then, with the help of mutations introduced in advance in the Fc region (specifically the CH3 region), heterodimers of different heavy chains can be assembled in vivo or in vitro. For example, the Duobody technology platform developed by Genmab introduces the classic F405L and K409R mutations in the Fc region.

[0003] However, exploring more mutation combinations in the Fc region to ensure high purity of parental antibody expression after mutation, and to achieve heterodimeric antibody assembly with success rate and purity comparable to or better than Duobody, remains a persistent goal in this field. Summary of the Invention

[0004] This disclosure relates to heterodimeric antibodies, methods for preparing such heterodimeric antibodies, and the use of such antibodies in the preparation of medicaments for treating tumors or autoimmune diseases. For example, bispecific antibodies can be assembled in vitro based on two monospecific antibodies as raw materials.

[0005] Heterodimeric antibodies

[0006] This disclosure provides 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 designation, 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. 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 as follows: 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. 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 heterodimeric antibodies, 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, and the amino acid substitution positions of the first CH3 region and the second CH3 region are different.

[0007] 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.

[0008] 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:

[0009] (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;

[0010] (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

[0011] (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.

[0012] 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,

[0013] (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; or

[0014] (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. In some embodiments, the heterodimeric antibody comprises an amino acid substitution that promotes the formation of the heterodimer, the amino acid substitution that promotes the formation of the heterodimer being selected from (1) and / or (2).

[0015] In some embodiments, the heterodimeric antibody comprises a first half-antibody and a second half-antibody, the first half-antibody comprising a first CH3 region and the second half-antibody comprising a second CH3 region, wherein, 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 CH3 region and the second CH3 region are different. 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.

[0016] In some embodiments, the heterodimeric antibody comprises a first half-antibody and a second half-antibody, the first half-antibody comprising a first CH3 region and the second half-antibody comprising a second CH3 region, wherein, 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 CH3 region and the second CH3 region are different. 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.

[0017] 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:

[0018] (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;

[0019] (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

[0020] (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.

[0021] 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,

[0022] (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

[0023] (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 heterodimeric antibody comprises an amino acid substitution that promotes the formation of the heterodimer, the amino acid substitution being selected from (1) and / or (2) above.

[0024] 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,

[0025] (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

[0026] (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 heterodimeric antibody comprises an amino acid substitution that promotes the formation of the heterodimer, the amino acid substitution being selected from (1) and / or (2) above.

[0027] 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 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 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: 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 position 366, and the second CH3 region has Thr, Ala, or Val at position 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, 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, 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.

[0028] 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 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. In some embodiments, the heterodimeric antibody comprises an amino acid substitution that promotes the formation of the heterodimer, wherein the amino acid substitution that promotes the formation of the heterodimer is: 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 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.

[0029] In some implementations, 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.

[0030] In some specific implementations, the first CH3 region and the second CH3 region have any of the exemplary mutation combinations in Table A.

[0031] Table A: Exemplary Mutation Combinations

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] In some implementations, the first and second haptens are IgG1 type haptens.

[0037] 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.

[0038] 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.

[0039] 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: 36. In some embodiments, compared to the sequence shown in SEQ ID NO: 36, 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: 36, 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.

[0040] 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.

[0041] In some embodiments, the first CH3 region and the second CH3 region comprise sequences shown as any one of SEQ ID NO:42 to SEQ ID NO:59, 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:42 to SEQ ID NO:46, SEQ ID NO:48, and SEQ ID NO:50 to SEQ ID NO:59, and the sequences of the first CH3 region and the second CH3 region are different.

[0042] 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:

[0043] (1) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0044] (2) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0045] (3) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0046] (4) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0047] (5) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0048] (6) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0049] (7) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0050] (8) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0051] (9) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0052] (10) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0053] (11) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0054] (12) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0055] (13) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0056] (14) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0057] (15) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0058] (16) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0059] (17) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0060] (18) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0061] (19) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0062] (20) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0063] (21) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0064] (22) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0065] (23) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0066] (24) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0067] (25) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0068] (26) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0069] (27) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0070] (28) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0071] (29) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0072] (30) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0073] (31) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0074] (32) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0075] (33) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0076] (34) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0077] (35) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0078] (36) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0079] (37) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0080] (38) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0081] (39) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0082] (40) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0083] (41) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0084] (42) The first CH3 region contains the sequence shown in SEQ ID NO:49, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0085] (43) The first CH3 region contains the sequence shown in SEQ ID NO:49, and the second CH3 region contains the sequence shown in SEQ ID NO:53; or

[0086] (44) The first CH3 region contains the sequence shown in SEQ ID NO:49, and the second CH3 region contains the sequence shown in SEQ ID NO:55.

[0087] 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:

[0088] (1) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0089] (2) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0090] (3) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0091] (4) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0092] (5) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0093] (6) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0094] (7) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0095] (8) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0096] (9) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0097] (10) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0098] (11) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0099] (12) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0100] (13) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0101] (14) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0102] (15) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0103] (16) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0104] (17) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0105] (18) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0106] (19) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0107] (20) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0108] (21) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0109] (22) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0110] (23) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0111] (24) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0112] (25) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0113] (26) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0114] (27) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0115] (28) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0116] (29) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0117] (30) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0118] (31) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0119] (32) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0120] (33) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0121] (34) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0122] (35) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:59; or

[0123] (36) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:59.

[0124] 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:

[0125] (1) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0126] (2) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0127] (3) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0128] (4) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0129] (5) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:52; or

[0130] (6) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:55.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] In some implementations, the heterodimeric antibody is divalent or multivalent (e.g., divalent, trivalent, tetravalent, etc.).

[0137] 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.

[0138] 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.

[0139] The heterodimeric antibodies disclosed herein include, but are not limited to, monoclonal antibodies, monospecific antibodies, bispecific antibodies, multispecific antibodies, and nanobodies. The heterodimeric antibodies disclosed herein 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, CD3, 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 (one, two, or three) targets selected from HER2, HER3, EGFR, ROR1, CLDN18.2, B7-H3, B7-H4, TROP-2, CD3, 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 EGFR and c-MET. In one specific embodiment, the heterodimeric antibody is a bispecific antibody targeting CD3 and CD20. In one specific embodiment, the heterodimeric antibody is a bispecific antibody targeting B7-H3 and B7-H4.

[0140] In some embodiments, the heterodimeric antibodies provided in this disclosure exhibit one or more of the following properties in combination:

[0141] (1) Combined with human EGFR;

[0142] (2) Combined with human c-MET;

[0143] (3) It has cytotoxic activity against tumor cells expressing EGFR and / or c-MET; and

[0144] (4) It has therapeutic effects on diseases related to EGFR and / or c-MET expression.

[0145] In some embodiments, the heterodimeric antibodies provided in this disclosure exhibit one or more of the following properties in combination:

[0146] (1) Combined with human CD3;

[0147] (2) Combined with human CD20;

[0148] (3) It has killing activity against tumor cells expressing CD3 and / or CD20; and

[0149] (4) It has therapeutic effects on diseases related to CD3 and / or CD20 expression.

[0150] In some embodiments, the heterodimeric antibodies provided in this disclosure exhibit one or more of the following properties in combination:

[0151] (1) Combined with human B7-H3;

[0152] (2) Combined with human B7-H4;

[0153] (3) It has killing activity against tumor cells expressing B7-H3 and / or B7-H4; and

[0154] (4) It has therapeutic effects on diseases related to B7-H3 and / or B7-H4 expression.

[0155] In some embodiments, the heterodimer antibody provided in this disclosure can be assembled in vitro with high assembly efficiency (e.g., higher than 80%, higher than 85%, higher than 90%, or even higher than 95%) through a first CH3 region and a second CH3 region containing a specific site mutation combination, to obtain the target heterodimer with high purity, low molecular weight and low molecular weight impurities, exhibiting good target antigen binding properties (can simultaneously bind to two target antigens or two epitopes of the same target antigen with good binding affinity), good thermal stability, plasma stability and freeze-thaw stability, can efficiently activate ADCC effect to achieve strong cytotoxicity, and exhibits good in vivo PK properties in animals.

[0156] The heterodimers provided in this disclosure can be assembled in vitro. Specifically, antibodies containing heterodimer Fc can be effectively prepared based on antibodies containing homodimer Fc. For example, two monospecific antibodies can be used as raw materials, and after reduction, specific mutant combinations can be introduced into the CH3 region to form bispecific antibodies. The antibodies have high purity and stability. The heterodimer antibodies provided in this disclosure achieve excellent efficacy and / or safety. The heterodimer antibodies provided in this disclosure achieve excellent antitumor effects and / or safety. In some embodiments, the heterodimer antibodies have good in vivo efficacy. In some embodiments, the heterodimer antibodies have good in vivo antitumor activity. In some embodiments, the heterodimer antibodies have excellent safety. In some embodiments, the heterodimer antibodies are not prone to aggregation. In some embodiments, the heterodimer antibodies have better solubility (e.g., water solubility).

[0157] Preparation method

[0158] This disclosure provides a method for preparing heterodimeric antibodies, which includes the following steps:

[0159] (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;

[0160] (2) 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.

[0161] (3) The first homodimeric antibody and the second homodimeric antibody were incubated together under conditions sufficient to allow the reduction of disulfide bonds in the hinge region; and

[0162] (4) Obtain the heterodimer antibody.

[0163] In some embodiments, the preparation method is in vitro.

[0164] 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.

[0165] 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.

[0166] In some embodiments of the method, step (4) includes subjecting the reaction solution obtained in step (3) to oxidative conditions sufficient to allow cysteine ​​in the antibody to be oxidized to interchain disulfide bonds. In other embodiments of the method, step (4) 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.

[0167] In one embodiment, the reduction conditions of step (3) include the addition of a reducing agent 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, 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.

[0168] The reduction temperature is 10-30℃, 15-30℃, 20-30℃, 25-30℃, etc., specifically 10℃, 15℃, 20℃, 25℃, 30℃, etc.

[0169] 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.

[0170] After reduction, the reducing agent in the system is removed, and after further incubation, heterodimeric antibodies are obtained. The reducing agent can be removed using conventional methods, such as buffer replacement. The incubation time is 1-24 hours, 2-24 hours, 4-24 hours, 6-24 hours, 8-24 hours, 10-24 hours, 12-24 hours, etc., specifically for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, etc. The incubation temperature is 0-10℃, 1-10℃, 1-8℃, 1-6℃, 1-4℃, etc., specifically for 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, etc.

[0171] In some embodiments, according to the EU designation, 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.

[0172] In some embodiments, according to the EU designation, 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.

[0173] In some embodiments, according to the EU designation, 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.

[0174] 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.

[0175] 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, or Glu at position 405, and / 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. In some of these embodiments, the first and second homodimeric antibodies contain amino acid substitutions that promote heterodimer formation, said heterodimer-promoting amino acid substitutions being selected from the amino acid substitutions of the first and / or second CH3 regions described above.

[0176] In some implementation schemes, according to EU designation,

[0177] (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;

[0178] (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

[0179] (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.

[0180] In some implementation schemes, according to EU designation,

[0181] (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

[0182] (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.

[0183] 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,

[0184] (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

[0185] (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.

[0186] In some embodiments, according to the EU number, 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. 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 position 366, and the second CH3 region has Thr at position 407. In some specific embodiments, the first CH3 region has Met 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 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 heterodimer 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 Leu at position 366, the second CH3 region has 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 Leu at position 366, and the second CH3 region has Ala at position 407. In some specific implementations, the first CH3 region has Leu at position 366, the second CH3 region has Ala at position 407, and the heterodimeric antibody does not contain one or more amino acid substitutions from K409F, L351Y, D399C, and K409P.

[0187] In some embodiments, according to the EU number, 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. 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 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.

[0188] In some specific implementations, the first CH3 region and the second CH3 region have any of the exemplary mutation combinations in Table A.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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 embodiments, the Fc domain is an IgG4 Fc domain. In some specific embodiments, the Fc domain is a human IgG4 Fc domain. In some specific embodiments, the Fc domain is a human IgG1 Fc domain.

[0195] In some embodiments, the first and / or second CH3 regions contain the sequence illustrated in SEQ ID NO: 36, in addition to the specified amino acid substitutions. In some embodiments, compared to the sequence shown in SEQ ID NO: 36, 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: 36, 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.

[0196] 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.

[0197] In some embodiments, the first CH3 region and the second CH3 region comprise sequences shown as any one of SEQ ID NO:42 to SEQ ID NO:59, 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:42 to SEQ ID NO:46, SEQ ID NO:48, and SEQ ID NO:50 to SEQ ID NO:59, and the sequences of the first CH3 region and the second CH3 region are different.

[0198] 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:

[0199] (1) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0200] (2) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0201] (3) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0202] (4) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0203] (5) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0204] (6) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0205] (7) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0206] (8) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0207] (9) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0208] (10) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0209] (11) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0210] (12) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0211] (13) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0212] (14) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0213] (15) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0214] (16) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0215] (17) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0216] (18) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0217] (19) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0218] (20) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0219] (21) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0220] (22) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0221] (23) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0222] (24) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0223] (25) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0224] (26) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0225] (27) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0226] (28) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0227] (29) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0228] (30) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0229] (31) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0230] (32) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0231] (33) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0232] (34) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0233] (35) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0234] (36) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0235] (37) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0236] (38) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0237] (39) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0238] (40) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0239] (41) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0240] (42) The first CH3 region contains the sequence shown in SEQ ID NO:49, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0241] (43) The first CH3 region contains the sequence shown in SEQ ID NO:49, and the second CH3 region contains the sequence shown in SEQ ID NO:53; or

[0242] (44) The first CH3 region contains the sequence shown in SEQ ID NO:49, and the second CH3 region contains the sequence shown in SEQ ID NO:55.

[0243] 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:

[0244] (1) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0245] (2) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0246] (3) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0247] (4) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0248] (5) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0249] (6) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0250] (7) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0251] (8) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:52;

[0252] (9) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0253] (10) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0254] (11) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0255] (12) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:53;

[0256] (13) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0257] (14) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0258] (15) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0259] (16) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:54;

[0260] (17) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0261] (18) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0262] (19) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0263] (20) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:55;

[0264] (21) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0265] (22) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0266] (23) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0267] (24) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:56;

[0268] (25) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0269] (26) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0270] (27) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0271] (28) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:57;

[0272] (29) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0273] (30) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0274] (31) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0275] (32) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:58;

[0276] (33) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0277] (34) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:59;

[0278] (35) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:59; or

[0279] (36) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:59.

[0280] 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:

[0281] (1) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0282] (2) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0283] (3) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:50;

[0284] (4) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:51;

[0285] (5) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:52; or

[0286] (6) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:55.

[0287] 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.

[0288] 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.

[0289] 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.

[0290] In some embodiments, the first antigen-binding portion and the second antigen-binding portion are each independently in the form of Fab, ScFv, VHH, or ScFab. In some embodiments, both the first and second antigen-binding portions are Fab. In some embodiments, the first homodimeric antibody comprises two first antigen-binding portions. In some embodiments, the second homodimeric antibody comprises two second antigen-binding portions.

[0291] 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.

[0292] In some implementations, the first and / or homodimeric antibody is divalent or multivalent (e.g., divalent, trivalent, tetravalent, etc.).

[0293] 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.

[0294] In some implementations, the first homodimeric antibody and / or the second homodimeric antibody include a hinge region.

[0295] In some embodiments, the first homodimeric antibody does not contain the Cys-Pro-Pro-Cys sequence in the hinge region. In some embodiments, the second homodimeric antibody does not contain the Cys-Pro-Pro-Cys sequence in the hinge region. In some embodiments, both the first and second homodimeric antibodies do not contain the Cys-Pro-Pro-Cys sequence in the hinge region. In some embodiments, the first homodimeric antibody contains the Cys-Pro-Pro-Cys sequence in the hinge region. In some embodiments, both the first and second homodimeric antibodies contain the Cys-Pro-Pro-Cys sequence in the hinge region. In some embodiments, the first and / or second haploidentical antibody contains a hinge region containing the sequence shown in SEQ ID NO:2.

[0296] 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.

[0297] 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, CD3, 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, CD3, 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 heterodimeric antibody is a bispecific antibody targeting EGFR and c-MET. In one specific embodiment, the heterodimeric antibody is a bispecific antibody targeting CD3 and CD20. In one specific embodiment, the first homodimer targets B7-H3, and the second homodimer targets B7-H4.

[0298] In some embodiments, the heterodimeric antibodies prepared by the methods of this disclosure exhibit one or more combinations of the following properties:

[0299] (5) Combined with human EGFR;

[0300] (6) Combined with human c-MET;

[0301] (7) It has cytotoxic activity against tumor cells expressing EGFR and / or c-MET; and

[0302] (8) It has therapeutic effects on diseases related to EGFR and / or c-MET expression.

[0303] In some embodiments, the heterodimeric antibodies prepared by the methods of this disclosure exhibit one or more combinations of the following properties:

[0304] (5) Combined with human CD3;

[0305] (6) Combined with human CD20;

[0306] (7) It has killing activity against tumor cells expressing CD3 and / or CD20; and

[0307] (8) It has therapeutic effects on diseases related to CD3 and / or CD20 expression.

[0308] In some embodiments, the heterodimeric antibodies prepared by the methods of this disclosure exhibit one or more combinations of the following properties:

[0309] (1) Combined with human B7-H3;

[0310] (2) Combined with human B7-H4;

[0311] (3) It has killing activity against tumor cells expressing B7-H3 and / or B7-H4; and

[0312] (4) It has therapeutic effects on diseases related to B7-H3 and / or B7-H4 expression.

[0313] The heterodimeric antibody prepared by the method disclosed herein:

[0314] (1) It has achieved excellent efficacy and / or safety;

[0315] (2) It achieved excellent anti-tumor effects and / or safety;

[0316] (3) It has good in vivo efficacy;

[0317] (4) It has good in vivo antitumor activity;

[0318] (5) It has excellent safety features;

[0319] (6) Not easy to accumulate; and / or

[0320] (7) It has better solubility (e.g., water solubility).

[0321] The method disclosed herein can assemble high-purity target heterodimers in vitro with high assembly efficiency, resulting in products with low content of both low-molecular-weight and high-molecular-weight impurities.

[0322] Pharmaceutical Composition

[0323] In one aspect, this disclosure provides a pharmaceutical composition comprising the heterodimeric antibody of this disclosure. In some embodiments, this disclosure provides a pharmaceutical composition comprising the heterodimeric antibody of this disclosure, and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other reagents.

[0324] use

[0325] This disclosure provides for the use of the heterodimeric antibodies of this disclosure. This disclosure also provides for the use of the pharmaceutical compositions of this disclosure. Uses may include the treatment of tumors or autoimmune diseases.

[0326] In one aspect, this disclosure provides the use of the heterodimeric antibody of this disclosure in the preparation of a medicament for treating diseases expressing an antigen bound to said heterodimeric antibody. In some embodiments, this disclosure provides the use of the heterodimeric antibody 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 heterodimeric antibody of this disclosure in the preparation of a medicament for treating diseases expressing B7-H3 and / or B7-H4, EGFR and / or c-MET, CD3 and / or CD20.

[0327] 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 B7-H3 and / or B7-H4, EGFR and / or c-MET, CD3 and / or CD20.

[0328] In one aspect, this disclosure provides the use of the heterodimeric antibody of this disclosure, and one or more other therapeutic agents, in the preparation of a medicament for treating diseases expressing an antigen bound to said heterodimeric antibody. In some embodiments, this disclosure provides the use of the heterodimeric antibody of this disclosure, and one or more other therapeutic agents, in the preparation of a medicament for treating tumors or autoimmune diseases. In some embodiments, this disclosure provides the use of the heterodimeric antibody of this disclosure, and one or more other therapeutic agents, in the preparation of a medicament for treating diseases expressing B7-H3 and / or B7-H4, EGFR and / or c-MET, CD3 and / or CD20.

[0329] 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 antibody. 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 B7-H3 and / or B7-H4, EGFR and / or c-MET, CD3 and / or CD20.

[0330] In one aspect, this disclosure provides a method for treating a disease expressing an antigen bound by the heterodimeric antibody, the method comprising administering to a subject the heterodimeric antibody of this disclosure or a pharmaceutical composition of this disclosure. In some embodiments, this disclosure provides a method for treating a tumor or an autoimmune disease, the method comprising administering to a subject the heterodimeric antibody of this disclosure or a pharmaceutical composition of this disclosure. In some embodiments, this disclosure provides a method for treating a disease expressing B7-H3 and / or B7-H4, EGFR and / or c-MET, CD3 and / or CD20, the method comprising administering to a subject the heterodimeric antibody of this disclosure or a pharmaceutical composition of this disclosure. In some embodiments, the heterodimeric antibody of this disclosure or a pharmaceutical composition of this disclosure is administered to a subject in a therapeutically effective amount.

[0331] In one aspect, this disclosure provides a method for treating a disease expressing an antigen bound by the heterodimeric antibody, the method comprising administering to a subject the heterodimeric antibody of this 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 the heterodimeric antibody of this 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, EGFR and / or c-MET, CD3 and / or CD20, the method comprising administering to a subject the heterodimeric antibody of this disclosure, and one or more additional therapeutic agents. In some embodiments, the heterodimeric antibody of this disclosure, and one or more additional therapeutic agents are administered to a subject in a therapeutically effective amount.

[0332] 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 B7-H3 and / or B7-H4, EGFR and / or c-MET, CD3 and / or CD20, 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.

[0333] In some embodiments, the tumor is a tumor expressing B7-H3 and / or B7-H4, EGFR and / or c-MET, CD3 and / or CD20. In some embodiments, the disease expressing B7-H3 and / or B7-H4, EGFR and / or c-MET, CD3 and / or CD20 is a tumor expressing B7-H3 and / or B7-H4, EGFR and / or c-MET, CD3 and / or CD20. In some embodiments, the additional therapeutic agent may be a tumor therapeutic agent known in the art.

[0334] In some embodiments, the method includes contacting tumor cells with the heterodimeric antibody or the pharmaceutical composition to kill tumor cells or inhibit tumor cell growth.

[0335] In some embodiments, the method includes contacting tumor cells with the heterodimeric antibody or the pharmaceutical composition, and simultaneously or sequentially contacting the tumor cells with one or more additional therapeutic agents, thereby killing the tumor cells or inhibiting tumor cell growth.

[0336] In some embodiments, administering the heterodimeric antibody or the pharmaceutical composition disclosed herein to a subject can kill tumor cells or inhibit tumor cell growth.

[0337] In some embodiments, administration of the disclosed heterodimeric antibody, 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 disclosed pharmaceutical composition, along with one or more other therapeutic agents, to a subject can kill tumor cells or inhibit tumor cell growth.

[0338] In one aspect, this disclosure provides a heterodimeric antibody of the present disclosure 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 a heterodimeric antibody of the present disclosure or a pharmaceutical composition of the present disclosure for treating tumors or autoimmune diseases.

[0339] In one aspect, this disclosure provides the use of the heterodimeric antibody of this disclosure 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 heterodimeric antibody of this disclosure or the pharmaceutical composition of this disclosure for treating tumors or autoimmune diseases.

[0340] 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.

[0341] Reagent test kit

[0342] This disclosure provides a kit comprising the heterodimeric antibody of this disclosure or the pharmaceutical composition of this disclosure.

[0343] This disclosure describes kits comprising the heterodimeric antibody or the pharmaceutical composition described herein. The kits can be used to implement the heterodimeric antibody or the pharmaceutical composition provided herein for other purposes. In some embodiments, the kit may include the heterodimeric antibody or the pharmaceutical composition 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

[0344] Figures 1A-1I are CEX overlays of heterodimeric antibodies and their parental monoclonal antibodies prepared by in vitro assembly. Figure 1A shows DV1-DV3 and their parental monoclonal antibodies, Figure 1B shows CH3-1 and its parental monoclonal antibody, Figure 1C shows CH3-2 and its parental monoclonal antibody, Figure 1D shows CH8 and its parental monoclonal antibody, Figure 1E shows CH51 and its parental monoclonal antibody, Figure 1F shows CH59 and its parental monoclonal antibody, Figure 1G shows CH75 and its parental monoclonal antibody, Figure 1H shows CH83 and its parental monoclonal antibody, and Figure 1I shows CH107 and its parental monoclonal antibody.

[0345] Figures 2A-2C show the mass spectrometry analysis results of the heterodimeric antibody samples. Figure 2A shows the molecular weight of EC-1, Figure 2B shows the molecular weight of EC-2, Figure 2C shows the molecular weight of EC-3, Figure 2D shows the molecular weight of EC-9, Figure 2E shows the molecular weight of EC-11, Figure 2F shows the molecular weight of EC-30, Figure 2G shows the molecular weight of CH3-1, Figure 2H shows the molecular weight of CH3-2, and Figure 2I shows the molecular weight of CH8.

[0346] Figures 3A and 3B show the binding activity of the B7H3×B7H4 heterodimer antibody on MX-1 and OVCAR-3 cells, respectively.

[0347] Figure 4 shows the binding activity of the EGFR×cMet heterodimer antibody to A549 cells.

[0348] Figure 5 shows the ADCC activity of EGFR×cMet heterodimer antibody on A549 cells.

[0349] Figure 6 shows the time-dependent stability curve of the EGFR×cMet heterodimer antibody in human plasma.

[0350] Figure 7 shows the time-dependent stability curve of the EGFR×cMet heterodimer antibody in mouse plasma.

[0351] Figure 8 shows the time-dependent stability curve of the EGFR×cMet heterodimer antibody in monkey plasma.

[0352] Figure 9 shows the pharmacokinetic curves of the EGFR×cMet heterodimer antibody in mice.

[0353] Explanation and definition

[0354] 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.

[0355] 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.

[0356] The term "thiol" refers to the -SH group.

[0357] The term "treatment" means administering the compounds (e.g., heterodimeric antibodies) 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:

[0358] (i) Suppress the disease or disease state, that is, curb its development;

[0359] (ii) Relieve the disease or disease state, even if the disease or disease state subsides;

[0360] (iii) Reduce any direct or indirect pathological consequences of disease or disease state.

[0361] The term "therapeutic effective amount" means 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 prevent or 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.

[0362] 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.

[0363] The term "pharmaceutically acceptable salt" refers to a salt of a compound (such as the antibody disclosed herein) that is safe and effective when used in mammals and has the intended biological activity. For example, it may 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.

[0364] The term "excipient" refers to any component other than the active ingredient (e.g., the antibody of this disclosure). The selection of excipients will depend to a great extent 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.

[0365] The term "solvent" refers to a substance formed by the association of a compound with solvent molecules.

[0366] 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 native sequence Fc (e.g., human IgG1 Fc, human IgG4 Fc) and variant Fc. The C-terminal lysine residue (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. One of the “Fc polypeptides” used herein 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.

[0367] 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.

[0368] 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.

[0369] 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.

[0370] 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.

[0371] 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.

[0372] 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.

[0373] The term "scFv" includes the VH and VL domains of an immunoglobulin, wherein these domains are present in 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.

[0374] 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.

[0375] 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.

[0376] 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.

[0377] "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.

[0378] 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.

[0379] 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.

[0380] 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, as a specific example, a half-antibody contains an Fc polypeptide, and the two Fc polypeptides of two half-antibodies associate to form a dimer antibody. For example, as 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 (i.e., one heavy chain and one light chain) of a complete monoclonal antibody. 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, the half-antibody contains a linked VHH antigen-binding moiety and an Fc polypeptide. As used herein, the term "EC" is used... 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.

[0381] “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.

[0382] The term "identity," also known as consistency, refers to the percentage of amino acid residues in the sequence to be aligned that are identical to those in the specific amino acid sequence shown herein, after aligning the sequence to be aligned with it and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. Amino acid sequence alignment for identity can be performed using various methods within the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the aligned sequences, including any algorithm required to achieve maximum alignment across the full length of the compared sequences.

[0383] 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.

[0384] 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”.

[0385] 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.

[0386] In this document, unless the context clearly indicates otherwise, singular terms encompass plural referents, and vice versa. Detailed Implementation

[0387] This disclosure also provides the following specific implementation schemes, but the scope of protection of this disclosure is not limited thereto:

[0388] Implementation Scheme 1. A heterodimeric antibody, wherein the heterodimeric antibody comprises a first half-antibody and a second half-antibody, the first half-antibody comprising a first CH3 region, and the second half-antibody comprising a second CH3 region, wherein, according to 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 CH3 region and the second CH3 region are different.

[0389] Implementation Scheme 2. The heterodimeric antibody according to Implementation Scheme 1, wherein the heterodimeric antibody comprises amino acid substitutions that promote the formation of the heterodimer, the amino acid substitutions that promote the formation of the heterodimer being as follows: 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, Glu or Gly 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.

[0390] Implementation Scheme 3. A heterodimeric antibody, wherein the heterodimeric antibody comprises a first half-antibody and a second half-antibody, the first half-antibody comprising a first CH3 region, and the second half-antibody comprising a second CH3 region, wherein, according to EU designation,

[0391] (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

[0392] (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.

[0393] Implementation Scheme 4. The heterodimeric antibody according to Implementation Scheme 3, wherein the heterodimeric antibody comprises an amino acid substitution that promotes the formation of the heterodimer, the amino acid substitution that promotes the formation of the heterodimer being selected from (1) and / or (2).

[0394] Implementation Scheme 5. The heterodimeric antibody according to Implementation Scheme 3 or 4, wherein the first CH3 region has Met or Leu at position 366, and the second CH3 region has Thr or Ala at position 407.

[0395] Implementation Scheme 6. The heterodimeric antibody according to Implementation Scheme 5, 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.

[0396] Implementation Scheme 7. The heterodimeric antibody according to Implementation Scheme 3 or 4, 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.

[0397] Implementation Scheme 8. The heterodimeric antibody according to Implementation Scheme 7, 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 ... The first CH3 region has Lys at bit 394; the second CH3 region has Ser at bit 405 and Trp at bit 394; the first CH3 region has Ser at bit 405 and Phe at bit 394; the first CH3 region has Ser at bit 405 and Tyr at bit 394; the first CH3 region has Ser at bit 405 and Lys at bit 394; the first CH3 region has Asp at bit 405 and Trp at bit 394; the first CH3 region has Asp at bit 405 and Phe at bit 394; the first CH3 region has Asp at bit 405 and Tyr at bit 394; or, the first CH3 region has Asp at bit 405 and Lys at bit 394.

[0398] Implementation Scheme 9. The heterodimeric antibody according to any one of Implementation Schemes 1-8, wherein the first CH3 region is the CH3 region of IgG, preferably the CH3 region of human IgG.

[0399] Implementation Scheme 10. The heterodimeric antibody according to any one of Implementation Schemes 1-9, 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.

[0400] Implementation Scheme 11. The heterodimeric antibody according to any one of Implementation Schemes 1-10, wherein the second CH3 region is the CH3 region of IgG, preferably the CH3 region of human IgG.

[0401] Implementation Scheme 12. The heterodimeric antibody according to any one of Implementation Schemes 1-11, 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.

[0402] Implementation Scheme 13. The heterodimeric antibody according to any one of Implementation Schemes 1-8, wherein the first CH3 region and the second CH3 region are both CH3 regions of human IgG1 or both CH3 regions of human IgG4.

[0403] Implementation Scheme 14. The heterodimeric antibody according to any one of Implementation Schemes 1-13, wherein the sequences of the first CH3 region and the second CH3 region are different, and 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.

[0404] Implementation Scheme 15. The heterodimeric antibody according to any one of Implementation Schemes 1-14, 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.

[0405] Implementation Scheme 16. The heterodimeric antibody according to Implementation Scheme 15, 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.

[0406] Implementation Scheme 17. The heterodimeric antibody 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.

[0407] Implementation Scheme 18. The heterodimeric antibody according to any one of Implementation Schemes 1-17, wherein the first half-antibody comprises a first antigen-binding portion and a first Fc polypeptide, and the second half-antibody comprises a second antigen-binding portion and a second Fc polypeptide.

[0408] Implementation Scheme 19. The heterodimeric antibody according to Implementation Scheme 17 or 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.

[0409] Implementation Scheme 20. The heterodimeric antibody 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.

[0410] Implementation Scheme 21. The heterodimeric antibody according to any one of Implementation Schemes 1-20, wherein the heterodimeric antibody contains a Cys-Pro-Pro-Cys sequence in the hinge region.

[0411] Implementation Scheme 22. The heterodimeric antibody according to any one of Implementation Schemes 1-21, 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, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, PD-1 and PD-L1.

[0412] Implementation Scheme 23. A method for preparing heterodimeric antibodies, comprising the following steps:

[0413] (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;

[0414] (2) 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.

[0415] (3) The first homodimeric antibody and the second homodimeric antibody were incubated together under conditions sufficient to allow the reduction of disulfide bonds in the hinge region;

[0416] (4) Obtain the heterodimer antibody.

[0417] Implementation Scheme 24. The method according to Implementation Scheme 23, wherein step (4) includes subjecting the reaction solution obtained in step (3) to oxidative conditions sufficient to allow cysteine ​​in the antibody to be oxidized to interchain disulfide bonds; or step (4) includes removing a reducing agent from the reaction system.

[0418] Implementation Scheme 25. The method according to Implementation Scheme 23, wherein the reduction conditions of step (3) include the addition of a reducing agent, the reducing agent being 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.

[0419] Implementation Scheme 26. The method according to Implementation Scheme 23, 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, 394, 405 and / or 407; 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.

[0420] Implementation Scheme 27. The method according to any one of Implementation Schemes 23-26, according to EU designation,

[0421] (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

[0422] (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.

[0423] Implementation Scheme 28. The method according to any one of Implementation Schemes 23-27, 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.

[0424] Implementation Scheme 29. The method according to Implementation Scheme 28, 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.

[0425] Implementation Scheme 30. The method according to any one of Implementation Schemes 23-27, 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.

[0426] Implementation Scheme 31. According to the method of Implementation Scheme 30, 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.

[0427] Implementation Scheme 32. The method according to any one of Implementation Schemes 23-31, wherein the first CH3 region is the CH3 region of IgG, preferably the CH3 region of human IgG.

[0428] Implementation Scheme 33. The method according to any one of Implementation Schemes 23-32, 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.

[0429] Implementation Scheme 34. The method according to any one of Implementation Schemes 23-33, wherein the second CH3 region is the CH3 region of IgG, preferably the CH3 region of human IgG.

[0430] Implementation Scheme 35. The method according to any one of Implementation Schemes 23-34, 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.

[0431] Implementation Scheme 36. The method according to any one of Implementation Schemes 23-35, wherein the first CH3 region and the second CH3 region are both CH3 regions of human IgG1 or both CH3 regions of human IgG4.

[0432] Implementation Scheme 37. The method according to any one of Implementation Schemes 23-36, wherein the sequences of the first CH3 region and the second CH3 region are different, and 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 respectively.

[0433] Implementation Scheme 38. The method according to any one of Implementation Schemes 23-37, wherein the first homodimeric antibody comprises a first Fc polypeptide, the first Fc polypeptide comprises a first CH3 region, the second homodimeric antibody comprises a second Fc polypeptide, the second Fc polypeptide comprises a second CH3 region, the two first Fc polypeptides constitute a first Fc domain, and the two second Fc polypeptides constitute a second Fc domain.

[0434] Implementation Scheme 39. The method according to Implementation Scheme 38, 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.

[0435] Implementation Scheme 40. The method according to any one of Implementation Schemes 23-39, wherein the first homodimeric antibody comprises a first antigen-binding portion, and the second homodimeric antibody comprises a second antigen-binding portion.

[0436] Implementation Scheme 41. The method according to any one of Implementation Schemes 23-40, 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.

[0437] Implementation Scheme 42. The method according to Implementation Scheme 40 or 41, 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.

[0438] Implementation Scheme 43. The method according to any one of Implementation Schemes 40-42, 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.

[0439] Implementation Scheme 44. The method according to any one of Implementation Schemes 23-43, wherein the first homodimeric antibody and the second homodimeric antibody both contain a Cys-Pro-Pro-Cys sequence in the hinge region.

[0440] Implementation Scheme 45. The method according to any one of Implementation Schemes 23-44, wherein the homodimer antibody targets HER2, HER3, EGFR, ROR1, CLDN18.2, B7-H3, B7-H4, TROP-2, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, PD-1, or PD-L1.

[0441] Implementation Scheme 46. The method according to any one of Implementation Schemes 23-45, wherein the concentration of the reducing agent is 1-150mM, 50-150mM, 50-100mM, 60-80mM, 60-75mM, 70-80mM or 75-80mM; and / or the reduction temperature is 10-30℃, 15-30℃, 20-30℃ or 25-30℃.

[0442] Implementation Scheme 47. According to any of the preceding embodiments, the first CH3 region and the second CH3 region have the amino acid substitutions described therein compared to the CH3 region sequence shown in SEQ ID NO: 36.

[0443] Implementation Scheme 48. A pharmaceutical composition comprising a heterodimeric antibody according to any one of Implementation Schemes 1-22.

[0444] Implementation Scheme 49. Use of the heterodimeric antibody according to any one of Implementation Schemes 1-22, or the pharmaceutical composition according to Implementation Scheme 48, in the preparation of a medicament for treating tumors or autoimmune diseases.

[0445] Implementation Scheme 50. Use of the heterodimeric antibody according to any one of Implementation Schemes 1-22, or the pharmaceutical composition according to Implementation Scheme 48, and one or more other therapeutic agents in the preparation of a medicament for treating tumors or autoimmune diseases.

[0446] Implementation Scheme 51. The use according to Implementation Scheme 49 or 50, 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.

[0447] Implementation Scheme 52. 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 1-22, or the pharmaceutical composition described in Implementation Scheme 48.

[0448] Implementation Scheme 53. The method according to Implementation Scheme 52, the method comprising contacting tumor cells with the heterodimeric antibody of any one of Implementation Schemes 1-22 or the pharmaceutical composition of Implementation Scheme 48, thereby killing tumor cells or inhibiting tumor cell growth.

[0449] Implementation Scheme 54. The method according to Implementation Scheme 52 or 53, wherein the method further comprises administering one or more additional therapeutic agents; preferably, the additional therapeutic agents are tumor therapeutic agents.

[0450] Implementation Scheme 55. The method according to any one of Implementation Schemes 52-54, 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.

[0451] Implementation Scheme 56. A kit comprising the heterodimeric antibody of any one of Implementation Schemes 1-22, or the pharmaceutical composition of Implementation Scheme 48.

[0452] Example

[0453] 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.

[0454] Example 1: Fc region mutation design

[0455] This embodiment details the design of Fc region mutations (or amino acid substitutions) of parental antibodies, as shown in Table 1-1 below.

[0456] 1. Application of mutation design in purity verification of anti-B7-H3 and anti-B7-H4 antibodies.

[0457] Table 1-1: Mutation design of the Fc region of parental antibodies (based on EU designations)

[0458] To verify whether the parental antibodies designed with the above mutations could be successfully assembled into heterodimeric bispecific antibodies, anti-B7-H3 antibody and anti-B7-H4 antibody (both IgG1 subtypes, with their Fc region sequences shown in SEQ ID NO:1 and hinge region sequences shown in SEQ ID NO:2) were used as two parental antibodies. Mutations were performed at one or more of the above sites on each antibody (the CH3 region of each antibody contains the sequences shown in SEQ 40-64). GenScript was commissioned to express and purify all antibodies. Based on the antibody report issued by GenScript and the preferred mutation design in Table 1-1, the SEC analysis of each mutated parental antibody showed high purity.

[0459] Table 1-2 shows the purity verification results of partial mutation designs for anti-B7-H3 and anti-B7-H4 antibodies.

[0460] 2. Application of mutation design in the purity validation of anti-EGFR and anti-cMet antibodies.

[0461] Anti-EGFR antibody and anti-cMet antibody (both IgG1 subtypes, with their Fc region sequences shown in SEQ ID NO:1) were used as two parental antibodies. Mutations were performed on the Fc region of each antibody, and expression and purification of all antibodies were performed by GenScript. SEC analysis of each mutated parental antibody showed high purity. The sequences of each antibody are shown in Table S1.

[0462] Table 1-3 Results of purity validation for mutation design in anti-EGFR and anti-cMet antibodies (based on EU designations)

[0463] 3. Application of mutation design in the purity verification of anti-CD20 and anti-CD3 antibodies.

[0464] Anti-CD20 antibody and anti-CD3 antibody (both IgG1 subtypes, with their Fc region sequences shown in SEQ ID NO:1) were used as two parental antibodies. Mutations were performed on the Fc region of each antibody, and expression and purification of all antibodies were performed by GenScript. SEC analysis of each mutated parental antibody showed high purity, exceeding that of the duobody mutant design. The sequences of each antibody are shown in Table S1.

[0465] Table 1-4 Results of purity validation for mutation design in anti-CD20 and anti-CD3 antibodies (based on EU designations)

[0466] Example 2: In vitro assembly of heterodimeric antibodies

[0467] This embodiment details a method for assembling heterodimeric antibodies in vitro. Two different parental antibodies with mutated Fc regions obtained in Example 1 were mixed in pairs at a 1:1 molar ratio and added to PBS solution. A reducing agent, 2-mercaptoethylamine·HCl (2-MEA) (Aladdin, catalog number C406662), was added to a final concentration of 75 mM, resulting in a final antibody concentration of 2 mg / mL. The reaction mixture was incubated at 30°C for 4 hours. After incubation, the mixture was buffer-displaced to remove 2-MEA using PBS without 2-MEA in an ultrafiltration centrifuge tube (Millipore, catalog number UFC901024). After the displacement, the reaction system was incubated overnight at 4°C to allow the antibodies in the mixture to oxidize naturally. The 2-MEA used here selectively cleaves the disulfide bonds in the antibody hinge region while preserving the disulfide bonds between the heavy and light chains.

[0468] Table 2-1: Assembled B7H3×B7H4 bispecific antibody samples and their parental monoclonal antibodies

[0469] Table 2-2: Assembled EGFR×cMET bispecific antibody samples and their parental monoclonal antibodies

[0470] Table 2-3: Assembled CD20×CD3 bispecific antibody samples and their parental monoclonal antibodies

[0471] Example 3: Evaluation of Heterodimeric Antibodies

[0472] This embodiment evaluates the results of in vitro assembly of the parental antibodies designed with mutations as described herein. The samples prepared in vitro in Example 2 were analyzed from different perspectives using size exclusion chromatography (SEC), cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), capillary electrophoresis (CE-SDS), and / or mass spectrometry (MS) to determine the purity, molecular weight distribution, and precise molecular weight of the target heterodimeric antibodies. Samples assembled using Duobody mutation techniques (as described in PCT / EP2011 / 056388 and PCT / US2013 / 071288) – specifically DV1-DV3 (B7H3-405L×B7H4-409R), EC-1 (EGFR-405L×cMet-409R), and EE-1 (CD20-405L×CD3-409R) – served as references.

[0473] SEC assessment

[0474] Size exclusion chromatography (SUC) was used to separate the sample components. A neutral pH buffer was used as the mobile phase for elution, and the components were eluted sequentially in descending order of molecular weight. An ACQUITY UPLC Protein BEH SEC Column was used. A 1.7 μm, 4.6*300 mm gel chromatographic column (Waters, catalog number 186005226) was used. The mobile phase was 50 mmol / L phosphate buffer-200 mmol / L sodium chloride, pH 7.0. A precise 20 μg sample was injected into the HPLC system and detected at 280 nm. The flow rate was 0.3 mL / min, with isocratic elution for 15 min. The results were quantified using the area normalization method. The peak area percentages of aggregates, immunoglobulin monomers, and low molecular weight impurities were calculated, with the peak before the main peak representing aggregates (HMW), the main peak representing immunoglobulin monomers, and the peak after the main peak representing low molecular weight impurities (LMW).

[0475] Tables 3-1 to 3-3 show the SEC test results for each assembled sample.

[0476] Table 3-1: SEC test results of assembled B7H3×B7H4 bispecific antibody samples

[0477] Table 3-2: SEC test results of assembled EGFR×cMET bispecific antibody samples

[0478] Table 3-3: SEC detection results of assembled CD20×CD3 bispecific antibody samples

[0479] CEX assessment and HIC testing

[0480] CEX assessment:

[0481] The sample components were separated using a non-porous thin-film resin packing material with bonded sulfonic acid functional groups. The pH of the mobile phase was adjusted to allow the protein surface to carry varying amounts of charges opposite to those on the bonded phase. The protein components were then eluted by adjusting the ionic strength of the mobile phase, with acidic components eluted first and basic components eluted later. A MAbPac SCX-10, 10 μm, 4*250 mm column (Thermo, catalog number 074625) was used at a column temperature of 30 °C. Mobile phase A consisted of 20 mM HEPES at pH 7.3. Mobile phase B consisted of 20 mM HEPES-500 mM NaCl at pH 7.3. The sample was diluted to 1 mg / mL with ultrapure water, and 50 μg of sample was accurately injected into the liquid chromatograph for detection at 280 nm. The flow rate was 1.0 mL / min, and gradient elution was performed for 50 min (0-2.0 min: 100% A; 2.0-32.0 min: 100% A increased to 50% A plus 50% B; 32.0-40.0 min: 50% A plus 50% B increased to 100% B; 40.0-40.1 min: 100% B increased to 100% A; 40.1-50.0 min: 100% A). The results were quantitatively analyzed using the area normalization method, calculating the peak area percentages of the acidic peak (acidic region), the main peak, and the basic peak (basic region). The CEX detection results of the assembled samples are shown in Tables 4-1 to 4-3. The CEX peak diagrams of the assembled samples and their parental proteins were overlaid; Figures 1A-1I show the overlay results of some assembled samples. The main peaks of the samples were all located between their parental monoclonal antibodies, indicating that each mutant combination was successfully assembled into a bispecific antibody molecule.

[0482] HIC testing:

[0483] The hydrophobicity of the bispecific products was analyzed by hydrophobic interaction chromatography (HIC) of the in vitro assembled samples. A non-porous polystyrene / divinylbenzene (PS / DVB) packing material with bonded ethyl groups was used to separate the sample components. A neutral, high-salt mobile phase was used to enhance the hydrophobicity of protein molecules, thereby enabling them to bind with hydrophobic bonds in the column. Substances were then eluted by gradually decreasing the salt concentration, with less hydrophobic substances eluted first and more hydrophobic substances eluted later. The column specifications were Proteomix HIC Ethyl-NP5, 4.6*100mm, 5μm (Sepax, #432NP5-4610), and the column temperature was 30℃. Mobile phase A was 50 mmol / L phosphate buffer-1.5 mol / L ammonium sulfate, pH 7.0 (3.04 g of disodium hydrogen phosphate dihydrate, 12.35 g of sodium dihydrogen phosphate dodecahydrate, and 198 g of ammonium sulfate were weighed, added to approximately 800 mL of ultrapure water, stirred until fully dissolved, adjusted to pH 7.0 with 2 M NaOH, and brought to a final volume of 1 L. After mixing, the solution was filtered through a 0.22 μm filter membrane). Mobile phase B was 50 mmol / L phosphate buffer, pH 7.0 (3.04 g of disodium hydrogen phosphate dihydrate and 12.35 g of sodium dihydrogen phosphate dodecahydrate were weighed, added to approximately 800 mL of ultrapure water, stirred until fully dissolved, and brought to a final volume of 1 L. After mixing, the solution was filtered through a 0.22 μm filter membrane). The sample was diluted 1-fold with a blank solution (mobile phase A and mobile phase B mixed at a 1:1 volume ratio) to prepare the test solution. The injection volume was adjusted according to the sample concentration, and 20 μg of protein was injected. Detection was performed at 280 nm. The flow rate was 0.5 mL / min, with gradient elution for 37 min (0-2 min 100% A, 2-22 min from 100% A to 100% B, 22-27 min 100% B, 27.1-37 min to 100% A). The data were processed, and the retention time of the main peak was read from the experimental data. The HIC peak diagrams of the assembled sample and its parental protein were overlaid. It was found that the main peaks of the sample were all located between their parental monoclonal antibodies, indicating that each mutant combination was successfully assembled into a bispecific antibody molecule.

[0484] Table 4-1: CEX test results and assembly results of assembled B7H3×B7H4 bispecific antibody samples

[0485] Table 4-2: CEX test results and assembly results of assembled EGFR×cMET bispecific antibody samples

[0486] Table 4-3: CEX detection results and assembly results of assembled CD20×CD3 bispecific antibody samples

[0487] Table 5: HIC test results of some assembled samples

[0488] CE-SDS Assessment

[0489] For assembled bispecific antibody samples (including Duobody mutant references), HT PROTEIN EXPRESS & PICO LABCHIP (Revity) and Protein Express Reagent Kit (Revity) were also used. CE-SDS analysis under both reducing and non-reducing conditions was performed using a GXII Touch (Revvity, Waltham MA, USA) system. Sample preparation buffer was supplemented with 1% SDS and 250 mM IAM, and the detection mode was HT Protein Express 200 in High Sensitivity mode. Results were processed using LabChip GX Reviewer version 5.10.172.0, and each peak is expressed as a percentage of the total peak area.

[0490] Table 6-1 summarizes the CE-SDS test results of some assembled samples.

[0491] Table 6-1: CE-SDS test results of assembled samples

[0492] MS Evaluation

[0493] For the assembled bispecific antibody samples containing CH3-1, CH3-2, CH8, EC-1, EC-2, EC-3, EC-9, EC-11, and EC-30, additional mass spectrometry analysis was performed to determine the molecular weight. First, 50 μg of sample was taken, and 1 μL of glycosidase PNGase F (Reno Biotech, China) was added, and incubated at 37°C for 2 hours. Using a Xevo G2-XS (Waters, USA) high-resolution mass spectrometer, the sample concentration was adjusted to 5 μM, and MabPAc was used for further analysis. TM Elution was performed using an RP 4μm, 2.1*50mm column, and mass spectrometry data were acquired in positive ion mode. The acquired mass spectrometry data were analyzed using MassLynx V4.1 software (Waters, USA). Based on the molecular weight of the antibody used for in vitro assembly in this experiment, the bispecific antibody could be distinguished from the original antibody. For the peak of the bispecific antibody, the area under the curve was determined and divided by the total area under the curve to calculate the percentage of bispecific antibody in each sample. The molecular weight detection results of some samples are shown in Figures 2A-2I. The molecular weights of all samples prepared by in vitro assembly met expectations, and the mass spectrometry results showed no residual parental monoclonal antibody.

[0494] Assembly efficiency

[0495] The assembly efficiency of the bispecific antibody was calculated based on the proportion of the target peak. The results are shown in the table below. The assembly efficiency of the multi-mutation method of this invention is superior to that of the DUOBODY mutation.

[0496] Table 6-2 Assembly efficiency of dual-antibody drugs

[0497] Example 4: Tumor cell binding activity of heterodimeric antibodies

[0498] This embodiment details the determination of the tumor cell binding activity of heterodimer antibodies.

[0499] 1. Assay for the binding activity of B7H3×B7H4 bispecific antibodies

[0500] The binding of heterodimer antibodies assembled in vitro to tumor cell lines MX-1 (low B7H3 expression, high B7H4 expression) and OVCAR-3 (high B7H3 expression, low B7H4 expression) was analyzed by flow cytometry.

[0501] In short, flow cytometry was used to measure the binding of bispecific antibodies to cells expressing human B7H3 and B7H4, followed by detection using phycoerythrin (PE)-labeled anti-human IgG antibodies. Specifically, a starting concentration of 200 nM of bispecific antibody was used, followed by 5-fold serial dilutions, resulting in 10 concentration gradients, and incubated at 4°C at 2 × 10⁻⁶ ppm. 5 Cells were incubated per well for 1 hour. After incubation, cells were washed twice with flow cytometry buffer (MACS, catalog number 130-091-221), and PE-labeled anti-human IgG secondary antibody was added to the cells followed by incubation for another 30 minutes. After incubation, cells were washed with the same flow cytometry buffer, resuspended in 200 μL of flow cytometry buffer, and analyzed by flow cytometry using a Sartorius IQUE3 flow cytometer.

[0502] The results are shown in Figures 3A-3B. In terms of the binding activity of the assembled heterodimer antibodies to MX-1 and OVCAR-3 cells, the bispecific antibodies CH3-1, CH3-2, and CH8 assembled by the tested mutant combinations were comparable to the bispecific antibodies DV1-DV3 assembled by the Duobody mutants.

[0503] 2. Assay for the binding activity of EGFR×cMet bispecific antibodies

[0504] The binding ability of the in vitro assembled EGFR×cMet sample to the tumor cell line A549 was determined by flow cytometry.

[0505] In short, flow cytometry was used to measure the binding of bispecific antibodies to cells expressing human EGFR and cMet, followed by detection using phycoerythrin (PE)-labeled anti-human IgG antibodies. Specifically, a bispecific antibody at an initial concentration of 200 nM was serially diluted 5-fold to a total of 10 concentration gradients, and then incubated at 4°C at a rate of 2 × 10⁻⁶. 5 Cells were incubated per well for 1 hour. After incubation, cells were washed twice with flow cytometry buffer (MACS, catalog number 130-091-221), and PE-labeled anti-human IgG secondary antibody was added to the cells followed by incubation for another 30 minutes. After incubation, cells were washed with the same flow cytometry buffer, resuspended in 200 μL of flow cytometry buffer, and analyzed by flow cytometry. As shown in Figure 4, regarding the binding activity of the assembled heterodimeric antibodies to A549 cells, the bispecific antibodies EC-2, EC-3, EC-9, EC-11, and EC-30 assembled with the mutant combination were comparable to the bispecific antibody EC-1 assembled with the Duobody mutant.

[0506] Example 5: Thermal stability of heterodimeric antibodies

[0507] One method to determine protein conformational stability is to track its unfolding over a temperature gradient. Increasing sample temperature causes proteins to transition from a folded to an unfolded state. The temperature at which this transition occurs, Tm, is used as a proxy parameter for protein thermal stability. Using a protein stability analyzer (NANO Temper, #Prometheus NT.48), the Tm and aggregation temperatures (Tagg values) of samples were analyzed using micro-differential fluorescence scanning and back-reflection light. In short, after confirming the detection area was clean, the sample was mixed, centrifuged at 14000g for 15 min, and the sample was aspirated using a capillary tube. Two replicates of each sample were placed sequentially into the detection area. The heating rate was set to 1.0℃ / min, and the temperature range was 20.0℃–95.0℃. After the run, the Tm and Tagg values ​​were read from the experimental data, and the results are summarized in Table 7. It can be seen that the thermal stability of the heterodimeric antibody assembled from the tested mutant combination is comparable to that of the bispecific antibody assembled from the Duobody mutant.

[0508] Table 7: Tm / Tagg test results of assembled samples

[0509] Example 6: Antibody-dependent cell-mediated cytotoxicity (ADCC) of heterodimer antibodies in in vitro cell lines

[0510] Human non-small cell lung cancer cell line A549 was used as target cells, and hPBMCs (from Oribiotech) were used as effector cells. ADCC was measured using a ratio of 1 target cell to 10 effector cells. Target cells were digested with trypsin and resuspended in McCoy'5A and 2% FBS (both from Gibco). Combined target cells (2.5 × 10⁻⁶) 4 (1 cell) and effector cells (2.5 × 10) 5 150 μL of the cell mixture was added to the wells of a 96-well Thermo plate. Another 50 μL of serially diluted pre-concentrated antibody was added, starting at 10 nM and serially diluted 5-fold for a total of 10 concentration gradients. The 96-well plate was incubated at 37°C and 5% CO2 for 24 hours. Lysis buffer was then added to the target cell maximum release group and the lysis buffer group, and incubation continued for 45 minutes. The 96-well plate was centrifuged at 200 g for 5 minutes. A total of 50 μL of supernatant was removed from each well, and 50 μL of lactate dehydrogenase assay reagent (Promega) was added. The plate was incubated at 37°C for 30 minutes to determine cell lysis. The absorbance was measured at 492 nm using a TECAN microplate reader. The absorbance values ​​of all experimental wells, spontaneous LDH release from target cells, and spontaneous LDH release from effector cells were subtracted from the mean absorbance of the culture medium background. The absorbance value of the maximum LDH release control from target cells was subtracted from the mean absorbance of the volume-corrected control. The corrected values ​​were then substituted into the following formula: (Experimental release - Spontaneous release from target cells - Spontaneous release from effector cells) / (Maximum release from target cells - Spontaneous release from target cells) × 100% to calculate the percentage of cytotoxicity. The data were then fitted to a four-parameter logistic curve using software. The ADCC results of different assembled bispecific antibody samples are shown in Figure 5. EC 50 The values ​​are shown in Table 8.

[0511] Table 8: ADCC activity of assembled bispecific antibody samples on A549 cells

[0512] Example 7: Plasma stability of heterodimeric antibodies

[0513] Plasma stability was tested on EGFR×cMet bispecific antibody samples prepared by in vitro assembly. The samples were mixed with blank plasma from humans (Oribiotech, batch number SP000332), mice (Huizhiheyuan, batch number B0125C009), and monkeys (Oribiotech, batch number SP000521), respectively, vortexed for 30 seconds, and dispensed into 0.5 mL sterile EP tubes (0.1 mL per tube, final concentration of 500 μg / mL). The tubes were statically incubated in a 37℃ incubator (Shanghai Zhicheng, ZXSD-B1090). The EP tubes were removed at 0, 1, 4, 7, 14, and 21 days, and the samples were stored at -80℃ for batch testing.

[0514] The binding ability of antibody samples to EGFR / cMet protein was compared using a double antigen ELISA. Human EGFR-Fc tag (ARCO, EGR-H5252) was diluted with PBS to a final concentration of 2 μg / mL. 100 μL was added to each well of a 96-well microplate (Costar, 9018) according to the pre-defined plate pattern. After sealing, the plate was incubated at 2–8°C for 12–18 hours. The coating solution was discarded, and 350 μL of PBST washing buffer (containing 0.05% Tween-20) was added to each well. The plate was soaked for 1 minute, then patted dry. This washing process was repeated 3 times. 200 μL of 1% BSA-PBST (0.05% Tween-20) was added to each well as blocking buffer, and the plate was blocked at 25±2°C for 1 hour. The plate was washed 3 times using the same method. The antibody samples to be tested were then soaked in 1% PBST. BSA-PBST was diluted 25-fold, and 100 μL of sample was added to each well. The plate was incubated at 25±2℃ with shaking (200 rpm) for 2 hours±10 minutes, followed by washing three times with PBST wash buffer. Human cMet-His tag (ARCO, MET-H5227) was diluted with 1% BSA-PBST to a final concentration of 2 μg / mL, and 100 μL was added to each well. The plate was incubated at 25±2℃ in the dark for 1 hour±10 minutes, followed by washing three times with PBST wash buffer. Anti-His-HRP (Sino Biological, 105327-MM02T-H) was diluted with 1% BSA-PBST. Dilute BSA-PBST to 0.2 μg / mL, add 100 μL to each well, and incubate at 25 ± 2 °C in the dark for 30 min ± 2 min. Wash the plate 3 times with PBST washing buffer. Add 100 μL of TMB chromogenic substrate (Sigma-Aldrich, T0440) to each well at room temperature, and incubate in the dark for 2–15 min. Then, stop the reaction by adding 50 μL of 1M H2SO4. Measure the absorbance at 450 nm (main wavelength) and 620 nm (reference wavelength) using a microplate reader (TECAN, Infinite) within 30 minutes. The results are shown in Figures 6-8.

[0515] Example 8: Mouse PK Experiment of Heterodimeric Antibodies

[0516] Antibodies against EC-1, EC-2, EC-3, EC-9, EC-11, and EC-30 were administered intravenously to ICR mice at a single dose of 10 mg / kg. Blood samples were collected from mice at 0 min before administration and at 0.5 h, 2 h (±1 min), 8 h (±1 min), 24 h (±2 min), 48 h (±2 min), 96 h (±2 min), 168 h (±5 min), 336 h (±5 min), and 504 h (±5 min). Serum samples were obtained after centrifugation and quantitative detection was performed using ELISA. Blood drug concentration-time curves were plotted based on the detection results, and the following pharmacokinetic parameters were evaluated using WinNonlin non-compartmental model analysis (NCA): T max (Peak time), C max (Peak concentration), AUC (Area under curve at time), t 1 / 2 (Terminal elimination half-life), MRT (mean residence time of drug), CL (clearance rate). The results are shown in Figure 9 and Table 9.

[0517] Table 9: In vivo pharmacokinetic data in EGFR+cMet antibody ICR mice

[0518] Example 9: Stability test of heterodimer

[0519] The stability of the EGFR×cMet bispecific antibody samples prepared by in vitro assembly was determined as follows:

[0520] Stability testing was performed on the in vitro assembled EGFR×cMet bispecific antibody samples EC1, EC3, and EC9. Samples were aliquoted into 0.5 mL sterile EP tubes. The control group was stored at -80°C, while the remaining samples were statically incubated at 4°C or 25°C. EP tubes were retrieved at 3, 7, 10, and 14 days, and then stored at -80°C for batch testing. For samples tested for freeze-thaw stability, they were placed at -80°C overnight, then thawed at room temperature, and this freeze-thaw cycle was repeated five times. All samples were subjected to SEC evaluation according to the method described in Example 3.

[0521] The results showed that freeze-thaw cycles had a significant impact on the samples. After five freeze-thaw cycles, the sample purity decreased and the amount of polymeric impurities increased. In comparison, EC3 / EC9 exhibited better freeze-thaw stability than duobody.

[0522] Example 10: Design combinations of other F405 mutations and T394 mutations

[0523] Referring to Examples 1-3, other Fc mutations of the parental antibodies shown in Table 11 were designed (where the CH3 region sequences of DN1-36, DN3-21, DN3-34, and DN3-36 are shown in SEQ ID NO: 49, 52, 53, and 55), and parental antibodies containing the mutations were synthesized. The purity of the parental antibodies was detected by SEC. Heterodimeric antibodies were assembled in vitro, and the purity, molecular weight distribution, and precise molecular weight heterodimers of the target heterodimeric antibodies were analyzed from different perspectives using size exclusion chromatography (SEC), cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), capillary electrophoresis (CE-SDS), and / or mass spectrometry (MS).

[0524] Table 11: Other F405 and T394 mutation design combinations (based on EU numbers)

[0525] Table 12-1: Results of purity verification of mutation design for anti-B7-H3 and anti-B7-H4 antibodies

[0526] Table 12-2: Results of purity validation for mutation design in anti-CD20 and anti-CD3 antibodies

[0527] Table 13-1: Bispecific antibody samples assembled based on B7H3 and B7H4 targets and their parental monoclonal antibodies

[0528] Table 13-2: Bispecific antibody samples assembled based on CD20 and CD3 targets and their parental monoclonal antibodies

[0529] Table 14-1: SEC test results of assembled B7H3×B7H4 bispecific antibody samples

[0530] Table 14-2: SEC detection results of assembled CD20×CD3 bispecific antibody samples

[0531] Table 15-1: CEX test results and assembly results of assembled B7H3×B7H4 bispecific antibody samples

[0532] Table 15-2: CEX test results and assembly results of assembled CD20×CD3 bispecific antibody samples

[0533] Table 16: Assembly efficiency of CD20×CD3 bispecific antibody samples

[0534] Example 11: Design combination of T366 / K409 mutation and L368 mutation

[0535] Referring to Examples 1-3, other Fc mutations of the parental antibodies shown in Table 17 were designed, and parental antibodies containing these mutations were synthesized. The purity of the parental antibodies was determined by size exclusion chromatography (SEC). Heterodimeric antibodies were assembled in vitro and analyzed by size exclusion chromatography (SEC). The results showed that the T366Q / K409R+L368F mutation could not effectively assemble bispecific antibodies in vitro.

[0536] Table 17: Other mutation designs for the Fc region of parental antibodies (based on EU designations)

[0537] Table 18: Results of purity validation for mutation design in anti-EGFR and anti-cMet antibodies

[0538] Table 19: Assembled EGFR×c-Met bispecific antibody samples and their parental monoclonal antibodies

[0539] Table 20: SEC detection results of in vitro assembled EGFR×c-Met bispecific antibody samples

[0540] To verify whether the above-mentioned mutation design could successfully assemble into a heterodimeric bispecific antibody in CHO cells, GenScript was commissioned to perform bispecific antibody expression and affinity chromatography purification in CHO cells. Based on the antibody report provided by GenScript, the purity of the bispecific antibody according to SEC analysis is as follows, indicating that the T366Q / K409R+L368F mutation can effectively assemble the bispecific antibody in vivo.

[0541] Table 21: Results of purity validation for mutation design in anti-EGFR and anti-cMet antibodies

[0542] For purposes of description and disclosure, all patents, patent applications, and other identified publications are expressly incorporated herein by reference. These publications are provided solely because their publications predate the filing date of this disclosure. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or contents of these documents. Furthermore, any reference to these publications herein in any country does not constitute an endorsement that such publications are part of the general knowledge in the art.

[0543] Although this disclosure has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of this disclosure are all within the scope of protection claimed by this disclosure.

[0544] The sequence information disclosed herein is summarized in Table S1 below.

[0545] Table S1. Sequence Information

Claims

1. A heterodimeric antibody, wherein, The heterodimeric antibody comprises a first half-antibody and a second half-antibody. The first half-antibody comprises a first CH3 region, and the second half-antibody comprises a second CH3 region. 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, and / or the amino acid Thr, Ala, or Val at position 407. The amino acid substitution positions of the first CH3 region and the second CH3 region are different.

2. The heterodimeric antibody 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; 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.

3. The heterodimeric antibody according to claim 1 or 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.

4. The heterodimeric antibody 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.

5. The heterodimeric antibody according to any one of claims 1-4, 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; 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.

6. The heterodimeric antibody according to any one of claims 1-5, wherein, The first CH3 region and the second CH3 region are both CH3 regions of human IgG1 or both CH3 regions of human IgG4.

7. The heterodimeric antibody according to any one of claims 1-6, 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:42, and the second CH3 region contains the sequence shown in SEQ ID NO:50; (2) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:51; (3) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:50; (4) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:51; (5) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:52; (6) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:52; (7) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:52; (8) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:52; (9) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:52; (10) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:53; (11) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:53; (12) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:53; (13) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:53; (14) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:53; (15) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:54; (16) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:54; (17) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:54; (18) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:54; (19) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:55; (20) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:55; (21) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:55; (22) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:55; (23) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:55; (24) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:56; (25) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:56; (26) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:56; (27) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:56; (28) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:57; (29) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:57; (30) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:57; (31) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:57; (32) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:58; (33) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:58; (34) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:58; (35) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:58; (36) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:58; (37) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:59; (38) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:59; (39) The first CH3 region contains the sequence shown in SEQ ID NO:46, and the second CH3 region contains the sequence shown in SEQ ID NO:59; (40) The first CH3 region contains the sequence shown in SEQ ID NO:47, and the second CH3 region contains the sequence shown in SEQ ID NO:59; (41) The first CH3 region contains the sequence shown in SEQ ID NO:48, and the second CH3 region contains the sequence shown in SEQ ID NO:59; (42) The first CH3 region contains the sequence shown in SEQ ID NO:49, and the second CH3 region contains the sequence shown in SEQ ID NO:52; (43) The first CH3 region contains the sequence shown in SEQ ID NO:49, and the second CH3 region contains the sequence shown in SEQ ID NO:53; or (44) The first CH3 region contains the sequence shown in SEQ ID NO:49, and the second CH3 region contains the sequence shown in SEQ ID NO:55; preferably 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:42, and the second CH3 region contains the sequence shown in SEQ ID NO:50; (2) The first CH3 region contains the sequence shown in SEQ ID NO:42, and the second CH3 region contains the sequence shown in SEQ ID NO:51; (3) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:50; (4) The first CH3 region contains the sequence shown in SEQ ID NO:43, and the second CH3 region contains the sequence shown in SEQ ID NO:51; (5) The first CH3 region contains the sequence shown in SEQ ID NO:44, and the second CH3 region contains the sequence shown in SEQ ID NO:52; or (6) The first CH3 region contains the sequence shown in SEQ ID NO:45, and the second CH3 region contains the sequence shown in SEQ ID NO:

55.

8. The heterodimeric antibody according to any one of claims 1-7, 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.

9. The heterodimeric antibody according to any one of claims 1-8, 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.

10. The heterodimeric antibody according to claim 9, 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.

11. The heterodimeric antibody according to any one of claims 1-10, wherein, The heterodimeric antibody contains a Cys-Pro-Pro-Cys sequence in its hinge region.

12. The heterodimeric antibody according to any one of claims 1-11, 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, CD3, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, PD-1, and PD-L1.

13. A method for preparing the heterodimeric antibody according to any one of claims 1-12, 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) 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. (3) The first homodimeric antibody and the second homodimeric antibody were incubated together under conditions sufficient to allow the reduction of disulfide bonds in the hinge region; (4) Obtain the heterodimer antibody.

14. The method according to claim 13, wherein, Step (4) includes subjecting the reaction solution obtained in step (3) to oxidative conditions sufficient to allow cysteine ​​in the antibody-conjugate to be oxidized to interchain disulfide bonds; or step (4) includes removing a reducing agent from the reaction system.

15. The method according to claim 13 or 14, wherein, The reduction conditions in step (3) include the addition of a reducing agent 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.

16. A pharmaceutical composition comprising a heterodimeric antibody according to any one of claims 1-12.

17. Use of the heterodimeric antibody according to any one of claims 1-12, or the pharmaceutical composition according to claim 46, in the preparation of a medicament for treating tumors or autoimmune diseases.

18. Use of the heterodimeric antibody according to any one of claims 1-12, or the pharmaceutical composition according to claim 46, and one or more other therapeutic agents in the preparation of a medicament for treating tumors or autoimmune diseases.

19. The use according to claim 17 or 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.

20. A kit comprising the heterodimeric antibody of any one of claims 1-12, or the pharmaceutical composition of claim 16.

Images