Method for preparing heteromultimers by recombination reaction

Abstract

The present disclosure relates to methods of making heteromultimers by recombination reactions. In particular, the present disclosure relates to methods of making heteromultimers, especially multispecific antibodies, and altering amino acids of CH3 domains to facilitate formation of heteromultimers, especially multispecific antibodies.

Description

Method for preparing heteromultimers by recombination reaction

[0001] This application claims priority to Chinese patent application CN 202311544684.7 filed on November 20, 2023 and Chinese patent application CN 202410093537.0 filed on January 23, 2024. Technical Field

[0002] The present disclosure relates to a method for preparing heteromultimers (especially multispecific antibodies) and changing the amino acids of the CH3 domain to promote the formation of heteromultimers (especially multispecific antibodies). Background Art

[0003] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0004] Multispecific antibodies, such as bispecific antibodies, are antibody molecules that can simultaneously and specifically bind to two antigens or two epitopes. Compared to monoclonal antibodies, bispecific antibodies have a unique mechanism of action and offer significant advantages over monoclonal antibodies.

[0005] Among the various bispecific antibody platforms developed by many drug research and development companies (Brinkmann U, Kontermann R E. The making of bispecific antibodies [C] / / MAbs. Taylor & Francis, 2017, 9 (2): 182-212.), the DuoBody platform has unique mechanisms and advantages in antibody engineering.

[0006] Human IgG is divided into four subtypes: IgG1, IgG2, IgG3, and IgG4, of which IgG4 accounts for approximately 5%. IgG4 possesses unique biological properties, allowing for dynamic heavy chain exchange in vivo, also known as Fab-arm exchange (FAE), leading to the formation of half-antibody molecules or two half-antibody molecules that further form bispecific antibodies (Van Der Neut Kolfschoten M, Schuurman J, Losen M, et al. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange. Science, 2007, 317(5844):1554-1557). Genmab leverages this FAE property of IgG4 to develop bispecific antibodies, achieving controllable FAE. Genmab's research has shown that dissociation between non-covalent CH3 regions is a key rate-limiting step in FAE, and that the R409 residue of IgG4 reduces CH3-CH3 interactions. Dutch researchers found that mutating the lysine at position 409 of IgG1 to arginine (IgG1-K409R) can give IgG1 the ability to FAE (Labrijn AF, Rispens T, Meesters J, et al. Species-specific determinants in the IgG CH3 domain enable Fab-arm exchange by affecting the noncovalent CH3–CH3 interaction strength. The Journal of Immunology, 2011, 187(6): 3238-3246.); introducing corresponding mutation sites (L368 / K370 / D399 / F405 / Y407) in the CH3 region of another antibody, the results of multiple combinations of FAE showed that the combination of IgG1-K409R and IgG1-F405L can improve the efficiency of FAE (Labrijn AF, Meesters JI, de Goeij BECG, et al. Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. Proceedings of the National Academy of Sciences). of Sciences,2013,110(13):5145-5150.).

[0007] The DuoBody platform is relatively simple to use for preparing bispecific antibodies. It only requires introducing K409R and F405L mutation sites into the CH3 of the Fc region of two IgG1 antibodies, mixing the two target antibodies in proportion, and completing FAE under specific reducing conditions to form a bispecific antibody. The DuoBody platform has multiple advantages: minimal engineering modifications are required, requiring only one mutation site in each of the two parent antibodies. The resulting bispecific antibody is very close to natural IgG1 in size and conformation, theoretically possessing similar physicochemical properties and drugability to natural monoclonal antibodies, with a low risk of immunogenicity, while retaining natural IgG1 Fc functions (ADCC, ADCP, CDC, etc.) and pharmacokinetics similar to natural IgG1.

[0008] In recent years, three DuoBody-based bispecific antibodies have been approved for marketing by the U.S. FDA, including Amivantamab, Teclistamab, and Talquetamab. Among them, Amivantamab (JNJ-61186372) is a bispecific antibody targeting EGFR and c-Met developed based on the DuoBody platform. It was approved for marketing by the U.S. FDA on May 21, 2021, for the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer carrying EGFR exon 20 insertion mutations who have progressed during or after platinum-based chemotherapy. Teclistamab and Talquetamab are CD3×BCMA and CD3×GPRC5D bispecific T-Cell-Engagers (TCEs), respectively, and are used for adult patients with relapsed or refractory multiple myeloma (MM) who have previously received at least multiple lines of therapy.

[0009] The present disclosure found that when using Genmab's DuoBody platform to prepare bispecific antibodies, some parent antibodies carrying the F405L mutation often had an SEC purity of less than 90% after affinity purification with Protein A, indicating that F405L may reduce the intrinsic physicochemical stability of the parent antibody. In addition, the results of evaluating the recombinant efficiency using HPLC-IEC showed that the efficiency of the FAE of the IgG1-K409R and IgG1-F405L combination was around 95%, and the reaction efficiency needs to be improved. In addition, the K409R mutation used by Duobody is derived from human IgG4, while the F405L mutation is derived from rhesus monkey IgG4, so in theory the stability of the bispecific antibody molecules produced by this platform is still comparable to that of IgG4 class antibodies, but inferior to human wild-type IgG1. Bispecific antibodies prepared using the Duobody platform may not be suitable for the preparation of certain types of antibody-drug conjugates (ADCs). Because some ADCs use cysteine-directed site-specific conjugation technology, the two pairs of disulfide bonds in the antibody hinge region are reduced and conjugated to the drug molecule. If the interaction between the CH3 domains of the antibody Fc is not strong enough, the ADC molecule may undergo FAE in vivo, which may pose a risk to clinical application. The CH3 domain of the bispecific antibody prepared in this disclosure may have better physical and chemical stability than Duobody, resulting in better drugability and wider application. Summary of the Invention

[0010] The present disclosure provides a method for preparing a heteromultimer (e.g., a heterodimer), comprising the following steps:

[0011] a) providing a dimer comprising two first polypeptides;

[0012] b) providing a dimer comprising two second polypeptides; and

[0013] c) incubating the dimer comprising the two first polypeptides and the dimer comprising the two second polypeptides together under reducing conditions;

[0014] d) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide (e.g., a heterodimer comprising one first polypeptide and one second polypeptide);

[0015] wherein the first polypeptide and the second polypeptide each comprise a CH3 domain;

[0016] In the CH3 domain of the first polypeptide and / or the second polypeptide, at least one amino acid mutation is selected from the group consisting of: 347, 349, 351, 354, 356, 357, 364, 366, 368, 394, 405, 407, 409, 411 and 439; the CH3 domains of the first polypeptide and the second polypeptide differ in at least one mutation site, and the CH3 domain mutation sites are represented by EU numbering.

[0017] The present disclosure provides a method for preparing a heteromultimer, comprising the following steps:

[0018] a) providing a molecule comprising a first polypeptide homomer;

[0019] b) providing a molecule comprising a second polypeptide homomer; and

[0020] c) a step of incubating the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer under reducing conditions;

[0021] The first polypeptide and the second polypeptide each comprise a CH3 domain, and the CH3 domains of the first polypeptide and the second polypeptide have at least one different mutation site, and the CH3 domain mutation site is represented by EU numbering.

[0022] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items further comprises the step d) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide (e.g., a heterodimer comprising one first polypeptide and one second polypeptide). In some embodiments, step c) adding a reducing agent to the mixture and incubating the mixture can result in dissociation between homopolymeric (e.g., first polypeptide homopolymer and second polypeptide homopolymer) monomers and recombination of heteropolymeric (e.g., one first polypeptide and one second polypeptide) monomers, thereby forming a heterodimer comprising one dissociated first polypeptide monomer and one dissociated second polypeptide monomer.

[0023] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer bind to different antigens or epitopes.

[0024] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide and / or the second polypeptide comprises at least one amino acid mutation selected from: 347, 349, 351, 354, 356, 357, 364, 366, 368, 394, 405, 407, 409, 411 and 439.

[0025] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide and / or the second polypeptide comprises at least one amino acid mutation selected from: positions 349, 351, 354, 356, 364, 366, 368, 394, 405, 409, 411 and 439.

[0026] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain one or more mutations that promote heteromerization formation. In some embodiments, the mutations that promote heteromerization formation refer to a class of amino acid mutations that can promote the dissociation of homopolymers (e.g., homodimers) into monomers, and / or promote the formation of heteromultimers (e.g., heterodimers) between two heteromonomers. In some embodiments, the mutations that promote heteromerization formation are amino acid mutations at the CH3 interaction interface. In some embodiments, the mutations that promote heteromerization formation are amino acid mutations that carry a charge at the CH3 interaction interface. In some embodiments, the mutations that promote heteromerization formation are amino acid mutations that carry an opposite charge at the CH3 interaction interface. In some embodiments, hydrogen bonds, electrostatic interactions, or salt bridges are formed between the CH3 domains of the first polypeptide and the second polypeptide.

[0027] In some embodiments, the mutation promoting heterologous formation is any mutation described in the present disclosure. In some embodiments, the mutation promoting heterologous formation is one or more amino acid mutations selected from positions 347, 349, 351, 354, 356, 357, 364, 366, 368, 394, 405, 407, 409, 411, and 439. In some embodiments, the mutation promoting heterologous formation is one or more amino acid mutations selected from positions 349, 351, 354, 356, 364, 366, 368, 394, 405, 409, 411, and 439.

[0028] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide and / or the second polypeptide comprises at least one amino acid mutation selected from the following combinations:

[0029] The amino acid at position 356 is mutated to Lys (K), Arg (R) or His (H); and / or

[0030] The amino acid at position 439 is mutated to Glu (E) or Asp (D); and / or

[0031] The amino acid at position 349 is mutated to Leu (L), Phe (F), Ser (S), Cys (C), Ala (A), Val (V), Thr (T) or Gly (G); and / or

[0032] The amino acid at position 351 is mutated to Cys (C), Val (V), Thr (T), Ile (I), Phe (F), or Met (M); and / or

[0033] The amino acid at position 364 is mutated to Ala (A), Val (V), Thr (T), Leu (L), Tyr (Y) or Phe (F); and / or

[0034] The amino acid at position 366 is mutated to Gly (G), Ser (S), Ala (A), Val (V), Leu (L), His (H) or Ile (I); and / or

[0035] The amino acid at position 368 is mutated to Val (V), Ile (I), Met (M) or Ala (A); and / or

[0036] The amino acid at position 394 is mutated to Phe (F), Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); and / or

[0037] The amino acid at position 405 is mutated to Thr (T), Leu (L) or Tyr (Y); and / or

[0038] The amino acid at position 407 is mutated to Cys (C), Val (V), Leu (L), His (H) or Phe (F); and / or

[0039] The amino acid at position 409 is mutated to Gln (Q), Arg (R) or Asp (D); and / or

[0040] The amino acid at position 411 is mutated to Asn (N), Tyr (Y) or Leu (L); and / or

[0041] The amino acid at position 354 is mutated to Tyr (Y), Cys (C), Phe (F) or Trp (W); and / or

[0042] The amino acid at position 357 is mutated to Cys (C); and / or

[0043] The amino acid at position 347 was mutated to Glu (E).

[0044] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0045] (a1) amino acid residues 356 and 351 according to EU numbering;

[0046] (a2) amino acid residues 356 and 364 according to EU numbering;

[0047] (a3) amino acid residues 356 and 366 according to EU numbering;

[0048] (a4) amino acid residues 356 and 368 according to EU numbering;

[0049] (a5) amino acid residues 356 and 394 according to EU numbering;

[0050] (a6) amino acid residues 356 and 405 according to EU numbering;

[0051] (a7) amino acid residues 356 and 409 according to EU numbering;

[0052] (a8) amino acid residues 356 and 411 according to EU numbering;

[0053] (b1) amino acid residues 356 and 349 according to EU numbering;

[0054] (c1) amino acid residue 405 according to EU numbering; or

[0055] (c2) amino acid residue 349 according to EU numbering;

[0056] The CH3 domain of the second polypeptide has a structure selected from the group consisting of (d1) to (i2):

[0057] (d1) amino acid residues 439 and 351 according to EU numbering;

[0058] (d2) amino acid residues 439 and 364 according to EU numbering;

[0059] (d3) amino acid residues 439 and 366 according to EU numbering;

[0060] (d4) amino acid residues 439 and 368 according to EU numbering;

[0061] (d5) amino acid residues 439 and 394 according to EU numbering;

[0062] (d6) amino acid residues 439 and 405 according to EU numbering;

[0063] (d7) amino acid residues 439 and 409 according to EU numbering;

[0064] (d8) amino acid residues 439 and 411 according to EU numbering;

[0065] (e1) amino acid residues 439 and 354 according to EU numbering;

[0066] (f1) amino acid residues 439, 354, and 351 according to EU numbering;

[0067] (f2) amino acid residues 439, 354, and 366 according to EU numbering;

[0068] (f3) amino acid residues 439, 354, and 368 according to EU numbering;

[0069] (g1) amino acid residue 394 according to EU numbering;

[0070] (h) amino acid residues 354 and 364 according to EU numbering;

[0071] (i1) amino acid residue 364 according to EU numbering; or

[0072] (i2) Amino acid residue at position 354 according to EU numbering.

[0073] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0074] (a1) amino acid residues 356 and 351 according to EU numbering;

[0075] (a5) amino acid residues 356 and 394 according to EU numbering;

[0076] (a8) amino acid residues 356 and 411 according to EU numbering;

[0077] (b1) amino acid residues 356 and 349 according to EU numbering; or

[0078] (c2) amino acid residue 349 according to EU numbering;

[0079] The CH3 domain of the second polypeptide has a structure selected from the group consisting of the following (d1) to (i1):

[0080] (d1) amino acid residues 439 and 351 according to EU numbering;

[0081] (d5) amino acid residues 439 and 394 according to EU numbering;

[0082] (d8) amino acid residues 439 and 411 according to EU numbering;

[0083] (f1) amino acid residues 439, 354, and 351 according to EU numbering;

[0084] (h) amino acid residues 354 and 364 according to EU numbering; or

[0085] (i1) Amino acid residue at position 364 according to EU numbering.

[0086] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0087] (a1) amino acid residues 356 and 351 according to EU numbering;

[0088] (b1) amino acid residues 356 and 349 according to EU numbering; or

[0089] (c2) amino acid residue 349 according to EU numbering;

[0090] The CH3 domain of the second polypeptide has a structure selected from the group consisting of the following (d1) to (i1):

[0091] (d1) amino acid residues 439 and 351 according to EU numbering;

[0092] (f1) amino acid residues 439, 354, and 351 according to EU numbering;

[0093] (h) amino acid residues 354 and 364 according to EU numbering; or

[0094] (i1) Amino acid residue at position 364 according to EU numbering.

[0095] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0096] (a1) amino acid residues 356K and 351I according to EU numbering; or

[0097] amino acid residues 356K and 351T according to EU numbering;

[0098] (a2) amino acid residues 356K and 364A represented by EU numbering;

[0099] (a3) amino acid residues 356K and 366A represented by EU numbering;

[0100] (a4) amino acid residues 356K and 368I represented by EU numbering;

[0101] (a5) amino acid residues 356K and 394A according to EU numbering; or

[0102] amino acid residues 356K and 394S according to EU numbering;

[0103] (a6) amino acid residues 356K and 405Y represented by EU numbering;

[0104] (a7) amino acid residues 356K and 409Q represented by EU numbering;

[0105] (a8) amino acid residues 356K and 411Y represented by EU numbering;

[0106] (b1) amino acid residues 356K and 349C according to EU numbering; or

[0107] amino acid residues 356K and 349S according to EU numbering;

[0108] (c1) the amino acid residue 405T according to EU numbering; or

[0109] (c2) amino acid residue 349C, 349S or 349G according to EU numbering;

[0110] The CH3 domain of the second polypeptide has a structure selected from the group consisting of (d1) to (i2):

[0111] (d1) amino acid residues 439E and 351I according to EU numbering; or

[0112] amino acid residues 439E and 351T according to EU numbering;

[0113] (d2) amino acid residues 439E and 364A according to EU numbering;

[0114] (d3) amino acid residues 439E and 366A according to EU numbering;

[0115] (d4) amino acid residues 439E and 368I represented by EU numbering;

[0116] (d5) amino acid residues 439E and 394A according to EU numbering; or

[0117] amino acid residues 439E and 394S according to EU numbering;

[0118] (d6) amino acid residues 439E and 405Y represented by EU numbering;

[0119] (d7) amino acid residues 439E and 409Q represented by EU numbering;

[0120] (d8) amino acid residues 439E and 411Y according to EU numbering;

[0121] (e1) amino acid residues 439E and 354Y represented by EU numbering;

[0122] (f1) amino acid residues 439E, 354C, and 351I according to EU numbering;

[0123] (f2) amino acid residues 439E, 354C, and 366A according to EU numbering;

[0124] (f3) amino acid residues 439E, 354C, and 368I according to EU numbering;

[0125] (g1) amino acid residue 394F represented by EU numbering;

[0126] (h) amino acid residues 354C and 364Y according to EU numbering; or

[0127] amino acid residues 354C and 364F according to EU numbering;

[0128] (i1) the amino acid residue 364Y according to EU numbering; or

[0129] (i2) Amino acid residue 354W represented by EU numbering.

[0130] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0131] (a1) amino acid residues 356K and 351I represented by EU numbering;

[0132] (a5) amino acid residues 356K and 394S represented by EU numbering;

[0133] (a8) amino acid residues 356K and 411Y represented by EU numbering;

[0134] (b1) amino acid residues 356K and 349C according to EU numbering; or

[0135] (c2) amino acid residue 349S or 349C represented by EU numbering;

[0136] The CH3 domain of the second polypeptide has a structure selected from the group consisting of the following (d1) to (i1):

[0137] (d1) amino acid residues 439E and 351I represented by EU numbering;

[0138] (d5) amino acid residues at positions 439E and 394S according to EU numbering;

[0139] (d8) amino acid residues 439E and 411Y according to EU numbering;

[0140] (f1) amino acid residues 439E, 354C, and 351I according to EU numbering;

[0141] (h) amino acid residues 354C and 364Y according to EU numbering; or

[0142] amino acid residues 354C and 364F according to EU numbering;

[0143] (i1) Amino acid residue 364Y represented by EU numbering.

[0144] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0145] (a1) amino acid residues 356K and 351I represented by EU numbering;

[0146] (a5) amino acid residues 356K and 349C according to EU numbering; or

[0147] (c2) amino acid residue 349S or 349C represented by EU numbering;

[0148] The CH3 domain of the second polypeptide has a structure selected from the group consisting of the following (d1) to (i1):

[0149] (d1) amino acid residues 439E and 351I represented by EU numbering;

[0150] (f1) amino acid residues 439E, 354C, and 351I according to EU numbering;

[0151] (h) amino acid residues 354C and 364Y according to EU numbering; or

[0152] (i1) Amino acid residue 364Y represented by EU numbering.

[0153] Method for preparing heteromultimers containing first type mutation combination

[0154] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise one identical or different amino acid mutation selected from positions 349, 351, 364, 366, 368, 394, 405, 407, 409 and 411.

[0155] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409 and 411.

[0156] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 351, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 351.

[0157] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid mutation selected from 349L, 349F, 351C, 351V, 351T, 351I, 351F, Amino acid mutations in 351M, 364A, 364V, 364T, 364L, 366G, 366S, 366A, 366V, 366L, 366H, 366I, 368V, 368I, 368A, 368M, 394A, 394S, 394C, 394V, 394N, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411N, 411L and 411Y.

[0158] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid mutation selected from 349L, 351C, 351V, 351T, 351I, Amino acid mutations in 351F, 351M, 364A, 364T, 364L, 366G, 366A, 366V, 366L, 366H, 366I, 368V, 368I, 368A, 368M, 394A, 394S, 394C, 394V, 394N, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411L and 411Y.

[0159] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid selected from 349L, 351C, 351V, 351T, 351I, 351F, 351M , 364A, 364T, 364L, 366G, 366A, 366V, 366L, 366H, 366I, 368V, 368I, 368A, 368M, 394A, 394S, 394C, 394V, 394N, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411L and 411Y amino acid mutations.

[0160] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0161] The CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409 and 411, wherein,

[0162] mutating amino acid 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I), Phe (F), or Met (M); or

[0163] mutating amino acid position 364 (EU numbering) to Ala (A), Val (V), Thr (T) or Leu (L); or

[0164] mutating amino acid position 366 (EU numbering) to Ser (S), Ala (A), Val (V), Leu (L), His (H), or Ile (I); or

[0165] mutating amino acid position 368 (EU numbering) to Val (V), Ile (I), Met (M) or Ala (A); or

[0166] mutating amino acid position 394 (EU numbering) to Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[0167] mutating amino acid position 405 (EU numbering) to Leu (L) or Tyr (Y); or

[0168] mutating amino acid position 409 according to EU numbering to Gln (Q); or

[0169] The amino acid at position 411 represented by EU numbering is mutated to Asn (N), Tyr (Y), or Leu (L).

[0170] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0171] The CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409 and 411, wherein,

[0172] mutating position 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I) or Phe (F); or

[0173] mutating position 364 (EU numbering) to Ala (A), Val (V), Thr (T) or Leu (L); or

[0174] mutating position 366 (EU numbering) to Ser (S), Ala (A), Val (V), Leu (L) or His (H); or

[0175] mutating position 368 (EU numbering) to Val (V), Ile (I), or Met (M); or

[0176] mutating position 394 (EU numbering) to Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[0177] Mutating position 405 (EU numbering) to Leu (L) or Tyr (Y); or

[0178] mutating position 409 (EU numbering) to Gln (Q) or Arg (R); or

[0179] Position 411 (EU numbering) was mutated to Asn (N) or Tyr (Y).

[0180] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from 351T, 351I, 364A, 366A, 368I, 394A, 394S, 405Y, 409Q and 411Y.

[0181] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 351I, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 351I.

[0182] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0183] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y349L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y349L; or

[0184] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351C; or

[0185] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351V, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351V; or

[0186] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351T, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351T; or

[0187] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351I, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351I; or

[0188] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351F, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351F; or

[0189] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351M, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351M; or

[0190] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and S364A, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and S364A; or

[0191] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and S364T, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and S364T; or

[0192] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and S364L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and S364L; or

[0193] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366G, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366G; or

[0194] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366A, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366A; or

[0195] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366V, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366V; or

[0196] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366L; or

[0197] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366H, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366H; or

[0198] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366I, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366I; or

[0199] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L368V, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L368V; or

[0200] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L368I, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L368I; or

[0201] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L368A, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L368A; or

[0202] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L368A, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L368A; or

[0203] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T394A, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394A; or

[0204] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T394S, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394S; or

[0205] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T394C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394C; or

[0206] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T394V, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394V; or

[0207] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T394N, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394N; or

[0208] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and F405L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and F405L; or

[0209] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and F405Y, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and F405Y; or

[0210] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407C; or

[0211] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407V, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407V; or

[0212] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407L; or

[0213] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407H, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407H; or

[0214] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407F, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407F; or

[0215] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and K409Q, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and K409Q; or

[0216] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and K409R, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and K409R; or

[0217] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T411L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T411L; or

[0218] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T411Y, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T411Y.

[0219] The present disclosure provides a method for preparing a heteromultimer, comprising the following steps:

[0220] a) providing a molecule comprising a first polypeptide homomer, wherein the first polypeptide comprises a CH3 domain, and the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351I;

[0221] b) providing a molecule comprising a second polypeptide homomer, wherein the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351I; and

[0222] c) a step of incubating the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer under reducing conditions;

[0223] The CH3 domain mutation sites are represented by EU numbering.

[0224] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a charged amino acid mutation (e.g., a positively charged amino acid, a negatively charged amino acid), and the amino acid mutation in the CH3 domain of the first polypeptide and the amino acid mutation in the CH3 domain of the second polypeptide have opposite charges. Furthermore, the CH3 domains of the first polypeptide and the second polypeptide each contain an identical amino acid mutation, wherein the mutation is located at the CH3 interaction interface, which can weaken the interaction between the CH3 domains of the first polypeptide homomer and / or the second polypeptide homomer, making the first polypeptide homomer and / or the second polypeptide homomer more easily dissociated into first polypeptide monomers and second polypeptide monomers under reducing conditions, thereby promoting the production of a heteromultimer comprising one first polypeptide and one second polypeptide.

[0225] Preparation method of heterodimer containing second type mutation combination

[0226] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to iv) below:

[0227] i)349;

[0228] ii) 354 and 351;

[0229] ⅲ)354 and 366; and

[0230] iv)354 and 368.

[0231] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from positions 351, 366 and 368.

[0232] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 354, and 351.

[0233] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R, and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of the following i) to iv):

[0234] ⅰ)349C;

[0235] ii) 354C and 351I;

[0236] iii) 354C and 366A; and

[0237] iv) 354C and 368I.

[0238] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 351I, 366A and 368I.

[0239] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 354C, and 351I.

[0240] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0241] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E, S354C and L351I; or

[0242] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E, S354C and T366A; or

[0243] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E, S354C and L368I.

[0244] The present disclosure provides a method for preparing a heteromultimer, comprising the following steps:

[0245] a) providing a molecule comprising a first polypeptide homomer, wherein the first polypeptide comprises a CH3 domain, and the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C;

[0246] b) providing a molecule comprising a second polypeptide homomer, wherein the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C, and 351I; and

[0247] c) a step of incubating the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer under reducing conditions;

[0248] The CH3 domain mutation sites are represented by EU numbering.

[0249] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items comprises: the CH3 domains of the first and second polypeptides each comprising a charged amino acid mutation (e.g., a positively charged amino acid, a negatively charged amino acid), and the amino acid mutation in the CH3 domain of the first polypeptide and the amino acid mutation in the CH3 domain of the second polypeptide have opposite charges. Furthermore, the CH3 domains of the first and second polypeptides each further comprise a cysteine ​​mutation, which can form a disulfide bond. Furthermore, the CH3 domain of the first or second polypeptide further comprises an amino acid mutation located at the CH3 interaction interface, which can weaken the interaction between the CH3 domains of the first or second polypeptide homomers, making the first or second polypeptide homomers more easily dissociated into first and second polypeptide monomers under reducing conditions, thereby promoting the production of heteromultimers comprising one first polypeptide and one second polypeptide.

[0250] Preparation method of heteromultimer containing third type mutation combination

[0251] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0252] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364 or 354; or

[0253] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394; or

[0254] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 366, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 405 or 407.

[0255] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0256] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364 or 354; or

[0257] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394.

[0258] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364.

[0259] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0260] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 364Y, 354F, 354W and 354Y; or

[0261] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W; or

[0262] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349A, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 354F, 354Y and 354W; or

[0263] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349V, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or

[0264] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or

[0265] The CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or

[0266] The CH3 domain of the first polypeptide comprises an amino acid mutation of 366H, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 405L or 407L.

[0267] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0268] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y; or

[0269] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W; or

[0270] The CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F.

[0271] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y.

[0272] The present disclosure provides a method for preparing a heteromultimer, comprising the following steps:

[0273] a) providing a molecule comprising a first polypeptide homomer, wherein the first polypeptide comprises a CH3 domain, and the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S;

[0274] b) providing a molecule comprising a second polypeptide homomer, wherein the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y; and

[0275] c) a step of incubating the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer under reducing conditions;

[0276] The CH3 domain mutation sites are represented by EU numbering.

[0277] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0278] The CH3 domain of the first polypeptide comprises an amino acid mutation from a small-volume amino acid residue to a large-volume amino acid residue, which can generate a protrusion in the CH3 interface, and the CH3 domain of the second polypeptide comprises an amino acid mutation from a large-volume amino acid residue to a small-volume amino acid residue, which can generate a cavity in the CH3 interface, thereby promoting the production of a heteromultimer comprising one first polypeptide and one second polypeptide; or

[0279] The CH3 domain of the first polypeptide comprises an amino acid mutation from a bulky amino acid residue to a small bulky amino acid residue, which can generate a cavity in the CH3 interface, and the CH3 domain of the second polypeptide comprises an amino acid mutation from a small bulky amino acid residue to a bulky amino acid residue, which can generate a protrusion in the CH3 interface, thereby promoting the production of a heterologous multimer comprising a first polypeptide and a second polypeptide.

[0280] Preparation method of heteromultimer containing fourth type mutation combination

[0281] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0282] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 354, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from positions 351, 364, 366, 368, and 405; or

[0283] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 347 and 357.

[0284] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 354 and 364.

[0285] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0286] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 351I, 364Y, 364F, 366A, 368I and 405Y; or

[0287] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 347E and 357C.

[0288] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation of 364Y or 364F.

[0289] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 364Y.

[0290] The present disclosure provides a method for preparing a heteromultimer, comprising the following steps:

[0291] a) providing a molecule comprising a first polypeptide homomer, wherein the first polypeptide comprises a CH3 domain, and the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C;

[0292] b) providing a molecule comprising a second polypeptide homomer, wherein the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations 354C and 364Y; and

[0293] c) a step of incubating the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer under reducing conditions;

[0294] The CH3 domain mutation sites are represented by EU numbering.

[0295] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation, which can form a disulfide bond; further, the CH3 domains of the first polypeptide or the second polypeptide each further contain an amino acid mutation, wherein the mutation is located at the CH3 interaction interface, which can weaken the interaction between the CH3 domains of the first polypeptide homomer and / or the second polypeptide homomer, making the first polypeptide homomer and / or the second polypeptide homomer more easily dissociated into first polypeptide monomers and second polypeptide monomers under reducing conditions, thereby promoting the production of a heteromultimer comprising a first polypeptide and a second polypeptide.

[0296] Preparation method of heterologous multimer containing fifth type mutation combination

[0297] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to vii) below:

[0298] i)349;

[0299] ii)354;

[0300] iii)357;

[0301] iv)366;

[0302] ⅴ)394;

[0303] ⅵ) 405; and

[0304] ⅶ)407.

[0305] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0306] The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at position 439, wherein the CH3 domain of the second polypeptide further comprises amino acid mutations at position 354 or 357; or

[0307] The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 405, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 394; or

[0308] The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 366, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation at position 405 or 407; or

[0309] The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 407, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 366.

[0310] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354.

[0311] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R, and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to vii) below:

[0312] i) 349S or 349C;

[0313] ii) 354Y or 354C;

[0314] iii) 357C;

[0315] iv)366H;

[0316] ⅴ)394F;

[0317] vi) 405T or 405L; and

[0318] ⅶ)407L or 407H.

[0319] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0320] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or

[0321] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354C; or

[0322] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 357C; or

[0323] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394F; or

[0324] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366H, and the CH3 domain of the second polypeptide comprises amino acid mutation 439E, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 405L, 407L and 407H; or

[0325] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; or

[0326] The CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 407H, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 366H.

[0327] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 354Y.

[0328] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0329] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y349S, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and S354Y; or

[0330] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and S354C; or

[0331] The CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and Y349C, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and E357C; or

[0332] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and F405T, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394F; or

[0333] The CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and T366H, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and F405L; or

[0334] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366H, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407L; or

[0335] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366H, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407H; or

[0336] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366H; or

[0337] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407H, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366H.

[0338] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domains of the first polypeptide and the second polypeptide each comprise a charged amino acid mutation (e.g., a positively charged amino acid, a negatively charged amino acid), and the amino acid mutation in the CH3 domain of the first polypeptide and the amino acid mutation in the CH3 domain of the second polypeptide have opposite charges. Furthermore, the CH3 domains of the first polypeptide and the second polypeptide further comprise amino acid mutations selected from any one of the following groups:

[0339] The CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation, which can form a disulfide bond; or

[0340] The CH3 domains of the first polypeptide and the second polypeptide each further comprise a different amino acid mutation, wherein the amino acid mutation in the CH3 domain of the first polypeptide is from a small-volume amino acid residue to a large-volume amino acid residue, which can generate a protrusion in the CH3 interface, and the amino acid mutation in the CH3 domain of the second polypeptide is from a large-volume amino acid residue to a small-volume amino acid residue, which can generate a cavity in the CH3 interface; or

[0341] The CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation, which can form a disulfide bond; further, the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation, wherein the amino acid mutation in the CH3 domain of the first polypeptide mutates from a bulky amino acid residue to a small amino acid residue, which can generate a cavity in the CH3 interface, and the amino acid mutation in the CH3 domain of the second polypeptide mutates from a small amino acid residue to a bulky amino acid residue, which can generate a protrusion in the CH3 interface;

[0342] This promotes the production of heteromultimers comprising a first polypeptide and a second polypeptide.

[0343] Preparation method of heteromultimer containing sixth type mutation combination

[0344] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 357.

[0345] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 357C.

[0346] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation, which can form a disulfide bond.

[0347] Preparation method of the seventh type of mutation combination

[0348] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 354, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 349, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from i) or ii):

[0349] i)405;

[0350] ⅱ)394.

[0351] In some embodiments, the method for preparing a heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations at positions 354 and 405, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 349 and 394.

[0352] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 354C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 349C, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from i) or ii):

[0353] ⅰ)405T;

[0354] ⅱ)394F.

[0355] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 354C and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations of 349C and 394F.

[0356] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein:

[0357] The CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation, which can form a disulfide bond; further, the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation, wherein the amino acid mutation in the CH3 domain of the first polypeptide mutates from a small-volume amino acid residue to a large-volume amino acid residue, which can generate a protrusion in the CH3 interface, and the amino acid mutation in the CH3 domain of the second polypeptide mutates from a large-volume amino acid residue to a small-volume amino acid residue, which can generate a cavity in the CH3 interface; or

[0358] The CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation, which can form a disulfide bond; further, the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation, wherein the amino acid mutation in the CH3 domain of the first polypeptide mutates from a bulky amino acid residue to a small amino acid residue, which can generate a cavity in the CH3 interface, and the amino acid mutation in the CH3 domain of the second polypeptide mutates from a small amino acid residue to a bulky amino acid residue, which can generate a protrusion in the CH3 interface;

[0359] This promotes the production of heteromultimers comprising a first polypeptide and a second polypeptide.

[0360] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the heterodimeric interaction between the CH3 domains of the first polypeptide and the second polypeptide is stronger than the homodimeric interaction between the CH3 domains of the first polypeptide and the second polypeptide.

[0361] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the first polypeptide and the second polypeptide have a difference in isoelectric point. In some embodiments, the heteromultimer as described in any of the preceding items, wherein the amino acid mutations disclosed herein confer or increase the difference in isoelectric point between the first polypeptide and the second polypeptide. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the first polypeptide and the second polypeptide further comprise additional amino acid mutations to confer or increase the difference in isoelectric point between the first polypeptide and the second polypeptide. In some embodiments, the heteromultimer as described in any of the preceding items, wherein the first polypeptide and the second polypeptide further comprise additional amino acid mutations to confer or increase the difference in isoelectric point between the first polypeptide and the second polypeptide.

[0362] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items comprises the following steps:

[0363] a) providing a molecule comprising a first polypeptide homomer;

[0364] b) providing a molecule comprising a second polypeptide homomer;

[0365] c) a step of incubating the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer under reducing conditions; and

[0366] d) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide;

[0367] The CH3 domains of the first polypeptide and the second polypeptide differ in at least one mutation site, and the CH3 domain mutation sites are represented by EU numbering;

[0368] Wherein the first polypeptide and the second polypeptide have a difference in isoelectric point, and the first polypeptide and the second polypeptide further introduce additional amino acid mutations to confer or increase the difference in isoelectric point between the first polypeptide and the second polypeptide. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the first polypeptide and the second polypeptide further comprise a variable region. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the first polypeptide and the second polypeptide further comprise a VH and a VL. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the VH of the first polypeptide and / or the second polypeptide further comprises an amino acid mutation selected from Q105E, Q105R, and Q105K, wherein the mutation site is represented by KABAT numbering. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the VL of the first polypeptide and / or the second polypeptide further comprises an amino acid mutation of K42E, wherein the mutation site is represented by KABAT numbering.

[0369] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the reducing conditions can cause cysteine ​​in the hinge region to cause disulfide bond isomerization. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the reducing conditions are sufficient to allow the reduction of interchain disulfide bonds in the hinge region. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the reducing conditions include but are not limited to adding one or more reducing agents selected from 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione (GSH), tris (2-carboxyethyl) phosphine (TCEP), L-cysteine, D-cysteine ​​and β-mercapto-ethanol and its chemical derivatives. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the reducing agent is selected from one or more of 2-MEA, glutathione, L-cysteine, dithiothreitol, β-mercaptoethanol and TCEP. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the reducing agent is 2-MEA.

[0370] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the final concentration of the reducing agent is 0.1 mM to 1 M. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the final concentration of the reducing agent is 1 mM to 1 M. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the final concentration of the reducing agent is 5 mM to 500 mM. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, the final concentration of the reducing agent is 5 mM to 200 mM. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, the final concentration of the reducing agent is 25 mM to 100 mM. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, the final concentration of the reducing agent is 60 mM to 90 mM. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, the final concentration of the reducing agent is 70 mM to 80 mM. In some embodiments, in the method for preparing a heteromultimer as described in any of the preceding items, the final concentration of the reducing agent is 75 mM.

[0371] In some embodiments, in the method for preparing a heteromultimer as described in any of the preceding items, the final concentration of the reducing agent is about 0.1 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 80 mM, about 85 mM, about 90 mM, about 100 mM, about 15 ... about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 110mM, about 120mM, about 130mM, about 140mM, about 150mM, about 160mM, about 170mM, about 180mM, about 190mM, about 200mM, about 210mM, about 220mM, about 230mM, about 240mM M, about 250mM, about 260mM, about 270mM, about 280mM, about 290mM, about 300mM, about 310mM, about 320mM, about 330mM, about 340mM, about 350mM, about 360mM, about 370mM, about 380mM, about 390mM, about 400mM, about 410mM, about 420mM, about 430mM In some embodiments, the present invention relates to a method for preparing an aqueous phase of the present invention that is at least about 1 M, about 440 mM, about 450 mM, about 460 mM, about 470 mM, about 480 mM, about 490 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, about 800 mM, about 850 mM, about 900 mM, about 950 mM or about 1 M, or any range therebetween.

[0372] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the heteromultimer is a multispecific antibody or an Fc fusion protein.

[0373] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the heteromultimer is a heterodimer. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the heterodimer comprises a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide and / or the second polypeptide is a polypeptide structure constituting a heterodimer, which comprises at least a CH3 domain. In some embodiments, the first polypeptide is an Fc region (first Fc region), the second polypeptide is an Fc region (second Fc region), and the first Fc region and / or the second Fc region comprise CH2 and CH3 domains. In some embodiments, the first polypeptide and / or the second polypeptide comprise CH1, CH2, and CH3 domains.

[0374] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the heteromultimer is a bispecific antibody. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the bispecific antibody comprises a first half antibody and a second half antibody. In some embodiments, the first half antibody comprises a first polypeptide, and the second half antibody comprises a second polypeptide. The first polypeptide and / or the second polypeptide is a polypeptide structure constituting the bispecific antibody, which comprises at least a CH3 domain. In some embodiments, the first polypeptide is an Fc region (first Fc region) and the second polypeptide is an Fc region (second Fc region). In some embodiments, the first Fc region and / or the second Fc region comprise CH2 and CH3 domains. In some embodiments, the first polypeptide and / or the second polypeptide comprise CH1, CH2, and CH3 domains. In some embodiments, the first half antibody binds to a first antigen and the second half antibody binds to a second antigen. In some embodiments, the first half antibody comprises a first antigen-binding domain and the second half antibody comprises a second antigen-binding domain. In some embodiments, the structures of the first and second antigen binding domains are selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, dAb, and VHH.

[0375] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the heteromultimer is a trispecific antibody. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the trispecific antibody comprises a first half antibody and a second half antibody. In some embodiments, the first half antibody binds to a first antigen, and the second half antibody binds to a second antigen and a third antigen. In some embodiments, the first half antibody comprises a first antigen-binding domain, and the second half antibody comprises a second antigen-binding domain and a third antigen-binding domain. In some embodiments, the structures of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, dAb, and VHH.

[0376] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are selected from an Fc region, an antibody, a fusion protein comprising an Fc region (e.g., an Fc region fused to a receptor, cytokine, or hormone), and an Fc region conjugated to a drug (peptide or toxin). In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the molecule comprising a first polypeptide homomer is a first parent antibody, and the molecule comprising a second polypeptide homomer is a second parent antibody. In some embodiments, the first parent antibody is a first parent monoclonal antibody and the second parent antibody is a second parent monoclonal antibody.

[0377] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the first polypeptide homomer is a homomer composed of two first polypeptides, and the second polypeptide homomer is a homomer composed of two second polypeptides.

[0378] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the first polypeptide homomer and the second polypeptide homomer both comprise a Cys-Pro-Pro-Cys sequence in the hinge region.

[0379] When describing "first polypeptide" in this disclosure, it may also refer to each first polypeptide in the first polypeptide homopolymer. When describing "second polypeptide" in this disclosure, it may also refer to each second polypeptide in the second polypeptide homopolymer. In some embodiments, the first polypeptide and / or the second polypeptide is a polypeptide structure constituting a heterodimer, which comprises at least a CH3 domain. In some embodiments, the first polypeptide is an Fc region (first Fc region), the second polypeptide is an Fc region (second Fc region), and the first Fc region and / or the second Fc region comprise CH2 and CH3 domains. In some embodiments, the first polypeptide and / or the second polypeptide comprise CH1, CH2 and CH3 domains.

[0380] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the first polypeptide is an antibody heavy chain, and / or the second polypeptide is an antibody heavy chain. In some embodiments, the heteromultimer further comprises one or more antibody light chains.

[0381] In some embodiments, the first polypeptide and the second polypeptide sequences are different. For example, a molecule comprising two first polypeptides is a first maternal antibody, and a molecule comprising two second polypeptides is a second maternal antibody. In the present disclosure, the first maternal antibody further comprises one or more polypeptides (e.g., light chains that form half antibodies with the two first polypeptides, respectively). In the present disclosure, the second maternal antibody further comprises one or more polypeptides (e.g., light chains that form half antibodies with the two second polypeptides, respectively).

[0382] The first polypeptide, second polypeptide, first parent antibody, or second parent antibody are used only to distinguish the amino acid sequence and do not limit the positional relationship between the polypeptides and proteins. For example, in a heterodimer composed of a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide are two polypeptides with different amino acid sequences. When either polypeptide is the first polypeptide, the other polypeptide is the second polypeptide.

[0383] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the amino acids in the core hinge region of the heteromultimer form a disulfide bond.

[0384] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain is derived from IgG. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain is derived from IgG1, IgG2, or IgG3. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domain is derived from IgG1. In some embodiments, the CH3 domain is derived from human IgG1. In some embodiments, the human IgG1 has the amino acid sequence set forth in SEQ ID NO: 39, 40, or 41.

[0385] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the heteromultimer comprises at least one replaced Fab comprising a titin chain and an obscurin chain capable of forming a dimer. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the replaced Fab comprises an original CH1 and CL of the Fab replaced by an obscurin chain and a titin chain, respectively, or the replaced Fab comprises an original CH1 and CL of the Fab replaced by a titin chain and an obscurin chain, respectively. In some embodiments, the titin chain has the amino acid sequence set forth in SEQ ID NO: 6, and the obscurin chain has the amino acid sequence set forth in SEQ ID NO: 5.

[0386] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein more than 70% (e.g., more than 75%, more than 80%, more than 85%, more than 88%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99%) of the total product is the desired heteromultimer (compared to other products in the total product, such as half antibodies or homomers).

[0387] In some embodiments, the heteromultimer has good thermal stability.

[0388] In some embodiments, the thermal stability of the heteromultimer is higher than the thermal stability of the heteromultimer comprising Fc region mutations in the prior art. In some embodiments, the thermal stability comprises a thermodynamic index.

[0389] In some embodiments, the parent antibody of the heteromultimer has good thermal stability.

[0390] In some embodiments, the thermal stability of the heteromultimer is greater than the thermal stability of the parent antibody (eg, the first parent antibody, the second parent antibody).

[0391] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein: step a) and step b) further comprise a step of purifying the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer. In some embodiments, the purification method includes but is not limited to protein A or protein G chromatography, affinity chromatography based on antigen binding, affinity chromatography based on anti-idiotypic antibodies, ion exchange, hydrophobic interaction chromatography, mixed chromatography (such as hydroxyapatite), immobilized metal affinity chromatography, thiophilic adsorption chromatography and size exclusion chromatography (SEC), etc. In some embodiments, the purification method is affinity chromatography. In some embodiments, the purification method is protein A chromatography.

[0392] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein step c) is followed by a step of removing the reducing agent. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, the reducing agent is removed by, but not limited to, dialysis, precipitation, chromatography, or filtration. In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, the reducing agent is removed by dialysis.

[0393] In some embodiments, the step for removing the reducing agent can be any method that results in or is capable of separating the two without damaging the heteromultimer. Such methods include, but are not limited to, dialysis, precipitation, chromatography, or filtration. The step for removing the reducing agent can be performed as a continuous process or it can be performed as a batch process.

[0394] In some embodiments, the content of heteromultimers in the product obtained in step c) is more than 70% (e.g., more than 75%, more than 80%, more than 85%, more than 88%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99%).

[0395] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein step d) further comprises a method for purifying the product obtained from step c). In some embodiments, the purification method includes but is not limited to protein A or protein G chromatography, affinity chromatography based on antigen binding, affinity chromatography based on anti-idiotypic antibodies, ion exchange, hydrophobic interaction chromatography, mixed chromatography (such as hydroxyapatite), immobilized metal affinity chromatography, thiophilic adsorption chromatography, and size exclusion chromatography (SEC). In some embodiments, the purification method is affinity chromatography. In some embodiments, the purification method is size exclusion chromatography (SEC).

[0396] In some embodiments, the following methods can be cited: a method in which cell lines producing molecules comprising a first polypeptide homopolymer and molecules comprising a second polypeptide homopolymer are separately cultured, the culture supernatant is purified, and then a purified antibody is used to induce an FAE (Fab arm exchange) reaction; a method in which cell lines producing molecules comprising a first polypeptide homopolymer and molecules comprising a second polypeptide homopolymer are separately cultured, the culture supernatant is not purified but mixed, an FAE reaction is induced in the mixed culture supernatant, and then purification is performed; a method in which a cell line producing a molecule comprising a first polypeptide homopolymer is mixed with a cell line producing a molecule comprising a second polypeptide homopolymer and cultured, the culture supernatant is purified, and then a FAE reaction is induced using a purified antibody; a method in which a cell line producing a molecule comprising a first polypeptide homopolymer is mixed with a cell line producing a molecule comprising a second polypeptide homopolymer and cultured, an FAE reaction is induced in the culture supernatant, and then purification is performed.

[0397] In some embodiments, the present disclosure provides a method for preparing a heteromultimer, the method comprising the following steps a) to c):

[0398] a) separately culturing a cell line that produces a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer;

[0399] b) purifying the culture supernatant of each cell line to obtain molecules comprising the first polypeptide homomer and molecules comprising the second polypeptide homomer, and incubating the molecules comprising the first polypeptide homomer and the molecules comprising the second polypeptide homomer together in the presence of a reducing agent; and

[0400] c) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide.

[0401] In some embodiments, the present disclosure provides a method for preparing a heteromultimer, the method comprising the following steps a) to c):

[0402] a) mixing a cell line producing a molecule comprising a first polypeptide homomer with a cell line producing a molecule comprising a second polypeptide homomer;

[0403] b) a step of incubating the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer in the culture supernatant in the presence of a reducing agent; and

[0404] c) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide.

[0405] In some embodiments, the present disclosure provides a method for preparing a heteromultimer, the method comprising the following steps a) to c):

[0406] a) separately culturing a cell line that produces a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer;

[0407] b) mixing the culture supernatants of the cell lines, and incubating the molecules comprising the first polypeptide homomer and the molecules comprising the second polypeptide homomer together in the presence of a reducing agent; and

[0408] c) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide.

[0409] In some embodiments, the present disclosure provides a method for preparing a heteromultimer, the method comprising the following steps:

[0410] a) providing a first nucleic acid construct encoding a molecule comprising a first polypeptide homomer,

[0411] b) providing a second nucleic acid construct encoding a molecule comprising a second polypeptide homomer,

[0412] wherein the first polypeptide and the second polypeptide have different sequences, and the heterodimeric interaction between the first polypeptide and the second polypeptide is stronger than the homodimeric interaction between the first polypeptide and the second polypeptide;

[0413] c) co-expressing the first and second nucleic acid constructs in a host cell, and

[0414] d) obtaining the heteromultimer from cell culture.

[0415] In another aspect, the present disclosure provides a heteromultimer prepared according to the method as described in any of the preceding items.

[0416] In another aspect, the present disclosure provides a heteromultimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise a CH3 domain, wherein the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide comprise amino acid mutations, wherein the amino acid mutations comprise at least one amino acid mutation selected from: positions 347, 349, 351, 354, 356, 357, 364, 366, 368, 394, 405, 407, 409, 411 and 439; wherein the CH3 domains of the first polypeptide and the second polypeptide differ in at least one mutation site, and the CH3 domain mutation sites are represented by EU numbering.

[0417] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the amino acid mutation comprises at least one amino acid mutation selected from: 349, 351, 354, 356, 364, 366, 368, 394, 405, 409, 411 and 439.

[0418] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain comprises one or more mutations that promote heteromerization formation. The mutation that promotes heteromerization formation, in the present disclosure, refers to a class of amino acid mutations that can promote the dissociation of homopolymers (e.g., homodimers) into monomers, and / or promote the formation of heteromultimers (e.g., heterodimers) by two heteromonomers. In some embodiments, the mutation that promotes heteromerization formation is an amino acid mutation at the CH3 interaction interface. In some embodiments, the mutation that promotes heteromerization formation is an amino acid mutation with a charge at the CH3 interaction interface. In some embodiments, the mutation that promotes heteromerization formation is an amino acid mutation with an opposite charge at the CH3 interaction interface.

[0419] In some embodiments, the mutation that promotes heteromer formation is any mutation described in the present disclosure.

[0420] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide and / or the second polypeptide comprises at least one amino acid mutation selected from the following combinations:

[0421] The amino acid at position 356 is mutated to Lys (K), Arg (R) or His (H); and / or

[0422] The amino acid at position 439 is mutated to Glu (E) or Asp (D); and / or

[0423] The amino acid at position 349 is mutated to Leu (L), Phe (F), Ser (S), Cys (C), Ala (A), Val (V), Thr (T) or Gly (G); and / or

[0424] The amino acid at position 351 is mutated to Cys (C), Val (V), Thr (T), Ile (I), Phe (F), or Met (M); and / or

[0425] The amino acid at position 364 is mutated to Ala (A), Val (V), Thr (T), Leu (L), Tyr (Y) or Phe (F); and / or

[0426] The amino acid at position 366 is mutated to Gly (G), Ser (S), Ala (A), Val (V), Leu (L), His (H) or Ile (I); and / or

[0427] The amino acid at position 368 is mutated to Val (V), Ile (I), Met (M) or Ala (A); and / or

[0428] The amino acid at position 394 is mutated to Phe (F), Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); and / or

[0429] The amino acid at position 405 is mutated to Thr (T), Leu (L) or Tyr (Y); and / or

[0430] The amino acid at position 407 is mutated to Cys (C), Val (V), Leu (L), His (H) or Phe (F); and / or

[0431] The amino acid at position 409 is mutated to Gln (Q), Arg (R) or Asp (D); and / or

[0432] The amino acid at position 411 is mutated to Asn (N), Tyr (Y) or Leu (L); and / or

[0433] The amino acid at position 354 is mutated to Tyr (Y), Cys (C), Phe (F) or Trp (W); and / or

[0434] The amino acid at position 357 is mutated to Cys (C); and / or

[0435] The amino acid at position 347 was mutated to Glu (E).

[0436] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0437] (a1) amino acid residues 356 and 351 according to EU numbering;

[0438] (a2) amino acid residues 356 and 364 according to EU numbering;

[0439] (a3) amino acid residues 356 and 366 according to EU numbering;

[0440] (a4) amino acid residues 356 and 368 according to EU numbering;

[0441] (a5) amino acid residues 356 and 394 according to EU numbering;

[0442] (a6) amino acid residues 356 and 405 according to EU numbering;

[0443] (a7) amino acid residues 356 and 409 according to EU numbering;

[0444] (a8) amino acid residues 356 and 411 according to EU numbering;

[0445] (b1) amino acid residues 356 and 349 according to EU numbering;

[0446] (c1) amino acid residue 405 according to EU numbering; or

[0447] (c2) amino acid residue 349 according to EU numbering;

[0448] The CH3 domain of the second polypeptide has a structure selected from the group consisting of (d1) to (i2):

[0449] (d1) amino acid residues 439 and 351 according to EU numbering;

[0450] (d2) amino acid residues 439 and 364 according to EU numbering;

[0451] (d3) amino acid residues 439 and 366 according to EU numbering;

[0452] (d4) amino acid residues 439 and 368 according to EU numbering;

[0453] (d5) amino acid residues 439 and 394 according to EU numbering;

[0454] (d6) amino acid residues 439 and 405 according to EU numbering;

[0455] (d7) amino acid residues 439 and 409 according to EU numbering;

[0456] (d8) amino acid residues 439 and 411 according to EU numbering;

[0457] (e1) amino acid residues 439 and 354 according to EU numbering;

[0458] (f1) amino acid residues 439, 354, and 351 according to EU numbering;

[0459] (f2) amino acid residues 439, 354, and 366 according to EU numbering;

[0460] (f3) amino acid residues 439, 354, and 368 according to EU numbering;

[0461] (g1) amino acid residue 394 according to EU numbering;

[0462] (h) amino acid residues 354 and 364 according to EU numbering;

[0463] (i1) amino acid residue 364 according to EU numbering; or

[0464] (i2) Amino acid residue at position 354 according to EU numbering.

[0465] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0466] (a1) amino acid residues 356 and 351 according to EU numbering;

[0467] (a5) amino acid residues 356 and 394 according to EU numbering;

[0468] (a8) amino acid residues 356 and 411 according to EU numbering;

[0469] (b1) amino acid residues 356 and 349 according to EU numbering; or

[0470] (c2) amino acid residue 349 according to EU numbering;

[0471] The CH3 domain of the second polypeptide has a structure selected from the group consisting of the following (d1) to (i1):

[0472] (d1) amino acid residues 439 and 351 according to EU numbering;

[0473] (d5) amino acid residues 439 and 394 according to EU numbering;

[0474] (d8) amino acid residues 439 and 411 according to EU numbering;

[0475] (f1) amino acid residues 439, 354, and 351 according to EU numbering;

[0476] (h) amino acid residues 354 and 364 according to EU numbering; or

[0477] (i1) Amino acid residue at position 364 according to EU numbering.

[0478] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0479] (a1) amino acid residues 356 and 351 according to EU numbering;

[0480] (b1) amino acid residues 356 and 349 according to EU numbering; or

[0481] (c2) amino acid residue 349 according to EU numbering;

[0482] The CH3 domain of the second polypeptide has a structure selected from the group consisting of the following (d1) to (i1):

[0483] (d1) amino acid residues 439 and 351 according to EU numbering;

[0484] (f1) amino acid residues 439, 354, and 351 according to EU numbering;

[0485] (h) amino acid residues 354 and 364 according to EU numbering; or

[0486] (i1) Amino acid residue at position 364 according to EU numbering.

[0487] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0488] (a1) amino acid residues 356K and 351I according to EU numbering; or

[0489] amino acid residues 356K and 351T according to EU numbering;

[0490] (a2) amino acid residues 356K and 364A represented by EU numbering;

[0491] (a3) amino acid residues 356K and 366A represented by EU numbering;

[0492] (a4) amino acid residues 356K and 368I represented by EU numbering;

[0493] (a5) amino acid residues 356K and 394A according to EU numbering; or

[0494] amino acid residues 356K and 394S according to EU numbering;

[0495] (a6) amino acid residues 356K and 405Y represented by EU numbering;

[0496] (a7) amino acid residues 356K and 409Q represented by EU numbering;

[0497] (a8) amino acid residues 356K and 411Y represented by EU numbering;

[0498] (b1) amino acid residues 356K and 349C according to EU numbering; or

[0499] amino acid residues 356K and 349S according to EU numbering;

[0500] (c1) the amino acid residue 405T according to EU numbering; or

[0501] (c2) amino acid residue 349C, 349S or 349G according to EU numbering;

[0502] The CH3 domain of the second polypeptide has a structure selected from the group consisting of (d1) to (i2):

[0503] (d1) amino acid residues 439E and 351I according to EU numbering; or

[0504] amino acid residues 439E and 351T according to EU numbering;

[0505] (d2) amino acid residues 439E and 364A according to EU numbering;

[0506] (d3) amino acid residues 439E and 366A according to EU numbering;

[0507] (d4) amino acid residues 439E and 368I represented by EU numbering;

[0508] (d5) amino acid residues 439E and 394A according to EU numbering; or

[0509] amino acid residues 439E and 394S according to EU numbering;

[0510] (d6) amino acid residues 439E and 405Y represented by EU numbering;

[0511] (d7) amino acid residues 439E and 409Q represented by EU numbering;

[0512] (d8) amino acid residues 439E and 411Y according to EU numbering;

[0513] (e1) amino acid residues 439E and 354Y represented by EU numbering;

[0514] (f1) amino acid residues 439E, 354C, and 351I according to EU numbering;

[0515] (f2) amino acid residues 439E, 354C, and 366A according to EU numbering;

[0516] (f3) amino acid residues 439E, 354C, and 368I according to EU numbering;

[0517] (g1) amino acid residue 394F represented by EU numbering;

[0518] (h) amino acid residues 354C and 364Y according to EU numbering; or

[0519] amino acid residues 354C and 364F according to EU numbering;

[0520] (i1) the amino acid residue 364Y according to EU numbering; or

[0521] (i2) Amino acid residue 354W represented by EU numbering.

[0522] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0523] (a1) amino acid residues 356K and 351I represented by EU numbering;

[0524] (a5) amino acid residues 356K and 394S represented by EU numbering;

[0525] (a8) amino acid residues 356K and 411Y represented by EU numbering;

[0526] (b1) amino acid residues 356K and 349C according to EU numbering; or

[0527] (c2) amino acid residue 349S or 349C represented by EU numbering;

[0528] The CH3 domain of the second polypeptide has a structure selected from the group consisting of the following (d1) to (i1):

[0529] (d1) amino acid residues 439E and 351I represented by EU numbering;

[0530] (d5) amino acid residues at positions 439E and 394S according to EU numbering;

[0531] (d8) amino acid residues 439E and 411Y according to EU numbering;

[0532] (f1) amino acid residues 439E, 354C, and 351I according to EU numbering;

[0533] (h) amino acid residues 354C and 364Y according to EU numbering; or

[0534] amino acid residues 354C and 364F according to EU numbering;

[0535] (i1) Amino acid residue 364Y represented by EU numbering.

[0536] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide has an amino acid mutation selected from the group consisting of the following (a1) to (c2):

[0537] (a1) amino acid residues 356K and 351I represented by EU numbering;

[0538] (a5) amino acid residues 356K and 349C according to EU numbering; or

[0539] (c2) amino acid residue 349S or 349C represented by EU numbering;

[0540] The CH3 domain of the second polypeptide has a structure selected from the group consisting of the following (d1) to (i1):

[0541] (d1) amino acid residues 439E and 351I represented by EU numbering;

[0542] (f1) amino acid residues 439E, 354C, and 351I according to EU numbering;

[0543] (h) amino acid residues 354C and 364Y according to EU numbering; or

[0544] (i1) Amino acid residue 364Y represented by EU numbering.

[0545] The first type of mutation combination

[0546] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise one identical or different amino acid mutation selected from positions 349, 351, 364, 366, 368, 394, 405, 407, 409 and 411.

[0547] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409 and 411.

[0548] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 351, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 351.

[0549] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R, and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domains of the first polypeptide and the second polypeptide further each comprise an identical or different amino acid mutation selected from 349L, 349F, 351C, 351V, 351T, 351I, 351F, 351D, Amino acid mutations in 1M, 364A, 364V, 364T, 364L, 366G, 366S, 366A, 366V, 366L, 366H, 366I, 368V, 368I, 368A, 368M, 394A, 394S, 394C, 394V, 394N, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411N, 411L and 411Y.

[0550] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid mutation selected from 349L, 351C, 351V, 351T, 351I, 351D, 351E, 351F, 351I ... Amino acid mutations in 1F, 351M, 364A, 364T, 364L, 366G, 366A, 366V, 366L, 366H, 366I, 368V, 368I, 368A, 368M, 394A, 394S, 394C, 394V, 394N, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411L and 411Y.

[0551] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 of the first polypeptide The CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide further each comprise one identical or different amino acid mutation selected from 349L, 351C, 351V, 351T, 351I, 351F, 351M, 364A, 364T, 364L, 366G, 366A, 366V, 366L, 366H, 366I, 368V, 368I, 368A, 368M, 394A, 394S, 394C, 394V, 394N, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411L and 411Y.

[0552] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0553] The CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R, and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409, and 411, wherein:

[0554] mutating amino acid 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I), Phe (F), or Met (M); or

[0555] mutating amino acid position 364 (EU numbering) to Ala (A), Val (V), Thr (T) or Leu (L); or

[0556] mutating amino acid position 366 (EU numbering) to Ser (S), Ala (A), Val (V), Leu (L), His (H), or Ile (I); or

[0557] mutating amino acid position 368 (EU numbering) to Val (V), Ile (I), Met (M) or Ala (A); or

[0558] mutating amino acid position 394 (EU numbering) to Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[0559] mutating amino acid position 405 (EU numbering) to Leu (L) or Tyr (Y); or

[0560] mutating amino acid position 409 according to EU numbering to Gln (Q); or

[0561] The amino acid at position 411 represented by EU numbering is mutated to Asn (N), Tyr (Y), or Leu (L).

[0562] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0563] The CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409, and 411, wherein:

[0564] mutating position 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I) or Phe (F); or

[0565] mutating position 364 (EU numbering) to Ala (A), Val (V), Thr (T) or Leu (L); or

[0566] mutating position 366 (EU numbering) to Ser (S), Ala (A), Val (V), Leu (L) or His (H); or

[0567] mutating position 368 (EU numbering) to Val (V), Ile (I), or Met (M); or

[0568] mutating position 394 (EU numbering) to Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[0569] Mutating position 405 (EU numbering) to Leu (L) or Tyr (Y); or

[0570] mutating position 409 (EU numbering) to Gln (Q) or Arg (R); or

[0571] Position 411 (EU numbering) was mutated to Asn (N) or Tyr (Y).

[0572] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from 351T, 351I, 364A, 366A, 368I, 394A, 394S, 405Y, 409Q and 411Y.

[0573] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 351I, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 351I.

[0574] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0575] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y349L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y349L; or

[0576] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351C; or

[0577] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351V, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351V; or

[0578] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351T, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351T; or

[0579] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351I, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351I; or

[0580] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351F, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351F; or

[0581] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L351M, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L351M; or

[0582] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and S364A, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and S364A; or

[0583] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and S364T, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and S364T; or

[0584] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and S364L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and S364L; or

[0585] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366G, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366G; or

[0586] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366A, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366A; or

[0587] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366V, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366V; or

[0588] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366L; or

[0589] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366H, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366H; or

[0590] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366I, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366I; or

[0591] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L368V, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L368V; or

[0592] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L368I, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L368I; or

[0593] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L368A, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L368A; or

[0594] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T394A, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394A; or

[0595] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T394S, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394S; or

[0596] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T394C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394C; or

[0597] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T394V, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394V; or

[0598] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T394N, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394N; or

[0599] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and F405L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and F405L; or

[0600] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and F405Y, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and F405Y; or

[0601] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407C; or

[0602] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407V, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407V; or

[0603] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407L; or

[0604] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407H, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407H; or

[0605] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407F, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407F; or

[0606] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and K409Q, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and K409Q; or

[0607] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and K409R, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and K409R; or

[0608] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T411L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T411L; or

[0609] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T411Y, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T411Y.

[0610] In some embodiments, the method for preparing a heteromultimer as described in any of the preceding items, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a charged amino acid mutation (e.g., a positively charged amino acid, a negatively charged amino acid), and the amino acid mutation in the CH3 domain of the first polypeptide and the amino acid mutation in the CH3 domain of the second polypeptide have opposite charges. Furthermore, the CH3 domains of the first polypeptide and the second polypeptide each contain an identical amino acid mutation, wherein the mutation is located at the CH3 interaction interface, which can weaken the interaction between the CH3 domains of the first polypeptide homomer and / or the second polypeptide homomer, making the first polypeptide homomer and / or the second polypeptide homomer more easily dissociated into first polypeptide monomers and second polypeptide monomers under reducing conditions, thereby promoting the production of a heteromultimer comprising one first polypeptide and one second polypeptide.

[0611] The second type of mutation combination

[0612] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to iv) below:

[0613] i)349;

[0614] ii) 354 and 351;

[0615] ⅲ)354 and 366; and

[0616] iv)354 and 368.

[0617] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354, wherein the CH3 domain region of the second polypeptide further comprises an amino acid mutation selected from positions 351, 366 and 368.

[0618] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 354, and 351.

[0619] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R, and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to iv) below:

[0620] ⅰ)349C;

[0621] ii) 354C and 351I;

[0622] iii) 354C and 366A; and

[0623] iv) 354C and 368I.

[0624] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 351I, 366A and 368I.

[0625] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 354C, and 351I.

[0626] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0627] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E, S354C and L351I; or

[0628] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E, S354C and T366A; or

[0629] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E, S354C and L368I.

[0630] In some embodiments, the heteromultimer described in any of the preceding items comprises a CH3 domain of each of the first and second polypeptides comprising a charged amino acid mutation (e.g., a positively charged amino acid, a negatively charged amino acid), and the amino acid mutation in the CH3 domain of the first polypeptide and the amino acid mutation in the CH3 domain of the second polypeptide have opposite charges. Furthermore, the CH3 domains of the first and second polypeptides each comprise a cysteine ​​mutation, which can form a disulfide bond. Furthermore, the CH3 domain of the first or second polypeptide further comprises an amino acid mutation located at the CH3 interaction interface, which can weaken the interaction between the CH3 domains of the first or second polypeptide homomers, making the first or second polypeptide homomers more easily dissociated into first polypeptide monomers and second polypeptide monomers under reducing conditions, thereby promoting the production of heteromultimers comprising one first polypeptide and one second polypeptide.

[0631] The third type of mutation combination

[0632] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0633] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364 or 354; or

[0634] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394; or

[0635] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 366, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 405 or 407.

[0636] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0637] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364 or 354; or

[0638] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394.

[0639] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364.

[0640] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0641] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 364Y, 354F, 354W and 354Y; or

[0642] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W; or

[0643] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349A, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 354F, 354Y and 354W; or

[0644] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349V, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or

[0645] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or

[0646] The CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or

[0647] The CH3 domain of the first polypeptide comprises an amino acid mutation of 366H, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 405L or 407L.

[0648] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0649] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y; or

[0650] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W; or

[0651] The CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F.

[0652] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y.

[0653] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0654] The CH3 domain of the first polypeptide comprises an amino acid mutation from a small-volume amino acid residue to a large-volume amino acid residue, which can generate a protrusion in the CH3 interface, and the CH3 domain of the second polypeptide comprises an amino acid mutation from a large-volume amino acid residue to a small-volume amino acid residue, which can generate a cavity in the CH3 interface; or

[0655] The CH3 domain of the first polypeptide comprises an amino acid mutation from a bulky amino acid residue to a small bulky amino acid residue, which can generate a cavity in the CH3 interface, and the CH3 domain of the second polypeptide comprises an amino acid mutation from a small bulky amino acid residue to a bulky amino acid residue, which can generate a protrusion in the CH3 interface;

[0656] This promotes the production of heteromultimers comprising a first polypeptide and a second polypeptide.

[0657] The fourth type of mutation combination

[0658] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0659] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 354, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from positions 351, 364, 366, 368, and 405; or

[0660] The CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 347 and 357.

[0661] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 354 and 364.

[0662] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0663] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 351I, 364Y, 364F, 366A, 368I and 405Y; or

[0664] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 347E and 357C.

[0665] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation of 364Y or 364F.

[0666] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 364Y.

[0667] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation, which can form a disulfide bond; further, the CH3 domain of the first polypeptide or the second polypeptide each further contains an amino acid mutation, wherein the mutation is located at the CH3 interaction interface, which can weaken the interaction between the CH3 domains of the first polypeptide homomer and / or the second polypeptide homomer, making the first polypeptide homomer and / or the second polypeptide homomer more easily dissociated into first polypeptide monomers and second polypeptide monomers under reducing conditions, thereby promoting the production of a heterologous multimer comprising a first polypeptide and a second polypeptide.

[0668] The fifth type of mutation combination

[0669] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to vii) below:

[0670] i)349;

[0671] ii)354;

[0672] iii)357;

[0673] iv)366;

[0674] ⅴ)394;

[0675] ⅵ) 405; and

[0676] ⅶ)407.

[0677] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0678] The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at position 439, wherein the CH3 domain of the second polypeptide further comprises amino acid mutations at position 354 or 357; or

[0679] The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 405, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 394; or

[0680] The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 366, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation at position 405 or 407; or

[0681] The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 407, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 366.

[0682] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354.

[0683] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R, and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to vii) below:

[0684] i) 349S or 349C;

[0685] ii) 354Y or 354C;

[0686] iii) 357C;

[0687] iv)366H;

[0688] ⅴ)394F;

[0689] vi) 405T or 405L; and

[0690] ⅶ)407L or 407H.

[0691] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0692] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or

[0693] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354C; or

[0694] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 357C; or

[0695] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394F; or

[0696] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366H, and the CH3 domain of the second polypeptide comprises amino acid mutation 439E, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 405L, 407L and 407H; or

[0697] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; or

[0698] The CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 407H, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 366H.

[0699] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 354Y.

[0700] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0701] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y349S, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and S354Y; or

[0702] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and S354C; or

[0703] The CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and Y349C, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and E357C; or

[0704] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and F405T, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T394F; or

[0705] The CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and T366H, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and F405L; or

[0706] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366H, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407L; or

[0707] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and T366H, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Y407H; or

[0708] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407L, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366H; or

[0709] The CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Y407H, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and T366H.

[0710] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domains of the first polypeptide and the second polypeptide each comprise a charged amino acid mutation (e.g., a positively charged amino acid, a negatively charged amino acid), and the amino acid mutation in the CH3 domain of the first polypeptide and the amino acid mutation in the CH3 domain of the second polypeptide have opposite charges. Furthermore, the CH3 domains of the first polypeptide and the second polypeptide further comprise amino acid mutations selected from any one of the following groups:

[0711] The CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation, which can form a disulfide bond; or

[0712] The CH3 domains of the first polypeptide and the second polypeptide each further comprise a different amino acid mutation, wherein the amino acid mutation in the CH3 domain of the first polypeptide is from a small-volume amino acid residue to a large-volume amino acid residue, which can generate a protrusion in the CH3 interface, and the amino acid mutation in the CH3 domain of the second polypeptide is from a large-volume amino acid residue to a small-volume amino acid residue, which can generate a cavity in the CH3 interface; or

[0713] The CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation, which can form a disulfide bond; further, the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation, wherein the amino acid mutation in the CH3 domain of the first polypeptide mutates from a bulky amino acid residue to a small amino acid residue, which can generate a cavity in the CH3 interface, and the amino acid mutation in the CH3 domain of the second polypeptide mutates from a small amino acid residue to a bulky amino acid residue, which can generate a protrusion in the CH3 interface;

[0714] This promotes the production of heteromultimers comprising a first polypeptide and a second polypeptide.

[0715] Class VI mutation combination

[0716] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 357.

[0717] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 357C.

[0718] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation and can form a disulfide bond.

[0719] Class 7 mutation combination

[0720] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 354, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 349, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from i) or ii):

[0721] i)405;

[0722] ⅱ)394.

[0723] In some embodiments, the heterologous multimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations at positions 354 and 405, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 349 and 394.

[0724] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 354C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 349C, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from i) or ii):

[0725] ⅰ)405T;

[0726] ⅱ)394F.

[0727] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 354C and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations of 349C and 394F.

[0728] In some embodiments, the heteromultimer as described in any of the preceding items, wherein:

[0729] The CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation, which can form a disulfide bond; further, the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation, wherein the amino acid mutation in the CH3 domain of the first polypeptide mutates from a small-volume amino acid residue to a large-volume amino acid residue, which can generate a protrusion in the CH3 interface, and the amino acid mutation in the CH3 domain of the second polypeptide mutates from a large-volume amino acid residue to a small-volume amino acid residue, which can generate a cavity in the CH3 interface; or

[0730] The CH3 domains of the first polypeptide and the second polypeptide each contain a cysteine ​​mutation, which can form a disulfide bond; further, the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation, wherein the amino acid mutation in the CH3 domain of the first polypeptide mutates from a bulky amino acid residue to a small amino acid residue, which can generate a cavity in the CH3 interface, and the amino acid mutation in the CH3 domain of the second polypeptide mutates from a small amino acid residue to a bulky amino acid residue, which can generate a protrusion in the CH3 interface;

[0731] This promotes the production of heteromultimers comprising a first polypeptide and a second polypeptide.

[0732] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the first polypeptide and the second polypeptide have a difference in isoelectric point. In some embodiments, the heteromultimer as described in any of the preceding items, wherein the amino acid mutations described herein confer or increase the difference in isoelectric point between the first polypeptide and the second polypeptide.

[0733] In some embodiments, the heteromultimer described in any of the preceding items further comprises an additional amino acid mutation to confer or increase a difference in isoelectric point between the first and second polypeptides. In some embodiments, the heteromultimer described in any of the preceding items further comprises an additional amino acid mutation to confer or increase a difference in isoelectric point between the first and second polypeptides. In some embodiments, the heteromultimer described in any of the preceding items further comprises an additional amino acid mutation to confer a difference in isoelectric point between the first and second polypeptides. In some embodiments, the heteromultimer described in any of the preceding items further comprises a variable region. In some embodiments, the heteromultimer described in any of the preceding items further comprises a VH and a VL. In some embodiments, the heteromultimer described in any of the preceding items further comprises an amino acid mutation selected from Q105E, Q105R, and Q105K, wherein the mutation site is represented by KABAT numbering. In some embodiments, the heteromultimer described in any of the preceding items further comprises an amino acid mutation of K42E, wherein the mutation site is represented by KABAT numbering.

[0734] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the heteromultimer is a multispecific antibody or an Fc fusion protein.

[0735] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the heteromultimer is a heterodimer. In some embodiments, the heteromultimer as described in any of the preceding items, wherein the heterodimer comprises a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide and / or the second polypeptide is a polypeptide structure constituting a heterodimer, which comprises at least a CH3 domain. In some embodiments, the first polypeptide is an Fc region (first Fc region), the second polypeptide is an Fc region (second Fc region), and the first Fc region and / or the second Fc region comprise CH2 and CH3 domains. In some embodiments, the first polypeptide and / or the second polypeptide comprise CH1, CH2, and CH3 domains.

[0736] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the heteromultimer is a bispecific antibody. In some embodiments, the heteromultimer as described in any of the preceding items, wherein the bispecific antibody comprises a first half antibody and a second half antibody. In some embodiments, the first half antibody comprises a first polypeptide, and the second half antibody comprises a second polypeptide. The first polypeptide and / or the second polypeptide is a polypeptide structure constituting the bispecific antibody, which comprises at least a CH3 domain. In some embodiments, the first polypeptide is an Fc region (first Fc region) and the second polypeptide is an Fc region (second Fc region). In some embodiments, the first Fc region and / or the second Fc region comprise CH2 and CH3 domains. In some embodiments, the first polypeptide and / or the second polypeptide comprise CH1, CH2, and CH3 domains. In some embodiments, the first half antibody binds to a first antigen and the second half antibody binds to a second antigen. In some embodiments, the first half antibody comprises a first antigen-binding domain and the second half antibody comprises a second antigen-binding domain. In some embodiments, the structures of the first and second antigen binding domains are selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, dAb, and VHH.

[0737] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the heteromultimer is a trispecific antibody. In some embodiments, the heteromultimer as described in any of the preceding items, wherein the trispecific antibody comprises a first half antibody and a second half antibody. In some embodiments, the first half antibody binds to a first antigen, and the second half antibody binds to a second antigen and a third antigen. In some embodiments, the first half antibody comprises a first antigen binding domain, and the second half antibody comprises a second antigen binding domain and a third antigen binding domain. In some embodiments, the structures of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain are selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, dAb, and VHH.

[0738] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the first polypeptide is an antibody heavy chain, and / or the second polypeptide is an antibody heavy chain. In some embodiments, the heteromultimer further comprises one or more antibody light chains.

[0739] In some embodiments, the first polypeptide and the second polypeptide have different sequences. The first polypeptide or the second polypeptide is used only to distinguish the amino acid sequence and does not limit the positional relationship between the polypeptides and the protein. For example, in a heterodimer consisting of a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide are two polypeptides with different amino acid sequences. When either polypeptide is the first polypeptide, the other polypeptide is the second polypeptide.

[0740] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the amino acids in the core hinge region of the heteromultimer form a disulfide bond.

[0741] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domains of the first and second polypeptides are derived from IgG. In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domains of the first and second polypeptides are derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domains are derived from IgG1, IgG2, or IgG3. In some embodiments, the heteromultimer as described in any of the preceding items, wherein the CH3 domains of the first and second polypeptides are derived from IgG1. In some embodiments, wherein the CH3 domains of the first and second polypeptides are derived from human IgG1. In some embodiments, the human IgG1 has the amino acid sequence set forth in SEQ ID NO: 39, 40, or 41.

[0742] In some embodiments, the heteromultimer as described in any of the preceding items, wherein the heteromultimer has at least one replaced Fab comprising a titin chain and an obscurin chain capable of forming a dimer. In some embodiments, the heteromultimer as described in any of the preceding items, wherein the replaced Fab comprises an original CH1 and CL of the Fab replaced by an obscurin chain and a titin chain, respectively, or the replaced Fab comprises an original CH1 and CL of the Fab replaced by a titin chain and an obscurin chain, respectively. In some embodiments, the titin chain has the amino acid sequence shown in SEQ ID NO: 6, and the obscurin chain has the amino acid sequence shown in SEQ ID NO: 5.

[0743] In another aspect, the present disclosure provides a heteromultimer comprising a first polypeptide and a second polypeptide, wherein:

[0744] The first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351I,

[0745] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351I, and the mutation sites are represented by EU numbering.

[0746] In some embodiments, the heteromultimer as described above, wherein the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises amino acid mutations D356K and L351I,

[0747] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L351I, and the mutation sites are represented by EU numbering; or

[0748] wherein the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises amino acid mutations E356K and L351I,

[0749] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L351I, and the mutation sites are represented by EU numbering.

[0750] In another aspect, the present disclosure provides a heteromultimer comprising a first polypeptide and a second polypeptide, wherein:

[0751] The first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C,

[0752] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C and 351I, and the mutation sites are represented by EU numbering.

[0753] In some embodiments, the heteromultimer as described above, wherein the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises amino acid mutations D356K and Y349C,

[0754] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations K439E, S354C and L351I, and the mutation sites are represented by EU numbering; or

[0755] wherein the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises amino acid mutations E356K and Y349C,

[0756] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations of K439E, S354C and L351I, and the mutation sites are represented by EU numbering.

[0757] In another aspect, the present disclosure provides a heteromultimer comprising a first polypeptide and a second polypeptide, wherein:

[0758] The first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S,

[0759] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y, and the mutation site is represented by EU numbering.

[0760] In some embodiments, the heteromultimer as described above, wherein the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of Y349S,

[0761] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises an amino acid mutation of S364Y, and the mutation site is represented by EU numbering.

[0762] In another aspect, the present disclosure provides a heteromultimer prepared by an extracellular Fab-arm exchange reaction (extracellular half-antibody recombination reaction), comprising a first polypeptide and a second polypeptide, wherein:

[0763] The first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S,

[0764] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y, and the mutation site is represented by EU numbering.

[0765] In some embodiments, the heterologous multimer prepared by the extracellular Fab-arm exchange reaction (extracellular half-antibody recombination reaction) as described above, wherein the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of Y349S,

[0766] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises an amino acid mutation of S364Y, and the mutation site is represented by EU numbering.

[0767] In another aspect, the present disclosure provides a heteromultimer comprising a first polypeptide and a second polypeptide, wherein:

[0768] The first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C,

[0769] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations 354C and 364Y, and the mutation sites are represented by EU numbering.

[0770] In some embodiments, the heterologous multimer as described above, wherein the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of Y349C,

[0771] The second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations S354C and S364Y, and the mutation sites are represented by EU numbering.

[0772] In another aspect, the present disclosure provides a pharmaceutical composition comprising the heteromultimer as described in any of the preceding items and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0773] In another aspect, the present disclosure provides an immunoconjugate comprising: a heteromultimer as described in any of the preceding items and an effector molecule, wherein the effector molecule is conjugated to the heteromultimer. In some embodiments, the effector molecule is selected from an anti-tumor agent, an immunomodulator, a biological response modifier, a lectin, a cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof.

[0774] In another aspect, the present disclosure provides a method for preparing an immunoconjugate, comprising the steps of:

[0775] a) providing a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer;

[0776] b) mixing the molecules comprising the first polypeptide homomer and the molecules comprising the second polypeptide homomer to form a mixture; and

[0777] c) adding a reducing agent to the mixture and incubating;

[0778] d) obtaining an immunoconjugate comprising the first polypeptide and the second polypeptide;

[0779] wherein the first polypeptide and the second polypeptide each comprise a CH3 domain, and each of the CH3 domains comprises one or more mutations that promote heterologous formation;

[0780] wherein the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer comprise an Fc region conjugated to a prodrug, peptide, drug or toxin.

[0781] In some embodiments, the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer comprise an Fc region conjugated to a toxin.

[0782] In some embodiments, the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer are antibody drug conjugates.

[0783] In another aspect, the present disclosure provides a method for preparing an immunoconjugate, comprising:

[0784] The heteromultimer obtained using any of the methods for preparing heteromultimers described herein is conjugated to a toxin.

[0785] In another aspect, the present disclosure provides one or more isolated nucleic acids encoding the first polypeptide and / or the second polypeptide of the heteromultimer as described in any of the preceding items.

[0786] In another aspect, the present disclosure provides one or more vectors comprising one or more isolated nucleic acids as described in any of the preceding items.

[0787] In another aspect, the present disclosure provides one or more host cells comprising one or more isolated nucleic acids as described in any of the preceding items.

[0788] In another aspect, the present disclosure provides a method for preparing the heteromultimer as described in any of the preceding items, comprising expressing one or more isolated nucleic acids as described in any of the preceding items, or culturing one or more host cells as described in any of the preceding items, to produce the heteromultimer.

[0789] In another aspect, the present disclosure provides a method for preparing a heteromultimer as described in any of the preceding items, comprising: (a) a step of altering a nucleic acid encoding amino acid residues forming an interface between the polypeptides; (b) a step of culturing a host cell harboring the nucleic acid to express the polypeptide; (c) a step of recovering the polypeptide from the culture of the host cell; and (d) a step of incubating each polypeptide in the presence of a reducing agent to recover the desired heteromultimer.

[0790] In another aspect, the present disclosure provides a method for preparing a multispecific antibody by a recombination reaction, comprising the following steps:

[0791] a) providing a first parent antibody;

[0792] b) providing a second parent antibody;

[0793] c) incubating the first parent antibody and the second parent antibody together under reducing conditions sufficient to allow reduction of interchain disulfide bonds in the hinge region; and

[0794] d) a step of obtaining a multispecific antibody, wherein:

[0795] The CH3 domain of the first parent antibody and / or the second parent antibody comprises at least one amino acid mutation selected from the group consisting of: 347, 349, 351, 354, 356, 357, 364, 366, 368, 394, 397, 399, 405, 407, 409, 411 and 439;

[0796] The first parent antibody and the second parent antibody bind to different antigens or epitopes, and the CH3 domain mutation sites are represented by EU numbering.

[0797] The present disclosure provides a method for preparing a multispecific antibody by a recombination reaction, comprising the following steps:

[0798] a) providing a first parent antibody;

[0799] b) providing a second parent antibody;

[0800] c) incubating the first parent antibody and the second parent antibody together under reducing conditions sufficient to allow reduction of interchain disulfide bonds in the hinge region; and

[0801] d) a step of obtaining a multispecific antibody, wherein:

[0802] The CH3 domain of the first parent antibody and / or the second parent antibody comprises at least one amino acid mutation selected from the group consisting of: 349, 351, 354, 356, 357, 364, 366, 368, 394, 397, 405, 407, 409, 411 and 439;

[0803] The first parent antibody and the second parent antibody bind to different antigens or epitopes, and the CH3 domain mutation sites are represented by EU numbering.

[0804] In another aspect, the present disclosure provides a method for preparing a multispecific antibody by a recombination reaction, comprising the following steps:

[0805] a) providing a first parent antibody;

[0806] b) providing a second parent antibody;

[0807] c) incubating the first parent antibody and the second parent antibody together under reducing conditions sufficient to allow reduction of interchain disulfide bonds in the hinge region; and

[0808] d) a step of obtaining a multispecific antibody, wherein:

[0809] In the CH3 domain of the first parent antibody and / or the second parent antibody, at least one amino acid mutation is selected from the group consisting of: 347, 349, 351, 354, 356, 357, 364, 366, 368, 394, 399, 405, 407, 411 and 439;

[0810] The first parent antibody and the second parent antibody bind to different antigens or epitopes, and the CH3 domain mutation sites are represented by EU numbering;

[0811] The premise is that the following two situations are not included:

[0812] i) the CH3 domain of the first parent antibody contains only one amino acid mutation at position 356, and the CH3 domain of the second parent antibody contains only one amino acid mutation at position 439; and

[0813] ii) the CH3 domain of the first parent antibody contains only one amino acid mutation at position 405, and the CH3 domain of the second parent antibody contains only one amino acid mutation at position 409.

[0814] In another aspect, the present disclosure provides a method for preparing a multispecific antibody by a recombination reaction, comprising the following steps:

[0815] a) providing a first parent antibody;

[0816] b) providing a second parent antibody;

[0817] c) incubating the first parent antibody and the second parent antibody together under reducing conditions sufficient to allow reduction of interchain disulfide bonds in the hinge region; and

[0818] d) a step of obtaining a multispecific antibody, wherein:

[0819] The CH3 domain of the first parent antibody and / or the second parent antibody comprises at least one amino acid mutation selected from the group consisting of: 349, 351, 354, 356, 357, 364, 366, 368, 394, 397, 405, 407, 409, 411 and 439;

[0820] The first parent antibody and the second parent antibody bind to different antigens or epitopes, and the CH3 domain mutation sites are represented by EU numbering;

[0821] The premise is that the following two situations are not included:

[0822] i) the CH3 domain of the first parent antibody contains only one amino acid mutation at position 356, and the CH3 domain of the second parent antibody contains only one amino acid mutation at position 439; and

[0823] ii) the CH3 domain of the first parent antibody contains only one amino acid mutation at position 405, and the CH3 domain of the second parent antibody contains only one amino acid mutation at position 409.

[0824] In some embodiments, the method for preparing a multispecific antibody as described above, wherein the CH3 domain of the first parent antibody and / or the second parent antibody comprises at least one amino acid mutation selected from: 349, 351, 354, 356, 364, 366, 368, 394, 405, 409, 411 and 439.

[0825] In some embodiments, the method for preparing a multispecific antibody as described above, wherein the CH3 domain of the first parent antibody and / or the second parent antibody comprises at least one amino acid mutation selected from the following combinations:

[0826] mutating amino acid position 356 according to EU numbering to Lys (K); or

[0827] Mutating amino acid position 439 according to EU numbering to Glu (E); or

[0828] mutating amino acid position 349 (EU numbering) to Leu (L), Phe (F), Ser (S), Cys (C), Ala (A), Val (V), Thr (T) or Gly (G); or

[0829] mutating amino acid position 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I), Phe (F), or Met (M); or

[0830] mutating amino acid position 364 (EU numbering) to Ala (A), Val (V), Thr (T), Leu (L), Tyr (Y) or Phe (F); or

[0831] mutating amino acid position 366 (EU numbering) to Gly (G), Ser (S), Ala (A), Val (V), Leu (L), His (H), or Ile (I); or

[0832] mutating amino acid position 368 (EU numbering) to Val (V), Ile (I), Met (M), or Ala (A); or

[0833] mutating amino acid position 394 (EU numbering) to Phe (F), Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[0834] mutating the amino acid at position 397 as represented by EU numbering to Thr (T), Ile (I) or Leu (L); or mutating the amino acid at position 405 as represented by EU numbering to Thr (T), Leu (L) or Tyr (Y); or

[0835] mutating amino acid position 407 (EU numbering) to Cys (C), Val (V), Leu (L), His (H) or Phe (F); or

[0836] mutating amino acid position 409 (EU numbering) to Gln (Q), Arg (R) or Asp (D); or

[0837] mutating amino acid position 411 according to EU numbering to Asn (N), Tyr (Y) or Leu (L); or

[0838] mutating amino acid position 354 (EU numbering) to Tyr (Y), Cys (C), Phe (F) or Trp (W); or

[0839] mutating amino acid position 357 (EU numbering) to Cys (C); or

[0840] Mutating amino acid position 347 according to EU numbering to Glu (E); or

[0841] The amino acid at position 399 represented by EU numbering was mutated to Lys (K).

[0842] In some embodiments, the method for preparing a multispecific antibody as described above, wherein the CH3 domain of the first parent antibody and / or the second parent antibody comprises at least one amino acid mutation selected from the following combinations:

[0843] Mutating position 356 (EU numbering) to Lys (K); or

[0844] Mutating position 439 (EU numbering) to Glu (E); or

[0845] mutating position 349 (EU numbering) to Leu (L), Phe (F), Ser (S) or Cys (C); or

[0846] mutating position 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I) or Phe (F); or

[0847] mutating position 364 (EU numbering) to Ala (A), Val (V), Thr (T) or Leu (L); or

[0848] mutating position 366 (EU numbering) to Gly (G), Ser (S), Ala (A), Val (V), Leu (L) or His (H); or

[0849] mutating position 368 (EU numbering) to Val (V), Ile (I), or Met (M); or

[0850] mutating position 394 (EU numbering) to Phe (F), Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[0851] mutating position 397 (EU numbering) to Thr (T), Ile (I) or Leu (L); or

[0852] mutating position 405 (EU numbering) to Thr (T), Leu (L) or Tyr (Y); or

[0853] mutating position 407 (EU numbering) to Cys (C), Val (V), Leu (L) or His (H); or

[0854] mutating position 409 (EU numbering) to Gln (Q) or Arg (R); or

[0855] Mutating position 411 (EU numbering) to Asn (N) or Tyr (Y); or

[0856] Mutating position 354 (EU numbering) to Tyr (Y) or Cys (C); or

[0857] Position 357, as represented by EU numbering, is mutated to Cys (C). In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[0858] 1) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation at position 354 or 357; or

[0859] 2) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 405, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 394; or

[0860] 3) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 356, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439, wherein the CH3 domains of the first and second parent antibodies each further comprise one identical or different amino acid mutation selected from the group consisting of positions 349, 351, 364, 366, 368, 394, 397, 405, 407, 409, and 411; or

[0861] 4) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439 and 354, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation selected from positions 351, 366, and 368; or

[0862] 5) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 405, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439 and 394; or

[0863] 6) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 349, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 357; or

[0864] 7) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 349, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 354, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation selected from positions 351, 364, 366, 368, and 405; or

[0865] 8) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 349, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 364 or 354; or

[0866] 9) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 366, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 405 or 407; or

[0867] 10) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 349, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 347 and 357; or

[0868] 11) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 366, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation at position 405 or 407; or

[0869] 12) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 407, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439 and 366; or

[0870] 13) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 354 and 405, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 349 and 394; or

[0871] 14) The CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 409, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439 and 399.

[0872] In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[0873] i) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation selected from position 354 or 357; or

[0874] ii) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 405, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 394; or

[0875] iii) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 356, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439, wherein the CH3 domains of the first and second parent antibodies each further comprise one identical or different amino acid mutation selected from the group consisting of positions 349, 351, 364, 366, 368, 394, 397, 405, 407, 409 and 411; or

[0876] iv) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439 and 354, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation selected from positions 351, 366 and 368; or

[0877] v) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 405, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439 and 394; or

[0878] vi) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 349, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 357; or

[0879] vii) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 349, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 354, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation selected from positions 366, 368 and 405.

[0880] In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[0881] 1) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439 and 354; or

[0882] 2) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 405, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 394; or

[0883] 3) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 356, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439, wherein the CH3 domains of the first and second parent antibodies each further comprise the same amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409, and 411; or

[0884] 4) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439, 354, and 351; or

[0885] 5) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 349, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 354 and 364; or

[0886] 6) The CH3 domain of the first parent antibody comprises an amino acid mutation at position 349, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 364 or 354.

[0887] In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[0888] i) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439 and 354; or

[0889] ii) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 405, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 394; or

[0890] iii) the CH3 domain of the first parent antibody comprises an amino acid mutation at position 356, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439, wherein the CH3 domains of the first and second parent antibodies each further comprise the same amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409 and 411; or

[0891] iv) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439 and 354, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation at position 351. In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[0892] The CH3 domain of the first parent antibody comprises an amino acid mutation of 356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 439E, wherein the CH3 domains of the first and second parent antibodies each further comprise an identical amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409 and 411, wherein,

[0893] mutating amino acid 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I), Phe (F), or Met (M); or

[0894] mutating amino acid position 364 (EU numbering) to Ala (A), Val (V), Thr (T) or Leu (L); or

[0895] mutating amino acid position 366 (EU numbering) to Ser (S), Ala (A), Val (V), Leu (L), His (H), or Ile (I); or

[0896] mutating amino acid position 368 (EU numbering) to Val (V), Ile (I), Met (M), or Ala (A); or

[0897] mutating amino acid position 394 (EU numbering) to Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[0898] mutating amino acid position 405 (EU numbering) to Leu (L) or Tyr (Y); or

[0899] mutating amino acid position 407 according to EU numbering to Phe(F); or

[0900] mutating amino acid position 409 according to EU numbering to Gln (Q); or

[0901] The amino acid at position 411 represented by EU numbering is mutated to Asn (N), Tyr (Y), or Leu (L).

[0902] In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[0903] The CH3 domain of the first parent antibody comprises an amino acid mutation at 356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation at 439E, wherein the CH3 domains of the first and second parent antibodies each further comprise an identical amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409 and 411, wherein,

[0904] mutating position 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I) or Phe (F); or

[0905] mutating position 364 (EU numbering) to Ala (A), Val (V), Thr (T) or Leu (L); or

[0906] mutating position 366 (EU numbering) to Ser (S), Ala (A), Val (V), Leu (L) or His (H); or

[0907] mutating position 368 (EU numbering) to Val (V), Ile (I), or Met (M); or

[0908] mutating position 394 (EU numbering) to Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[0909] Mutating position 405 (EU numbering) to Leu (L) or Tyr (Y); or

[0910] mutating position 409 (EU numbering) to Gln (Q) or Arg (R); or

[0911] The 411 position represented by EU numbering is mutated to Asn (N) or Tyr (Y). In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[0912] 1) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 349S, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 354Y; or

[0913] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 357C; or

[0914] 2) the CH3 domain of the first parent antibody comprises an amino acid mutation of 405T, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 394F; or

[0915] 3) the CH3 domain of the first parent antibody comprises an amino acid mutation of 356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 439E, wherein the CH3 domains of the first and second parent antibodies each further comprise an identical or different amino acid sequence selected from 349L, 351C, 351V, 351T, 351I, 351F, 351M, 364A, 364T, 364L, 366G, 366A, 366V, 366L, 366H, 366I, 368V, 368I, amino acid mutations of 368A, 394A, 394S, 394C, 394V, 394N, 397I, 397L, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411L, and 411Y; or

[0916] 4) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 354C, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation selected from 351I, 366A, and 368I; or

[0917] 5) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 405T, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 394F; or

[0918] 6) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 357C; or

[0919] 7) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354C, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation selected from 351I, 364Y, 364F, 366A, 368I, and 405Y; or

[0920] 8) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349S, and the CH3 domain of the second parent antibody comprises an amino acid mutation selected from 364Y, 354F, 354W and 354Y; or

[0921] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349G, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354W; or

[0922] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349A, and the CH3 domain of the second parent antibody comprises an amino acid mutation selected from 354F, 354Y and 354W; or

[0923] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349V, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354F; or

[0924] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349T, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354F; or

[0925] 9) the CH3 domain of the first parent antibody comprises an amino acid mutation of 366H, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 405L or 407L; or

[0926] 10) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 347E and 357C; or

[0927] 11) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 366H, and the CH3 domain of the second parent antibody comprises an amino acid mutation 439E, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation selected from 405L, 407L, and 407H; or

[0928] 12) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 407L, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 366H; or

[0929] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 407H, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 366H; or

[0930] 13) the CH3 domain of the first parent antibody comprises amino acid mutations 354C and 405T, and the CH3 domain of the second parent antibody comprises amino acid mutations 349C and 394F; or

[0931] 14) The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 409D, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 399K.

[0932] In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[0933] i) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349S, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 354Y; or

[0934] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 357C; or

[0935] ii) the CH3 domain of the first parent antibody comprises an amino acid mutation of 405T, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 394F; or

[0936] iii) the CH3 domain of the first parent antibody comprises an amino acid mutation of 356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 439E, wherein the CH3 domains of the first and second parent antibodies each further comprise one identical or different amino acid mutation selected from the group consisting of 349L, 351C, 351V, 351T, 351I, 351F, 364A, 364T, 364L, 366G, 366A, 366V, 366L, 366H, 368V, 368I, 394A, 394S, 394C, 394V, 394N, 397I, 397L, 405L, 405Y, 407C, 407V, 407L, 407H, 409Q, 409R, and 411Y; or

[0937] iv) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 354C, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation selected from 351I, 366A and 368I; or

[0938] v) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 405T, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 394F; or

[0939] vi) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 357C; or

[0940] vii) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354C, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation selected from 366A, 368I and 405Y.

[0941] In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[0942] 1) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 349S, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 354Y; or

[0943] 2) the CH3 domain of the first parent antibody comprises an amino acid mutation of 405T, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 394F; or

[0944] 3) the CH3 domain of the first parent antibody comprises an amino acid mutation of 356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 439E, wherein the CH3 domains of the first and second parent antibodies each further comprise an identical amino acid mutation selected from the group consisting of 351T, 351I, 364A, 366A, 368I, 394A, 394S, 405Y, 409Q and 411Y; or

[0945] 4) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E, 354C, and 351I; or

[0946] 5) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354C, wherein the CH3 domain of the second parent antibody further comprises an amino acid mutation of 364Y or 364F; or

[0947] 6) the CH3 domain of the first parent antibody comprises an amino acid mutation at 349S, and the CH3 domain of the second parent antibody comprises an amino acid mutation at 364Y; or

[0948] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349G, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354W.

[0949] In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[0950] i) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349S, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 354Y; or

[0951] ii) the CH3 domain of the first parent antibody comprises an amino acid mutation of 405T, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 394F; or

[0952] iii) the CH3 domain of the first parent antibody comprises an amino acid mutation of 356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 439E, wherein the CH3 domains of the first and second parent antibodies each further comprise the same amino acid mutation selected from the group consisting of 351T, 351I, 364A, 366A, 368I, 394A, 394S, 405Y, 409Q and 411Y; or

[0953] iv) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E, 354C and 351I.

[0954] In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[0955] 1) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 349S, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 354Y; or

[0956] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 357C; or

[0957] 2) the CH3 domain of the first parent antibody comprises an amino acid mutation of 405T, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 394F; or

[0958] 3) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 349L, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 349L; or

[0959] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351C; or

[0960] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351V, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351V; or

[0961] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351T, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351T; or

[0962] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351I; or

[0963] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351F, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351F; or

[0964] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351M, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351M; or

[0965] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 364A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 364A; or

[0966] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 364T, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 364T; or

[0967] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 364L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 364L; or

[0968] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 366G, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 366G; or

[0969] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 366A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 366A; or

[0970] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 366V, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 366V; or

[0971] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 366L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 366L; or

[0972] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 366H, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 366H; or

[0973] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 366I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 366I; or

[0974] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 368V, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 368V; or

[0975] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 368I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 368I; or

[0976] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 368A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 368A; or

[0977] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394A; or

[0978] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394S, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394S; or

[0979] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394C; or

[0980] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394V, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394V; or

[0981] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394N, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394N; or

[0982] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 397I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 397I; or

[0983] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 397L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 397L; or

[0984] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 405L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 405L; or

[0985] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 405Y, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 405Y; or

[0986] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 407C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 407C; or

[0987] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 407V, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 407V; or

[0988] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 407L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 407L; or

[0989] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 407H, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 407H; or

[0990] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 407F, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 407F; or

[0991] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 409Q, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 409Q; or

[0992] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 409R, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 409R; or

[0993] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 411L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 411L; or

[0994] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 411Y, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 411Y; or

[0995] 4) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E, 354C, and 351I; or

[0996] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E, 354C and 366A; or

[0997] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E, 354C and 368I; or

[0998] 5) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 405T, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 394F; or

[0999] 6) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 357C; or

[1000] 7) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 351I; or

[1001] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 364Y; or

[1002] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 364F; or

[1003] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 366A; or

[1004] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 368I; or

[1005] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 405Y; or

[1006] 8) the CH3 domain of the first parent antibody comprises an amino acid mutation at 349S, and the CH3 domain of the second parent antibody comprises an amino acid mutation at 364Y; or

[1007] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349S, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354F; or

[1008] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349S, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354W; or

[1009] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349S, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354Y; or

[1010] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349G, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354W; or

[1011] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349A, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354F; or

[1012] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349A, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354Y; or

[1013] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349A, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354W; or

[1014] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349V, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354F; or

[1015] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349T, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354F; or

[1016] 9) the CH3 domain of the first parent antibody comprises an amino acid mutation of 366H, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 405L; or

[1017] The CH3 domain of the first parent antibody comprises an amino acid mutation of 366H, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 407L; or

[1018] 10) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 347E and 357C; or

[1019] 11) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 366H, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 405L; or

[1020] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 366H, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 407L; or

[1021] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 366H, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 407H; or

[1022] 12) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 407L, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 366H; or

[1023] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 407H, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 366H; or

[1024] 13) the CH3 domain of the first parent antibody comprises amino acid mutations 354C and 405T, and the CH3 domain of the second parent antibody comprises amino acid mutations 349C and 394F; or

[1025] 14) The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 409D, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 399K.

[1026] In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[1027] i) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349S, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 354Y; or

[1028] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 357C; or

[1029] ii) the CH3 domain of the first parent antibody comprises an amino acid mutation of 405T, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 394F; or

[1030] iii) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 349L; or

[1031] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351C; or

[1032] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351V, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351V; or

[1033] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351T, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351T; or

[1034] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351I; or

[1035] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351F, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351F; or

[1036] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 364A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 364A; or

[1037] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 364T, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 364T; or

[1038] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 364L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 364L; or

[1039] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 366G, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 366G; or

[1040] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 366A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 366A; or

[1041] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 366V, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 366V; or

[1042] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 366L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 366L; or

[1043] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 366H, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 366H; or

[1044] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 368V, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 368V; or

[1045] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 368I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 368I; or

[1046] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394A; or

[1047] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394S, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394S; or

[1048] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394C; or

[1049] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394V, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394V; or

[1050] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394N, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394N; or

[1051] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 397I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 397I; or

[1052] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 397L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 397L; or

[1053] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 405L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 405L; or

[1054] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 405Y, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 405Y; or

[1055] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 407C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 407C; or

[1056] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 407V, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 407V; or

[1057] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 407L, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 407L; or

[1058] The CH3 domain of the first parent antibody comprises amino acid mutations 356K and 407H, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 407H; or

[1059] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 409Q, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 409Q; or

[1060] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 409R, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 409R; or

[1061] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 411Y, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 411Y; or

[1062] iv) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E, 354C and 351I; or

[1063] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E, 354C and 366A; or

[1064] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E, 354C and 368I; or

[1065] v) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 405T, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 394F; or

[1066] vi) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 357C; or

[1067] vii) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 366A; or

[1068] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 368I; or

[1069] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 405Y.

[1070] In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[1071] 1) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 349S, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 354Y; or

[1072] 2) the CH3 domain of the first parent antibody comprises an amino acid mutation of 405T, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 394F; or

[1073] 3) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 351T, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E and 351T; or

[1074] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351I; or

[1075] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 364A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 364A; or

[1076] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 366A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 366A; or

[1077] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 368I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 368I; or

[1078] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394A; or

[1079] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394S, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394S; or

[1080] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 405Y, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 405Y; or

[1081] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 409Q, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 409Q; or

[1082] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 411Y, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 411Y; or

[1083] 4) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E, 354C, and 351I; or

[1084] 5) the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 364Y; or

[1085] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 364F; or

[1086] 6) the CH3 domain of the first parent antibody comprises an amino acid mutation at 349S, and the CH3 domain of the second parent antibody comprises an amino acid mutation at 364Y; or

[1087] The CH3 domain of the first parent antibody comprises an amino acid mutation of 349G, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 354W.

[1088] In some embodiments, the method for preparing a multispecific antibody as described above, wherein:

[1089] i) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349S, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 354Y; or

[1090] ii) the CH3 domain of the first parent antibody comprises an amino acid mutation of 405T, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 394F; or

[1091] iii) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351T, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351T; or

[1092] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351I; or

[1093] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 364A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 364A; or

[1094] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 366A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 366A; or

[1095] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 368I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 368I; or

[1096] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394A, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394A; or

[1097] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394S, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394S; or

[1098] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 405Y, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 405Y; or

[1099] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 409Q, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 409Q; or

[1100] The CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 411Y, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 411Y; or

[1101] iv) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E, 354C and 351I.

[1102] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349S, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 354Y.

[1103] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody comprises an amino acid mutation of 405T, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 394F.

[1104] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 351I, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 351I.

[1105] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 394S, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 394S.

[1106] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 411Y, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 411Y.

[1107] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody comprises an amino acid mutation of 349S, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 364Y.

[1108] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E, 354C, and 351I.

[1109] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 364Y.

[1110] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody comprises an amino acid mutation of 349C, and the CH3 domain of the second parent antibody comprises amino acid mutations of 354C and 364F.

[1111] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody only comprises amino acid mutations of 356K and 349S, and the CH3 domain of the second parent antibody only comprises amino acid mutations of 439E and 354Y.

[1112] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody only comprises the amino acid mutation 405T, and the CH3 domain of the second parent antibody only comprises the amino acid mutation 394F.

[1113] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody only comprises amino acid mutations of 356K and 351I, and the CH3 domain of the second parent antibody only comprises amino acid mutations of 439E and 351I.

[1114] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody only comprises amino acid mutations of 356K and 394S, and the CH3 domain of the second parent antibody only comprises amino acid mutations of 439E and 394S.

[1115] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody only comprises amino acid mutations of 356K and 411Y, and the CH3 domain of the second parent antibody only comprises amino acid mutations of 439E and 411Y.

[1116] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody only comprises the amino acid mutation 349S, and the CH3 domain of the second parent antibody only comprises the amino acid mutation 364Y.

[1117] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody only comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second parent antibody only comprises amino acid mutations of 439E, 354C, and 351I.

[1118] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody only comprises the amino acid mutation 349C, and the CH3 domain of the second parent antibody only comprises the amino acid mutations 354C and 364Y.

[1119] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the CH3 domain of the first parent antibody only comprises the amino acid mutation 349C, and the CH3 domain of the second parent antibody only comprises the amino acid mutations 354C and 364F.

[1120] In some embodiments, the method for preparing a multispecific antibody is as described above, wherein the first maternal antibody is a first maternal monoclonal antibody, and the second maternal antibody is a second maternal monoclonal antibody.

[1121] In some embodiments, the method for preparing a multispecific antibody as described above, wherein the CH3 domain is derived from IgG1. In some embodiments, the CH3 domain is derived from human IgG1.

[1122] In some embodiments, the method for preparing a multispecific antibody as described above, wherein the multispecific antibody comprises at least one replaced Fab comprising a titin chain and an obscurin chain capable of forming a dimer. In some embodiments, the method for preparing a multispecific antibody as described above, wherein the replaced Fab comprises an original CH1 and CL of the Fab replaced by an obscurin chain and a titin chain, respectively, or wherein the replaced Fab comprises an original CH1 and CL of the Fab replaced by a titin chain and an obscurin chain, respectively. In some embodiments, the titin chain has the amino acid sequence set forth in SEQ ID NO: 6, and the obscurin chain has the amino acid sequence set forth in SEQ ID NO: 5.

[1123] In some embodiments, the method for preparing a multispecific antibody as described above, wherein the reducing conditions include but are not limited to adding a reducing agent selected from 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, D-cysteine ​​and β-mercapto-ethanol and chemical derivatives thereof. In some embodiments, the method for preparing a multispecific antibody as described above, wherein the reducing agent is selected from 2-MEA, glutathione, L-cysteine, dithiothreitol, β-mercaptoethanol and TCEP. In some embodiments, the method for preparing a multispecific antibody as described above, wherein the reducing agent is 2-MEA.

[1124] In some embodiments, the method for preparing a multispecific antibody as described above, wherein the multispecific antibody is a bispecific antibody. In some embodiments, the method for preparing a multispecific antibody as described above, wherein more than 80% (e.g., more than 85%, more than 88%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99%) of the total product is the desired multispecific antibody. In some embodiments, the method for preparing a multispecific antibody as described above, wherein more than 92% (e.g., more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99%) of the total product is the desired multispecific antibody. In some embodiments, the method for preparing a multispecific antibody as described above, wherein more than 94% (e.g., more than 95%, more than 96%, more than 97%, more than 98% or more than 99%) of the total product is the desired multispecific antibody. In some embodiments, the method of making a multispecific antibody is as described above, wherein greater than 95% (eg, greater than 96%, greater than 97%, greater than 98%, or greater than 99%) of the total product is the desired multispecific antibody.

[1125] In some embodiments, the method for preparing a multispecific antibody as described above, wherein: step a) and step b) further comprise the step of purifying the first parent antibody and the second parent antibody. In some embodiments, the purification method includes but is not limited to protein A or protein G chromatography, affinity chromatography based on antigen binding, affinity chromatography based on anti-idiotypic antibodies, ion exchange, hydrophobic interaction chromatography, mixed chromatography (such as hydroxyapatite), immobilized metal affinity chromatography, thiophilic adsorption chromatography and size exclusion chromatography (SEC), etc.

[1126] In some embodiments, the method for preparing a multispecific antibody as described above, wherein: step c) further comprises a step of separating the multispecific antibody and the reducing agent. In some embodiments, the method for separating the multispecific antibody and the reducing agent includes, but is not limited to, any of the methods described below, such as dialysis, precipitation, chromatography, or filtration.

[1127] The method for separating the multispecific antibody from the reducing agent can, in principle, be any method that results in or is capable of separating the two without damaging the multispecific antibody. Such methods include, but are not limited to, dialysis, precipitation, chromatography, or filtration. Separation of the multispecific antibody from the reducing agent can be performed as a continuous process or as a batch process.

[1128] In some embodiments, the method for preparing a multispecific antibody as described above, wherein step d) further comprises a method for purifying the composition obtained from step c). In some embodiments, the purification method includes but is not limited to protein A or protein G chromatography, antigen binding-based affinity chromatography, anti-idiotypic antibody-based affinity chromatography, ion exchange, hydrophobic interaction chromatography, mixed-mode chromatography (such as hydroxyapatite), immobilized metal affinity chromatography, thiophilic adsorption chromatography, and size exclusion chromatography (SEC).

[1129] In some embodiments, the following methods can be cited: a method in which cell lines producing the first and second maternal antibodies are cultured separately, the culture supernatants are purified, and an FAE (Fab arm exchange) reaction is induced using the purified antibodies; a method in which cell lines producing the first and second maternal antibodies are cultured separately, the culture supernatants are mixed without purification, an FAE reaction is induced in the mixed culture supernatant, and then purification is performed; a method in which a cell line producing the first maternal antibody and a cell line producing the second maternal antibody are mixed and cultured, the culture supernatant is purified, and an FAE reaction is induced using the purified antibodies; a method in which a cell line producing the first maternal antibody and a cell line producing the second maternal antibody are mixed and cultured, an FAE reaction is induced in the culture supernatant, and then purification is performed.

[1130] In some embodiments, the present disclosure provides a method for preparing a multispecific antibody, the method comprising the following steps a) to c):

[1131] a) culturing cell lines producing the first and second parent antibodies, respectively;

[1132] b) purifying the culture supernatant of each cell line to obtain the first and second parent antibodies, and incubating the first parent antibody and the second parent antibody together to cause isomerization of the disulfide bond at the cysteine ​​in the hinge region; and

[1133] c) the step of obtaining a multispecific antibody comprising the first and second parent antibodies.

[1134] In some embodiments, the present disclosure provides a method for preparing a multispecific antibody, the method comprising the following steps a) to c):

[1135] a) mixing a cell line producing the first maternal antibody with a cell line producing the second maternal antibody;

[1136] b) incubating the first parent antibody and the second parent antibody together in the culture supernatant to isomerize the disulfide bond at the cysteine ​​in the hinge region; and

[1137] c) the step of obtaining a multispecific antibody comprising the first and second parent antibodies.

[1138] In some embodiments, the present disclosure provides a method for preparing a multispecific antibody, the method comprising the following steps a) to c):

[1139] a) culturing cell lines producing the first and second parent antibodies, respectively;

[1140] b) mixing the culture supernatants of the cell lines, and incubating the first parent antibody and the second parent antibody together to cause isomerization of the disulfide bond at the cysteine ​​in the hinge region; and

[1141] c) the step of obtaining a multispecific antibody comprising the first and second parent antibodies.

[1142] In another aspect, the present disclosure provides a multispecific antibody prepared according to the method described above. In some embodiments, the present disclosure provides a multispecific antibody comprising two polypeptides having CH3 domains that bind to each other, wherein amino acid mutations are introduced into the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide to promote the formation of a heterologous multispecific antibody, wherein the amino acid mutations include at least one selected from: amino acid mutations at positions 347, 349, 351, 354, 356, 357, 364, 366, 368, 394, 397, 399, 405, 407, 409, 411, and 439; wherein the first polypeptide and the second polypeptide bind to different antigens or epitopes, and the CH3 domain mutation sites are represented by EU numbering.

[1143] In some embodiments, the present disclosure provides a multispecific antibody comprising two polypeptides that bind to each other via CH3 domains, wherein amino acid mutations are introduced into the CH3 domains of the first polypeptide and the CH3 domains of the second polypeptide to promote the formation of a heterologous multispecific antibody, wherein the amino acid mutations comprise at least one amino acid mutation selected from: positions 349, 351, 354, 356, 357, 364, 366, 368, 394, 397, 405, 407, 409, 411, and 439; wherein the first polypeptide and the second polypeptide bind to different antigens or epitopes, and the CH3 domain mutation sites are represented by EU numbering.

[1144] In some embodiments, the multispecific antibody as described above, wherein the amino acid mutations in the CH3 domain comprise at least one amino acid mutation selected from: positions 349, 351, 354, 356, 364, 366, 368, 394, 405, 409, 411, and 439; wherein the first polypeptide and the second polypeptide bind to different antigens or epitopes, and the CH3 domain mutation sites are represented by EU numbering.

[1145] In some embodiments, the multispecific antibody as described above, wherein the amino acid mutation of the CH3 domain comprises at least one amino acid mutation selected from the following combinations:

[1146] mutating amino acid position 356 according to EU numbering to Lys (K); or

[1147] Mutating amino acid position 439 according to EU numbering to Glu (E); or

[1148] mutating amino acid position 349 (EU numbering) to Leu (L), Phe (F), Ser (S), Cys (C), Ala (A), Val (V), Thr (T) or Gly (G); or

[1149] mutating amino acid 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I), Phe (F), or Met (M); or

[1150] mutating amino acid position 364 (EU numbering) to Ala (A), Val (V), Thr (T), Leu (L), Tyr (Y) or Phe (F); or

[1151] mutating amino acid position 366 (EU numbering) to Gly (G), Ser (S), Ala (A), Val (V), Leu (L), His (H), or Ile (I); or

[1152] mutating amino acid position 368 (EU numbering) to Val (V), Ile (I), Met (M), or Ala (A); or

[1153] mutating amino acid position 394 (EU numbering) to Phe (F), Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[1154] mutating the amino acid at position 397 as represented by EU numbering to Thr (T), Ile (I) or Leu (L); or mutating the amino acid at position 405 as represented by EU numbering to Thr (T), Leu (L) or Tyr (Y); or

[1155] mutating amino acid position 407 (EU numbering) to Cys (C), Val (V), Leu (L), His (H) or Phe (F); or

[1156] mutating amino acid position 409 represented by EU numbering to Gln (Q) or Asp (D); or

[1157] mutating amino acid position 411 according to EU numbering to Asn (N), Tyr (Y) or Leu (L); or

[1158] mutating amino acid position 354 (EU numbering) to Tyr (Y), Cys (C), Phe (F) or Trp (W); or

[1159] mutating amino acid position 357 (EU numbering) to Cys (C); or

[1160] Mutating amino acid position 347 according to EU numbering to Glu (E); or

[1161] The amino acid at position 399 represented by EU numbering was mutated to Lys (K).

[1162] In some embodiments, the multispecific antibody as described above, wherein the amino acid mutation of the CH3 domain comprises at least one amino acid mutation selected from the following combinations:

[1163] Mutating position 356 (EU numbering) to Lys (K); or

[1164] Mutating position 439 (EU numbering) to Glu (E); or

[1165] mutating position 349 (EU numbering) to Leu (L), Phe (F), Ser (S) or Cys (C); or

[1166] mutating position 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I) or Phe (F); or

[1167] mutating position 364 (EU numbering) to Ala (A), Val (V), Thr (T) or Leu (L); or

[1168] mutating position 366 (EU numbering) to Gly (G), Ser (S), Ala (A), Val (V), Leu (L) or His (H); or

[1169] mutating position 368 (EU numbering) to Val (V), Ile (I), or Met (M); or

[1170] mutating position 394 (EU numbering) to Phe (F), Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[1171] mutating position 397 (EU numbering) to Thr (T), Ile (I) or Leu (L); or

[1172] mutating position 405 (EU numbering) to Thr (T), Leu (L) or Tyr (Y); or

[1173] mutating position 407 (EU numbering) to Cys (C), Val (V), Leu (L) or His (H); or

[1174] mutating position 409 (EU numbering) to Gln (Q) or Arg (R); or

[1175] Mutating position 411 (EU numbering) to Asn (N) or Tyr (Y); or

[1176] Mutating position 354 (EU numbering) to Tyr (Y) or Cys (C); or

[1177] Position 357 (EU numbering) was mutated to Cys (C).

[1178] In some embodiments, the multispecific antibody as described above, wherein:

[1179] 1) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation at position 354 or 357; or

[1180] 2) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394; or

[1181] 3) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid mutation selected from positions 349, 351, 364, 366, 368, 394, 397, 405, 407, 409, and 411; or

[1182] 4) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354, wherein the CH3 domain region of the second polypeptide further comprises an amino acid mutation selected from positions 351, 366, and 368; or

[1183] 5) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 405, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 394; or

[1184] 6) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 357; or

[1185] 7) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 354, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from positions 351, 364, 366, 368, and 405; or

[1186] 8) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364 or 354; or

[1187] 9) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 366, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 405 or 407; or

[1188] 10) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 347 and 357; or

[1189] 11) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 366, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation at position 405 or 407; or

[1190] 12) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 407, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 366; or

[1191] 13) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 354 and 405, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 349 and 394; or

[1192] 14) The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 409, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 399.

[1193] In some embodiments, the multispecific antibody as described above, wherein:

[1194] i) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from positions 354 or 357; or

[1195] ii) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394; or

[1196] iii) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise one identical or different amino acid mutation selected from positions 349, 351, 364, 366, 368, 394, 397, 405, 407, 409, and 411; or

[1197] iv) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354, wherein the CH3 domain region of the second polypeptide further comprises an amino acid mutation selected from positions 351, 366, and 368; or

[1198] v) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 405, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 394; or

[1199] vi) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 357; or

[1200] vii) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 354, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from positions 366, 368 and 405.

[1201] In some embodiments, the multispecific antibody as described above, wherein:

[1202] 1) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354; or

[1203] 2) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394; or

[1204] 3) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409, and 411; or

[1205] 4) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 354, and 351; or

[1206] 5) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 354 and 364; or

[1207] 6) The CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364 or 354.

[1208] In some embodiments, the multispecific antibody as described above, wherein:

[1209] i) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354; or

[1210] ii) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394; or

[1211] iii) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409 and 411; or

[1212] iv) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354, wherein the CH3 domain of the second polypeptide further comprises the same amino acid mutation selected from position 351.

[1213] In some embodiments, the multispecific antibody as described above, wherein:

[1214] The CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409, and 411, wherein,

[1215] mutating amino acid 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I), Phe (F), or Met (M); or

[1216] mutating amino acid position 364 (EU numbering) to Ala (A), Val (V), Thr (T) or Leu (L); or

[1217] mutating amino acid position 366 (EU numbering) to Ser (S), Ala (A), Val (V), Leu (L), His (H), or Ile (I); or

[1218] mutating amino acid position 368 (EU numbering) to Val (V), Ile (I), Met (M), or Ala (A); or

[1219] mutating amino acid position 394 (EU numbering) to Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[1220] mutating amino acid position 405 (EU numbering) to Leu (L) or Tyr (Y); or

[1221] mutating amino acid position 407 according to EU numbering to Phe(F); or

[1222] mutating amino acid position 409 according to EU numbering to Gln (Q); or

[1223] The amino acid at position 411 represented by EU numbering is mutated to Asn (N), Tyr (Y), or Leu (L).

[1224] In some embodiments, the multispecific antibody as described above, wherein:

[1225] The CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409, and 411, wherein,

[1226] mutating position 351 (EU numbering) to Cys (C), Val (V), Thr (T), Ile (I) or Phe (F); or

[1227] Mutating position 364 (EU numbering) to Ala (A), Val (V), Thr (T) or Leu (L); or

[1228] mutating position 366 (EU numbering) to Ser (S), Ala (A), Val (V), Leu (L) or His (H); or

[1229] mutating position 368 (EU numbering) to Val (V), Ile (I), or Met (M); or

[1230] mutating position 394 (EU numbering) to Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); or

[1231] Mutating position 405 (EU numbering) to Leu (L) or Tyr (Y); or

[1232] mutating position 409 (EU numbering) to Gln (Q) or Arg (R); or

[1233] Position 411 (EU numbering) was mutated to Asn (N) or Tyr (Y).

[1234] In some embodiments, the multispecific antibody as described above, wherein:

[1235] 1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or

[1236] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 357C; or

[1237] 2) the CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or

[1238] 3) the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise one identical or different amino acid mutation selected from 349L, 351C, 351V, 351T, 351I, 351F, 351M, 364A, 364T, 364L, 366G, 366A, 366V, 366L, 366H, 366I, 368V, 368I, 368A, 394A, 394S, 394C, 394V, 394N, 397I, 397L, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411L, and 411Y; or

[1239] 4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 351I, 366A, and 368I; or

[1240] 5) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394F; or

[1241] 6) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 357C; or

[1242] 7) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 351I, 364Y, 364F, 366A, 368I, and 405Y; or

[1243] 8) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 364Y, 354F, 354W and 354Y; or

[1244] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W; or

[1245] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349A, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 354F, 354Y and 354W; or

[1246] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349V, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or

[1247] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or

[1248] 9) the CH3 domain of the first polypeptide comprises an amino acid mutation of 366H, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 405L or 407L; or

[1249] 10) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 347E and 357C; or

[1250] 11) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366H, and the CH3 domain of the second polypeptide comprises an amino acid mutation 439E, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 405L, 407L, and 407H; or

[1251] 12) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; or

[1252] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407H, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; or

[1253] 13) the CH3 domain of the first polypeptide comprises amino acid mutations 354C and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations 349C and 394F; or

[1254] 14) The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 409D, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 399K.

[1255] In some embodiments, the multispecific antibody as described above, wherein:

[1256] i) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or

[1257] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 357C; or

[1258] ii) the CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or

[1259] iii) the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise one identical or different amino acid mutation selected from the group consisting of 349L, 351C, 351V, 351T, 351I, 351F, 364A, 364T, 364L, 366G, 366A, 366V, 366L, 366H, 368V, 368I, 394A, 394S, 394C, 394V, 394N, 397I, 397L, 405L, 405Y, 407C, 407V, 407L, 407H, 409Q, 409R, and 411Y; or

[1260] iv) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 351I, 366A and 368I; or

[1261] v) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394F; or

[1262] vi) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 357C; or

[1263] vii) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 366A, 368I and 405Y.

[1264] In some embodiments, the multispecific antibody as described above, wherein:

[1265] 1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or

[1266] 2) the CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or

[1267] 3) the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from 351T, 351I, 364A, 366A, 368I, 394A, 394S, 405Y, 409Q, and 411Y; or

[1268] 4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C, and 351I; or

[1269] 5) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation of 364Y or 364F; or

[1270] 6) the CH3 domain of the first polypeptide comprises an amino acid mutation at 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation at 364Y; or

[1271] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W.

[1272] In some embodiments, the multispecific antibody as described above, wherein:

[1273] i) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or

[1274] ii) the CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or

[1275] iii) the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from 351T, 351I, 364A, 366A, 368I, 394A, 394S, 405Y, 409Q, 411Y; or

[1276] iv) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 354C and 351I.

[1277] In some embodiments, the multispecific antibody as described above, wherein:

[1278] 1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or

[1279] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 357C; or

[1280] 2) the CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or

[1281] 3) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 349L; or

[1282] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351C; or

[1283] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351V, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351V; or

[1284] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351T; or

[1285] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351I; or

[1286] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351F, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351F; or

[1287] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351M, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351M; or

[1288] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 364A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 364A; or

[1289] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 364T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 364T; or

[1290] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 364L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 364L; or

[1291] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366G, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366G; or

[1292] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366A; or

[1293] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366V, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366V; or

[1294] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366L; or

[1295] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366H, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; or

[1296] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366I; or

[1297] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368V, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368V; or

[1298] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368I; or

[1299] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368A; or

[1300] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394A; or

[1301] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394S; or

[1302] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394C; or

[1303] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394V, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394V; or

[1304] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394N, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394N;

[1305] or the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 397I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 397I; or

[1306] The CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 397L, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 397L; or the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 405L, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 405L; or

[1307] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405Y, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 405Y; or

[1308] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 407C; or

[1309] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407V, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 407V; or

[1310] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 407L; or

[1311] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407H, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 407H; or

[1312] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407F, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 407F; or

[1313] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 409Q, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 409Q; or

[1314] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 409R, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 409R; or

[1315] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 411L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 411L; or

[1316] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 411Y, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 411Y; or

[1317] 4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C, and 351I; or

[1318] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C, and 366A; or

[1319] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C, and 368I; or

[1320] 5) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394F; or

[1321] 6) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 357C; or

[1322] 7) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 351I; or

[1323] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 364Y; or

[1324] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 364F; or

[1325] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 366A; or

[1326] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 368I; or

[1327] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 405Y; or

[1328] 8) the CH3 domain of the first polypeptide comprises an amino acid mutation at 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation at 364Y; or

[1329] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or

[1330] The CH3 domain of the first polypeptide comprises an amino acid mutation at 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation at 354W; or

[1331] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354Y; or

[1332] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W; or

[1333] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349A, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or

[1334] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349A, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354Y; or

[1335] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349A, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W; or

[1336] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349V, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or

[1337] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or

[1338] 9) the CH3 domain of the first polypeptide comprises an amino acid mutation of 366H, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 405L; or

[1339] The CH3 domain of the first polypeptide comprises an amino acid mutation of 366H, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 407L; or

[1340] 10) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 347E and 357C; or

[1341] 11) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366H, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 405L; or

[1342] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366H, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 407L; or

[1343] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366H, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 407H; or

[1344] 12) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; or

[1345] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407H, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; or

[1346] 13) the CH3 domain of the first polypeptide comprises amino acid mutations 354C and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations 349C and 394F; or

[1347] 14) The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 409D, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 399K.

[1348] In some embodiments, the multispecific antibody as described above, wherein:

[1349] i) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or

[1350] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 357C; or

[1351] ii) the CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or

[1352] iii) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 349L; or

[1353] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351C; or

[1354] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351V, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351V; or

[1355] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351T; or

[1356] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351I; or

[1357] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351F, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351F; or

[1358] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 364A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 364A; or

[1359] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 364T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 364T; or

[1360] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 364L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 364L; or

[1361] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366G, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366G; or

[1362] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366A; or

[1363] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366V, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366V; or

[1364] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366L; or

[1365] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366H, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; or

[1366] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368V, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368V; or

[1367] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368I; or

[1368] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394A; or

[1369] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394S; or

[1370] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394C; or

[1371] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394V, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394V; or

[1372] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394N, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394N; or

[1373] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 397I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 397I; or

[1374] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 397L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 397L; or

[1375] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 405L; or

[1376] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405Y, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 405Y; or

[1377] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 407C; or

[1378] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407V, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 407V; or

[1379] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 407L; or

[1380] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407H, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 407H; or

[1381] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 409Q, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 409Q; or

[1382] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 409R, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 409R; or

[1383] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 411Y, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 411Y; or

[1384] iv) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C, and 351I; or

[1385] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C, and 366A; or

[1386] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C, and 368I; or

[1387] v) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394F; or

[1388] vi) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 357C; or

[1389] vii) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 366A; or

[1390] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 368I; or

[1391] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 405Y.

[1392] In some embodiments, the multispecific antibody as described above, wherein:

[1393] 1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or

[1394] 2) the CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or

[1395] 3) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351T; or

[1396] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351I; or

[1397] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 364A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 364A; or

[1398] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366A; or

[1399] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368I; or

[1400] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394A; or

[1401] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394S; or

[1402] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405Y, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 405Y; or

[1403] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 409Q, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 409Q; or

[1404] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 411Y, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 411Y; or

[1405] 4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C, and 351I; or

[1406] 5) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 364Y; or

[1407] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 364F; or

[1408] 6) the CH3 domain of the first polypeptide comprises an amino acid mutation at 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation at 364Y; or

[1409] The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W.

[1410] In some embodiments, the multispecific antibody as described above, wherein:

[1411] i) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or

[1412] ii) the CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or

[1413] iii) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351T; or

[1414] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351I; or

[1415] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 364A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 364A; or

[1416] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366A; or

[1417] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368I; or

[1418] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394A, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394A; or

[1419] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 394S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394S; or

[1420] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405Y, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 405Y; or

[1421] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 409Q, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 409Q; or

[1422] The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 411Y, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 411Y; or

[1423] iv) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 354C and 351I.

[1424] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 354Y.

[1425] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F.

[1426] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 351I, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 351I.

[1427] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 394S, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 394S.

[1428] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 411Y, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 411Y.

[1429] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y.

[1430] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 354C, and 351I.

[1431] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 364Y.

[1432] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 364F.

[1433] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide only comprises amino acid mutations of 356K and 349S, and the CH3 domain of the second polypeptide only comprises amino acid mutations of 439E and 354Y.

[1434] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide only comprises the amino acid mutation 405T, and the CH3 domain of the second polypeptide only comprises the amino acid mutation 394F.

[1435] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide only comprises amino acid mutations of 356K and 351I, and the CH3 domain of the second polypeptide only comprises amino acid mutations of 439E and 351I.

[1436] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide only comprises amino acid mutations of 356K and 394S, and the CH3 domain of the second polypeptide only comprises amino acid mutations of 439E and 394S.

[1437] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide only comprises amino acid mutations of 356K and 411Y, and the CH3 domain of the second polypeptide only comprises amino acid mutations of 439E and 411Y.

[1438] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide only comprises the amino acid mutation 349S, and the CH3 domain of the second polypeptide only comprises the amino acid mutation 364Y.

[1439] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide only comprises amino acid mutations of 356K and 349C, and the CH3 domain of the second polypeptide only comprises amino acid mutations of 439E, 354C, and 351I.

[1440] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide only comprises the amino acid mutation 349C, and the CH3 domain of the second polypeptide only comprises the amino acid mutations 354C and 364Y.

[1441] In some embodiments, the multispecific antibody as described above, wherein the CH3 domain of the first polypeptide only comprises the amino acid mutation 349C, and the CH3 domain of the second polypeptide only comprises the amino acid mutations 354C and 364F.

[1442] In some embodiments, the multispecific antibody as described above, wherein the CH3 domains of the first polypeptide and the second polypeptide are derived from IgG1. In some embodiments, wherein the CH3 domains of the first polypeptide and the second polypeptide are derived from human IgG1.

[1443] In some embodiments, the multispecific antibody described above comprises at least one replaced Fab comprising a titin chain and an obscurin chain capable of forming a dimer. In some embodiments, the multispecific antibody described above comprises at least one replaced Fab wherein the CH1 and CL of the original Fab are replaced by an obscurin chain and a titin chain, respectively, or wherein the CH1 and CL of the original Fab are replaced by a titin chain and an obscurin chain, respectively. In some embodiments, the titin chain has the amino acid sequence set forth in SEQ ID NO: 6, and the obscurin chain has the amino acid sequence set forth in SEQ ID NO: 5.

[1444] In another aspect, the present disclosure provides a pharmaceutical composition comprising the multispecific antibody as described above and one or more pharmaceutically acceptable carriers, diluents or excipients.

[1445] In another aspect, the present disclosure provides an immunoconjugate comprising: a multispecific antibody as described above and an effector molecule, wherein the effector molecule is conjugated to the multispecific antibody. In some embodiments, the effector molecule is selected from an anti-tumor agent, an immunomodulator, a biological response modifier, a lectin, a cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof.

[1446] In another aspect, the present disclosure provides an isolated nucleic acid encoding a multispecific antibody as described above.

[1447] In another aspect, the present disclosure provides a vector comprising the isolated nucleic acid as described above.

[1448] In another aspect, the present disclosure provides a host cell comprising the isolated nucleic acid as described above.

[1449] In another aspect, the present disclosure provides a method for preparing the multispecific antibody as described above, comprising culturing the host cell as described above, and recovering the multispecific antibody from the host cell culture.

[1450] In another aspect, the present disclosure provides a method for preparing the multispecific antibody as described above, comprising: (a) a step of altering a nucleic acid encoding amino acid residues forming an interface between the polypeptides, etc.; (b) a step of culturing a host cell harboring the nucleic acid to express the polypeptide; (c) a step of recovering the polypeptide from the culture of the host cell; and (d) a step of incubating each polypeptide under reducing conditions to recover the desired multispecific antibody.

[1451] In some embodiments, the multispecific antibody as described above, wherein the first polypeptide is an antibody heavy chain, and / or the second polypeptide is an antibody heavy chain.

[1452] In some embodiments, the multispecific antibody described above further comprises one or ...

Claims

1. A method for preparing a heteromultimer, comprising the following steps: a) providing a molecule comprising a first polypeptide homomer; b) providing a molecule comprising a second polypeptide homomer; c) a step of incubating the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer under reducing conditions; and d) a step of obtaining a heteromultimer comprising the first polypeptide and the second polypeptide; The first polypeptide and the second polypeptide each comprise a CH3 domain, and the CH3 domains of the first polypeptide and the second polypeptide have at least one different mutation site, and the CH3 domain mutation site is represented by EU numbering; wherein: 1) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid mutation selected from positions 351, 349, 364, 366, 368, 394, 405, 407, 409 and 411; Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409 and 411; More preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 351, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 351; or 2) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 354, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from positions 364, 351, 366, 368 and 405; or The CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 347 and 357; Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 354 and 364; or 3) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to iv) below: ⅰ)349； ii) 354 and 351; ⅲ)354 and 366; and iv) 354 and 368; Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and The CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from positions 351, 366 and 368; More preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 354 and 351; or 4) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364 or 354; or The CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394; or The CH3 domain of the first polypeptide comprises an amino acid mutation at position 366, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 405 or 407; Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364 or 354; or The CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394; More preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364; or 5) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to vi) below: ⅰ)349； ⅱ)354； ⅲ)357； ⅳ)366； ⅴ)394； vi) 405; and ⅶ)407； Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutation at position 439, wherein the CH3 domain of the second polypeptide further comprises amino acid mutation at position 354 or 357; or The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 405, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 394; or The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 366, and the CH3 domain of the second polypeptide comprises amino acid mutation at position 439, wherein the CH3 domain of the second polypeptide further comprises amino acid mutation at position 405 or 407; or The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 407, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 366; Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354; or 6) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 357; or 7) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 354, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 349, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from i) or ii): ⅰ)405； ⅱ)394； Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 354 and 405, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 349 and 394; Optionally, wherein: The amino acid at position 356 is mutated to Lys (K), Arg (R) or His (H); and / or The amino acid at position 439 is mutated to Glu (E) or Asp (D); and / or The amino acid at position 351 is mutated to Ile (I), Cys (C), Val (V), Thr (T), Phe (F), Met (M); and / or The amino acid at position 349 is mutated to Leu (L), Phe (F), Ser (S), Cys (C), Ala (A), Val (V), Thr (T) or Gly (G); and / or The amino acid at position 364 is mutated to Ala (A), Val (V), Thr (T), Leu (L), Tyr (Y) or Phe (F); and / or The amino acid at position 366 is mutated to Gly (G), Ser (S), Ala (A), Val (V), Leu (L), His (H) or Ile (I); and / or The amino acid at position 368 is mutated to Val (V), Ile (I), Met (M) or Ala (A); and / or The amino acid at position 394 is mutated to Phe (F), Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); and / or The amino acid at position 405 is mutated to Thr (T), Leu (L) or Tyr (Y); and / or The amino acid at position 407 is mutated to Cys (C), Val (V), Leu (L), His (H) or Phe (F); and / or The amino acid at position 409 is mutated to Gln (Q), Arg (R) or Asp (D); and / or The amino acid at position 411 is mutated to Asn (N), Tyr (Y) or Leu (L); and / or The amino acid at position 354 is mutated to Tyr (Y), Cys (C), Phe (F) or Trp (W); and / or The amino acid at position 357 is mutated to Cys (C); and / or The amino acid at position 347 was mutated to Glu (E).

2. The method according to claim 1, wherein: 1) the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domains of the first polypeptide and the second polypeptide further each comprise an identical or different amino acid mutation selected from 351I, 351C, 351V, 351T, 351F, 351M, 349L, 364A, 364T, 364L, 366G, 366A, 366V, 366L, 366H, 366I, 368V, 368I, 368A, 368M, 394A, 394S, 394C, 394V, 394N, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411L, and 411Y; Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid mutation selected from 351I, 351C, 351V, 351T, 351F, 351M, 349L, 364A, 364T, 364L, 366G, 366A, 366V, 366L, 366H, 366I, 368V, 368I, 368A, 368M, 394A, 394S, 394C, 394V, 394N, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411L and 411Y; Further preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from 351I, 351T, 364A, 366A, 368I, 394A, 394S, 405Y, 409Q and 411Y; More preferably, the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351I; or 2) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 364Y, 364F, 351I, 366A, 368I and 405Y; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 347E and 357C; Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation of 364Y or 364F; More preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 364Y; or 3) the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to iv) below: ⅰ)349C； ii) 354C and 351I; iii) 354C and 366A; and iv) 354C and 368I; Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 351I, 366A and 368I; More preferably, the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C and 351I; or 4) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 364Y, 354F, 354W and 354Y; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349A, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 354F, 354Y and 354W; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349V, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 366H, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 405L or 407L; or Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; More preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y; or 5) the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to vi) below: i) 349S or 349C; ii) 354Y or 354C; ⅲ)357C； ⅳ)366H； ⅴ)394F； vi) 405T or 405L; and ⅶ) 407L or 407H; Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354C; or The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 357C; or The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394F; or The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366H, and the CH3 domain of the second polypeptide comprises amino acid mutation 439E, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 405L, 407L and 407H; or The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; or The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407H, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; More preferably, the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or 6) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 357C; or 7) the CH3 domain of the first polypeptide comprises an amino acid mutation of 354C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 349C, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from i) or ii): ⅰ)405T； ⅱ)394F； Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations of 354C and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations of 349C and 394F.

3. The method according to claim 1 or 2, wherein the isoelectric points of the first polypeptide and the second polypeptide are different; preferably, the first polypeptide and the second polypeptide further comprise additional amino acid mutations so that the isoelectric points of the first polypeptide and the second polypeptide are different.

4. The method according to any one of claims 1 to 3, wherein the reducing conditions comprise adding one or more reducing agents selected from 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, D-cysteine ​​and β-mercapto-ethanol and chemical derivatives thereof; preferably, the reducing agent is selected from one or more of 2-MEA, glutathione, L-cysteine, dithiothreitol, β-mercaptoethanol and TCEP; more preferably, the reducing agent is 2-MEA. The method according to any one of claims 1 to 4, wherein the heteromultimer is a multispecific antibody or an Fc fusion protein.

6. The method of claim 1, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are selected from an Fc region, an antibody, a fusion protein comprising an Fc region (e.g., an Fc region fused to a receptor, a cytokine, or a hormone), and an Fc region conjugated to a drug (e.g., a peptide or a toxin); Preferably, said molecule comprising a first polypeptide homomer is a first parent antibody, and said molecule comprising a second polypeptide homomer is a second parent antibody.

7. A heteromultimer prepared according to the method of any one of claims 1 to 6.

8. A heteromultimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise a CH3 domain, wherein: 1) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid mutation selected from positions 351, 349, 364, 366, 368, 394, 405, 407, 409 and 411; Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from positions 351, 364, 366, 368, 394, 405, 409 and 411; or More preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 351, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 351; or 2) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 354, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from positions 364, 351, 366, 368 and 405; or The CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 347 and 357; or Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 354 and 364; or 3) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the first polypeptide and / or the second polypeptide The CH3 domain further comprises at least one amino acid mutation selected from the group consisting of the following i) to iv): ⅰ)349； ii) 354 and 351; ⅲ)354 and 366; and iv) 354 and 368; Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354, wherein the CH3 domain region of the second polypeptide further comprises an amino acid mutation selected from positions 351, 366 and 368; More preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 354 and 351; or 4) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364 or 354; or The CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394; or The CH3 domain of the first polypeptide comprises an amino acid mutation at position 366, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 405 or 407; Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364 or 354; or The CH3 domain of the first polypeptide comprises an amino acid mutation at position 405, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 394; More preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 364; or 5) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to vi) below: ⅰ)349； ⅱ)354； ⅲ)357； ⅳ)366； ⅴ)394； vi) 405; and ⅶ)407； Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutation at position 439, wherein the CH3 domain of the second polypeptide further comprises amino acid mutation at position 354 or 357; or A first polypeptide and a second polypeptide wherein the CH3 domain of the first polypeptide comprises amino acids at positions 356 and 405 The CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 394; or The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 366, and the CH3 domain of the second polypeptide comprises amino acid mutation at position 439, wherein the CH3 domain of the second polypeptide further comprises amino acid mutation at position 405 or 407; or The CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 407, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 366; Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 349, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 354; or 6) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 349, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 357; or 7) the CH3 domain of the first polypeptide comprises an amino acid mutation at position 354, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 349, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from i) or ii): ⅰ)405； ⅱ)394； Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations at positions 354 and 405, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 349 and 394; Optionally, wherein: The amino acid at position 356 is mutated to Lys (K), Arg (R) or His (H); and / or The amino acid at position 439 is mutated to Glu (E) or Asp (D); and / or The amino acid at position 351 is mutated to Ile (I), Cys (C), Val (V), Thr (T), Phe (F), Met (M); and / or The amino acid at position 349 is mutated to Leu (L), Phe (F), Ser (S), Cys (C), Ala (A), Val (V), Thr (T) or Gly (G); and / or The amino acid at position 364 is mutated to Ala (A), Val (V), Thr (T), Leu (L), Tyr (Y) or Phe (F); and / or The amino acid at position 366 is mutated to Gly (G), Ser (S), Ala (A), Val (V), Leu (L), His (H) or Ile (I); and / or The amino acid at position 368 is mutated to Val (V), Ile (I), Met (M) or Ala (A); and / or The amino acid at position 394 is mutated to Phe (F), Ala (A), Ser (S), Cys (C), Val (V) or Asn (N); and / or The amino acid at position 405 is mutated to Thr (T), Leu (L) or Tyr (Y); and / or The amino acid at position 407 is mutated to Cys (C), Val (V), Leu (L), His (H) or Phe (F); and / or The amino acid at position 409 is mutated to Gln (Q), Arg (R) or Asp (D); and / or The amino acid at position 411 is mutated to Asn (N), Tyr (Y) or Leu (L); and / or The amino acid at position 354 is mutated to Tyr (Y), Cys (C), Phe (F) or Trp (W); and / or The amino acid at position 357 is mutated to Cys (C); and / or The amino acid at position 347 mutated to Glu(E) first polypeptide and second polypeptide; The CH3 domains of the first polypeptide and the second polypeptide have at least one different mutation site, and the CH3 domain mutation site is represented by EU numbering.

9. The heteromultimer according to claim 8, wherein: 1) the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical or different amino acid selected from 351I, 351C, 351V, 351T, 351F, 351M, 349L, 364A , 364T, 364L, 366G, 366A, 366V, 366L, 366H, 366I, 368V, 368I, 368A, 368M, 394A, 394S, 394C, 394V, 394N, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411L, and 411Y amino acid mutations; Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide further each comprise an identical amino acid mutation selected from 351I, 351C, 351V, 351T, 351F, 351M, 349L, 364A, 364T, 364L, 366G, 366A, 366V, 366L, 366H, 366I, 368V, 368I, 368A, 368M, 394A, 394S, 394C, 394V, 394N, 405L, 405Y, 407C, 407V, 407L, 407H, 407F, 409Q, 409R, 411L and 411Y; Further preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domains of the first polypeptide and the second polypeptide each further comprise an identical amino acid mutation selected from 351I, 351T, 364A, 366A, 368I, 394A, 394S, 405Y, 409Q and 411Y; More preferably, the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351I, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351I; or 2) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 364Y, 364F, 351I, 366A, 368I and 405Y; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 347E and 357C; Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation of 364Y or 364F; More preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 364Y; or 3) the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to iv) below: ⅰ)349C； ii) 354C and 351I; iii) 354C and 366A; and iv) 354C and 368I; Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354C, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 351I, 366A and 368I; More preferably, the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C and 351I; or 4) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 364Y, 354F, 354W and 354Y; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349A, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 354F, 354Y and 354W; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349V, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354F; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 366H, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 405L or 407L; Preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 349G, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 354W; or The CH3 domain of the first polypeptide comprises an amino acid mutation of 405T, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 394F; More preferably, the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y; or 5) the CH3 domain of the first polypeptide comprises an amino acid mutation selected from 356K, 356R and 356H, and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from 439E and 439D, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from the group consisting of i) to vi) below: i) 349S or 349C; ii) 354Y or 354C; ⅲ)357C； ⅳ)366H； ⅴ)394F； vi) 405T or 405L; and ⅶ) 407L or 407H; Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354C; or The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 357C; or a first polypeptide and a second polypeptide, wherein the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 394F; or The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 366H, and the CH3 domain of the second polypeptide comprises amino acid mutation 439E, wherein the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from 405L, 407L and 407H; or The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407L, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; or The CH3 domain of the first polypeptide comprises amino acid mutations 356K and 407H, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 366H; More preferably, the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349S, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 354Y; or 6) the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 357C; or 7) the CH3 domain of the first polypeptide comprises an amino acid mutation of 354C, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 349C, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises at least one amino acid mutation selected from i) or ii): ⅰ)405T； ⅱ)394F； Preferably, the CH3 domain of the first polypeptide comprises amino acid mutations 354C and 405T, and the CH3 domain of the second polypeptide comprises amino acid mutations 349C and 394F.

10. The heterologous multimer according to any one of claims 8 or 9, wherein the isoelectric points of the first polypeptide and the second polypeptide are different; preferably, the first polypeptide and the second polypeptide further comprise additional amino acid mutations so that the isoelectric points of the first polypeptide and the second polypeptide are different. The heteromultimer according to any one of claims 8 to 10, wherein the heteromultimer is a multispecific antibody or an Fc fusion protein.

12. A heteromultimer prepared by an extracellular Fab-arm exchange reaction, comprising a first polypeptide and a second polypeptide; wherein: (1) the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 351I, and the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 351I, and the mutation sites are represented by EU numbering; Preferably, the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises amino acid mutations D356K and L351I, and the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L351I, and the mutation sites are represented by EU numbering; or The first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises amino acid mutations E356K and L351I, and the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L351I, and the mutation sites are represented by EU numbering; or (2) the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349C, and the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations of 354C and 364Y, and the mutation sites are represented by EU numbering; Preferably, the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of Y349C, and the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations of S354C and S364Y, and the mutation sites are represented by EU numbering; or (3) the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 349C, and the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 354C and 351I, and the mutation sites are represented by EU numbering; Preferably, the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises amino acid mutations D356K and Y349C, and the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises amino acid mutations K439E, S354C and L351I, and the mutation sites are represented by EU numbering; or The first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises E356K and Y349C amino acid mutations, the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises K439E, S354C and L351I amino acid mutations, and the mutation sites are represented by EU numbering; or (4) the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 349S, and the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises an amino acid mutation of 364Y, and the mutation sites are represented by EU numbering; Preferably, the first polypeptide comprises a CH3 domain, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of Y349S, and the second polypeptide comprises a CH3 domain, wherein the CH3 domain of the second polypeptide comprises an amino acid mutation of S364Y, and the mutation sites are represented by EU numbering.

13. A pharmaceutical composition comprising the heteromultimer according to any one of claims 8 to 12 and one or more pharmaceutically acceptable carriers, diluents or excipients.

14. An immunoconjugate comprising: a heteromultimer according to any one of claims 8 to 12 and an effector molecule, wherein the effector molecule is conjugated to the heteromultimer; preferably, the effector molecule is selected from antitumor agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

15. One or more isolated nucleic acids encoding the first polypeptide and / or the second polypeptide of the heteromultimer of any one of claims 8 to 12.

16. One or more host cells comprising one or more isolated nucleic acids of claim 15.

17. A method for preparing a heteromultimer as claimed in any one of claims 8 to 12, comprising expressing one or more isolated nucleic acids as claimed in claim 15, or culturing one or more host cells as claimed in claim 16, to produce the heteromultimer.

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