Method for preparing stable hetero-oligomer and use thereof

Abstract

The present invention relates to a method for preparing a stable hetero-oligomer and a use thereof, and specifically relates to a method for preparing a hetero-oligomer and a use thereof, and a hetero-oligomer, the formation of which is promoted by modifying amino acids in a CH3 domain.

Description

A method for preparing stable heteropolymers and its application

[0001] This application claims priority to Chinese Patent Application No. 202510381528.6, filed on March 28, 2025; Chinese Patent Application No. 202510713026.9, filed on May 30, 2025; and Chinese Patent Application No. 202610274847.1, filed on March 6, 2026. Technical Field

[0002] This disclosure pertains to the field of biotechnology, specifically relating to a method for preparing heteropolymers (e.g., multispecific antibodies) and their applications, and to altering the amino acids in the CH3 domain to promote the formation of heteropolymers (e.g., multispecific antibodies). Background Technology

[0003] The statements herein are provided only as background information in connection with this 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 can exert a unique mechanism of action, giving them a significant advantage.

[0005] Among the numerous bispecific antibody platforms developed by pharmaceutical companies, the DuoBody platform possesses a unique antibody engineering principle. Genmab developed the DuoBody bispecific antibody preparation platform (WO2011131746A2 and WO2013060867A2) based on the Fab-arm Exchange (FAE) capability of IgG4. Genmab's research shows that mutating lysine at position 409 of IgG1 to arginine (IgG1-K409R) endows IgG1 with FAE capability, and mutating phenylalanine at position 405 of IgG1 to leucine (IgG1-F405L) also endows IgG1 with FAE capability. IgG1-K409R and IgG1-F405L can undergo FAE under specific reducing conditions to form bispecific antibodies. Nevertheless, the K409R mutation used in DuoBody originates from human IgG4, and the F405L mutation originates from rhesus monkey IgG4; therefore, the stability of the bispecific antibody molecules produced by this platform is inferior to that of wild-type human IgG1.

[0006] There is still a need for multispecific antibody molecules with higher recombination efficiency and more stable physicochemical properties to meet the needs of antibody engineering development and clinical applications. Summary of the Invention

[0007] This disclosure provides a method for preparing heteropolymers (e.g., heterodimers), comprising the following steps:

[0008] a) The step of providing a dimer comprising two first polypeptides;

[0009] b) Providing a dimer comprising two second polypeptides; and

[0010] c) The step of incubating the dimer containing two first polypeptides and the dimer containing two second polypeptides together under reducing conditions;

[0011] d) Obtain a heteropolymer containing a first polypeptide and a second polypeptide (e.g., a heterodimer containing one first polypeptide and one second polypeptide);

[0012] The first polypeptide and the second polypeptide both contain (for example, one each) a CH3 domain, and the first polypeptide and the second polypeptide have at least one different mutation site in their CH3 domains.

[0013] This disclosure provides a method for preparing heteropolymers, comprising the following steps:

[0014] a) The step of providing a molecule comprising a first polypeptide homopolymer;

[0015] b) the step of providing a molecule comprising a second polypeptide homopolymer; and

[0016] c) The step of incubating the molecule containing the first polypeptide homopolymer and the molecule containing the second polypeptide homopolymer together under reducing conditions;

[0017] The first polypeptide and the second polypeptide both contain (for example, one each) a CH3 domain, and the first polypeptide and the second polypeptide have at least one different mutation site in their CH3 domains.

[0018] In some embodiments, the method for preparing heteropolymers as described in any of the preceding embodiments further includes the step of d) obtaining a heteropolymer 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 it can lead to dissociation between homopolymer monomers (e.g., a first polypeptide homopolymer and a second polypeptide homopolymer) and recombination of heteromonomers (e.g., one first polypeptide and one second polypeptide), thereby forming a heterodimer comprising one dissociated first polypeptide monomer and one dissociated second polypeptide monomer.

[0019] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molecule comprising a first polypeptide homopolymer and the molecule comprising a second polypeptide homopolymer bind the same or different antigens or epitopes. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molecule comprising a first polypeptide homopolymer and the molecule comprising a second polypeptide homopolymer bind different antigens or epitopes.

[0020] In some embodiments, the method for preparing heteropolymers as described in any of the preceding embodiments, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain one or more mutations that promote heterologation. In some embodiments, the mutation promoting heterologation refers to an amino acid mutation capable of promoting the dissociation of a homopolymer (e.g., a homodimer) into monomers, and / or promoting the formation of a heteropolymer (e.g., a heterodimer) from two heteromonomers. In some embodiments, the mutation promoting heterologation is an amino acid mutation at the CH3 interaction interface. In some embodiments, the mutation promoting heterologation is a charged amino acid mutation at the CH3 interaction interface. In some embodiments, the mutation promoting heterologation is an amino acid mutation at the CH3 interaction interface that is mutated to a charged amino acid. In some embodiments, the mutation promoting heterologation is an amino acid mutation at the CH3 interaction interface that is mutated to an amino acid with the opposite charge. In some embodiments, the mutation promoting heterologation is an amino acid mutation at the CH3 interaction interface that is mutated to an amino acid with the opposite charge. In some embodiments, hydrogen bonds, electrostatic interactions, or salt bridges are formed between the CH3 domains of the first polypeptide and the second polypeptide.

[0021] In some embodiments, the mutation that promotes heterologization is any of the mutations described in this disclosure. In some embodiments, the mutation that promotes heterologization is one or more amino acid mutations selected from positions 347, 349, 351, 354, 356, 360, 364, 366, 394, 405, 409, 411, and 439.

[0022] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domains of the first polypeptide and / or the second polypeptide each contain one or more charged amino acid mutations. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein one or more amino acids in the CH3 domains of the first polypeptide and / or the second polypeptide are mutated to charged amino acids.

[0023] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domains of the first polypeptide and / or the second polypeptide each contain one or more amino acid mutations with opposite charges (e.g., positively charged amino acids, negatively charged amino acids). In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein one or more amino acids in the CH3 domains of the first polypeptide and / or the second polypeptide are mutated to amino acids with opposite charges (e.g., positively charged amino acids, negatively charged amino acids).

[0024] In some embodiments, in the method for preparing heteropolymers as described in any of the preceding claims, the CH3 domains of the first polypeptide and the second polypeptide each contain one or more charged amino acid mutations (e.g., positively charged amino acids, negatively charged amino acids), and the amino acid mutations in the CH3 domains of the first polypeptide and the second polypeptide carry opposite charges. In some embodiments, in the method for preparing heteropolymers as described in any of the preceding claims, one or more amino acids in the CH3 domains of the first polypeptide and the second polypeptide are mutated to charged amino acids (e.g., positively charged amino acids, negatively charged amino acids), and the amino acids in the CH3 domains of the first polypeptide and the second polypeptide are mutated to amino acids with opposite charges.

[0025] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, one or more amino acids in the CH3 domains of the first and second polypeptides are mutated to charged amino acids (e.g., positively charged amino acids, negatively charged amino acids), and the amino acids in the CH3 domains of the first and second polypeptides at the same position are mutated to carry opposite charges.

[0026] In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the negatively charged amino acid is selected from glutamic acid (E) and aspartic acid (D), and the positively charged amino acid is selected from lysine (K), arginine (R), and histidine (H). In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the negatively charged amino acid is selected from glutamic acid (E) and aspartic acid (D), and the positively charged amino acid is selected from lysine (K) and arginine (R). In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains one or more amino acid mutations selected from lysine (K), arginine (R), and histidine (H), and the second polypeptide contains one or more amino acid mutations selected from glutamic acid (E) and aspartic acid (D).

[0027] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide and / or the second polypeptide comprises one or more amino acid mutations selected from positions 347, 360, 351, 364, 366, 394, 405, 411, 349, 354, 409, 356, and 439. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide and / or the second polypeptide comprises one or more amino acid mutations selected from positions 347, 360, 351, 356, and 439.

[0028] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains an amino acid mutation at position 347, and / or the CH3 domain of the second polypeptide contains amino acid mutations at positions 347 and 360.

[0029] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0030] The first polypeptide has a mutated amino acid at position 347 of its CH3 domain to Lys(K), Arg(R), or His(H); and / or

[0031] The CH3 domain of the second polypeptide is mutated to Glu(E) or Asp(D) at positions 347 and 360.

[0032] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains an amino acid mutation of 347K, and the CH3 domain of the second polypeptide contains amino acid mutations of 347E and 360E. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains an amino acid mutation of 347R, and the CH3 domain of the second polypeptide contains amino acid mutations of 347E and 360E. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains an amino acid mutation of 347R, and the CH3 domain of the second polypeptide contains amino acid mutations of 347D and 360D.

[0033] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises one or more amino acid mutations selected from the group consisting of i) to vi):

[0034] i) 351, 364, 366, 394, 405 or 411;

[0035] ii) 356 and 351;

[0036] iii) 439 and 351;

[0037] ⅳ)349;

[0038] (v)354;

[0039] ⅵ)409;

[0040] (vii)356;

[0041] ⅷ)439.

[0042] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises one or more amino acid mutations selected from the group consisting of i) to vi):

[0043] i) 351, 364, 366, 394, 405 or 411;

[0044] ii) 356 and 351;

[0045] iii) 439 and 351;

[0046] ⅳ)349;

[0047] v)354; and

[0048] ⅵ)409.

[0049] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises one or more amino acid mutations selected from the group consisting of i-1), ii), and iii):

[0050] i-1)351;

[0051] ⅱ)356 and 351; and

[0052] ⅲ)439 and 351.

[0053] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains an amino acid mutation at position 347, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347 and 360, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further contains at least one identical or different amino acid mutation selected from positions 351, 364, 366, 394, 405, 411, 349, 354, 409, 356, and 439.

[0054] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0055] 1) The CH3 domain of the first polypeptide contains an amino acid mutation at position 347, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347 and 360, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain an identical amino acid mutation selected from positions 351, 364, 366, 394, 405, and 411; or

[0056] 2) The CH3 domain of the first polypeptide contains amino acid mutations at positions 439, 347, and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 356, 347, 360, and 351; or

[0057] 3) The CH3 domain of the first polypeptide contains an amino acid mutation at position 347, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347 and 360, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation selected from positions 349, 354, 364, 405 and 409.

[0058] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0059] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 351, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 351; or

[0060] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 364, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 364; or

[0061] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 366, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 366; or

[0062] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 394, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 394; or

[0063] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 405, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 405; or

[0064] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 411, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 411.

[0065] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0066] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 351, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 351.

[0067] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains amino acid mutations at positions 439, 347, and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 356, 347, 360, and 351.

[0068] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0069] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 349, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 354; or

[0070] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 349, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 364; or

[0071] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 405, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 409.

[0072] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide and / or the second polypeptide contains at least one amino acid mutation selected from the following combinations:

[0073] The amino acid at position 347 is mutated to a residue selected from the following: Lys(K), Arg(R), Glu(E), and Asp(D); and / or

[0074] The amino acid at position 360 is mutated to a residue selected from Glu (E) and Asp (D); and / or

[0075] The amino acid mutation at position 351 is selected from the following residues: Met (M), Ile (I), Cys (C), Val (V), Thr (T), and Phe (F); and / or

[0076] The amino acid at position 364 is mutated to a residue selected from the following: Ala (A), Tyr (Y), Val (V), Thr (T), Leu (L), and Phe (F); and / or

[0077] The amino acid at position 366 is mutated to a residue selected from the following: Ala (A), Gly (G), Ser (S), Val (V), Leu (L), His (H), and Ile (I); and / or

[0078] The amino acid mutation at position 394 is selected from the following residues: Ala (A), Ser (S), Phe (F), Cys (C), Val (V), and Asn (N); and / or

[0079] The amino acid at position 405 is mutated to a residue selected from Tyr(Y), Leu(L), and Thr(T); and / or

[0080] The amino acid at position 411 is mutated to a residue selected from the following: Tyr (Y), Asn (N), and Leu (L); and / or

[0081] The amino acid mutation at position 349 is selected from the following residues: Ser(S), Leu(L), Phe(F), Cys(C), Ala(A), Val(V), Thr(T), and Gly(G); and / or

[0082] The amino acid at position 354 is mutated to a residue selected from the following: Tyr (Y), Cys (C), Phe (F), and Trp (W); and / or

[0083] The amino acid at position 409 is mutated to a residue selected from the following: Arg(R), Gln(Q), and Asp(D); and / or

[0084] The amino acid at position 356 is mutated to a residue selected from the following: Lys(K), Arg(R), and His(H); and / or

[0085] The amino acid at position 439 is mutated to residues selected from the following: Glu (E) and Asp (D).

[0086] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0087] 1) The CH3 domain of the first polypeptide contains an amino acid mutation at 347K, and the CH3 domain of the second polypeptide contains amino acid mutations at 347E and 360E, wherein the CH3 domains of the first and second polypeptides each contain an identical amino acid mutation selected from 351M, 351I, 364A, 366A, 394A, 394S, 405Y, and 411Y; or

[0088] The first polypeptide has a CH3 domain containing amino acid mutations of 347R and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations of 347E, 360E, and 351I; or

[0089] The first polypeptide has a CH3 domain containing amino acid mutations of 347R and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations of 347D, 360D, and 351I; or

[0090] 2) The CH3 domain of the first polypeptide contains amino acid mutations at 439E, 347K, and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 356K, 347E, 360E, and 351M; or

[0091] The first polypeptide has a CH3 domain containing amino acid mutations at 439E, 347K, and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 356K, 347E, 360E, and 351I; or

[0092] 3) The CH3 domain of the first polypeptide contains an amino acid mutation of 347K, and the CH3 domain of the second polypeptide contains amino acid mutations of 347E and 360E, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation selected from 349S, 354Y, 364Y, 405L and 409R.

[0093] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0094] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 351M, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 351M; or

[0095] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 351I; or

[0096] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 364A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 364A; or

[0097] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 366A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 366A; or

[0098] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 394A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 394A; or

[0099] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 394S, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 394S; or

[0100] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 405Y, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 405Y; or

[0101] The first polypeptide has a CH3 domain containing amino acid mutations of 347K and 411Y, and the second polypeptide has a CH3 domain containing amino acid mutations of 347E, 360E, and 411Y.

[0102] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains amino acid mutations of 347K and 351I, and the CH3 domain of the second polypeptide contains amino acid mutations of 347E, 360E, and 351I.

[0103] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains amino acid mutations at 347K and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 347E, 360E, and 351M.

[0104] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0105] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 349S, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 354Y; or

[0106] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 349S, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 364Y; or

[0107] The first polypeptide has a CH3 domain containing amino acid mutations of 347K and 405L, and the second polypeptide has a CH3 domain containing amino acid mutations of 347E, 360E, and 409R.

[0108] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0109] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 351I; or

[0110] The first polypeptide has a CH3 domain containing amino acid mutations of 439E, 347K, and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations of 356K, 347E, 360E, and 351I.

[0111] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0112] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 351M, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 351M; or

[0113] The first polypeptide has a CH3 domain containing amino acid mutations at 439E, 347K, and 351M, and the second polypeptide has a CH3 domain containing amino acid mutations at 356K, 347E, 360E, and 351M.

[0114] In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the CH3 domain is derived from the CH3 domain of IgG. In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the CH3 domain is derived from the CH3 domain of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the CH3 domain is derived from the CH3 domain of IgG1. In some embodiments, the CH3 domain is derived from the CH3 domain of human IgG1. In some embodiments, the human IgG1 has the amino acid sequence shown in SEQ ID NO: 11, 12, or 13.

[0115] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the amino acid mutation in the CH3 domain of the first and second polypeptides is a mutation based on the CH3 domain of natural (or wild-type) IgG1, IgG2, IgG3, or IgG4. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the amino acid mutation in the CH3 domain of the first and second polypeptides is a mutation based on the CH3 domain of natural (or wild-type) human IgG1. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the amino acid mutation in the CH3 domain of the first and second polypeptides is a mutation based on SEQ ID NO: 11, 12, or 13. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the amino acid sequence of the natural (or wild-type) human IgG1 is as shown in SEQ ID NO: 11, 12, or 13. In some embodiments, the method for preparing heteropolymers as described above, wherein the sequence of the CH3 domain of the natural (or wild-type) human IgG1 is as shown in SEQ ID NO: 14 or 15.

[0116] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0117] 1) The CH3 domain of the first polypeptide contains an amino acid mutation of Q347K, and the CH3 domain of the second polypeptide contains amino acid mutations of Q347E and K360E, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain an identical amino acid mutation selected from L351M, L351I, S364A, T366A, T394A, T394S, F405Y, and T411Y; or

[0118] The first polypeptide has a CH3 domain containing amino acid mutations of Q347R and L351I, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and L351I; or

[0119] The first polypeptide has a CH3 domain containing amino acid mutations of Q347R and L351I, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347D, K360D, and L351I; or

[0120] 2) The CH3 domain of the first polypeptide contains amino acid mutations of K439E, Q347K, and L351M, and the CH3 domain of the second polypeptide contains amino acid mutations of D / E356K, Q347E, K360E, and L351M; or

[0121] The first polypeptide has a CH3 domain containing amino acid mutations of K439E, Q347K, and L351I, and the second polypeptide has a CH3 domain containing amino acid mutations of D / E356K, Q347E, K360E, and L351I; or

[0122] 3) The CH3 domain of the first polypeptide contains an amino acid mutation of Q347K, and the CH3 domain of the second polypeptide contains amino acid mutations of Q347E and K360E, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation selected from Y349S, S354Y, S364Y, F405L and K409R.

[0123] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0124] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and L351M, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and L351M; or

[0125] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and L351I, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and L351I; or

[0126] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and S364A, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and S364A; or

[0127] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and T366A, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and T366A; or

[0128] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and T394A, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and T394A; or

[0129] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and T394S, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and T394S; or

[0130] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and F405Y, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and F405Y; or

[0131] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and T411Y, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and T411Y.

[0132] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains amino acid mutations of Q347K and L351I, and the CH3 domain of the second polypeptide contains amino acid mutations of Q347E, K360E, and L351I.

[0133] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains amino acid mutations of Q347K and L351M, and the CH3 domain of the second polypeptide contains amino acid mutations of Q347E, K360E, and L351M.

[0134] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains amino acid mutations of K439E, Q347K, and L351I, and the CH3 domain of the second polypeptide contains amino acid mutations of D / E356K, Q347E, K360E, and L351I.

[0135] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, wherein the CH3 domain of the first polypeptide comprises amino acid mutations of K439E, Q347K, and L351M, and the CH3 domain of the second polypeptide comprises amino acid mutations of D / E356K, Q347E, K360E, and L351M.

[0136] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0137] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and Y349S, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and S354Y; or

[0138] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and Y349S, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and S364Y; or

[0139] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and F405L, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and K409R.

[0140] In some embodiments, the CH3 domain mutation sites are designated by EU numbers. In this disclosure, unless otherwise stated, CH3 domain mutation sites are designated by EU numbers.

[0141] In some embodiments, such as the method for preparing heterodimers as described in any of the preceding embodiments, the heterodimer interaction between the CH3 domains of the first polypeptide and the second polypeptide is stronger than the homodimer interaction between the respective CH3 domains of the first polypeptide and the second polypeptide.

[0142] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the first polypeptide and the second polypeptide have different isoelectric points. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the amino acid mutations disclosed herein are used to impart or increase the difference in isoelectric points between the first polypeptide and the second polypeptide. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the first polypeptide and the second polypeptide further comprise additional amino acid mutations to impart or increase the difference in isoelectric points between the first polypeptide and the second polypeptide. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the first polypeptide and the second polypeptide further comprise additional amino acid mutations such that the isoelectric points between the first polypeptide and the second polypeptide differ.

[0143] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the first polypeptide and the second polypeptide further comprise a variable region. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the first polypeptide and the second polypeptide further comprise VH and VL. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the VH of the first polypeptide and / or the second polypeptide further comprises an amino acid mutation selected from Q105E, Q105R, and Q105K, the mutation site being indicated by KABAT numbers. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the VL of the first polypeptide and / or the second polypeptide further comprises an amino acid mutation of K42E, the mutation site being indicated by KABAT numbers.

[0144] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein both the first polypeptide homopolymer and the second polypeptide homopolymer contain a hinge region (e.g., a core hinge region). In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein both the first polypeptide homopolymer and the second polypeptide homopolymer contain a Cys-Pro-Pro-Cys sequence in their hinge regions (e.g., core hinge regions).

[0145] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the reduction conditions allow for disulfide bond isomerization of cysteine ​​within the hinge region. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the reduction conditions are sufficient to allow reduction of interchain disulfide bonds in the hinge region. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the reduction conditions include, but are not limited to, the addition of 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-mercaptoethanol and their chemical derivatives. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the reduction conditions include the addition of one or more reducing agents selected from 2-MEA, glutathione, L-cysteine, dithiothreitol, β-mercaptoethanol, and TCEP. In some embodiments, the method for preparing heteropolymers as described in any of the preceding embodiments, wherein the reduction conditions include the addition of a reducing agent 2-MEA.

[0146] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the final concentration of the reducing agent is 0.1 mM to 1 M. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the final concentration of the reducing agent is 1 mM to 1 M. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the final concentration of the reducing agent is 5 mM to 500 mM. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the final concentration of the reducing agent is 5 mM to 200 mM. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the final concentration of the reducing agent is 25 mM to 100 mM. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the final concentration of the reducing agent is 60 mM to 90 mM. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the final concentration of the reducing agent is 70 mM to 80 mM. In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding embodiments, the final concentration of the reducing agent is 75 mM.

[0147] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, 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. Approximately 75mM, approximately 80mM, approximately 85mM, approximately 90mM, approximately 95mM, approximately 100mM, approximately 110mM, approximately 120mM, approximately 130mM, approximately 140mM, approximately 150mM, approximately 160mM, approximately 170mM, approximately 180mM, approximately 190mM, approximately 200mM, approximately 210mM, approximately 220mM, approximately 230mM, approximately 240m M, approximately 250mM, approximately 260mM, approximately 270mM, approximately 280mM, approximately 290mM, approximately 300mM, approximately 310mM, approximately 320mM, approximately 330mM, approximately 340mM, approximately 350mM, approximately 360mM, approximately 370mM, approximately 380mM, approximately 390mM, approximately 400mM, approximately 410mM, approximately 420mM, approximately 430m M, approximately 440 mM, approximately 450 mM, approximately 460 mM, approximately 470 mM, approximately 480 mM, approximately 490 mM, approximately 500 mM, approximately 550 mM, approximately 600 mM, approximately 650 mM, approximately 700 mM, approximately 750 mM, approximately 800 mM, approximately 850 mM, approximately 900 mM, approximately 950 mM, or approximately 1 M, or any range between these values. In some embodiments, in the method for preparing heteropolymers as described in any of the preceding claims, the final concentration of the reducing agent is approximately 75 mM.

[0148] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the concentration of the molecule comprising the first polypeptide homopolymer or the molecule comprising the second polypeptide homopolymer ranges from 3.45 μM to 690 μM. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the concentration of the molecule comprising the first polypeptide homopolymer or the molecule comprising the second polypeptide homopolymer ranges from 3.45 μM to 345 μM. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the concentration of the molecule comprising the first polypeptide homopolymer or the molecule comprising the second polypeptide homopolymer ranges from 6.9 μM to 69 μM.

[0149] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, the concentration of the mole comprising the first polypeptide homopolymer or the mole comprising the second polypeptide homopolymer is about 3.45 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 6.6 μM, about 6.7 μM, about 6.8 μM, about 6.9 μM, about 7 μM, about 7.1 μM, about 7.2 μM, about 7.3 μM, about 7.4 μM, about 7.5 μM, about 8 μM, about 8.5 μM, or about 9 μM. M, approximately 9.5μM, approximately 10μM, approximately 15μM, approximately 20μM, approximately 25μM, approximately 30μM, approximately 35μM, approximately 40μM, approximately 45μM, approximately 50μM, approximately 55μM, approximately 60μM, approximately 65μM, approximately 66μM, approximately 67μM, approximately 68μM, approximately 69μM, approximately 70μM, approximately 71μM, approximately 72μM, approximately 73μM, approximately 74μM, approximately 75μM, approximately 80μM, approximately 85μM, approximately 90μM, approximately 95μM, approximately 100μM, approximately 110μM, approximately 120μM, approximately 130μM, approximately 140μM, approximately 1 50μM, approximately 160μM, approximately 170μM, approximately 180μM, approximately 190μM, approximately 200μM, approximately 210μM, approximately 220μM, approximately 230μM, approximately 240μM, approximately 250μM, approximately 260μM, approximately 270μM, approximately 280μM, approximately 290μM, approximately 300μM, approximately 310μM, approximately 320μM, approximately 330μM, approximately 340μM, approximately 345μM, approximately 350μM, approximately 360μM, approximately 370μM, approximately 380μM, approximately 390μM, approximately 400μM, approximately 410μM, approximately 420μM, approximately 430 μM, approximately 440 μM, approximately 450 μM, approximately 460 μM, approximately 470 μM, approximately 480 μM, approximately 490 μM, approximately 500 μM, approximately 510 μM, approximately 520 μM, approximately 530 μM, approximately 540 μM, approximately 550 μM, approximately 560 μM, approximately 570 μM, approximately 580 μM, approximately 590 μM, approximately 600 μM, approximately 610 μM, approximately 620 μM, approximately 630 μM, approximately 640 μM, approximately 650 μM, approximately 660 μM, approximately 670 μM, approximately 680 μM, or approximately 690 μM, or any range between these point values.

[0150] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the concentration of the molecule comprising the first polypeptide homopolymer or the molecule comprising the second polypeptide homopolymer is from 0.5 mg / mL to 100 mg / mL. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the concentration of the molecule comprising the first polypeptide homopolymer or the molecule comprising the second polypeptide homopolymer is from 0.5 mg / mL to 50 mg / mL. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the concentration of the molecule comprising the first polypeptide homopolymer or the molecule comprising the second polypeptide homopolymer is from 1 mg / mL to 10 mg / mL.

[0151] In some embodiments, in the method for preparing heteropolymers as described in any of the preceding claims, the concentration of the molecule comprising the first polypeptide homopolymer or the molecule comprising the second polypeptide homopolymer is about 0.5 mg / mL, about 1 mg / mL, about 1.5 mg / mL, about 2 mg / mL, about 2.5 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, about 5 mg / mL, about 5.5 mg / mL, about 6 mg / mL, about 6.5 mg / mL, about 7 mg / mL, about 7.5 mg / mL, about 8 mg / mL, or about 8.5 mg / mL. mg / mL, about 9 mg / mL, about 9.5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, or about 100 mg / mL, or any range between these values.

[0152] In some embodiments, in the method for preparing heteropolymers as described in any of the preceding claims, the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is about 1:2 to about 2:1. In some embodiments, in the method for preparing heteropolymers as described in any of the preceding claims, the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is about 1:2 to about 1:1. In some embodiments, in the method for preparing heteropolymers as described in any of the preceding claims, the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is about 1:1 to about 2:1. In some embodiments, in the method for preparing heteropolymers as described in any of the preceding claims, the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is about 1:1.5 to about 1.5:1. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is about 1:1.4 to about 1.4:1. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is about 1:1.3 to about 1.3:1. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is about 1:1.2 to about 1.2:1. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is about 1:1.1 to about 1.1:1.

[0153] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is approximately 1:1. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is approximately 1.05:1. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is approximately 1:1.03 to approximately 1:2. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is approximately 1:1.1 to approximately 1:1.5. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is from about 1:1.1 to about 1:1.4. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is from about 1:1.5 to about 1:1.35. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molar concentration ratio of the molecules comprising the first polypeptide homopolymer to the molecules comprising the second polypeptide homopolymer is from about 1:1.2 to about 1:1.3.

[0154] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, wherein the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer have the same molar concentration.

[0155] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation temperature for the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer is 15°C to 40°C. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation temperature for the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer is 20°C to 40°C. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation temperature for the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer is 25°C to 40°C. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation temperature for the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer is 30°C to 40°C. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation temperature for the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer is 35°C to 40°C.

[0156] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, the incubation temperature of the molecule comprising the first polypeptide homopolymer and the molecule comprising the second polypeptide homopolymer is about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C, or any range between these values.

[0157] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation temperature of the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer is about 37°C. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation temperature of the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer is 37°C.

[0158] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molecules comprising a first polypeptide homopolymer and the molecules comprising a second polypeptide homopolymer are incubated for at least 10 minutes. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molecules comprising a first polypeptide homopolymer and the molecules comprising a second polypeptide homopolymer are incubated for at least 10 minutes to 30 hours. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the molecules comprising a first polypeptide homopolymer and the molecules comprising a second polypeptide homopolymer are incubated for at least 10 minutes to 24 hours. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation time for the molecules comprising a first polypeptide homopolymer and the molecules comprising a second polypeptide homopolymer is 0.5 hours to 3 hours. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation time for the molecules comprising a first polypeptide homopolymer and the molecules comprising a second polypeptide homopolymer is 1 hour to 3 hours. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation time for the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer is 2 to 3 hours. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation time for the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer is 2.5 hours.

[0159] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, the incubation time for the molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer is about 10 minutes, about 20 minutes, about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, or about 7.5 hours. The incubation time may be approximately 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, or 30 hours, or any range between these values. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims, wherein the incubation time for the molecule comprising the first polypeptide homopolymer and the molecule comprising the second polypeptide homopolymer is approximately 2.5 hours.

[0160] In some embodiments, in the method of preparing heteropolymers as described in any of the preceding claims, the molecule comprising a first polypeptide homopolymer comprises at least two identical first polypeptides. In some embodiments, in the method of preparing heteropolymers as described in any of the preceding claims, the molecule comprising a second polypeptide homopolymer comprises at least two identical second polypeptides. In some embodiments, in the method of preparing heteropolymers as described in any of the preceding claims, wherein the molecule comprising the first polypeptide homopolymer and the molecule comprising the second polypeptide homopolymer comprise 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 (e.g., a peptide or toxin).

[0161] In some embodiments, as described in any of the preceding methods for preparing heteropolymers, the molecule comprising the first polypeptide homopolymer and the molecule comprising the second polypeptide homopolymer, in addition to the Fc region, comprise one or more or all of the other regions of the antibody, namely the CH1 region, VH region, CL region, and / or VL region. In one embodiment, the molecule comprising the first polypeptide homopolymer is a full-length antibody. In another embodiment, the molecule comprising the second polypeptide homopolymer is a full-length antibody.

[0162] In key embodiments, both the molecule containing the first polypeptide homomer and the molecule containing the second polypeptide homomer are antibodies, preferably full-length antibodies, and bind to different epitopes. In such embodiments, the resulting heteropolymer is a bispecific antibody. The epitopes may be located on different antigens or the same antigen.

[0163] However, in other embodiments, only the molecule containing the first polypeptide isomer is a full-length antibody, while the other molecule containing the second polypeptide isomer is not a full-length antibody, such as an Fc region without a variable region, which is expressed together with another protein or peptide sequence, such as a receptor, cytokine, or hormone, or conjugated to a prodrug, peptide, drug, or toxin. In yet another embodiment, neither the molecule containing the first polypeptide isomer nor the molecule containing the second polypeptide isomer is a full-length antibody. For example, both molecules may be Fc regions fused to another protein or peptide sequence (e.g., a receptor, cytokine, or hormone) or conjugated to a prodrug, peptide, drug, or toxin.

[0164] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, the molecule comprising a first polypeptide homopolymer and the molecule comprising a second polypeptide homopolymer are independently selected from monoclonal antibodies, monospecific antibodies, or bispecific antibodies.

[0165] In some embodiments, such as the method for preparing heteropolymers as described in the preceding one, wherein:

[0166] When each of the molecules containing the first polypeptide homomer and the molecules containing the second polypeptide homomer is a monospecific antibody or a monoclonal antibody, the heteropolymer is a bispecific antibody; or

[0167] When the molecule containing the first polypeptide homopolymer and the molecule containing the second polypeptide homopolymer are respectively a monospecific antibody and a bispecific antibody, the heteropolymer is a trispecific antibody; or

[0168] When the molecule containing the first polypeptide homomer and the molecule containing the second polypeptide homomer are respectively a monoclonal antibody and a bispecific antibody, the heteropolymer is a trispecific antibody.

[0169] In some embodiments, the method for preparing a heteropolymer as described in any of the preceding embodiments is used, wherein the heteropolymer is a multispecific antibody or a heteroFc fusion protein.

[0170] In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the heteropolymer is a heterodimer. In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, 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 the heterodimer, which at least contains 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 contains CH2 and CH3 domains. In some embodiments, the first polypeptide and / or the second polypeptide contains CH1, CH2, and CH3 domains.

[0171] In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the heteropolymer is a bispecific antibody. In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the bispecific antibody comprises a first hapten and a second hapten. In some embodiments, the first hapten comprises a first polypeptide, and the second hapten comprises a second polypeptide. The first polypeptide and / or the second polypeptide is a polypeptide structure constituting the bispecific antibody, which at least includes 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 includes CH2 and CH3 domains. In some embodiments, the first polypeptide and / or the second polypeptide includes CH1, CH2, and CH3 domains. In some embodiments, the first hapten binds to a first antigen, and the second hapten binds to a second antigen. In some embodiments, the first hapten includes a first antigen-binding domain, and the second hapten includes a second antigen-binding domain. In some implementations, the structures of the first antigen-binding domain and the second antigen-binding domain are selected from Fab, Fab′, F(ab')2, Fd, Fv, scFv, dsFv, dAb, and VHH.

[0172] In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the heteropolymer is a trispecific antibody. In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the trispecific antibody comprises a first hapten and a second hapten. In some embodiments, the first hapten binds to a first antigen, and the second hapten binds to a second and a third antigen. In some embodiments, the first hapten includes a first antigen-binding domain, and the second hapten includes a second antigen-binding domain and a third antigen-binding domain. In some embodiments, the structures of the first, second, and third antigen-binding domains are selected from Fab, Fab′, F(ab')2, Fd, Fv, scFv, dsFv, dAb, and VHH.

[0173] In some embodiments, in the method for preparing heteropolymers as described in any of the preceding claims, the molecule comprising a first polypeptide homopolymer is a first parent antibody, and the molecule comprising a second polypeptide homopolymer 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. In some embodiments, in the method for preparing heteropolymers as described in any of the preceding claims, the first parent antibody is a first parent monospecific antibody, and the second parent antibody is a second parent monospecific antibody or a bispecific antibody. In some embodiments, in the method for preparing heteropolymers as described above, the first parent antibody is a first parent monoclonal antibody, and the second parent antibody is a second parent monoclonal antibody or a bispecific antibody.

[0174] In some embodiments, such as the method for preparing heteropolymers as described in any of the preceding claims, wherein the first polypeptide homopolymer is a homopolymer composed of two first polypeptides, and the second polypeptide homopolymer is a homopolymer composed of two second polypeptides.

[0175] In this disclosure, the term "first polypeptide" may also refer to each of the first polypeptide homomers. Similarly, the term "second polypeptide" in this disclosure may refer to each of the second polypeptide homomers.

[0176] In some embodiments, the first polypeptide and / or the second polypeptide are polypeptide structures constituting a heterodimer, which contain at least a CH3 domain.

[0177] 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 contains CH2 and CH3 domains. In some embodiments, the first polypeptide and / or the second polypeptide contains CH1, CH2, and CH3 domains. In some embodiments, the first polypeptide and the second polypeptide also contain hinge regions (e.g., core hinge regions).

[0178] In some embodiments, the method for preparing a heteropolymer as described in any of the preceding embodiments, wherein the first polypeptide is an antibody heavy chain, and / or the second polypeptide is an antibody heavy chain. In some embodiments, the heteropolymer further comprises one or more antibody light chains.

[0179] In some embodiments, the sequences of the first polypeptide and the second polypeptide are different. For example, a molecule containing two first polypeptides is a first parent antibody, and a molecule containing two second polypeptides is a second parent antibody. In this disclosure, the first parent antibody also contains one or more polypeptides (e.g., light chains that form haptens with the two first polypeptides, respectively). In this disclosure, the second parent antibody also contains one or more polypeptides (e.g., light chains that form haptens with the two second polypeptides, respectively).

[0180] The terms "first polypeptide," "second polypeptide," "first parent antibody," or "second parent antibody" are used only to distinguish amino acid sequences and do not restrict the positional relationship between the polypeptide and the protein. 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 one is the first polypeptide, the other is the second polypeptide.

[0181] In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the heteropolymer comprises a core hinge region. In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the core hinge region of the heteropolymer comprises a Cys-Pro-Pro-Cys sequence. In some embodiments, the method for preparing a heteropolymer as described in any of the preceding claims, wherein the amino acids in the core hinge region of the heteropolymer form disulfide bonds.

[0182] In some embodiments, the method for preparing heteropolymers as described in any of the preceding embodiments, 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 heteropolymer (compared to other products in the total product, such as haptens or homopolymers).

[0183] In some implementations, the heteropolymer exhibits good thermal stability.

[0184] In some embodiments, the thermal stability includes a thermodynamic property. In some embodiments, the thermodynamic property is a Tm value.

[0185] In some embodiments, the heteropolymer exhibits higher thermal stability than wild-type IgG4. In some embodiments, the heteropolymer has a Tm that is at least 1 °C / K higher than that of the CH3 segment of wild-type IgG4 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 °C / K).

[0186] In some implementations, the thermal stability of the heteropolymer is comparable to that of wild-type IgG1.

[0187] In some implementations, the heteropolymer exhibits superior thermal stability compared to wild-type IgG1 (e.g., Tm2).

[0188] In some implementations, the heteropolymer maternal antibody exhibits good thermal stability.

[0189] In some embodiments, the method for preparing heteropolymers as described in any of the preceding embodiments, wherein steps a) and b) further comprise the step of purifying the molecule containing the first polypeptide homopolymer and the molecule containing the second polypeptide homopolymer. In some embodiments, the purification method includes, but is not limited to, protein A or protein G chromatography, antigen-binding-based affinity chromatography, anti-idiotype 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). In some embodiments, the purification method is affinity chromatography. In some embodiments, the purification method is protein A chromatography.

[0190] In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims further includes a step of removing the reducing agent after step c). In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims removes the reducing agent by, but not limited to, dialysis, precipitation, chromatography, or filtration. In some embodiments, the method for preparing heteropolymers as described in any of the preceding claims removes the reducing agent by dialysis.

[0191] In some embodiments, the step for removing the reducing agent can, in principle, be any method that causes or enables the separation of the two without impairing the heteropolymer. Such methods include, but are not limited to, dialysis, precipitation, chromatography, or filtration. The step for removing the reducing agent can be carried out as a continuous process or it can be carried out as a batch process.

[0192] In some embodiments, the product obtained in step c) contains more than 70% heteropolymer (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%).

[0193] In some embodiments, the method for preparing heteropolymers as described in any of the preceding embodiments further includes step d) 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, antigen-binding-based affinity chromatography, anti-idiotype 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). In some embodiments, the purification method is affinity chromatography. In some embodiments, the purification method is size exclusion chromatography (SEC).

[0194] In some embodiments, the following methods can be cited: culturing cell lines that produce molecules containing a first polypeptide homomer and molecules containing a second polypeptide homomer separately, purifying the culture supernatant, and inducing a FAE (Fab arm exchange) reaction using a purification antibody; culturing cell lines that produce molecules containing a first polypeptide homomer and molecules containing a second polypeptide homomer separately, mixing them without purifying the culture supernatant, inducing a FAE reaction in the mixed culture supernatant, and then purifying; mixing and culturing cell lines that produce molecules containing a first polypeptide homomer with cell lines that produce molecules containing a second polypeptide homomer, purifying the culture supernatant, and inducing a FAE reaction using a purification antibody; mixing and culturing cell lines that produce molecules containing a first polypeptide homomer with cell lines that produce molecules containing a second polypeptide homomer, inducing a FAE reaction in the culture supernatant, and then purifying.

[0195] In some embodiments, this disclosure provides a method for preparing heteropolymers, the method comprising the steps of a) to c) below:

[0196] a) The step of culturing cell lines that produce molecules containing a first polypeptide homomer and molecules containing a second polypeptide homomer, respectively;

[0197] b) The step of purifying the culture supernatant of each cell line to obtain molecules containing a first polypeptide homomer and molecules containing a second polypeptide homomer, and incubating the molecules containing the first polypeptide homomer and the molecules containing the second polypeptide homomer together in the presence of a reducing agent; and

[0198] c) Obtain a heteropolymer containing the first polypeptide and the second polypeptide.

[0199] In some embodiments, this disclosure provides a method for preparing heteropolymers, the method comprising the steps of a) to c) below:

[0200] a) The step of mixing a cell line that produces a molecule containing a first polypeptide homopolymer with a cell line that produces a molecule containing a second polypeptide homopolymer.

[0201] b) The step of incubating the molecules containing the first polypeptide homomer and the molecules containing the second polypeptide homomer together in the culture supernatant in the presence of a reducing agent; and

[0202] c) Obtain a heteropolymer containing the first polypeptide and the second polypeptide.

[0203] In some embodiments, this disclosure provides a method for preparing heteropolymers, the method comprising the steps of a) to c) below:

[0204] a) The step of culturing cell lines that produce molecules containing a first polypeptide homomer and molecules containing a second polypeptide homomer, respectively;

[0205] b) The step of mixing the culture supernatants of each cell line and incubating the molecules containing the first polypeptide homomer and the molecules containing the second polypeptide homomer together in the presence of a reducing agent; and

[0206] c) Obtain a heteropolymer containing the first polypeptide and the second polypeptide.

[0207] In some embodiments, this disclosure provides a method for preparing heteropolymers, the method comprising the following steps:

[0208] a) Provide a first nucleic acid construct encoding a molecule containing a first polypeptide homopolymer.

[0209] b) Provide a second nucleic acid construct encoding a molecule containing a second polypeptide homopolymer.

[0210] The first polypeptide and the second polypeptide have different sequences, and the heterodimer interaction between the first polypeptide and the second polypeptide is stronger than the homodimer interaction between the first polypeptide and the second polypeptide.

[0211] c) Co-express the first and second nucleic acid constructs in host cells, and

[0212] d) Obtain the heteropolymer from cell culture.

[0213] In another aspect, this disclosure provides a heteropolymer prepared according to the method described in any of the preceding claims. In yet another aspect, this disclosure provides a composition comprising a heteropolymer prepared as described in any of the preceding claims, and a reducing agent. In some embodiments, the composition as described in any of the preceding claims contains 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 desired heteropolymer (compared to other products in the total product, such as haptens or homopolymers).

[0214] In another aspect, this disclosure provides a mixture comprising a molecule comprising a first polypeptide homopolymer as described in any of the preceding claims and a molecule comprising a second polypeptide homopolymer.

[0215] In some embodiments, the molar concentration ratio of the mixture containing molecules of the first polypeptide isomer to molecules containing molecules of the second polypeptide isomer is about 1:2 to about 2:1. In some embodiments, the molar concentration ratio of the mixture containing molecules of the first polypeptide isomer to molecules containing molecules of the second polypeptide isomer is about 1:2 to about 1:1. In some embodiments, the molar concentration ratio of the mixture containing molecules of the first polypeptide isomer to molecules containing molecules of the second polypeptide isomer is about 1:1 to about 2:1. In some embodiments, the molar concentration ratio of the mixture containing molecules of the first polypeptide isomer to molecules containing molecules of the second polypeptide isomer is about 1:1.5 to about 1.5:1. In some embodiments, the molar concentration ratio of the mixture containing molecules of the first polypeptide isomer to molecules containing molecules of the second polypeptide isomer is about 1:1.4 to about 1.4:1. In some embodiments, the molar concentration ratio of the mixture containing molecules of the first polypeptide isomer to molecules containing molecules of the second polypeptide isomer is about 1:1.3 to about 1.3:1. In some embodiments, the molar concentration ratio of the molecules containing the first polypeptide isomer to the molecules containing the second polypeptide isomer in the mixture is about 1:1.2 to about 1.2:1. In some embodiments, the molar concentration ratio of the molecules containing the first polypeptide isomer to the molecules containing the second polypeptide isomer in the mixture is about 1:1.1 to about 1.1:1.

[0216] In some embodiments, the molar concentration ratio of the molecules containing the first polypeptide isomer to the molecules containing the second polypeptide isomer in the mixture is about 1:1. In some embodiments, the molar concentration ratio of the molecules containing the first polypeptide isomer to the molecules containing the second polypeptide isomer in the mixture is about 1.05:1. In some embodiments, the molar concentration ratio of the molecules containing the first polypeptide isomer to the molecules containing the second polypeptide isomer in the mixture is about 1:1.03 to about 1:2. In some embodiments, the molar concentration ratio of the molecules containing the first polypeptide isomer to the molecules containing the second polypeptide isomer in the mixture is about 1:1.1 to about 1:1.5. In some embodiments, the molar concentration ratio of the molecules containing the first polypeptide isomer to the molecules containing the second polypeptide isomer in the mixture is about 1:1.1 to about 1:1.4. In some embodiments, the molar concentration ratio of the molecules containing the first polypeptide isomer to the molecules containing the second polypeptide isomer in the mixture is about 1:1.5 to about 1:1.35. In some embodiments, the molar concentration ratio of the molecules containing the first polypeptide isomer to the molecules containing the second polypeptide isomer in the mixture is about 1:1.2 to about 1:1.3.

[0217] In some embodiments, the mixture contains molecules of the first polypeptide isomer and molecules of the second polypeptide isomer at the same molar concentration.

[0218] In some embodiments, the mixture also contains a reducing agent.

[0219] In another aspect, this disclosure provides a heteropolymer comprising a first polypeptide and a second polypeptide, wherein both the first polypeptide and the second polypeptide comprise (e.g., one each) a CH3 domain, wherein:

[0220] The first polypeptide has a CH3 domain containing an amino acid mutation at position 347, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 360, wherein the CH3 domains of the first polypeptide and / or the second polypeptide further contain at least one identical or different amino acid mutation selected from positions 351, 364, 366, 394, 405, 411, 349, 354, 409, 356, and 439.

[0221] In some embodiments, such as the heteropolymer described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains an amino acid mutation at position 347, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347 and 360, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further contains at least one amino acid mutation selected from the group consisting of i) to vii):

[0222] i) 351, 364, 366, 394, 405 or 411;

[0223] ii) 356 and 351;

[0224] iii) 439 and 351;

[0225] ⅳ)349;

[0226] (v)354;

[0227] ⅵ)409;

[0228] (vii)356; and

[0229] ⅷ)439.

[0230] In some embodiments, such as the heteropolymer described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains an amino acid mutation at position 347, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347 and 360, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further contains at least one amino acid mutation selected from the group consisting of i) to vi):

[0231] i) 351, 364, 366, 394, 405 or 411;

[0232] ii) 356 and 351;

[0233] iii) 439 and 351;

[0234] ⅳ)349;

[0235] v)354; and

[0236] ⅵ)409.

[0237] In some embodiments, such as the heteropolymer described in any of the preceding claims, wherein the CH3 domain of the first polypeptide contains an amino acid mutation at position 347, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347 and 360, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further contains at least one amino acid mutation selected from the group consisting of i-1), ii), and iii):

[0238] i-1)351;

[0239] ⅱ)356 and 351; and

[0240] ⅲ)439 and 351.

[0241] In some implementations, such as the heteropolymer described in the preceding one, wherein:

[0242] 1) The CH3 domain of the first polypeptide contains an amino acid mutation at position 347, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347 and 360, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain an identical amino acid mutation selected from positions 351, 364, 366, 394, 405, and 411; or

[0243] 2) The CH3 domain of the first polypeptide contains amino acid mutations at positions 439, 347, and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 356, 347, 360, and 351; or

[0244] 3) The CH3 domain of the first polypeptide contains an amino acid mutation at position 347, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347 and 360, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation selected from positions 349, 354, 364, 405 and 409.

[0245] In some implementations, such as the heteropolymer described in the preceding one, wherein:

[0246] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 351, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 351; or

[0247] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 364, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 364; or

[0248] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 366, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 366; or

[0249] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 394, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 394; or

[0250] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 405, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 405; or

[0251] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 411, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 411.

[0252] In some implementations, such as the heteropolymer described in the preceding one, wherein:

[0253] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 351, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 351.

[0254] In some embodiments, such as the heteropolymer described in any of the preceding embodiments, wherein the CH3 domain of the first polypeptide contains amino acid mutations at positions 439, 347, and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 356, 347, 360, and 351.

[0255] In some implementations, such as the heteropolymer described in the preceding one, wherein:

[0256] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 349, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 354; or

[0257] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 349, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 364; or

[0258] The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 405, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 409.

[0259] In some embodiments, such as the heteropolymers described in any of the preceding claims, the CH3 domain of the first polypeptide and / or the second polypeptide contains at least one amino acid mutation selected from the following combinations:

[0260] The amino acid at position 347 is mutated to a residue selected from the following: Lys(K), Arg(R), Glu(E), and Asp(D); and / or

[0261] The amino acid at position 360 is mutated to a residue selected from Glu (E) and Asp (D); and / or

[0262] The amino acid mutation at position 351 is selected from the following residues: Met (M), Ile (I), Cys (C), Val (V), Thr (T), and Phe (F); and / or

[0263] The amino acid at position 364 is mutated to a residue selected from the following: Ala (A), Tyr (Y), Val (V), Thr (T), Leu (L), and Phe (F); and / or

[0264] The amino acid at position 366 is mutated to a residue selected from the following: Ala (A), Gly (G), Ser (S), Val (V), Leu (L), His (H), and Ile (I); and / or

[0265] The amino acid mutation at position 394 is selected from the following residues: Ala (A), Ser (S), Phe (F), Cys (C), Val (V), and Asn (N); and / or

[0266] The amino acid at position 405 is mutated to a residue selected from Tyr(Y), Leu(L), and Thr(T); and / or

[0267] The amino acid at position 411 is mutated to a residue selected from the following: Tyr (Y), Asn (N), and Leu (L); and / or

[0268] The amino acid mutation at position 349 is selected from the following residues: Ser(S), Leu(L), Phe(F), Cys(C), Ala(A), Val(V), Thr(T), and Gly(G); and / or

[0269] The amino acid at position 354 is mutated to a residue selected from the following: Tyr (Y), Cys (C), Phe (F), and Trp (W); and / or

[0270] The amino acid at position 409 is mutated to a residue selected from the following: Arg(R), Gln(Q), and Asp(D); and / or

[0271] The amino acid at position 356 is mutated to a residue selected from the following: Lys(K), Arg(R), and His(H); and / or

[0272] The amino acid at position 439 is mutated to residues selected from the following: Glu (E) and Asp (D).

[0273] In some implementations, such as the heteropolymer described in the preceding one, wherein:

[0274] 1) The CH3 domain of the first polypeptide contains an amino acid mutation at 347K, and the CH3 domain of the second polypeptide contains amino acid mutations at 347E and 360E, wherein the CH3 domains of the first and second polypeptides each contain an identical amino acid mutation selected from 351M, 351I, 364A, 366A, 394A, 394S, 405Y, and 411Y; or

[0275] The first polypeptide has a CH3 domain containing amino acid mutations of 347R and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations of 347E, 360E, and 351I; or

[0276] The first polypeptide has a CH3 domain containing amino acid mutations of 347R and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations of 347D, 360D, and 351I; or

[0277] 2) The CH3 domain of the first polypeptide contains amino acid mutations at 439E, 347K, and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 356K, 347E, 360E, and 351M; or

[0278] The first polypeptide has a CH3 domain containing amino acid mutations at 439E, 347K, and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 356K, 347E, 360E, and 351I; or

[0279] 3) The CH3 domain of the first polypeptide contains an amino acid mutation of 347K, and the CH3 domain of the second polypeptide contains amino acid mutations of 347E and 360E, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation selected from 349S, 354Y, 364Y, 405L and 409R.

[0280] In some implementations, such as the heteropolymer described in the preceding one, wherein:

[0281] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 351M, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 351M; or

[0282] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 351I; or

[0283] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 364A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 364A; or

[0284] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 366A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 366A; or

[0285] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 394A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 394A; or

[0286] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 394S, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 394S; or

[0287] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 405Y, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 405Y; or

[0288] The first polypeptide has a CH3 domain containing amino acid mutations of 347K and 411Y, and the second polypeptide has a CH3 domain containing amino acid mutations of 347E, 360E, and 411Y.

[0289] In some embodiments, such as the heteropolymer described in any of the preceding claims, the CH3 domain of the first polypeptide contains amino acid mutations of 347K and 351I, and the CH3 domain of the second polypeptide contains amino acid mutations of 347E, 360E, and 351I.

[0290] In some embodiments, such as the heteropolymer described in any of the preceding claims, the CH3 domain of the first polypeptide contains amino acid mutations at 347K and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 347E, 360E, and 351M.

[0291] In some embodiments, such as the heteropolymer described in any of the preceding claims, the CH3 domain of the first polypeptide contains amino acid mutations of 439E, 347K, and 351I, and the CH3 domain of the second polypeptide contains amino acid mutations of 356K, 347E, 360E, and 351I.

[0292] In some embodiments, such as the heteropolymer described in any of the preceding claims, the CH3 domain of the first polypeptide contains amino acid mutations at 439E, 347K, and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 356K, 347E, 360E, and 351M.

[0293] In some implementations, such as the heteropolymer described in the preceding one, wherein:

[0294] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 349S, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 354Y; or

[0295] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 349S, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 364Y; or

[0296] The first polypeptide has a CH3 domain containing amino acid mutations of 347K and 405L, and the second polypeptide has a CH3 domain containing amino acid mutations of 347E, 360E, and 409R.

[0297] In some embodiments, as described in any of the preceding embodiments, the CH3 domains of the first and second polypeptides are derived from the CH3 domains of IgG. In some embodiments, as described in any of the preceding embodiments, the CH3 domains of the first and second polypeptides are derived from the CH3 domains of an isotype of IgG1, IgG2, IgG3, or IgG4. In some embodiments, as described in any of the preceding embodiments, the CH3 domains of the first and second polypeptides are derived from the CH3 domains of an isotype of IgG1, IgG2, or IgG3. In some embodiments, as described in any of the preceding embodiments, the CH3 domains of the first and second polypeptides are derived from the CH3 domains of IgG1. In some embodiments, as described in any of the preceding embodiments, the CH3 domains of the first and second polypeptides are derived from the CH3 domains of human IgG1. In some embodiments, the human IgG1 has the amino acid sequence shown in SEQ ID NO: 11, 12, or 13.

[0298] In some embodiments, as described in any of the preceding heteropolymers, the amino acid mutations in the CH3 domains of the first and second polypeptides are based on mutations in the CH3 domains of natural (or wild-type) IgG1, IgG2, IgG3, or IgG4. In some embodiments, as described in any of the preceding heteropolymers, the amino acid mutations in the CH3 domains of the first and second polypeptides are based on mutations in the CH3 domains of natural (or wild-type) human IgG1. In some embodiments, as described in any of the preceding heteropolymers, the amino acid mutations in the CH3 domains of the first and second polypeptides are based on mutations in SEQ ID NO: 11, 12, or 13. In some embodiments, as described in the preceding heteropolymers, the amino acid sequence of the natural (or wild-type) human IgG1 is as shown in SEQ ID NO: 11, 12, or 13. In some embodiments, as described in any of the preceding heteropolymers, the sequence of the CH3 domain of the natural (or wild-type) human IgG1 is as shown in SEQ ID NO: 14 or 15.

[0299] In some implementations, such as the heteropolymer described in the preceding one, wherein:

[0300] 1) The CH3 domain of the first polypeptide contains an amino acid mutation of Q347K, and the CH3 domain of the second polypeptide contains amino acid mutations of Q347E and K360E, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain an identical amino acid mutation selected from L351M, L351I, S364A, T366A, T394A, T394S, F405Y, and T411Y; or

[0301] The first polypeptide has a CH3 domain containing amino acid mutations of Q347R and L351I, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and L351I; or

[0302] The first polypeptide has a CH3 domain containing amino acid mutations of Q347R and L351I, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347D, K360D, and L351I; or

[0303] 2) The CH3 domain of the first polypeptide contains amino acid mutations of K439E, Q347K, and L351M, and the CH3 domain of the second polypeptide contains amino acid mutations of D / E356K, Q347E, K360E, and L351M; or

[0304] The first polypeptide has a CH3 domain containing amino acid mutations of K439E, Q347K, and L351I, and the second polypeptide has a CH3 domain containing amino acid mutations of D / E356K, Q347E, K360E, and L351I; or

[0305] 3) The CH3 domain of the first polypeptide contains an amino acid mutation of Q347K, and the CH3 domain of the second polypeptide contains amino acid mutations of Q347E and K360E, wherein the CH3 domains of the first polypeptide and the second polypeptide each contain a different amino acid mutation selected from Y349S, S354Y, S364Y, F405L and K409R.

[0306] In some implementations, such as the heteropolymer described in the preceding one, wherein:

[0307] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and L351M, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and L351M; or

[0308] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and L351I, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and L351I; or

[0309] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and S364A, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and S364A; or

[0310] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and T366A, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and T366A; or

[0311] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and T394A, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and T394A; or

[0312] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and T394S, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and T394S; or

[0313] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and F405Y, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and F405Y; or

[0314] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and T411Y, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and T411Y.

[0315] In some embodiments, such as the heteropolymer described in any of the preceding claims, the CH3 domain of the first polypeptide contains amino acid mutations of Q347K and L351I, and the CH3 domain of the second polypeptide contains amino acid mutations of Q347E, K360E, and L351I.

[0316] In some embodiments, such as the heteropolymer described in any of the preceding claims, the CH3 domain of the first polypeptide contains amino acid mutations of K439E, Q347K, and L351I, and the CH3 domain of the second polypeptide contains amino acid mutations of D / E356K, Q347E, K360E, and L351I.

[0317] In some embodiments, such as the heteropolymer described in any of the preceding claims, the CH3 domain of the first polypeptide contains amino acid mutations of Q347K and L351M, and the CH3 domain of the second polypeptide contains amino acid mutations of Q347E, K360E, and L351M.

[0318] In some embodiments, such as the heteropolymer described in any of the preceding embodiments, the CH3 domain of the first polypeptide contains amino acid mutations of K439E, Q347K, and L351M, and the CH3 domain of the second polypeptide contains amino acid mutations of D / E356K, Q347E, K360E, and L351M.

[0319] In some implementations, such as the heteropolymer described in the preceding one, wherein:

[0320] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and Y349S, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and S354Y; or

[0321] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and Y349S, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and S364Y; or

[0322] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and F405L, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and K409R.

[0323] In some implementations, the CH3 domain mutation sites are designated by EU numbers.

[0324] In some embodiments, such as the heteropolymer described in any of the preceding claims, the isoelectric points of the first polypeptide and the second polypeptide differ. In some embodiments, such as the heteropolymer described in any of the preceding claims, the amino acid mutations described herein are used to confer or increase the difference in isoelectric points between the first polypeptide and the second polypeptide.

[0325] In some embodiments, as described in any of the preceding heteropolymers, the first polypeptide and the second polypeptide further comprise additional amino acid mutations to impart or increase the isoelectric point difference between the first polypeptide and the second polypeptide. In some embodiments, as described in any of the preceding heteropolymers, the first polypeptide and the second polypeptide further comprise additional amino acid mutations such that a difference exists between the isoelectric points of the first polypeptide and the second polypeptide.

[0326] In some embodiments, as described in any of the preceding heteropolymers, the first polypeptide and the second polypeptide further comprise a variable region. In some embodiments, as described in any of the preceding heteropolymers, the first polypeptide and the second polypeptide further comprise VH and VL. In some embodiments, as described in any of the preceding heteropolymers, the VH of the first polypeptide and / or the second polypeptide further comprises an amino acid mutation selected from Q105E, Q105R, and Q105K, the mutation site being indicated by KABAT numbers. In some embodiments, as described in any of the preceding heteropolymers, the VL of the first polypeptide and / or the second polypeptide further comprises an amino acid mutation of K42E, the mutation site being indicated by KABAT numbers.

[0327] In some embodiments, the heteropolymer as described in any of the preceding embodiments is a multispecific antibody or a heteroFc fusion protein.

[0328] In some embodiments, the heteropolymer as described in any of the preceding claims is a heterodimer. In some embodiments, the heterodimer as described in any of the preceding claims comprises a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide and / or the second polypeptide is a polypeptide structure constituting the heterodimer, which includes 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 includes CH2 and CH3 domains. In some embodiments, the first polypeptide and / or the second polypeptide includes CH1, CH2, and CH3 domains.

[0329] In some embodiments, the heteropolymer as described in any of the preceding claims is a bispecific antibody. In some embodiments, the bispecific antibody comprises a first hapten and a second hapten. In some embodiments, the first hapten comprises a first polypeptide, and the second hapten comprises a second polypeptide. The first polypeptide and / or the second polypeptide is a polypeptide structure constituting the bispecific antibody, which includes 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 includes CH2 and CH3 domains. In some embodiments, the first polypeptide and / or the second polypeptide includes CH1, CH2, and CH3 domains. In some embodiments, the first hapten binds to a first antigen, and the second hapten binds to a second antigen. In some embodiments, the first hapten includes a first antigen-binding domain, and the second hapten includes a second antigen-binding domain. In some implementations, the structures of the first antigen-binding domain and the second antigen-binding domain are selected from Fab, Fab′, F(ab')2, Fd, Fv, scFv, dsFv, dAb, and VHH.

[0330] In some embodiments, the heteropolymer as described in any of the preceding claims is a trispecific antibody. In some embodiments, the heteropolymer as described in any of the preceding claims comprises a first hapten and a second hapten. In some embodiments, the first hapten binds to a first antigen, and the second hapten binds to a second and a third antigen. In some embodiments, the first hapten includes a first antigen-binding domain, and the second hapten includes a second antigen-binding domain and a third antigen-binding domain. In some embodiments, the structures of the first, second, and third antigen-binding domains are selected from Fab, Fab′, F(ab')2, Fd, Fv, scFv, dsFv, dAb, and VHH.

[0331] In some embodiments, the heteropolymer as described in any of the preceding embodiments, wherein the first polypeptide is an antibody heavy chain, and / or the second polypeptide is an antibody heavy chain. In some embodiments, the heteropolymer further comprises one or more antibody light chains.

[0332] In some embodiments, the first polypeptide and the second polypeptide have different sequences. The term "first polypeptide" or "second polypeptide" is used only to distinguish amino acid sequences and does not limit the positional relationship between the polypeptide and the protein. For example, in a heterodimer composed of one first polypeptide and one second polypeptide, the first polypeptide and the second polypeptide are two polypeptides with different amino acid sequences. When either one is the first polypeptide, the other is the second polypeptide. The ordinal numbers "first," "second," and "third," etc., used in this disclosure are only used to distinguish different technical features and do not indicate any difference in sequence, priority, or importance, nor do they limit the number of technical features.

[0333] In some embodiments, as described in any of the preceding embodiments, the first polypeptide and the second polypeptide comprise hinge regions (e.g., core hinge regions). In some embodiments, as described in any of the preceding embodiments, the first polypeptide and the second polypeptide both comprise Cys-Pro-Pro-Cys sequences in their hinge regions.

[0334] In some embodiments, the heteropolymer as described in any of the preceding claims further comprises a core hinge region. In some embodiments, the heteropolymer as described in any of the preceding claims, wherein the amino acids in the core hinge region of the heteropolymer form disulfide bonds.

[0335] In another aspect, this disclosure provides a heteropolymer comprising a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide comprising a CH3 domain, wherein:

[0336] The first polypeptide has a CH3 domain containing amino acid mutations at 439E, 347K, and 351M, and the second polypeptide has a CH3 domain containing amino acid mutations at 356K, 347E, 360E, and 351M; or

[0337] The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 351I; or

[0338] The first polypeptide has a CH3 domain containing amino acid mutations of 439E, 347K, and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations of 356K, 347E, 360E, and 351I.

[0339] In some implementations, such as the heteropolymer described in the preceding one, wherein:

[0340] The first polypeptide has a CH3 domain containing amino acid mutations of K439E, Q347K, and L351M, and the second polypeptide has a CH3 domain containing amino acid mutations of D / E356K, Q347E, K360E, and L351M; or

[0341] The first polypeptide has a CH3 domain containing amino acid mutations of Q347K and L351I, and the second polypeptide has a CH3 domain containing amino acid mutations of Q347E, K360E, and L351I; or

[0342] The first polypeptide has a CH3 domain containing amino acid mutations of K439E, Q347K, and L351I, and the second polypeptide has a CH3 domain containing amino acid mutations of D / E356K, Q347E, K360E, and L351I.

[0343] In another aspect, this disclosure provides a composition comprising a heteropolymer comprising a first polypeptide and a second polypeptide as described in any of the preceding claims, and a reducing agent. In some embodiments, the composition as described in any of the preceding claims, 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%) is the desired heteropolymer (compared to other products in the total product, such as haptens or homopolymers).

[0344] In another aspect, this disclosure provides a protein (e.g., a dimer) comprising two identical polypeptides, said polypeptides containing a CH3 domain, wherein:

[0345] 1) The CH3 domain contains amino acid mutations at 347 and 351; or

[0346] The CH3 domain contains amino acid mutations at 347, 360, and 351; or

[0347] The CH3 domain contains amino acid mutations at 347 and 364; or

[0348] The CH3 domain contains amino acid mutations at 347, 360, and 364; or

[0349] The CH3 domain contains amino acid mutations at 347 and 366; or

[0350] The CH3 domain contains amino acid mutations at 347, 360, and 366; or

[0351] The CH3 domain contains amino acid mutations at 347 and 394; or

[0352] The CH3 domain contains amino acid mutations at 347, 360, and 394; or

[0353] The CH3 domain contains amino acid mutations at 347 and 405; or

[0354] The CH3 domain contains amino acid mutations at 347, 360, and 405; or

[0355] The CH3 domain contains amino acid mutations at 347 and 411; or

[0356] The CH3 domain contains amino acid mutations at 347, 360, and 411; or

[0357] 2) The CH3 domain contains amino acid mutations at 439, 347, and 351; or

[0358] The CH3 domain contains amino acid mutations at 356, 347, 360, and 351; or

[0359] 3) The CH3 domain contains amino acid mutations at 347 and 349; or

[0360] The CH3 domain contains amino acid mutations at 347, 360, and 354; or

[0361] The CH3 domain contains amino acid mutations at 347, 360, and 409.

[0362] In some implementations, the protein (e.g., a dimer) as described above, wherein:

[0363] 1) The CH3 domain contains amino acid mutations at 347K and 351M; or

[0364] The CH3 domain contains amino acid mutations at 347E, 360E, and 351M; or

[0365] The CH3 domain contains amino acid mutations at 347K and 351I; or

[0366] The CH3 domain contains amino acid mutations at 347E, 360E, and 351I; or

[0367] The CH3 domain contains amino acid mutations at 347K and 364A; or

[0368] The CH3 domain contains amino acid mutations at 347E, 360E, and 364A; or

[0369] The CH3 domain contains amino acid mutations at 347K and 366A; or

[0370] The CH3 domain contains amino acid mutations at 347E, 360E, and 366A; or

[0371] The CH3 domain contains amino acid mutations at 347K and 394A; or

[0372] The CH3 domain contains amino acid mutations at 347E, 360E, and 394A; or

[0373] The CH3 domain contains amino acid mutations at 347K and 394S; or

[0374] The CH3 domain contains amino acid mutations at 347E, 360E, and 394S; or

[0375] The CH3 domain contains amino acid mutations at 347K and 405Y; or

[0376] The CH3 domain contains amino acid mutations at 347E, 360E, and 405Y; or

[0377] The CH3 domain contains amino acid mutations at 347K and 411Y; or

[0378] The CH3 domain contains amino acid mutations at 347E, 360E, and 411Y; or

[0379] The CH3 domain contains amino acid mutations at 347R and 351I; or

[0380] The CH3 domain contains amino acid mutations at 347D, 360D, and 351I; or

[0381] 2) The CH3 domain contains amino acid mutations at 439E, 347K, and 351M; or

[0382] The CH3 domain contains amino acid mutations at 356K, 347E, 360E, and 351M; or

[0383] The CH3 domain contains amino acid mutations at 439E, 347K, and 351I; or

[0384] The CH3 domain contains amino acid mutations at 356K, 347E, 360E, and 351I; or

[0385] 3) The CH3 domain contains amino acid mutations at 347K and 349S; or

[0386] The CH3 domain contains amino acid mutations at 347E, 360E, and 354Y; or

[0387] The CH3 domain contains amino acid mutations at 347E, 360E, and 364Y; or

[0388] The CH3 domain contains amino acid mutations at 347K and 405L; or

[0389] The CH3 domain contains amino acid mutations at 347E, 360E, and 409R.

[0390] In some embodiments, the protein (e.g., a dimer) as described in any of the preceding claims, wherein the CH3 domain is derived from the CH3 domain of IgG. In some embodiments, the protein (e.g., a dimer) as described in any of the preceding claims, wherein the CH3 domain is derived from the CH3 domain of an isotype of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the protein (e.g., a dimer) as described in any of the preceding claims, wherein the CH3 domain is derived from the CH3 domain of IgG1. In some embodiments, the protein (e.g., a dimer) as described in any of the preceding claims, wherein the CH3 domain is derived from the CH3 domain of human IgG1. In some embodiments, the human IgG1 has the amino acid sequence shown in SEQ ID NO: 11, 12, or 13.

[0391] In some embodiments, the protein (e.g., a dimer) as described in any of the preceding claims, wherein the amino acid mutation in the CH3 domain is a mutation based on the CH3 domain of natural (or wild-type) IgG1, IgG2, IgG3, or IgG4. In some embodiments, the protein (e.g., a dimer) as described in any of the preceding claims, wherein the amino acid mutation in the CH3 domain is a mutation based on the CH3 domain of natural (or wild-type) human IgG1. In some embodiments, the protein (e.g., a dimer) as described in any of the preceding claims, wherein the amino acid mutation in the CH3 domain is a mutation based on SEQ ID NO: 11, 12, or 13. In some embodiments, the protein (e.g., a dimer) as described above, wherein the amino acid sequence of the natural (or wild-type) human IgG1 is as shown in SEQ ID NO: 11, 12, or 13. In some embodiments, the protein (e.g., a dimer) as described in any of the preceding claims, wherein the sequence of the CH3 domain of the natural (or wild-type) human IgG1 is as shown in SEQ ID NO: 14 or 15.

[0392] In some implementations, the protein (e.g., a dimer) as described in any of the preceding embodiments, wherein:

[0393] 1) The CH3 domain contains amino acid mutations at Q347K and L351M; or

[0394] The CH3 domain contains amino acid mutations at Q347E, K360E, and L351M; or

[0395] The CH3 domain contains amino acid mutations at Q347K and L351I; or

[0396] The CH3 domain contains amino acid mutations at Q347E, K360E, and L351I; or

[0397] The CH3 domain contains amino acid mutations at Q347K and S364A; or

[0398] The CH3 domain contains amino acid mutations at Q347E, K360E, and S364A; or

[0399] The CH3 domain contains amino acid mutations at Q347K and T366A; or

[0400] The CH3 domain contains amino acid mutations at Q347E, K360E, and T366A; or

[0401] The CH3 domain contains amino acid mutations at Q347K and T394A; or

[0402] The CH3 domain contains amino acid mutations at Q347E, K360E, and T394A; or

[0403] The CH3 domain contains amino acid mutations at Q347K and T394S; or

[0404] The CH3 domain contains amino acid mutations at Q347E, K360E, and T394S; or

[0405] The CH3 domain contains amino acid mutations at Q347K and F405Y; or

[0406] The CH3 domain contains amino acid mutations at Q347E, K360E, and F405Y; or

[0407] The CH3 domain contains amino acid mutations at Q347K and T411Y; or

[0408] The CH3 domain contains amino acid mutations of Q347E, K360E, and T411Y; or

[0409] 2) The CH3 domain contains amino acid mutations of K439E, Q347K, and L351M; or

[0410] The CH3 domain contains amino acid mutations at D / E356K, Q347E, K360E, and L351M; or

[0411] The CH3 domain contains amino acid mutations at K439E, Q347K, and L351I; or

[0412] The CH3 domain contains amino acid mutations at D / E356K, Q347E, K360E, and L351I; or

[0413] 3) The CH3 domain contains amino acid mutations at Q347K and Y349S; or

[0414] The CH3 domain contains amino acid mutations at Q347E, K360E, and S354Y; or

[0415] The CH3 domain contains amino acid mutations at Q347E, K360E, and S364Y; or

[0416] The CH3 domain contains amino acid mutations at Q347K and F405L; or

[0417] The CH3 domain contains amino acid mutations of Q347E, K360E, and K409R.

[0418] In some implementations, the CH3 domain mutation sites are designated by EU numbers.

[0419] In some embodiments, the protein (e.g., a dimer) as described in any of the preceding embodiments is an antibody heavy chain, wherein the polypeptide containing the CH3 domain is an antibody heavy chain.

[0420] In some embodiments, the protein as described in any of the preceding embodiments further comprises one or more different polypeptides. In some embodiments, the protein as described in any of the preceding embodiments further comprises one or more antibody light chains.

[0421] In some embodiments, the protein described in any of the preceding embodiments is an antibody. In some embodiments, the protein described in any of the preceding embodiments is a monospecific antibody or a bispecific antibody.

[0422] In another aspect, this disclosure provides a pharmaceutical composition comprising a heteropolymer as described in any of the preceding claims, a protein as described in any of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0423] In another aspect, this disclosure provides an immunoconjugate comprising: a heteropolymer or a protein as described in any of the preceding claims, and a payload, wherein the payload is coupled to the heteropolymer or the protein. In some embodiments, the payload is selected from antitumor agents, immunomodulators, bioresponse modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0424] In another aspect, this disclosure provides a method for preparing an immunoconjugate, comprising the following steps:

[0425] a) The step of providing a molecule comprising a first polypeptide homopolymer and a molecule comprising a second polypeptide homopolymer;

[0426] b) Mixing the molecules containing the first polypeptide homopolymer and the molecules containing the second polypeptide homopolymer to form a mixture; and

[0427] c) The step of incubating the molecule containing the first polypeptide homopolymer and the molecule containing the second polypeptide homopolymer together under reducing conditions;

[0428] d) Obtain an immunoconjugate containing the first polypeptide and the second polypeptide;

[0429] The first polypeptide and the second polypeptide each contain a CH3 domain, and each CH3 domain contains one or more mutations that promote heterologation.

[0430] The molecule containing the first polypeptide isomer and the molecule containing the second polypeptide isomer contain an Fc region conjugated to a prodrug, peptide, drug, or toxin.

[0431] In some embodiments, the molecule containing the first polypeptide isomer and the molecule containing the second polypeptide isomer contain an Fc region conjugated to a toxin.

[0432] In some embodiments, the molecule containing the first polypeptide isomer and the molecule containing the second polypeptide isomer are antibody-drug conjugates.

[0433] In another aspect, this disclosure provides a method for preparing an immunoconjugate, comprising:

[0434] The heteropolymer obtained using any of the methods for preparing heteropolymers described in this disclosure is conjugated with a toxin.

[0435] In another aspect, this disclosure provides one or more nucleic acids (e.g., isolated nucleic acids) that encode heteropolymers as described in any of the preceding claims.

[0436] In another aspect, this disclosure provides one or more vectors that contain one or more nucleic acids (e.g., isolated nucleic acids) as described in any of the preceding claims.

[0437] In another aspect, this disclosure provides one or more host cells that contain one or more nucleic acids (e.g., isolated nucleic acids) as described in any of the preceding claims.

[0438] In another aspect, this disclosure provides a method for preparing a heteropolymer as described in any of the preceding claims, comprising expressing one or more nucleic acids (e.g., isolated nucleic acids) as described in any of the preceding claims, or culturing one or more host cells as described in any of the preceding claims to produce the heteropolymer.

[0439] In another aspect, this disclosure provides a method for preparing heteropolymers as described in any of the preceding claims, comprising: (a) altering a nucleic acid encoding amino acid residues forming the interface between the polypeptides; (b) culturing a host cell having the nucleic acid to express the polypeptide; (c) recovering the polypeptide from the culture of the host cell; and (d) incubating each polypeptide in the presence of a reducing agent to recover the desired heteropolymer.

[0440] Unless otherwise stated, in this disclosure, the positions of amino acids in the variable region are defined according to the Kabat numbering rules, and the positions of amino acids in the constant region are defined according to the Eu index. Attached Figure Description

[0441] Figure 1A shows the differential scanning calorimetry curve of IgG1-Fc.

[0442] Figure 1B shows the differential scanning calorimetry curve of IgG4-Fc.

[0443] Figure 1C shows the differential scanning calorimetry curve of Fc-F405L*Fc-K409R.

[0444] Figure 1D shows the differential scanning calorimetry curve of Fc-D356K+L351I*Fc-K439E+L351I.

[0445] Figure 1E shows the differential scanning calorimetry curve of Fc-Q347K+L351I*Fc-Q347E+K360E+L351I.

[0446] Figure 1F shows the differential scanning calorimetry curve of Fc-K439E+Q347K+L351I*Fc-D356K+Q347E+K360E+L351I.

[0447] Figure 1G shows the differential scanning calorimetry curves of Fc-K439E+Q347K+L351M*Fc-D356K+Q347E+K360E+L351M. Detailed Implementation

[0448] the term

[0449] To facilitate understanding of this disclosure, certain technical and scientific terms are described below. Unless otherwise expressly defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0450] The singular forms “a,” “an,” and “the” used in the specification and claims include plural references unless the context clearly indicates otherwise.

[0451] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0452] The term "and / or" implies both "and" and "or". For example, the phrase "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0453] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0454] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0455] The term "amino acid mutation" encompasses amino acid substitution (also known as amino acid replacement), deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, provided that the final construct possesses the desired properties, such as altering the affinity between homomers. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide chain. A specific amino acid mutation can be an amino acid substitution. In one embodiment, an amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid that has a different structure and / or chemical properties. Amino acid substitution includes substitution by non-naturally occurring amino acids or by derivatives of 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering that alter amino acid side chain groups, such as chemical modification, are also expected to be available. Various names may be used herein to refer to the same amino acid mutation. In this document, the amino acid residue at a specific site can be represented using the format of position + amino acid residue. For example, 356K indicates that the amino acid residue at position 356 is K. D356K indicates that the amino acid residue at position 356 has mutated from D to K. D / E356K indicates that the amino acid residue at position 356 has mutated from D to K, or that the amino acid residue at position 356 has mutated from E to K. Other sequences follow the same pattern. It should be understood that when the amino acid sequence is defined using the format of position + residue in the claims, the amino acid before the mutation at that site does not constitute a limitation on the technical solution. In this document, "the Fc region contains amino acid mutations of 356K and 349S" means that the amino acid mutation in the Fc region includes a mutation at position 356 to lysine (K) and a mutation at position 349 to serine (S).

[0456] The terms “polypeptide” and “protein” are used interchangeably in this article.

[0457] The term "heteromeric multimer" refers to a protein multimer composed of multiple different polypeptides that can associate with each other. More specifically, a "heteromeric multimer" has at least a first polypeptide and a second polypeptide, wherein the second polypeptide is a molecule whose amino acid sequence differs from the first polypeptide by at least one amino acid residue. Those skilled in the art will understand that "difference" here refers only to differences in the primary amino acid sequence (by default, in N-to-C order), and does not consider cases where the amino acid residues are the same but modified differently. Furthermore, although not specifically limited, the heteromeric multimer can possess antigen-binding activity to at least two different ligands, antigens (or epitopes), receptors, or substrates. In addition to "heterodimers" formed by the first and second polypeptides, other types of polypeptides may also exist in the heteromeric multimer. That is, the "heteromeric multimer" disclosed herein is not limited to heterodimers, but also includes, for example, heterotrimers, heterotetramers, etc.

[0458] The term "homomer" refers to the associated state of polypeptide companions that have the same amino acid sequence.

[0459] In this disclosure, the terms "multispecific antibody" and "polyspecific antibody" have the same meaning, referring to an antibody capable of specifically binding to multiple different epitopes. That is, a multispecific antibody is specific to at least two different epitopes, including antibodies that recognize different antigens as well as antibodies that recognize different epitopes on the same antigen. For example, when the antigen is a heterologous receptor, a multispecific antibody recognizes different domains constituting the heterologous receptor; or when the antigen is a monomer, a multispecific antibody recognizes multiple sites on the monomeric antigen. Typically, such a molecule binds to two antigens (bispecific antibody, in this specification, has the same meaning as "dual-specific antibody," but can be specific to two or more antigens (e.g., three).

[0460] The term "antibody" is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), provided they exhibit the desired antigen-binding activity. In the antibodies disclosed herein, multiple allotropic sequences resulting from gene polymorphisms in the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, and this disclosure may include any of these sequences. As a sequence of human IgG1, the amino acid sequence at positions 356-358, represented by the EU number, can be either DEL or EEM. In particular, the amino acid at position 356, represented by the EU number, can be either D or E (Jefferis R, Lefranc M P. Human immunoglobulin allotypes: possible implications for immunogenicity / / MAbs. Taylor & Francis, 2009, 1(4): 332-338.).

[0461] "Natural antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain or heavy chain variable region, followed by a heavy chain constant region. Natural IgG (e.g., IgA, IgD, and IgG) heavy chain constant regions typically contain three constant domains (CH1, CH2, and CH3). Similarly, from the N to the C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0462] The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably in this document, referring to antibodies with a structure substantially similar to that of natural antibodies or with a heavy chain containing the Fc region as defined herein. The light chain of a natural intact antibody includes a variable region (VL) and a constant region (CL), with VL located at the amino terminus of the light chain. The constant region includes the κ and λ chains. The heavy chain includes a variable region (VH) and constant regions (CH1, CH2, and CH3), with VH located at the amino terminus of the heavy chain and the constant region located at the carboxyl terminus. CH3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0463] As described in this article, a "full-length antibody" can be an antibody that specifically binds to a single antigenic epitope, or it can be a bispecific antibody that can bind to two different antigenic epitopes simultaneously.

[0464] The antibody used in this disclosed method can be of any allotype. Allotypes are not expected to affect Fab-arm exchange. Antibody allotypes are associated with amino acid sequence variations at specific positions within the antibody constant region sequence.

[0465] The common wild-type human IgG1 heavy chain constant region sequences are shown below:

[0466] hIgG1-1

[0467] hIgG1-2

[0468] hIgG1-3

[0469] The term "bispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. Bispecific antibodies with various structures have been disclosed in the prior art. Based on the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies; based on the number of antigen-binding regions, they can be divided into bivalent, trivalent, tetravalent, or more; and based on whether the structure is symmetrical, they can be divided into symmetrical and asymmetrical bispecific antibodies. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by combining two or more Fab fragments into one molecule. These antibodies have low immunogenicity, small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab bispecific antibodies. IgG-like bispecific antibodies (e.g., those with an Fc fragment) have a relatively large molecular weight. The Fc fragment facilitates subsequent antibody purification and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody's serum half-life. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, and 1Fab-IgG. Bispecific antibodies such as TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, and F(ab)4-CrossMAb (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11; 2019: 4516041).

[0470] The term "maternal antibody" is an antibody that serves as the source of one or more antibody fragments. A maternal antibody may contain a natural or wild-type sequence. A maternal antibody may contain an amino acid sequence in which one or more amino acid residues are replaced by one or more cysteine ​​residues. Compared to other natural, wild-type, or modified forms of antibodies, a maternal antibody may have pre-existing amino acid sequence modifications (such as additions, deletions, and / or substitutions). A maternal antibody may target a specific antigen, such as a biologically important peptide. A maternal antibody may target a non-peptide antigen (such as the human CCR8 antigen; e.g., patent WO2023208182A1). Exemplary maternal antibodies include, but are not limited to, antibodies with affinity and selectivity for cell surface receptors and transmembrane receptors and tumor-associated antigens (TAAs).

[0471] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and engineered Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. Suitable Fc regions for the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may also vary, for example, by omitting the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or by omitting both the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). Unless otherwise stated, the Fc region is numbered according to the EU numbering system, also known as the EU index (Kabat EA et al., 1991. Sequences of Proteins of Immunological Interest. NIH). Although a numbering system (such as EU) is used to define amino acid residues in a specific implementation, the corresponding technical solutions of other numbering systems will be regarded as equivalent technical solutions.

[0472] The Fc region can be appropriately obtained by partially digesting IgG monoclonal antibodies with proteolytic enzymes such as pepsin, followed by eluting the components adsorbed on the protein A or protein G column. As the proteolytic enzyme, any enzyme capable of restrictively digesting full-length antibodies to produce Fab and F(ab')2 by appropriately setting the enzyme reaction conditions such as pH is acceptable; there is no particular limitation, and examples include pepsin and papain.

[0473] In this disclosure, the term "Fc region" or "Fc domain" refers to an antibody region that contains at least a CH2 domain and a CH3 domain. In this disclosure, the term "CH2 region" or "CH2 domain" is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering system (according to the IMGT website). However, the CH2 region can also be any other antibody isotype as described in this disclosure.

[0474] In this disclosure, the terms “CH3 region,” “CH3 domain,” or “CH3 structural domain” are intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system (according to the IMGT website). However, the CH3 region can also be any other antibody isotype as described in this disclosure.

[0475] The CH3 region sequence of common wild-type human IgG1 is shown below:

[0476] hIgG1-1-CH3

[0477] hIgG1-3-CH3

[0478] In this disclosure, peptide association refers to, for example, the interaction of multiple peptide regions.

[0479] In this disclosure, “regulated association” means regulating to a desired association state, and more specifically, means preventing the formation of undesirable associations within the polypeptide.

[0480] In this disclosure, "interface" generally refers to the association surface during association (interaction). The amino acid residues forming the interface generally refer to one or more amino acid residues contained in the polypeptide region undergoing association, more preferably amino acid residues that are close to each other during association and participate in the interaction. This interaction specifically includes: the formation of hydrogen bonds, electrostatic interactions, salt bridges, etc., between amino acid residues that are close to each other during association.

[0481] In this disclosure, "amino acid residues forming the interface" specifically refers to the amino acid residues contained in the polypeptide region constituting the interface. Examples of polypeptide regions constituting the interface include polypeptide regions in antibodies, ligands, receptors, substrates, etc., which perform selective bonding within or between molecules. In this disclosure, it can also refer to amino acid residues at the interface where two CH3 regions interact.

[0482] The term "variable region" or "variable domain" in an antibody refers to the domain in the antibody heavy or light chain involved in antibody binding to the antigen. In this paper, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0483] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. The correspondence between various numbering systems is well known to those skilled in the art and is exemplarily shown in Table 1A below.

[0484] Table 1A. Relationship between CDR numbering systems

[0485] Unless otherwise stated, the variable regions and CDRs in this disclosure embodiment are governed by the "Kabat" numbering rule. Although the Kabat numbering rule is used in specific implementations to define amino acid residues, corresponding technical solutions using other numbering systems are considered equivalent.

[0486] The term "antibody fragment" refers to a molecule that is distinct from the intact antibody but contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, dsFv, Fab, Fab′, Fab′-SH, F(ab′)2, Fd, single-domain antibodies (sdAb, such as VH, VL, VHH, or VHH), single-chain Fab (scFab), biantibodies, linear antibodies, single-chain antibodies (such as scFv, sc(Fv)2); and multispecific antibodies formed from antibody fragments.

[0487] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0488] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0489] The terms "human antibody," "humanized antibody," "fully human antibody," and "completely human antibody" are used interchangeably, referring to antibodies whose variable and constant regions are human sequences. This term encompasses antibodies derived from human genes but with sequence alterations, such as reduced potential immunogenicity, increased affinity, or the elimination of cysteine ​​or glycosylation sites that might cause undesirable folding. This term also covers antibodies recombined in non-human cells (which may confer glycosylations not characteristic of human cells). The term also includes antibodies produced in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The term "human antibody" explicitly excludes humanized antibodies.

[0490] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art, including those described herein.

[0491] As used herein, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. For example, it can be measured using a biosensing system such as a system for measuring surface plasmon resonance (e.g., Biacore), or by measuring affinity in solution using solution equilibrium titration (SET).

[0492] The term “surface plasmon resonance” refers to the optical phenomenon of analyzing real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0493] The term "effector function" refers to biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region with amino acid sequence mutations) and that vary across antibody isotypes. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0494] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibodies typically comprise a variety of different antibodies with different amino acid sequences in their variable domains, and they generally specifically target different epitopes. "Monoclonal" should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0495] The term "antigen" refers to a molecule or molecular part that can be selectively bound by antigen-binding proteins, including, for example, antibodies. An antigen may have one or more epitopes that can interact with different antigen-binding proteins, such as antibodies.

[0496] The term "epitope" refers to a region on an antigen that is capable of specifically binding to an antibody or its antigen-binding fragment. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain non-continuous amino acids (conformal epitopes), such as those spatially close due to antigen folding. The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0497] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies are present in quantities of approximately 1 × 10⁻⁷ M or smaller (e.g., approximately 1 × 10⁻⁷ M). -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11The equilibrium dissociation constant (KD) of an antibody (M or less) binds to an antigen or its epitope. In some embodiments, the KD of antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by... The surface plasmon resonance assay is used to measure this. However, antibodies that specifically bind to an antigen or its epitope do not preclude cross-reactivity with other related antigens, such as cross-reactivity with corresponding antigens from other species (homologous) (e.g., humans or monkeys, such as the cynomolgus (cyno), the chimpanzee (chimp), or the common marmoset (marmoset)).

[0498] The terms “antibody-dependent cell cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to mechanisms that induce cell death that rely on the interaction between antibody-coated target cells and lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies described herein can be assessed in vitro using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) from lysed cells.

[0499] The term "antibody-dependent phagocytosis (ADCP)" refers to the mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells).

[0500] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q. C1q then activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0501] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs).

[0502] "Separated" nucleic acids refer to nucleic acid molecules that have been separated from their components in their natural environment. Separated nucleic acids encoding polypeptides refer to one or more nucleic acid molecules encoding polypeptides, including one or more such nucleic acid molecules in a single vector or separate vectors, and one or more such nucleic acid molecules present at one or more locations in the host cell. Unless otherwise stated, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues.

[0503] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions when optimally aligned; gaps may be introduced, where necessary, to obtain the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0504] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which an additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming host cells and containing a nucleic acid sequence that directs and / or controls (alongside the host cell) the expression of one or more heterologous coding regions operatively linked to it. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, influence RNA splicing of coding regions operatively linked to them.

[0505] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. Mutant progeny are included herein, which have the same function or biological activity as cells screened or selected in the initial transformed cells. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. Kluyveromyces polymorpha, Kluyveromyces lactis, Candida albicans, Aspergillus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.

[0506] The host cells disclosed herein cannot develop into plants or animal individuals.

[0507] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0508] The term "pharmaceutical composition" refers to a mixture containing one or more of the multispecific antibodies described herein, along with other chemical components, such as physiological / pharmaceutical carriers and excipients.

[0509] The term "pharmaceutically acceptable carrier, diluent, or excipient" refers to a component in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers, diluents, or excipients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0510] The terms “subject” or “individual” include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms “patient” or “subject” are used interchangeably herein. In some embodiments, the individual or subject is a human being.

[0511] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0512] The term "sample" refers to a collection (such as fluid, cells, or tissue) separated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues or organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as culture media (including conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0513] "Treatment" and "treatment" (and their grammatical variations) refer to a clinical intervention on the individual being treated, and may be administered for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, the antibodies disclosed herein are used to delay the onset of disease or slow its progression.

[0514] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or mitigate damage caused by or associated with a disease state (e.g., lung disease). In some embodiments, an effective amount is a therapeutically effective amount or a preventatively effective amount.

[0515] A “therapeutic effective dose” is a dose sufficient to treat a disease state or symptom, especially a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A “preventive effective dose” is a dose that, when administered to a subject, will have a predetermined preventive effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. Complete treatment or prevention may not occur after a single dose, but may occur after a series of doses. Therefore, a therapeutic or preventive effective dose may be administered in a single or multiple-dose manner. Both “therapeutic effective doses” and “preventive effective doses” can vary depending on a variety of factors, such as an individual’s disease state, age, sex, and weight, and the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status in the patient.

[0516] Combination with isoelectric point changing technology, etc.

[0517] As a further preferred embodiment of this disclosure, by introducing amino acid variations that alter the isoelectric point (pI value) of the polypeptide into the polypeptide disclosed herein, polypeptide polymers containing the target first to fourth polypeptides can be purified or prepared with higher purity and efficiency (WO2007114325, US20130171095). As an amino acid variation introduced to promote polypeptide association, methods can also be used, such as Protein Eng. 1996 Jul; 9(7):617-21., Protein Eng Des Sel. 2010 Apr; 23(4):195-202., J Biol Chem. 2010 Jun 18; 285(25):19637-46., WO2009080254, US20130195849, etc., which heterologously associate polypeptides containing two heavy chain constant regions by changing the CH3 domain of the heavy chain constant region; and methods for promoting association of specific combinations of heavy and light chains, such as WO2009080251, WO2009080252, WO2009080253, etc.

[0518] Combination with other constant region and / or variable region changing technologies

[0519] As a non-limiting embodiment of this disclosure, an example of a combination with the following modified technology is a constant region modification technology (WO2013047752) aimed at enhancing the binding with FcγR.

[0520] As another combination of this disclosure and other constant region modification techniques, examples include combinations with techniques for controlling complement binding. As complement, any complement component can be used as long as it is a polypeptide that forms a complement cascade, but preferred complement components include C1q, C1r, or C1s complement components involved in opsonin binding. An Fc region with higher complement binding activity than the native Fc region can be created by altering the amino acids of the native Fc region. The native Fc region referred to herein is the Fc region represented by human IgG1, IgG2, IgG3, or IgG4. Whether the complement binding activity of the Fc region is higher than that of the native Fc region can be appropriately determined using known immunological methods such as FACS and ELISA. "Amino acid alteration" or "amino acid modification" of the Fc region includes changing it to an amino acid sequence different from the amino acid sequence of the starting Fc region. Any Fc region can be used as the starting domain as long as it binds to complement within a neutral pH range. Furthermore, an Fc region that has already been modified and used as the starting Fc region, along with an Fc region that has been further modified, can also be suitably used as the Fc region disclosed herein. The starting Fc region refers to the polypeptide itself, a composition containing the starting Fc region, or the amino acid sequence encoding the starting Fc region. The starting Fc region may contain the Fc region of a known IgG antibody generated through recombinant synthesis as outlined in the antibody project. There are no particular limitations on the source of the starting Fc region; it can be obtained from any non-human animal organism or from humans. Suitable examples of suitable organisms include: mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cattle, horses, camels, and non-human primates. In other embodiments, the starting Fc region can also be obtained from cynomolgus monkeys, marmosets, macaques, chimpanzees, or humans. Preferably, the starting Fc region can be obtained from human IgG1, but is not limited to a specific type of IgG. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be suitably used as the starting Fc region. This also means that, in this disclosure, the Fc region of any type or subclass of IgG from any of the aforementioned organisms can preferably be used as the starting Fc region. Examples of naturally occurring IgG variants or engineered models are described in known literature (Curr. Opin. Biotechnol. (2009) 20(6), 685-91, Curr. Opin. Immunol. (2008) 20(4), 460-470, Protein Eng. Des. Sel. (2010) 23(4), 195-202, WO2009086320, WO2008092117, WO2007041635, and WO2006105338), but are not limited to these.

[0521] Amino acid alterations can be made at any position as long as they result in or enhance complement-binding activity. When an antibody contains the Fc region of human IgG1 as the human Fc region, it is preferable to include alterations that produce complement-binding activity stronger than that of the initial Fc region of human IgG1. As an example of amino acids that can be used to alter complement binding activity, examples include: amino acids in the Fc region whose binding activity to C1q is altered, as reported in Duncan et al. (Nature (1988) 332, 738-740), Tao et al. (J. Exp. Med. (1993) 178, 661-667), Brekke et al. (Eur. J. Immunol. (1994) 24, 2542-2547), Xu et al. (Immunol. (1993) 150, 152A), WO1994029351, WO2000042072 and WO2011091078.

[0522] Examples of amino acids that can be modified to enhance the binding activity to C1q include, for example, at least one amino acid selected from positions 231 to 238 and 318 to 337 (represented by EU designations). As a non-limiting example of this amino acid, at least one amino acid selected from positions 235, 237, 318, 320, 322, 324, 327, 331, and 333 can be included. By modifying these amino acids, the binding of the Fc region of IgG immunoglobulins to complement can be enhanced.

[0523] Other combinations of this disclosure with other constant-region alteration technologies include: combinations with antibody alteration technologies such as FcRn binding enhancement at acidic pH (WO2002060919, WO2004035752, WO2000042072), FcRn binding enhancement at neutral pH (WO2011122011, WO2012133782), selective binding enhancement of inhibitory Fcγ receptors (WO2012115241, WO2013125667), selective binding enhancement of active Fcγ receptors (ADCC activity enhancement technology) (WO2013002362), and techniques that reduce the binding activity of rheumatoid factor (WO2013046704), used to regulate effector function through Fc mutation combinations (Liu R, Oldham RJ, Teal E, et al. Fc-engineering for modulated effector functions—improving antibodies for cancer). treatment[J].Antibodies,2020,9(4):64.).

[0524] The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; or it can be a shortened C-terminus, for example, in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. Thus, in some embodiments, the composition of a complete antibody may include an antibody population with all K447 residues and / or G446+K447 residues removed. In some embodiments, the composition of a complete antibody may include an antibody population without the removal of K447 residues and / or G446+K447 residues. In some embodiments, the composition of a complete antibody has an antibody population consisting of a mixture of antibodies with and without K447 residues and / or G446+K447 residues.

[0525] As a non-limiting combination of this disclosure with variable region modification technology, examples include combinations with modification technologies such as pH-dependent antibodies (WO2009125825) and calcium-dependent antibodies (WO2012073992).

[0526] Changes in nucleic acids

[0527] In another embodiment of the preparation method disclosed herein, the disclosure provides a method for preparing heteropolymers (e.g., multispecific antibodies), which is a method for preparing heteropolymers by varying the amino acid residues (e.g., at least one amino acid residue selected from positions 347, 349, 351, 354, 356, 357, 364, 366, 368, 394, 397, 399, 405, 407, 409, 411, and 439, indicated by EU numbers) to control the dissociation and / or association between polypeptides. The preparation method includes the following steps: (a) modifying the nucleic acid encoding the amino acid residues forming the polypeptide interface, etc., according to the original nucleic acid to control the dissociation and association between polypeptides; (b) culturing a host cell containing the nucleic acid to express the polypeptide; (c) recovering the polypeptide from the culture of the host cell; and (d) incubating each polypeptide under reducing conditions to recover the desired heteropolymer.

[0528] Furthermore, a method that includes the step of modifying the nucleic acid encoding the amino acid residues that form the interface between the polypeptides according to the original nucleic acid to inhibit polypeptide association by utilizing the dissociation and / or association control method disclosed above is also one of the preferred embodiments of the preparation method disclosed above.

[0529] In the methods disclosed herein, "altering nucleic acids" means altering nucleic acids to correspond to amino acid residues introduced through the "alteration" described herein. More specifically, it refers to changing the nucleic acid encoding the original (unaltered) amino acid residues to a nucleic acid encoding the amino acid residues introduced through the alteration. Typically, this refers to gene manipulation or mutagenesis involving the insertion, deletion, or substitution of at least one base in the original nucleic acid to form a codon encoding the target amino acid residue. That is, the codon encoding the original amino acid residue is replaced by the codon encoding the amino acid residue introduced through the alteration. Such alteration of nucleic acids can be appropriately carried out using techniques known to those skilled in the art, such as site-specific mutagenesis, PCR mutagenesis, etc.

[0530] Furthermore, the nucleic acids described in this disclosure are typically carried (inserted) into a suitable vector and introduced into host cells. There are no particular limitations on the vector, as long as it stably retains the inserted nucleic acid.

[0531] There are no particular restrictions on host cells; various host cells can be used depending on the purpose.

[0532] To induce the secretion of peptides expressed in host cells into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, appropriate secretion signals can be combined with the target peptide. These signals can be endogenous or heterologous to the target peptide.

[0533] Regarding the recovery of the peptides in the above preparation method, the culture medium is recovered when the disclosed peptides are secreted into the culture medium. When the disclosed peptides are produced intracellularly, the cells are first lysed, and then the peptides are recovered.

[0534] When purifying the disclosed polypeptides recovered from recombinant cell cultures, known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography can be used.

[0535] Recombination method

[0536] Heteromeric polymers (such as multispecific antibodies) can be generated using recombinant methods. For these methods, one or more isolated nucleic acids encoding the heteromeric polymer are provided.

[0537] In one embodiment, this disclosure provides isolated nucleic acids encoding heteropolymers as described in any of the preceding claims. Such nucleic acids may each independently encode any of the aforementioned polypeptide chains. In another aspect, this disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In yet another aspect, this disclosure provides host cells comprising such nucleic acids. In one embodiment, a method for preparing heteropolymers is provided, wherein the method comprises culturing host cells comprising nucleic acids encoding said heteropolymers under conditions suitable for expression, as provided above, and optionally recovering said protein from the host cells (or host cell culture medium).

[0538] To generate heteromultimers through recombination, nucleic acids encoding proteins are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be readily isolated and sequenced using standard procedures, or generated through recombination methods or obtained through chemical synthesis.

[0539] Suitable host cells for cloning or expressing vectors encoding heteropolymers include prokaryotic or eukaryotic cells as described herein. For example, they can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, the expression can be separated from the bacterial cell paste in a soluble fraction and further purified.

[0540] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains. Suitable host cells for antibody expression can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells; plant cell cultures can also be used as hosts, such as US5959177, US 6040498, US6420548, US 7125978, and US6417429; and vertebrate cells, such as mammalian cell lines adapted for growth in suspension, can also be used as hosts. Other examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293T cells); young hamster kidney cells (BHK); mouse seltoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0541] Measurement

[0542] The heteropolymers provided herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activities using a variety of assays known in the art. In one aspect, the activity of the heteropolymers disclosed herein can be tested, for example, by known methods such as flow cytometry, ELISA, Western blotting, SEC, IEC, mass spectrometry, etc.

[0543] Treatment methods and routes of administration

[0544] Any heteropolymers provided herein may be used for therapeutic purposes. In another aspect, this disclosure provides the use of heteropolymers in the manufacture or preparation of medicaments. In some embodiments, in one such embodiment, said use further includes administering to a subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents). The “subject” according to any of the above embodiments may be a human being.

[0545] In another aspect, pharmaceutical compositions comprising the heteropolymer are provided, for example, for any of the pharmaceutical uses or therapeutic methods described above. In one embodiment, the pharmaceutical composition comprises any heteropolymer provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0546] The heteropolymers (and any other therapeutic agents) disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0547] The heteropolymers disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner. The heteropolymers may be formulated with or without one or more agents currently used for the prevention or treatment of the stated condition. The effective amount of such other agents depends on the amount present in the pharmaceutical composition, the type of condition or treatment, and other factors. These are generally used at the same dosage and route of administration as described herein, or at about 1 to 99% of the dosage described herein, or at other dosages, and at any route determined empirically / clinically as appropriate.

[0548] For the prevention or treatment of disease, the appropriate dosage of the heteropolymer disclosed herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and duration of the disease, whether it is administered for preventive or therapeutic purposes, prior treatment, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is appropriately administered to the patient either once or after a series of treatments.

[0549] Products

[0550] In another aspect of this disclosure, an article of manufacture is provided comprising materials that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article of manufacture (e.g., a kit) comprises a container and a label or package insert on or in conjunction with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container contains a composition, alone or in combination with another composition, that is effective in treating, preventing, and / or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper puncturable by a hypodermic needle). At least one active agent in the composition is a heteropolymer of this disclosure. The label or package insert indicates that the use of the composition is for the treatment of a selected condition. Furthermore, the article of manufacture may comprise: (a) a first container containing a composition comprising the heteropolymer of this disclosure; and (b) a second container containing a composition comprising additional cytotoxic agents or other therapeutic agents. The article of manufacture in this embodiment of the present disclosure may further include a packaging insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer solution. From a commercial and user perspective, it may further include other materials as desired, including other buffers, diluents, filters, needles, and syringes.

[0551] Although the antibodies used in the embodiments target specific antigens, those skilled in the art, guided by the teachings of this disclosure, will understand that the technical effect is not dependent on a specific CDR sequence or a specific antigen sequence, but rather benefits from mutations in the amino acids of the CH3 domain (e.g., at least one amino acid selected from positions 347, 360, 351, 364, 366, 394, 405, 411, 349, 354, 409, 356, and 439) to promote the formation of multispecific antibodies (e.g., bispecific antibodies).

[0552] Example

[0553] The following examples further illustrate this disclosure and should not be construed as limiting the scope of this disclosure. The examples disclosed do not include detailed descriptions of conventional methods. Experimental methods without specific conditions are generally performed under standard conditions, as such methods are well known to those skilled in the art and described in numerous publications, such as Green MR, Sambrook J. *Molecular Cloning* (Cold Spring Harbor Laboratory, 4th, 2012) or Springer Protocols' *Antibody Engineering: Methods and Protocols* (Humana Press, 2018); or under conditions recommended by the raw material or commodity manufacturer; reagents without a specific source are commercially available.

[0554] Example 1.1: Source and preparation of anti-CTLA4 monoclonal antibody Ipilimumab and anti-CCR8 monoclonal antibody CP11H2

[0555] The heavy and light chain amino acid sequences of Ipilimumab are derived from sequences 113 and 114 in patent WO2016059602A2; the heavy and light chain amino acid sequences of CP11H2 are derived from sequences 46 and 47 in patent WO2023208182A1.

[0556] >Ipilimumab heavy chain amino acid sequence

[0557]

[0558] >Ipilimumab light chain amino acid sequence

[0559] >CP11H2 heavy chain amino acid sequence

[0560] >CP11H2 light chain amino acid sequence Note: Italics indicate the antibody constant region.

[0561] DNA encoding the variable regions of the aforementioned antibodies was synthesized. The variable regions of the Ipilimumab heavy chain (Ipilimumab-VH) and light chain (Ipilimumab-VL) were linked to the DNA of the human IgG1 heavy chain constant region and the human Kappa light chain constant region, respectively, to construct the full-length Ipilimumab heavy chain and light chain genes, named Ipilimumab-HC and Ipilimumab-LC, respectively. The variable regions of the CP11H2 heavy chain (CP11H2-VH) and light chain (CP11H2-VL) were linked to the DNA of the human IgG1 heavy chain constant region and the human Kappa light chain constant region, respectively, to construct the full-length CP11H2 heavy chain and light chain genes, named CP11H2-HC and CP11H2-LC, respectively. The heavy and light chain genes were cloned into the expression vector pcDNA3.4, and the expression vectors for both the heavy and light chains of each antibody were simultaneously transfected into Expi293F using PEI (Polyethylenimine). TM The antibody was expressed in cells (Thermo Fisher Scientific, catalog number: A14527). 293 cells were cultured in serum-free medium for approximately 5 days, and the cell supernatant was collected. The antibody was then purified using Protein A affinity chromatography.

[0562] The purification steps are described as follows: High-speed centrifugation was used to remove impurities from the cell culture supernatant. The MabSelect Sure (Cytiva, catalog number: 17543801) affinity chromatography column was washed with 0.2M NaOH, rinsed with pure water, and then equilibrated with PBS. The supernatant was allowed to flow through the column, and the column was washed with PBS until the A280 level returned to baseline. The target protein was eluted with 0.1M acetate buffer (pH 3.5), and the antibody solution was neutralized with 1M Tris-HCl (pH 8.0). The antibody solution was appropriately concentrated using ultrafiltration, and then further purified using a HiLoad Superdex 200 gel chromatography column (Cytiva, catalog number: 28989335). The antibody concentration was determined by UV spectrophotometry. The solution was filtered for sterilization and stored at 4°C. This method is used to purify related monoclonal antibodies, and it can also be used to purify other antibodies or recombinant proteins disclosed herein.

[0563] Example 1.2: Preparation of Ipilimumab and CP11H2 monoclonal mutants

[0564] Site-directed mutagenesis was used to introduce gene mutations into the coding regions of the CH3 domain of the heavy chains of Ipilimumab and CP11H2, mutating amino acid residues at specific positions to specific amino acids. The positions of the amino acid residues were identified using the Eu numbering scheme. Ipilimumab and CP11H2 with gene mutations were prepared according to the expression and purification methods described in Example 1.1. The resulting antibody samples were sterilized by filtration through a 0.2-μm filter, and the antibody concentration was determined using a NanoDrop micro-spectrophotometer. The naming rules for antibody mutants are as follows: for example, Ipilimumab-Q347E indicates that the Q at position 347 of the Ipilimumab heavy chain is mutated to E, and so on, as shown in Table 1B. In recombinant reactions, Ipilimumab and CP11H2 are abbreviated as Ipi and CP11, respectively.

[0565] Table 1B. Maternal monoclonal mutants and corresponding recombination reactions Note: * indicates a recombination reaction between two monoclonal antibodies.

[0566] Example 1.3: Recombinant reaction method for anti-CTLA4 / CCR8 bispecific antibody

[0567] Weigh a certain mass of 2-MEA (Sigma-Aldrich, catalog number / specification: 30078 / 100G), dissolve it in PBS, and adjust the pH to 7.4 so that the molar concentration of 2-MEA in the solution is approximately 750 mM, which serves as the 2-MEA stock solution. Adjust the concentrations of the Ipilimumab mutant and CP11H2 mutant prepared in the above examples to the same molar concentration (3.45 μM-690 μM; corresponding to a mass concentration range of approximately 0.5-100 mg / mL) using PBS. Take an equal volume of Ipilimumab mutant and the corresponding CP11H2 mutant, mix well, and then add an appropriate amount of the 2-MEA stock solution to the antibody mixture above, mixing thoroughly to achieve a final 2-MEA concentration of approximately 75 mM. Incubate this mixture (containing Ipilimumab mutant, CP11H2 mutant, and a certain concentration of 2-MEA) in a water bath at 37°C for approximately 2.5 hours. Transfer the mixture to Slide-A-Lyzer TMIn G3 Dialysis Cassettes (Thermo Fisher Scientific, catalog number: A52971), dialysis was performed in 5 L of PBS. After 5 hours, the PBS was replaced, and the sample was placed in a cold storage (4°C) for complete dialysis. Samples were collected after 18 hours of dialysis, and antibody concentrations were re-determined. The physicochemical properties of the maternal monoclonal antibody and the corresponding recombinant reaction products were determined using HPLC-SEC and IEC.

[0568] Example 1.4: Recombinant reaction efficiency of anti-CTLA4 / CCR8 bispecific antibody

[0569] The physicochemical property analysis methods used in this embodiment are described below.

[0570] 1. High-performance liquid chromatography-size exclusion chromatography (HPLC-SEC)

[0571] Antibodies are high-molecular-weight proteins with highly complex secondary and tertiary structures. Due to post-translational modifications, aggregation, and degradation, antibodies are heterogeneous in their biochemical and biophysical properties. When analyzing bispecific antibodies using separation techniques, variants, aggregates, and degradation fragments are commonly observed, and their presence may compromise safety and efficacy. Aggregates, degradation fragments, and incompletely assembled molecules are prone to occur during antibody production and storage. This disclosure uses HPLC-SEC to detect the levels of these impurities in samples. Aggregates have a larger molecular weight than monomers, resulting in shorter retention times for their corresponding peaks; degradation fragments or incompletely assembled molecules have smaller molecular weights than monomers, resulting in longer retention times for their corresponding peaks.

[0572] The HPLC-SEC instrument used was a Waters e2695-2489. The mobile phase was prepared as follows: Weigh 3.06 g NaH₂PO₄, 3.48 g Na₂HPO₄, and 17.53 g NaCl, dissolve them in an appropriate amount of purified water, and continue adding purified water to a total volume of 1 L. Adjust the pH to 6.8 with 2M NaOH solution. The chromatographic column was a Waters XBridge BEH 200A SEC, with dimensions of 7.8 × 300 mm and a diameter of 3.5 μm. The injection volume was set to 30 μg, the flow rate to 0.5 mL / min, the elution time to 30 min, the column temperature to 30 °C, the sample chamber temperature to 15 °C, and the detection wavelength to 280 nm.

[0573] 2. High-performance liquid chromatography-ion exchange chromatography (HPLC-IEC)

[0574] Many post-translational modifications of proteins (such as N-glycosylation, C-terminal lysine residue modification, N-terminal glutamine or glutamate cyclization, asparagine deamidation, aspartic acid isomerization, and amino acid residue oxidation) can directly or indirectly cause changes in the surface charge of antibodies, leading to charge heterogeneity. Charge variants can be separated and analyzed based on their charge, with commonly used analytical methods including cation exchange chromatography (CEX) and anion exchange chromatography (AEX). When analyzed using chromatographic methods, acidic and basic species are defined based on their retention times relative to the main peak. Acidic species are variants eluted from the main peak earlier than the CEX peak or later than the AEX peak, while basic species are variants eluted from the main peak later than the CEX peak or earlier than the AEX peak. The spectral peaks corresponding to acidic and basic species are called acidic peaks and basic peaks, respectively. Charge variants are easily generated during antibody production and storage. HPLC-IEC was used to analyze the charge heterogeneity of the samples.

[0575] The HPLC-IEC used an Agilent 1260 Infinity IIDAD BIO-LC. Mobile phase A was 20 mM MES, pH 6.0, and mobile phase B was 20 mM MES + 500 mM NaCl, pH 6.0. The mixing ratio of the two mobile phases was changed over time according to a pre-set program, with a flow rate of 0.4 mL / min. The column was a YMC BioPro SP-F, 4.6 × 100 mm 5 μm. The injection volume was set to 30 μg, the column temperature to 30 °C, the sample chamber temperature to 15 °C, and the detection wavelength to 280 nm.

[0576] The HPLC-IEC spectra of the parent monoclonal antibody and the corresponding recombinant reaction product are superimposed. If bispecific antibodies are formed in the recombinant reaction, a new peak corresponding to the bispecific antibody will be generated in the HPLC-IEC spectrum, and the peak at the position corresponding to the parent monoclonal antibody will be relatively reduced. The percentage of the peak area corresponding to the parent monoclonal antibody is the residual amount after the recombinant reaction. The formula for calculating the recombinant reaction efficiency is: 100% - (Percentage of the peak area corresponding to parent monoclonal antibody A + Percentage of the peak area corresponding to parent monoclonal antibody B).

[0577] Table 2. SEC purity and recombination efficiency of maternal monoclonal mutant combinations and recombinant reaction products.

[0578] Table 2 shows that the recombination reaction efficiency of CP11-Q347K*Ipi-Q347E+K360E was only 4.1%. The HPLC-IEC chromatograms of the recombination products showed obvious residual peaks corresponding to the parent monoclonal antibody that did not participate in the recombination reaction, indicating that these two combinations are unlikely to produce bispecific antibodies under the conditions disclosed in this paper. In Table 2, multiple mutant combinations with additional mutations introduced based on the CP11-Q347K and Ipi-Q347E+K360E mutants (including recombination reactions corresponding to numbers 2, 3, 4, 5, and 6 in Table 2) all had recombination reaction efficiencies higher than 95%. Furthermore, the recombination efficiencies of the following reactions are all higher than 95%: CP11-Q347R+L351I*Ipi-Q347E+K360E+L351I (recombination reaction corresponding to number 7 in Table 2), CP11-Q347R+L351I*Ipi-Q347D+K360D+L351I (recombination reaction corresponding to number 8 in Table 2), CP11-K439E+Q347K+L351I*Ipi-D356K+Q347E+K360E+L351I (recombination reaction corresponding to number 9 in Table 2), and CP11-K439E+Q347K+L351M*Ipi-D356K+Q347E+K360E+L351M (recombination reaction corresponding to number 10 in Table 2).

[0579] Example 2.1: Source and preparation of anti-HER2 monoclonal antibodies Trastuzumab and Pertuzumab

[0580] The heavy and light chain amino acid sequences of Trastuzumab are derived from sequences 10 and 11 in patent WO2014096051A1, while the heavy and light chain amino acid sequences of Pertuzumab are derived from sequences 24 and 15 in patent WO2006033700A2.

[0581] Trastuzumab heavy chain amino acid sequence

[0582] Trastuzumab light chain amino acid sequence

[0583] Pertuzumab heavy chain amino acid sequence

[0584] Pertuzumab light chain amino acid sequence Note: Italics indicate the antibody constant region.

[0585] DNA encoding the variable regions of the aforementioned antibodies was synthesized. The Trastuzumab heavy chain variable region (Trastuzumab-VH) and light chain variable region (Trastuzumab-VL) were linked to the human IgG1 heavy chain constant region and the human Kappa light chain constant region DNA, respectively, to construct full-length Trastuzumab heavy chain and light chain genes, named Trastuzumab-HC and Trastuzumab-LC, respectively. The Pertuzumab heavy chain variable region (Pertuzumab-VH) and light chain variable region (Pertuzumab-VL) were linked to the human IgG1 heavy chain constant region and the human Kappa light chain constant region DNA, respectively, to construct full-length Pertuzumab heavy chain and light chain genes, named Pertuzumab-HC and Pertuzumab-LC, respectively. The above heavy chain and light chain genes were cloned into the expression vector pcDNA3.4, respectively. The antibodies were expressed and purified using the method described in Example 1.1.

[0586] Example 2.2: Preparation of Trastuzumab and Pertuzumab monoclonal mutants

[0587] Site-directed mutagenesis was used to introduce gene mutations into the coding regions of the CH3 domain of the heavy chains of Trastuzumab and Pertuzumab, mutating amino acid residues at specific positions to specific amino acids. The positions of the amino acid residues were identified using the Eu numbering scheme. Mutated Trastuzumab and Pertuzumab were prepared according to the expression and purification methods described in Example 2.1. The resulting antibody samples were sterilized by filtration through a 0.2-μm filter, and the antibody concentration was determined using a NanoDrop micro-spectrophotometer. The naming rules for antibody mutants are as follows: for example, Trastuzumab-Q347K indicates that the Q at position 347 of the Trastuzumab heavy chain is mutated to K, and so on, as shown in Table 3. In recombinant reactions, Trastuzumab and Pertuzumab are abbreviated as Tra and Per, respectively.

[0588] Table 3. Maternal monoclonal mutants and corresponding recombination reactions Note: * indicates a recombination reaction between two maternal monoclonal mutants under specific conditions.

[0589] Example 2.3: Preparation method of anti-HER2 biepisode bispecific antibody

[0590] Weigh a certain mass of 2-MEA (Sigma-Aldrich, catalog number / specification: 30078 / 100G), dissolve it in PBS, and adjust the pH to 7.4 so that the molar concentration of 2-MEA in the solution is approximately 750 mM, which serves as the 2-MEA stock solution. Adjust the concentrations of the Trastuzumab mutant and Pertuzumab mutant prepared in the above examples to equal molar concentrations (3.45 μM-690 μM; corresponding to a mass concentration range of approximately 0.5-100 mg / mL) using PBS. Take equal volumes of Trastuzumab mutant and the corresponding Pertuzumab mutant, mix them thoroughly, and then add an appropriate amount of the 2-MEA stock solution to the antibody mixture above, mixing thoroughly to achieve a final 2-MEA concentration of 75 mM. Incubate this mixture (containing Trastuzumab mutant, Pertuzumab mutant, and a certain concentration of 2-MEA) in a water bath at 37°C for 2.5 h. Transfer the mixture to Slide-A-Lyzer TM In G3 Dialysis Cassettes (Thermo Fisher Scientific, catalog number: A52971), dialysis was performed in 5L of PBS. After 5 hours, the PBS was replaced and the sample was placed in a cold storage (4°C) for complete dialysis. After 18 hours of dialysis, the sample was collected and the antibody concentration was re-measured.

[0591] Example 2.4: Recombinant reaction efficiency of anti-HER2 biepisode bispecific antibody

[0592] In this embodiment, the physicochemical properties of the maternal monoclonal antibody and the above-mentioned recombinant reaction products were determined using HPLC-SEC and HPLC-IEC. The physicochemical analysis methods used here, such as HPLC-SEC, HPLC-IEC, and mass spectrometry, are as described in Example 1.4.

[0593] Since IEC cannot effectively separate and analyze multiple components in specific recombinant reaction products, reversed-phase liquid chromatography (HPLC-RP) was used to analyze specific recombinant reaction products, and the recombinant reaction efficiency was calculated based on the results. The experimental method of HPLC-RP is described as follows: the chromatograph used was an Agilent 1290UPLC DAD; the mobile phase preparation method was as follows: mobile phase A was 0.1% trifluoroacetic acid aqueous solution, mobile phase B was acetonitrile, gradient elution B%: 0-22 min (32%~42%); 25 min (80%); 25-28 min (80%); injection volume: 30 μg; chromatographic column: Sepax proteomix-RP-1000, 4.6×150 mm 5.0 μM; flow rate: 0.5 mL / min, elution time: 30 min; column temperature: 75℃, sample chamber temperature: 15℃; detection wavelength: 280 nm.

[0594] The HPLC-RP spectra of the parent monoclonal antibody and the corresponding recombinant reaction product are superimposed. If bispecific antibodies are formed in the recombinant reaction, a new peak corresponding to the bispecific antibody will be generated in the HPLC-RP spectrum, and the peak at the position corresponding to the parent monoclonal antibody will be relatively reduced. The percentage of the peak area corresponding to the parent monoclonal antibody is the residual amount after the recombinant reaction. The formula for calculating the recombinant reaction efficiency is: 100% - (Percentage of the peak area corresponding to parent monoclonal antibody A + Percentage of the peak area corresponding to parent monoclonal antibody B).

[0595] Table 4. SEC purity and recombination efficiency of maternal monoclonal mutant combinations and recombinant reaction products

[0596] Table 4 shows that the recombination reaction efficiency of Tra-Q347K*Per-Q347E+K360E was only 5.1%. The HPLC-IEC chromatograms of the recombination products showed obvious residual peaks corresponding to the parent monoclonal antibody that did not participate in the recombination reaction, indicating that these two combinations were unlikely to produce bispecific antibodies under the conditions described in Table 4. In Table 4, the recombination reaction efficiencies of the other mutant combinations introduced with additional mutations based on the Tra-Q347K and Per-Q347E+K360E mutants (including the recombination reactions corresponding to numbers 2, 3, 4, 5, 6, 7, 8, 9, and 10 in Table 4) were all higher than 95%. Furthermore, the recombination efficiencies of the recombination reactions Tra-Q347R+L351I*Per-Q347E+K360E+L351I (recognition reaction corresponding to serial number 11 in Table 4), Tra-Q347R+L351I*Per-Q347D+K360D+L351I (recognition reaction corresponding to serial number 12 in Table 4), Tra-Q347E+K360E+L351I*Per-Q347K+L351I (recognition reaction corresponding to serial number 13 in Table 4), and Tra-E356K+Q347E+K360E+L351I*Per-K439E+Q347K+L351I (recognition reaction corresponding to serial number 14 in Table 4) were all higher than 95%.

[0597] Example 3: DSC determination of the thermodynamic stability of the Fc segment

[0598] The heavy chain constant region of ipilimumab is human wild-type IgG1. The DNA sequence corresponding to the amino acid sequence of its hinge region and Fc segment was cloned into the expression vector pcDNA3.4. The K at position 447 of the Fc terminal was deleted by conventional molecular cloning methods (to reduce the charge heterogeneity of the product to facilitate purification and physicochemical analysis). The Fc fragment carrying the hinge region was expressed and purified using the method described in Example 1.1 and named IgG1-Fc (SEQ ID NO: 9).

[0599] >IgG1-Fc amino acid sequence carrying the hinge region

[0600] DNA encoding the hinge region and the human IgG4 Fc fragment was synthesized and cloned into the expression vector pcDNA3.4. The K at the C-terminus was deleted using conventional molecular cloning methods (to reduce the charge heterogeneity of the product to facilitate purification and physicochemical analysis). The Fc fragment carrying the hinge region was expressed and purified using the method described in Example 1.1 and named IgG4-Fc (SEQ ID NO: 10).

[0601] >IgG4-Fc amino acid sequence carrying the hinge region

[0602] A site-directed mutagenesis method was used to introduce a gene mutation into the CH3 domain of IgG1-Fc, mutating an amino acid residue at a specific position to a specific amino acid. The positions of the amino acid residues were identified using the Eu numbering scheme. Fc mutants carrying the specific mutation were prepared according to the expression and purification methods described in Example 1.1. Then, two different Fc mutants were subjected to a recombination reaction according to the methods described in the above examples to prepare recombinant Fc mutants (as shown in Table 5).

[0603] Differential scanning calorimetry (DSC) is an analytical technique used to study the thermal stability of proteins. This method measures the thermal behavior of proteins when heated, particularly the heat released or absorbed when their structure changes. DSC provides crucial information about protein folding and stability. DSC works by comparing the heat flow difference between a sample and a reference material at the same heating rate. When a protein denatures (e.g., changes from an ordered structure to a disordered structure), it absorbs heat. This endothermic process can be detected by DSC and used to calculate the protein's thermal stability parameters. Through DSC, scientists can determine the protein's melting point (Tm), the temperature at which the protein begins to denature. The melting point of a protein is directly related to its thermal stability; a higher melting point indicates better thermal stability. Here, DSC is used to determine the Tm of the aforementioned recombinant Fc mutant to assess its thermodynamic stability.

[0604] The DSC experimental method is described as follows: The DSC instrument used was a MicroCal VP-Capillary DSC; the injection volume was 500 μL, with the sterile filtered sample diluted to 1 mg / mL using PBS. The detection program settings were: initial temperature 25℃, final temperature 95℃, heating rate 1℃ / min; and injection rate 25 μL / min.

[0605] Table 5. Tm of Fc recombinant mutants Note: NA indicates that Tm2 does not exist.

[0606] Figures 1A to 1G and Table 5 show that IgG1-Fc exhibits good thermal stability, with TmOnset / Tm1 / Tm2 values ​​of 62.59 / 71.07 / 83.04, while only IgG4-Fc showed TmOnset / Tm1 values ​​of 59.96 / 68.44, and IgG4-Fc did not have a higher Tm2 value. The thermal stability of the preferred recombinant Fc mutant disclosed in this paper is significantly better than that of IgG4-Fc and the recombinant Fc-F405L*Fc-K409R. The thermal stability of Fc-D356K+L351I*Fc-K439E+L351I, Fc-Q347K+L351I*Fc-Q347E+K360E+L351I, Fc-K439E+Q347K+L351I*Fc-D356K+Q347E+K360E+L351I and Fc-K439E+Q347K+L351M*Fc-D356K+Q347E+K360E+L351M gradually increases, mainly reflected in the gradual increase of Tm2, ​​which corresponds to the melting point of the CH3 structural domain. The results show that the TmOnset / Tm1 / Tm2 of the recombinant Fc mutants Fc-K439E+Q347K+L351I*Fc-D356K+Q347E+K360E+L351I and Fc-K439E+Q347K+L351M*Fc-D356K+Q347E+K360E+L351M are very similar to, or even better than, IgG1-Fc, indicating that the recombinant Fc carrying these mutations has good thermal stability.

[0607] By directionally mutagenesis of specific amino acid sites in the CH3 domain of the immunoglobulin heavy chain constant region, polypeptide pairs with differentiated charges and interfacial interactions are constructed, thereby weakening the association tendency of homodimers and significantly promoting the efficient formation of heterodimers. Those skilled in the art should understand that although specific Ipilimumab, CP11H2, Trastuzumab, and Pertuzumab antibodies were tested in the examples, the technical effect does not depend on the specific sequence of the antibody variable region or the specific antigen targeted, but rather stems from the altered association preference resulting from the directional modification of the CH3 domain interface. Therefore, it is applicable to any antibody framework containing the CH3 domain.

[0608] Although the invention has been described in detail with the aid of accompanying drawings and examples for clarity of understanding, these descriptions and examples should not be construed as limiting the scope of this disclosure. All patent and scientific literature disclosures cited herein are clearly and fully incorporated by reference.

Claims

1. A method for preparing heteropolymers, comprising the following steps: a) The step of providing a molecule comprising a first polypeptide homopolymer; b) The step of providing a molecule comprising a second polypeptide homopolymer; c) The step of incubating the molecule containing the first polypeptide homopolymer and the molecule containing the second polypeptide homopolymer together under reducing conditions; and d) The step of obtaining a heteropolymer comprising the first polypeptide and the second polypeptide; Both the first polypeptide and the second polypeptide contain a CH3 domain, and the first polypeptide and the second polypeptide differ in at least one mutation site in their CH3 domains; wherein: 1) The CH3 domain of the first polypeptide contains amino acid mutations at positions 439, 347, and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 356, 347, 360, and 351; or 2) The CH3 domain of the first polypeptide contains amino acid mutations at positions 347 and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347, 360, and 351; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 364, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 364; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 366, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 366; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 394, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 394; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 405, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 405; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 411, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 411; or 3) The CH3 domain of the first polypeptide contains amino acid mutations at positions 347 and 349, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347, 360, and 354; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 349, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 364; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 405, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 409. Preferably, 1) The CH3 domain of the first polypeptide contains amino acid mutations at positions 439, 347, and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 356, 347, 360, and 351; or 2) The CH3 domain of the first polypeptide contains amino acid mutations at positions 347 and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347, 360, and 351. Optional, wherein: The amino acid at position 347 is mutated to a residue selected from the following: Lys(K), Arg(R), Glu(E), and Asp(D); and / or The amino acid at position 360 is mutated to a residue selected from Glu (E) and Asp (D); and / or The amino acid mutation at position 351 is selected from the following residues: Met (M), Ile (I), Cys (C), Val (V), Thr (T), and Phe (F); and / or The amino acid at position 364 is mutated to a residue selected from the following: Ala (A), Tyr (Y), Val (V), Thr (T), Leu (L), and Phe (F); and / or The amino acid at position 366 is mutated to a residue selected from the following: Ala (A), Gly (G), Ser (S), Val (V), Leu (L), His (H), and Ile (I); and / or The amino acid mutation at position 394 is selected from the following residues: Ala (A), Ser (S), Phe (F), Cys (C), Val (V), and Asn (N); and / or The amino acid at position 405 is mutated to a residue selected from Tyr(Y), Leu(L), and Thr(T); and / or The amino acid at position 411 is mutated to a residue selected from the following: Tyr (Y), Asn (N), and Leu (L); and / or The amino acid mutation at position 349 is selected from the following residues: Ser(S), Leu(L), Phe(F), Cys(C), Ala(A), Val(V), Thr(T), and Gly(G); and / or The amino acid at position 354 is mutated to a residue selected from the following: Tyr (Y), Cys (C), Phe (F), and Trp (W); and / or The amino acid at position 409 is mutated to a residue selected from the following: Arg(R), Gln(Q), and Asp(D); and / or The amino acid at position 356 is mutated to a residue selected from the following: Lys(K), Arg(R), and His(H); and / or The amino acid at position 439 is mutated to residues selected from the following: Glu (E) and Asp (D); The position of the amino acid was determined according to the EU numbering system.

2. The method for preparing heteropolymers according to claim 1, wherein: 1) The CH3 domain of the first polypeptide contains amino acid mutations at 439E, 347K, and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 356K, 347E, 360E, and 351M; or The first polypeptide has a CH3 domain containing amino acid mutations at 439E, 347K, and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 356K, 347E, 360E, and 351I; or 2) The CH3 domain of the first polypeptide contains amino acid mutations at 347K and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 347E, 360E, and 351M; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 351I; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 364A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 364A; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 366A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 366A; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 394A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 394A; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 394S, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 394S; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 405Y, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 405Y; or The first polypeptide has a CH3 domain containing amino acid mutations of 347K and 411Y, and the second polypeptide has a CH3 domain containing amino acid mutations of 347E, 360E, and 411Y; or 3) The CH3 domain of the first polypeptide contains amino acid mutations at 347K and 349S, and the CH3 domain of the second polypeptide contains amino acid mutations at 347E, 360E, and 354Y; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 349S, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 364Y; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 405L, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 409R. Preferably, 1) The CH3 domain of the first polypeptide contains amino acid mutations at 439E, 347K, and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 356K, 347E, 360E, and 351M; or The first polypeptide has a CH3 domain containing amino acid mutations at 439E, 347K, and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 356K, 347E, 360E, and 351I; or 2) The CH3 domain of the first polypeptide contains amino acid mutations at 347K and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 347E, 360E, and 351M; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 351I; or The position of the amino acid was determined according to the EU numbering system.

3. The method for preparing heteropolymers according to claim 1 or 2, wherein: The CH3 domains of the first and second polypeptides are each independently derived from the CH3 domains of IgG1, IgG2, IgG3, or IgG4. Preferably, the CH3 domains of the first and second polypeptides are derived from the CH3 domains of human IgG1.

4. The method for preparing heteropolymers according to any one of claims 1 to 3, wherein: The molecule containing the first polypeptide homopolymer is a first parent antibody; and / or The molecule containing the second polypeptide homopolymer is the second parent antibody.

5. The method for preparing heteropolymers according to any one of claims 1 to 4, wherein the reduction conditions comprise the addition of 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-mercaptoethanol and their chemical derivatives; Preferably, the reduction conditions include the addition of one or more reducing agents selected from 2-MEA, glutathione, L-cysteine, dithiothreitol, β-mercaptoethanol and TCEP; More preferably, the reduction conditions include the addition of reducing agent 2-MEA.

6. The method for preparing a heteropolymer according to any one of claims 1 to 5, wherein the heteropolymer comprises a core hinge region.

7. The method for preparing heteropolymers according to any one of claims 1 to 6, wherein the heteropolymer is a multispecific antibody or a heteroFc fusion protein.

8. A heteropolymer comprising a first polypeptide and a second polypeptide, wherein each of the first polypeptide and the second polypeptide comprises a CH3 domain, wherein: 1) The CH3 domain of the first polypeptide contains amino acid mutations at positions 439, 347, and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 356, 347, 360, and 351; or 2) The CH3 domain of the first polypeptide contains amino acid mutations at positions 347 and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347, 360, and 351; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 364, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 364; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 366, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 366; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 394, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 394; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 405, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 405; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 411, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 411; or 3) The CH3 domain of the first polypeptide contains amino acid mutations at positions 347 and 349, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347, 360, and 354; or The first polypeptide has a CH3 domain containing amino acid mutations at positions 347 and 349, and the second polypeptide has a CH3 domain containing amino acid mutations at positions 347, 360, and 364; or The CH3 domain of the first polypeptide contains amino acid mutations at positions 347 and 405, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347, 360, and 409. Preferably, 1) The CH3 domain of the first polypeptide contains amino acid mutations at positions 439, 347, and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 356, 347, 360, and 351; or 2) The CH3 domain of the first polypeptide contains amino acid mutations at positions 347 and 351, and the CH3 domain of the second polypeptide contains amino acid mutations at positions 347, 360, and 351. Optional, wherein: The amino acid at position 347 is mutated to a residue selected from the following: Lys(K), Arg(R), Glu(E), and Asp(D); and / or The amino acid at position 360 is mutated to a residue selected from Glu (E) and Asp (D); and / or The amino acid mutation at position 351 is selected from the following residues: Met (M), Ile (I), Cys (C), Val (V), Thr (T), and Phe (F); and / or The amino acid at position 364 is mutated to a residue selected from the following: Ala (A), Tyr (Y), Val (V), Thr (T), Leu (L), and Phe (F); and / or The amino acid at position 366 is mutated to a residue selected from the following: Ala (A), Gly (G), Ser (S), Val (V), Leu (L), His (H), and Ile (I); and / or The amino acid mutation at position 394 is selected from the following residues: Ala (A), Ser (S), Phe (F), Cys (C), Val (V), and Asn (N); and / or The amino acid at position 405 is mutated to a residue selected from Tyr(Y), Leu(L), and Thr(T); and / or The amino acid at position 411 is mutated to a residue selected from the following: Tyr (Y), Asn (N), and Leu (L); and / or The amino acid mutation at position 349 is selected from the following residues: Ser(S), Leu(L), Phe(F), Cys(C), Ala(A), Val(V), Thr(T), and Gly(G); and / or The amino acid at position 354 is mutated to a residue selected from the following: Tyr (Y), Cys (C), Phe (F), and Trp (W); and / or The amino acid at position 409 is mutated to a residue selected from the following: Arg(R), Gln(Q), and Asp(D); and / or The amino acid at position 356 is mutated to a residue selected from the following: Lys(K), Arg(R), and His(H); and / or The amino acid at position 439 is mutated to residues selected from the following: Glu (E) and Asp (D); The position of the amino acid was determined according to the EU numbering system.

9. The heteropolymer according to claim 8, wherein: 1) The CH3 domain of the first polypeptide contains amino acid mutations at 439E, 347K, and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 356K, 347E, 360E, and 351M; or The first polypeptide has a CH3 domain containing amino acid mutations at 439E, 347K, and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 356K, 347E, 360E, and 351I; or 2) The CH3 domain of the first polypeptide contains amino acid mutations at 347K and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 347E, 360E, and 351M; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 351I; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 364A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 364A; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 366A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 366A; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 394A, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 394A; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 394S, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 394S; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 405Y, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 405Y; or The first polypeptide has a CH3 domain containing amino acid mutations of 347K and 411Y, and the second polypeptide has a CH3 domain containing amino acid mutations of 347E, 360E, and 411Y; or 3) The CH3 domain of the first polypeptide contains amino acid mutations at 347K and 349S, and the CH3 domain of the second polypeptide contains amino acid mutations at 347E, 360E, and 354Y; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 349S, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 364Y; or The first polypeptide has a CH3 domain containing amino acid mutations at 347K and 405L, and the second polypeptide has a CH3 domain containing amino acid mutations at 347E, 360E, and 409R. Preferably, 1) The CH3 domain of the first polypeptide contains amino acid mutations at 439E, 347K, and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 356K, 347E, 360E, and 351M; or The first polypeptide has a CH3 domain containing amino acid mutations at 439E, 347K, and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations at 356K, 347E, 360E, and 351I; or 2) The CH3 domain of the first polypeptide contains amino acid mutations at 347K and 351M, and the CH3 domain of the second polypeptide contains amino acid mutations at 347E, 360E, and 351M; or The first polypeptide has a CH3 domain containing amino acid mutations of 347K and 351I, and the second polypeptide has a CH3 domain containing amino acid mutations of 347E, 360E, and 351I. The position of the amino acid was determined according to the EU numbering system.

10. The heteropolymer according to claim 8 or 9, wherein: The CH3 domains of the first and second polypeptides are each independently derived from the CH3 domains of IgG1, IgG2, IgG3, or IgG4. Preferably, the CH3 domains of the first and second polypeptides are derived from the CH3 domains of human IgG1.

11. The heteropolymer according to any one of claims 8 to 10, wherein the heteropolymer is a multispecific antibody or a heteroFc fusion protein.

12. A pharmaceutical composition comprising: The heteropolymer according to any one of claims 8 to 11; and One or more pharmaceutically acceptable carriers, diluents or excipients.

13. An immunoconjugate comprising: The heteropolymer according to any one of claims 8 to 11; and Payload, in, The payload is coupled to the heteropolymer.

14. A nucleic acid encoding a heteropolymer as described in any one of claims 8 to 11.

15. A host cell comprising the nucleic acid as described in claim 14.

16. A mixture comprising: The molecules comprising the first polypeptide homopolymer and the molecules comprising the second polypeptide homopolymer as described in any one of claims 1 to 4; Preferably, the mixture further comprises a reducing agent.

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