Method for preparing hetero-oligomer and application thereof

Abstract

The present disclosure relates to a method for preparing a hetero-oligomer and an application thereof, and further relates to modifying an amino acid of a CH3 domain so as to promote the formation of a hetero-oligomer.

Description

Method for preparing heteromultimer and use thereof

[0001] This application claims priority to Chinese patent application CN202411384454.3 filed on September 30, 2024 and Chinese patent application CN202411921693.8 filed on December 25, 2024. TECHNICAL FIELD

[0002] The present disclosure belongs to the field of biotechnology, and specifically, the present disclosure relates to a method for preparing heteromultimer (e.g., multispecific antibody) and use thereof, and changing amino acids of CH3 domain to facilitate the formation of heteromultimer (e.g., multispecific antibody). BACKGROUND

[0003] The statements herein are provided only to enhance understanding of the present disclosure and are not necessarily intended to constitute part of the prior art.

[0004] Multispecific antibodies, such as bispecific antibodies, are antibody molecules that can specifically bind two antigens or two epitopes at the same time. Compared with monoclonal antibodies, bispecific antibodies can exert unique mechanisms of action and have significant advantages over monoclonal antibodies.

[0005] Among the bispecific antibody platforms developed by many pharmaceutical companies, the DuoBody platform has a unique antibody engineering principle. Genmab developed the Duobody bispecific antibody preparation platform based on the characteristics of IgG4 that can undergo Fab-arm exchange (FAE) (WO2011131746A2 and WO2013060867A2). Genmab's research showed that mutating lysine at position 409 of IgG1 to arginine (IgG1-K409R) can confer the ability of FAE to IgG1, and mutating phenylalanine at position 405 of IgG1 to leucine (IgG1-F405L) can also confer the ability of FAE to IgG1. IgG1-K409R and IgG1-F405L can undergo FAE under certain reducing conditions to form bispecific antibodies. However, the K409R mutation used by Duobody is derived from human IgG4, and the F405L mutation is derived from rhesus macaque IgG4, and theoretically the stability of the bispecific antibody molecules produced by this platform may not be as good as that of wild-type IgG1 in humans.

[0006] Under normal conditions, mutations on certain CH3 domains are not sufficient to promote dissociation of a monoclonal antibody into half antibodies. For example, the 356 and 439 amino acid residues of the human IgGl CH3 domain are D / E (356 can be either D or E due to different antibody allotypes) and K, respectively, and the D / E356 and K439 between the two CH3 of an antibody form two salt bridges (electrostatic interactions) that can enhance the interaction between CH3. If the D / E at position 356 of IgGl is mutated to K, the resulting monoclonal antibody IgGl-D / E356K will lack two salt bridges; if the K at position 439 of IgGl is mutated to E, the resulting monoclonal antibody IgGl-K439E will also lack two salt bridges. In theory, if the above two monoclonal antibodies can undergo FAE, the two CH3 of the resulting bispecific antibody will reform two salt bridges, and the interaction between the newly formed heterodimerized CH3 will be comparable to that of the wild-type homodimerized CH3. However, the above two monoclonal antibodies cannot effectively undergo FAE between them under normal temperature and reducing conditions (with an efficiency of less than 5%) (WO2015046467A1), which indicates that the interaction between the CH3 carrying the above charge switch mutations is still strong and cannot promote dissociation of the parent monoclonal antibody into half antibodies under mild conditions, and thus FAE cannot effectively occur. SUMMARY

[0007] The disclosure provides a method of making a heteromultimer (e.g., a heterodimer), comprising the steps of:

[0008] a) providing a dimer comprising two first polypeptides and a dimer comprising two second polypeptides;

[0009] b) mixing the dimer comprising two first polypeptides and the dimer comprising two second polypeptides to form a mixture; and

[0010] c) adding a reducing agent and an organic solvent to the mixture and incubating;

[0011] d) obtaining a heteromultimer comprising a first polypeptide and a second polypeptide (e.g., a heterodimer comprising one first polypeptide and one second polypeptide);

[0012] wherein the first polypeptide and the second polypeptide each comprise (e.g., each comprise one) CH3 domain, which each comprises one or more mutations that promote heteromerization.

[0013] The disclosure provides a method of making a heteromultimer, comprising the steps of:

[0014] a) providing a molecule comprising a homomer of a first polypeptide and a molecule comprising a homomer of a second polypeptide;

[0015] b) mixing the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer to form a mixture; and

[0016] c) adding a reducing agent and an organic solvent to the mixture and incubating;

[0017] wherein the first polypeptide and the second polypeptide each comprise (e.g., each comprise one) CH3 domain, which each comprise one or more mutations that promote heteromerization.

[0018] A mutation that promotes heteromerization, in the present disclosure, refers to a class of amino acid mutations that are capable of promoting the dissociation of a homomer (e.g., a homodimer) into monomers, and / or promoting the formation of a heteromer (e.g., a heterodimer) from two heteromeric monomers. In some embodiments, the mutation that promotes heteromerization is an amino acid mutation at the CH3 interaction interface. In some embodiments, the mutation that promotes heteromerization is an amino acid mutation at the CH3 interaction interface that is charged. In some embodiments, the mutation that promotes heteromerization is an amino acid mutation at the CH3 interaction interface that is oppositely charged. In some embodiments, the mutation that promotes heteromerization is an amino acid mutation that is not sufficient to promote dissociation of a homomer in the absence of an organic solvent. In some embodiments, the mutation that promotes heteromerization is an amino acid mutation that is not sufficient to promote efficient dissociation of a homomer in the absence of an organic solvent.

[0019] In some embodiments, the mutation that promotes heteromerization is any one of the mutations described in the present disclosure. In some embodiments, the mutation that promotes heteromerization is one or more amino acid mutations selected from positions 347, 356, 360, 362, 368, 392, 398, 400, 411, and 439. In some embodiments, the mutation that promotes heteromerization is one or more amino acid mutations selected from positions 347, 356, 360, 362, 368, 400, and 439.

[0020] In some embodiments, the method of making a heteromer as previously described, further comprises the step of d) obtaining a heteromer (e.g., a heterodimer comprising one first polypeptide and one second polypeptide) comprising the first polypeptide and the second polypeptide. In some embodiments, the step of c) adding a reducing agent and an organic solvent to the mixture and incubating, results in dissociation between monomers of a homomer (e.g., a first polypeptide homomer and a second polypeptide homomer) and recombination of heteromeric monomers (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.

[0021] In some embodiments, the method of making a heteromultimer as previously described, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer bind the same or different antigen or epitope. In some embodiments, the method of making a heteromultimer as previously described, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer bind different antigens or epitopes.

[0022] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first and / or second polypeptide comprises one or more amino acid mutations selected from positions 347, 356, 360, 362, 368, 392, 398, 400, 411, and 439. In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first and / or second polypeptide comprises one or more amino acid mutations selected from positions 347, 356, 360, 362, 368, 400, and 439. In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first and / or second polypeptide comprises one or more amino acid mutations selected from positions 347, 360, 362, 368, 392, 398, 400, and 411.

[0023] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first and / or second polypeptide each comprises one or more amino acid mutations to a charged amino acid.

[0024] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first and / or second polypeptide each comprises one or more amino acid mutations to a charged amino acid.

[0025] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first and second polypeptide each comprises one or more amino acid mutations to a charged amino acid (e.g., a positively charged amino acid, a negatively charged amino acid), and the amino acid at the same position in the CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide are oppositely charged.

[0026] In some embodiments, the method of making a heteromultimer as previously described, wherein the negatively charged amino acid is selected from the group consisting of glutamic acid (E) and aspartic acid (D), and the positively charged amino acid is selected from the group consisting of lysine (K), arginine (R), and histidine (H). In some embodiments, the method of making a heteromultimer as previously described, wherein the negatively charged amino acid is selected from the group consisting of glutamic acid (E) and aspartic acid (D), and the positively charged amino acid is selected from the group consisting of lysine (K) and arginine (R). In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises one or more amino acid mutations selected from the group consisting of lysine (K), arginine (R), and histidine (H), and the second polypeptide comprises one or more amino acid mutations selected from the group consisting of glutamic acid (E) and aspartic acid (D).

[0027] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide and / or the second polypeptide comprises one or more amino acid mutations selected from the group consisting of positions 347, 356, 360, 362, 392, 398, 400, 411, and 439.

[0028] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and / or the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439. In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439.

[0029] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356 to a positively charged amino acid, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439 to a negatively charged amino acid.

[0030] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0031] the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356 to Lys (K), Arg (R), or His (H); and / or

[0032] the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439 to Glu (E) or Asp (D).

[0033] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E.

[0034] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0035] one or more amino acid mutations in the CH3 domain of the first polypeptide are to a charged amino acid,

[0036] one or more amino acid mutations in the CH3 domain of the second polypeptide are to a charged amino acid, and

[0037] the amino acid in the CH3 domain of the first polypeptide and the amino acid in the CH3 domain of the second polypeptide at the same position are oppositely charged, the same position being selected from the group consisting of amino acids at positions 347, 360, 362, 392, 398, 400, and 411.

[0038] In some embodiments, the method of making a heteromultimer as previously described, wherein the charged amino acid is a positively charged amino acid or a negatively charged amino acid, wherein:

[0039] the positively charged amino acid is selected from the group consisting of lysine (K), arginine (R), and histidine (H);

[0040] the negatively charged amino acid is selected from the group consisting of glutamic acid (E) and aspartic acid (D).

[0041] In some embodiments, the method of making a heteromultimer as previously described, 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 viii) below:

[0042] i) 368;

[0043] ii) 347;

[0044] iii) 347 and 360;

[0045] iv) 362 and 400;

[0046] v) 392 and 398;

[0047] vi) 398;

[0048] vii) 392 and 400;

[0049] viii) 400; and

[0050] ix) 400 and 411.

[0051] In some embodiments, the method of making a heteromultimer as previously described, the CH3 domain of the first polypeptide and / or the second polypeptide further comprises an amino acid mutation selected from the group consisting of i) to iii) below:

[0052] i) 368;

[0053] ii) 347;

[0054] iii) 347 and 360; and

[0055] iv) 362 and 400.

[0056] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide and the second polypeptide each comprise an amino acid mutation of group i). In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide and the second polypeptide each comprise an amino acid mutation at position 368. In some embodiments, the method of making a heteromultimer as previously described, wherein the amino acid at position 368 is mutated to Met (M).

[0057] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from any one of groups ii) to viii), and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from any one of groups ii) to viii).

[0058] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from any one of groups ii) to viii), and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from any one of groups ii) to viii), and the amino acid mutation of the CH3 domain of the first polypeptide is a positively charged amino acid, and the amino acid mutation of the CH3 domain of the second polypeptide is a negatively charged amino acid.

[0059] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from any one of groups ii) to viii), and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from any one of groups ii) to viii), and the amino acid mutation of the CH3 domain of the first polypeptide is a negatively charged amino acid, and the amino acid mutation of the CH3 domain of the second polypeptide is a positively charged amino acid.

[0060] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from any of Group ii) to iv), and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from any of Group ii) to iv).

[0061] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from any of Group ii) to iv), and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from any of Group ii) to iv), and the amino acid mutation of the CH3 domain of the first polypeptide is a positively charged amino acid, and the amino acid mutation of the CH3 domain of the second polypeptide is a negatively charged amino acid.

[0062] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from any of Group ii) to iv), and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from any of Group ii) to iv), and the amino acid mutation of the CH3 domain of the first polypeptide is a negatively charged amino acid, and the amino acid mutation of the CH3 domain of the second polypeptide is a positively charged amino acid.

[0063] In some embodiments, the method of making a heteromultimer as previously described, wherein the amino acid at position 347, 360, 362, 392, 398, 400, or 411 is mutated to Lys (K) or Arg (R); and / or

[0064] the amino acid at position 347, 360, 362, 392, 398, 400, or 411 is mutated to Glu (E) or Asp (D).

[0065] In some embodiments, the method of making a heteromultimer as previously described, the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or second polypeptide further comprises one or more same or different amino acid mutations selected from the group consisting of positions 347, 360, 362, 368, 392, 398, 400, and 411.

[0066] In some embodiments, the method of making a heteromultimer as previously described, the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or second polypeptide further comprises one or more same or different amino acid mutations selected from the group consisting of positions 347, 360, 362, 368, and 400.

[0067] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0068] (1) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 347, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 347, and 360; or

[0069] (2) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 347, and 360, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 347; or

[0070] (3) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 368, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 368; or

[0071] (4) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 362, and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 362, and 400.

[0072] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0073] (1) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 347, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 347, and 360; or

[0074] (2) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 347, and 360, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 347; or

[0075] (4) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 362, and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 362, and 400.

[0076] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0077] (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347D / E, and 360D / E; or

[0078] (2) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K, 347D / E, and 360D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 347K / R; or

[0079] (3) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 368M, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 368M; or

[0080] (4) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K, 362D / E, and 400D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 362K / R, and 400K / R; or

[0081] (5) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K, 362K / R, and 400K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 362D / E, and 400D / E.

[0082] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0083] (1) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 347K, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 347E, and 360E; or

[0084] (2) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K, 347E, and 360E, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 347K; or

[0085] (3) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 368M, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 368M; or

[0086] (4) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K, 362E, and 400E, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 362K, and 400K; or

[0087] (5) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K, 362K, and 400K, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 362E, and 400E.

[0088] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0089] (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347E and 360E; or

[0090] (2) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 347E and 360E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 347K; or

[0091] (3) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368M, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368M; or

[0092] (4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362E and 400E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362K and 400K.

[0093] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0094] (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347E and 360E; or

[0095] (2) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 347E and 360E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 347K; or

[0096] (4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362E and 400E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362K and 400K.

[0097] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0098] (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347E and 360E; or

[0099] (2) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 347E and 360E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 347K.

[0100] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0101] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 392, and 398, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 398; or

[0102] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 398, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 392, and 398; or

[0103] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 392, and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 400; or

[0104] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 392, and 400; or

[0105] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 400, and 411, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 400, and 411; or

[0106] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 362, and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 362, and 400.

[0107] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domains of the first and second polypeptides are derived from CH3 domains of IgG. In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domains of the first and second polypeptides are derived from CH3 domains of a native IgGl, IgG2, IgG3, or IgG4 isotype. In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domains of the first and second polypeptides are derived from CH3 domains of a native IgGl, IgG2, or IgG3 isotype. In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domains of the first and second polypeptides are derived from CH3 domains of IgGl. In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domains of the first and second polypeptides are derived from CH3 domains of human IgGl. In some embodiments, the human IgGl has an amino acid sequence as set forth in SEQ ID NO: 5, 6, or 7.

[0108] In some embodiments, the method of making a heteromultimer as previously described, wherein the amino acid mutations in the CH3 domains of the first and second polypeptides are mutations made to a CH3 domain of a native IgGl, IgG2, IgG3, or IgG4. In some embodiments, the method of making a heteromultimer as previously described, wherein the amino acid mutations in the CH3 domains of the first and second polypeptides are mutations made to a CH3 domain of a native human IgGl. In some embodiments, the method of making a heteromultimer as previously described, wherein the amino acid mutations in the CH3 domains of the first and second polypeptides are mutations made to SEQ ID NO: 5, 6, or 7. In some embodiments, the method of making a heteromultimer as previously described, wherein the amino acid sequence of the native human IgGl is as set forth in SEQ ID NO: 5, 6, or 7. In some embodiments, the method of making a heteromultimer as previously described, wherein the sequence of the CH3 domain of the native human IgGl is as set forth in SEQ ID NO: 32 or 33.

[0109] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0110] (1) the CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Q347K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E, Q347D / E, and K360D / E; or

[0111] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q347D / E, and K360D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K / R; or

[0112] (3) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L368M; or

[0113] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362D / E, and S400D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K / R, and S400K / R; or

[0114] (5) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362K / R, and S400K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362D / E, and S400D / E.

[0115] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0116] (1) the CH3 domain of the first polypeptide comprises amino acid mutation D / E356K, and the CH3 domain of the second polypeptide comprises amino acid mutation K439E; or

[0117] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0118] (3) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0119] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L368M; or

[0120] (5) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K; or

[0121] (6) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362K, and S400K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362E, and S400E.

[0122] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0123] (1) the CH3 domain of the first polypeptide comprises amino acid mutation D / E356K, and the CH3 domain of the second polypeptide comprises amino acid mutation K439E; or

[0124] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0125] (3) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0126] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L368M; or

[0127] (5) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K.

[0128] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0129] the CH3 domain of the first polypeptide comprises amino acid mutation D / E356K, and the CH3 domain of the second polypeptide comprises amino acid mutation K439E.

[0130] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0131] (1) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0132] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0133] (3) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L368M; or

[0134] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K.

[0135] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0136] (1) the CH3 domain of the first polypeptide comprises amino acid mutations D356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0137] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0138] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K.

[0139] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation D356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation K439E.

[0140] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0141] (1) the CH3 domain of the first polypeptide comprises amino acid mutations D356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0142] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0143] (3) the CH3 domain of the first polypeptide comprises amino acid mutations D356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L368M; or

[0144] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K.

[0145] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises amino acid mutation E356K, and the CH3 domain of the second polypeptide comprises amino acid mutation K439E.

[0146] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0147] (1) the CH3 domain of the first polypeptide comprises amino acid mutations E356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0148] (2) the CH3 domain of the first polypeptide comprises amino acid mutations E356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0149] (3) the CH3 domain of the first polypeptide comprises amino acid mutations E356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L368M; or

[0150] (4) the CH3 domain of the first polypeptide comprises amino acid mutations E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K.

[0151] In some embodiments, the CH3 domain mutation sites are denoted by EU numbering. In this disclosure, unless otherwise specified, CH3 domain mutation sites are denoted by EU numbering.

[0152] In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, wherein the first polypeptide homomer and the second polypeptide homomer each comprise a hinge region. In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, wherein the first polypeptide homomer and the second polypeptide homomer each comprise a Cys-Pro-Pro-Cys sequence in the hinge region.

[0153] In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, wherein the reducing agent is sufficient to permit reduction of an interchain disulfide bond in the hinge region.

[0154] In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, wherein the reducing agent is selected from one or more of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, D-cysteine, and β-mercapto-ethanol and chemical derivatives thereof. In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, wherein the reducing agent is selected from one or more of 2-MEA, glutathione, L-cysteine, dithiothreitol, β-mercaptoethanol, and TCEP. In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, wherein the reducing agent is 2-MEA.

[0155] In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, wherein the final concentration of the reducing agent is between 0.1 mM and 1 M. In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, wherein the final concentration of the reducing agent is between 1 mM and 1 M. In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, wherein the final concentration of the reducing agent is between 5 mM and 500 mM. In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, the final concentration of the reducing agent is between 5 mM and 200 mM. In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, the final concentration of the reducing agent is between 25 mM and 100 mM. In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, the final concentration of the reducing agent is between 60 mM and 90 mM. In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, the final concentration of the reducing agent is between 70 mM and 80 mM. In some embodiments, the method of making a heteromultimer as in any of the preceding clauses, the final concentration of the reducing agent is 75 mM.

[0156] In some embodiments, the method of preparing a heteromultimer as previously described, the final concentration of the reducing agent is about 0.1 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, about 400 mM, about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM, about 460 mM, about 470 mM, about 480 mM, about 490 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, about 800 mM, about 850 mM, about 900 mM, about 950 mM, or about 1 M, or any range between these point values.

[0157] In some embodiments, the method of preparing a heteromultimer as previously described, wherein the organic solvent is selected from one or more of a carboxylic acid, an alcohol, a ketone, an ether, an ester, an alkane, an amine, an aromatic hydrocarbon (e.g., benzene or cumene), and dimethyl sulfoxide. In some embodiments, the method of preparing a heteromultimer as previously described, wherein the carboxylic acid is selected from formic acid and acetic acid, the alcohol is selected from ethanol, 1-pentanol, 1-propanol, 2-propanol, n-butanol, 2-butanol, 2-methyl-1-propanol, and 3-methyl-1-butanol, the ketone is selected from acetone, methyl ethyl ketone, and methyl isobutyl ketone, the ether is selected from anisole, t-butyl methyl ether, and diethyl ether, the ester is selected from butyl acetate, isobutyl acetate, isopropyl acetate, methyl acetate, ethyl acetate, ethyl formate, and propyl acetate, the alkane is selected from heptane and pentane, and the aromatic hydrocarbon is cumene.

[0158] In some embodiments, the organic solvent described herein has amphiphilic properties. In some embodiments, the organic solvent described herein is infinitely miscible with water. In some embodiments, the organic solvent described herein can weaken the interaction between CH3 domains in a homomultimer. In some embodiments, the organic solvent described herein can facilitate the dissociation of a homomultimer.

[0159] In some embodiments, the method of making a heteromultimer as previously described, wherein the organic solvent is selected from the group consisting of polar organic solvents. In some embodiments, the method of making a heteromultimer as previously described, wherein the organic solvent is selected from one or more of a nitrile, an alcohol, a ketone, an ether, an amine, an amide, dimethylsulfoxide, carbon disulfide, 1,4-dioxane, and pyridine. In some embodiments, the method of making a heteromultimer as previously described, wherein the organic solvent is selected from a nitrile, an alcohol, a ketone, an ether, an amine, an amide, dimethylsulfoxide, carbon disulfide, 1,4-dioxane, and pyridine. In some embodiments, the method of making a heteromultimer as previously described, wherein the nitrile is selected from acetonitrile, propionitrile, and succinonitrile, the alcohol is selected from ethanol, propanol, isopropanol, and ethylene glycol, the ether is selected from tetrahydrofuran, diethyl ether, and ethylene glycol dimethyl ether, the ketone is selected from acetone and butanone, and the amide is selected from dimethylformamide and dimethylacetamide.

[0160] In some embodiments, the method of making a heteromultimer as previously described, wherein the organic solvent is selected from a nitrile and an alcohol. In some embodiments, the method of making a heteromultimer as previously described, wherein the nitrile is acetonitrile or propionitrile and the alcohol is ethanol, propanol, or isopropanol. In some embodiments, the method of making a heteromultimer as previously described, wherein the nitrile is selected from acetonitrile and the alcohol is selected from ethanol or isopropanol. In some embodiments, the method of making a heteromultimer as previously described, wherein the organic solvent is selected from one or more of acetonitrile, ethanol, and isopropanol. In some embodiments, the method of making a heteromultimer as previously described, wherein the organic solvent is acetonitrile, ethanol, or isopropanol. In some embodiments, the method of making a heteromultimer as previously described, wherein the organic solvent is acetonitrile.

[0161] In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is between 1% and 100%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is between 1% and 30%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is between 1% and 20%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is between 1% and 10%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is between 4% and 10%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is between 5% and 10%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is less than or equal to 10%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is greater than or equal to 4%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is greater than or equal to 6%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is between 6% and 10%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is between 8% and 10%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is between 5% and 7%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is between 6% and 8%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is about 6%, about 7%, about 8%, about 9%, about 10%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is about 6%, about 8%, about 10%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is about 6%, about 8%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is 6%, 8%, 10%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is 6%, 8%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is 6% ± 1%. In some embodiments, the method of making a heteromultimer as previously described, wherein the final volume fraction of the organic solvent is 6%.

[0162] In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of molecules comprising a first polypeptide homomer to molecules comprising a second polypeptide homomer in the mixture of step b) is about 1 :2 to about 2: 1. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of molecules comprising a first polypeptide homomer to molecules comprising a second polypeptide homomer in the mixture of step b) is about 1 :2 to about 1 : 1. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of molecules comprising a first polypeptide homomer to molecules comprising a second polypeptide homomer in the mixture of step b) is about 1 : 1 to about 2: 1. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of molecules comprising a first polypeptide homomer to molecules comprising a second polypeptide homomer in the mixture of step b) is about 1 : 1.5 to about 1.5: 1. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of molecules comprising a first polypeptide homomer to molecules comprising a second polypeptide homomer in the mixture of step b) is about 1 : 1.4 to about 1.4: 1. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of molecules comprising a first polypeptide homomer to molecules comprising a second polypeptide homomer in the mixture of step b) is about 1 : 1.3 to about 1.3: 1. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of molecules comprising a first polypeptide homomer to molecules comprising a second polypeptide homomer in the mixture of step b) is about 1 : 1.2 to about 1.2: 1. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of molecules comprising a first polypeptide homomer to molecules comprising a second polypeptide homomer in the mixture of step b) is about 1 : 1.1 to about 1.1: 1.

[0163] In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of the molecules comprising a first polypeptide homomer to the molecules comprising a second polypeptide homomer in the mixture of step b) is about 1 : 1. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of the molecules comprising a first polypeptide homomer to the molecules comprising a second polypeptide homomer in the mixture of step b) is about 1.05: 1. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of the molecules comprising a first polypeptide homomer to the molecules comprising a second polypeptide homomer in the mixture of step b) is from about 1 : 1.03 to about 1 :2. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of the molecules comprising a first polypeptide homomer to the molecules comprising a second polypeptide homomer in the mixture of step b) is from about 1 : 1.1 to about 1 : 1.5. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of the molecules comprising a first polypeptide homomer to the molecules comprising a second polypeptide homomer in the mixture of step b) is from about 1 : 1.1 to about 1 : 1.4. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of the molecules comprising a first polypeptide homomer to the molecules comprising a second polypeptide homomer in the mixture of step b) is from about 1 : 1.5 to about 1 : 1.35. In some embodiments, the method of making a heteromultimer as previously described, wherein the ratio of the molar concentration of the molecules comprising a first polypeptide homomer to the molecules comprising a second polypeptide homomer in the mixture of step b) is from about 1 : 1.2 to about 1 : 1.3.

[0164] In some embodiments, the method of making a heteromultimer as previously described, wherein the molar concentration of the molecules comprising a first polypeptide homomer and the molecules comprising a second polypeptide homomer in the mixture of step b) are the same.

[0165] In some embodiments, the method of making a heteromultimer as previously described, wherein the concentration of the molecules comprising a first polypeptide homomer or the molecules comprising a second polypeptide homomer ranges from 3.45 μΜ to 690 μΜ. In some embodiments, the method of making a heteromultimer as previously described, wherein the concentration of the molecules comprising a first polypeptide homomer or the molecules comprising a second polypeptide homomer ranges from 3.45 μΜ to 345 μΜ. In some embodiments, the method of making a heteromultimer as previously described, wherein the concentration of the molecules comprising a first polypeptide homomer or the molecules comprising a second polypeptide homomer ranges from 6.9 μΜ to 69 μΜ.

[0166] In some embodiments, the method of making a heteromultimer as previously described, wherein the concentration of the amount of substance of the molecule comprising a first multimer of polypeptides or the molecule comprising a second multimer of polypeptides is about 3.45 μΜ, about 3.5 μΜ, about 4 μΜ, about 4.5 μΜ, about 5 μΜ, about 5.5 μΜ, about 6 μΜ, about 6.5 μΜ, about 6.6 μΜ, about 6.7 μΜ, about 6.8 μΜ, about 6.9 μΜ, about 7 μΜ, about 7.1 μΜ, about 7.2 μΜ, about 7.3 μΜ, about 7.4 μΜ, about 7.5 μΜ, about 8 μΜ, about 8.5 μΜ, about 9 μΜ, about 9.5 μΜ, about 10 μΜ, about 15 μΜ, about 20 μΜ, about 25 μΜ, about 30 μΜ, about 35 μΜ, about 40 μΜ, about 45 μΜ, about 50 μΜ, about 55 μΜ, about 60 μΜ, about 65 μΜ, about 66 μΜ, about 67 μΜ, about 68 μΜ, about 69 μΜ, about 70 μΜ, about 71 μΜ, about 72 μΜ, about 73 μΜ, about 74 μΜ, about 75 μΜ, about 80 μΜ, about 85 μΜ, about 90 μΜ, about 95 μΜ, about 100 μΜ, about 110 μΜ, about 120 μΜ, about 130 μΜ, about 140 μΜ, about 150 μΜ, about 160 μΜ, about 170 μΜ, about 180 μΜ, about 190 μΜ, about 200 μΜ, about 210 μΜ, about 220 μΜ, about 230 μΜ, about 240 μΜ, about 250 μΜ, about 260 μΜ, about 270 μΜ, about 280 μΜ, about 290 μΜ, about 300 μΜ, about 310 μΜ, about 320 μΜ, about 330 μΜ, about 340 μΜ, about 345 μΜ, about 350 μΜ, about 360 μΜ, about 370 μΜ, about 380 μΜ, about 390 μΜ, about 400 μΜ, about 410 μΜ, about 420 μΜ, about 430 μΜ, about 440 μΜ, about 450 μΜ, about 460 μΜ, about 470 μΜ, about 480 μΜ, about 490 μΜ, about 500 μΜ, about 510 μΜ, about 520 μΜ, about 530 μΜ, about 540 μΜ, about 550 μΜ, about 560 μΜ, about 570 μΜ, about 580 μΜ, about 590 μΜ, about 600 μΜ, about 610 μΜ, about 620 μΜ, about 630 μΜ, about 640 μΜ, about 650 μΜ, about 660 μΜ, about 670 μΜ, about 680 μΜ, or about 690 μΜ, or any range between these point values.

[0167] In some embodiments, the method of making a heteromultimer as previously described, wherein the concentration of the molecule comprising a first multimer of polypeptides or the molecule comprising a second multimer of polypeptides is between 0.5 mg / mL and 100 mg / mL. In some embodiments, the method of making a heteromultimer as previously described, wherein the concentration of the molecule comprising a first multimer of polypeptides or the molecule comprising a second multimer of polypeptides is between 0.5 mg / mL and 50 mg / mL. In some embodiments, the method of making a heteromultimer as previously described, wherein the concentration of the molecule comprising a first multimer of polypeptides or the molecule comprising a second multimer of polypeptides is between 1 mg / mL and 10 mg / mL.

[0168] In some embodiments, the method of making a heteromultimer as previously described, wherein the concentration of the molecule comprising a first multimer of polypeptides or the molecule comprising a second multimer of polypeptides 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, about 8.5 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 point values.

[0169] In some embodiments, the method of making a heteromultimer as previously described, wherein the incubation temperature of the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer is between 15°C and 40°C. In some embodiments, the method of making a heteromultimer as previously described, wherein the incubation temperature of the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer is between 20°C and 40°C. In some embodiments, the method of making a heteromultimer as previously described, wherein the incubation temperature of the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer is between 25°C and 40°C. In some embodiments, the method of making a heteromultimer as previously described, wherein the incubation temperature of the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer is between 30°C and 40°C. In some embodiments, the method of making a heteromultimer as previously described, wherein the incubation temperature of the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer is between 35°C and 40°C.

[0170] In some embodiments, the method of making a heteromultimer as previously described, wherein the incubation temperature of the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer 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 point values.

[0171] In some embodiments, the method of making a heteromultimer as previously described, wherein the incubation temperature of the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer is about 37°C. In some embodiments, the method of making a heteromultimer as previously described, wherein the incubation temperature of the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer is 37°C.

[0172] In some embodiments, the method of making a heteromultimer as previously described, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are incubated for at least 10 minutes. In some embodiments, the method of making a heteromultimer as previously described, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are incubated for at least 10 minutes to 30 hours. In some embodiments, the method of making a heteromultimer as previously described, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are incubated for at least 10 minutes to 24 hours. In some embodiments, the method of making a heteromultimer as previously described, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are incubated for a time period of 0.5 hours to 3 hours. In some embodiments, the method of making a heteromultimer as previously described, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are incubated for a time period of 1 hour to 3 hours. In some embodiments, the method of making a heteromultimer as previously described, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are incubated for a time period of 2 hours to 3 hours. In some embodiments, the method of making a heteromultimer as previously described, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are incubated for a time period of 2.5 hours.

[0173] In some embodiments, the method of making a heteromultimer as previously described, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are incubated for a time period of 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, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours, or any range between these point values.

[0174] In some embodiments, the method of making a heteromultimer as previously described, the molecule comprising a first polypeptide homomer comprises at least two identical first polypeptides. In some embodiments, the method of making a heteromultimer as previously described, the molecule comprising a second polypeptide homomer comprises at least two identical second polypeptides.

[0175] In some embodiments, the method of making a heteromultimer as previously described, the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer comprise an Fc region, an antibody, a fusion protein comprising an Fc region (e.g., an Fc region fused to a receptor, a cytokine or a hormone), and an Fc region conjugated to a drug (e.g., a peptide or a toxin).

[0176] In some embodiments, the method of making a heteromultimer as previously described, the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer comprise one or more or all other regions of an antibody, i.e., a CHI region, a VH region, a CL region and / or a VL region, in addition to an Fc region. Thus, in one embodiment, the molecule comprising a first polypeptide homomer is a full-length antibody. In another embodiment, the molecule comprising a second polypeptide homomer is a full-length antibody.

[0177] In an important embodiment, the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are both antibodies, preferably full-length antibodies, and bind to different epitopes. In such embodiments, the heteromultimer produced is a bispecific antibody. The epitopes can be on different antigens or on the same antigen.

[0178] However, in other embodiments, only the molecule comprising a first polypeptide homomer is a full-length antibody, and the other molecule comprising a second polypeptide homomer is not a full-length antibody, such as an Fc region devoid of variable regions, expressed with another protein or peptide sequence such as a receptor, a cytokine or a hormone, or conjugated to a prodrug, a peptide, a drug or a toxin. In yet another embodiment, neither the molecule comprising a first polypeptide homomer nor the molecule comprising a second polypeptide homomer is a full-length antibody. For example, both molecules can be Fc regions fused to another protein or peptide sequence (e.g., a receptor, a cytokine or a hormone) or conjugated to a prodrug, a peptide, a drug or a toxin.

[0179] In some embodiments, the method of making a heteromultimer as previously described, the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are independently selected from a monoclonal antibody, a monospecific antibody or a bispecific antibody.

[0180] In some embodiments, the method of making a heteromultimer as previously described, wherein:

[0181] when each of the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer is a monospecific antibody or a monoclonal antibody, the heteromultimer is a bispecific antibody; or

[0182] when the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are a monospecific antibody and a bispecific antibody, respectively, the heteromultimer is a trispecific antibody; or

[0183] when the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are a monospecific antibody and a bispecific antibody, respectively, the heteromultimer is a trispecific antibody.

[0184] In some embodiments, the method of making a heteromultimer as previously described, wherein the heteromultimer is a multispecific antibody or a hetero-Fc fusion protein. In some embodiments, the method of making a heteromultimer as previously described, wherein the heteromultimer is a heterodimer. In some embodiments, the method of making a heteromultimer as previously described, wherein the heteromultimer is a bispecific antibody or a trispecific antibody. In some embodiments, the method of making a heteromultimer as previously described, wherein the heteromultimer is a bispecific antibody.

[0185] In some embodiments, the method of making a heteromultimer as previously described, wherein the molecule comprising a first polypeptide homomer is a first parent antibody, and the molecule comprising a second polypeptide homomer is a second parent antibody. In some embodiments, the method of making a heteromultimer as previously described, the first parent antibody is a first parent monoclonal antibody, and the second parent antibody is a second parent monoclonal antibody. In some embodiments, the method of making a heteromultimer as previously described, 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, the method of making a heteromultimer as previously described, 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.

[0186] In some embodiments, the method of making a heteromultimer as previously described, wherein the first polypeptide homomer is a homomer of two first polypeptides, and the second polypeptide homomer is a homomer of two second polypeptides.

[0187] When a “first polypeptide” is described in the present disclosure, it can also refer to each of the first polypeptides in a first polypeptide homomer. When a “second polypeptide” is described in the present disclosure, it can also refer to each of the second polypeptides in a second polypeptide homomer.

[0188] The first polypeptide and / or the second polypeptide is a polypeptide structure that constitutes a heterodimer, which comprises at least a CH3 domain.

[0189] 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 and / or second Fc region comprises a CH2 and CH3 domain. In some embodiments, the first polypeptide and the second polypeptide comprise a hinge region.

[0190] In some embodiments, the method of making a heteromultimer as previously described, wherein the first polypeptide is an antibody heavy chain, and / or the second polypeptide is an antibody heavy chain. In some embodiments, the heteromultimer further comprises one or more antibody light chains.

[0191] In some embodiments, the first polypeptide and the second polypeptide sequences are different. For example, a molecule comprising two first polypeptides is a first parent antibody, and a molecule comprising two second polypeptides is a second parent antibody. In the present disclosure, the first parent antibody further comprises one or more polypeptides (e.g., light chains that form half-antibodies with the two first polypeptides, respectively). In the present disclosure, the second parent antibody further comprises one or more polypeptides (e.g., light chains that form half-antibodies with the two first polypeptides, respectively).

[0192] The first polypeptide, the second polypeptide, the first parent antibody, or the second parent antibody are used only to distinguish the amino acid sequences, and do not limit the positional relationship of the polypeptides and proteins. 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 a first polypeptide, the other is a second polypeptide.

[0193] In some embodiments, the method of making a heteromultimer as previously described, wherein the heteromultimer comprises a core hinge region. In some embodiments, the method of making a heteromultimer as previously described, wherein the core hinge region of the heteromultimer comprises a Cys-Pro-Pro-Cys sequence. In some embodiments, the method of making a heteromultimer as previously described, wherein the amino acids of the core hinge region of the heteromultimer form a disulfide bond.

[0194] In some embodiments, the method of making a heteromultimer as in any of the preceding embodiments, wherein there is a difference in isoelectric point between the first polypeptide and the second polypeptide. In some embodiments, the method of making a heteromultimer as in any of the preceding embodiments, wherein the amino acid mutation of the disclosure is to confer or increase the difference in isoelectric point between the first polypeptide and the second polypeptide. In some embodiments, the method of making a heteromultimer as in any of the preceding embodiments, wherein the first polypeptide and second polypeptide further comprise additional amino acid mutations to confer or increase the difference in isoelectric point between the first polypeptide and the second polypeptide. In some embodiments, the method of making a heteromultimer as in any of the preceding embodiments, wherein the first polypeptide and second polypeptide further comprise additional amino acid mutations such that there is a difference in isoelectric point between the first polypeptide and the second polypeptide.

[0195] In some embodiments, the method of making a heteromultimer as in any of the preceding embodiments, wherein the first polypeptide and second polypeptide further comprise a variable region. In some embodiments, the method of making a heteromultimer as in any of the preceding embodiments, wherein the first polypeptide and second polypeptide further comprise a VH and a VL. In some embodiments, the method of making a heteromultimer as in any of the preceding embodiments, wherein the VH of the first polypeptide and / or second polypeptide further comprises an amino acid mutation selected from Q105E, Q105R, and Q105K, the mutation site is denoted by KABAT numbering. In some embodiments, the method of making a heteromultimer as in any of the preceding embodiments, wherein the VL of the first polypeptide and / or second polypeptide further comprises an amino acid mutation of K42E, the mutation site is denoted by KABAT numbering.

[0196] In some embodiments, the method of making a heteromultimer as in any of the preceding embodiments, wherein more than 30% (e.g., more than 35%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 85%, more than 88%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%) of the total product is the desired heteromultimer (compared to other products in the total product, e.g., a half-antibody or a homomultimer).

[0197] In some embodiments, the heteromultimer has a higher thermal stability than wild-type IgG1. In some embodiments, the heteromultimer has a Tm that is at least 1 °C / K (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 °C / K) higher than the Fc fragment of wild-type IgG1.

[0198] In some embodiments, the heteromultimer has a higher thermal stability than the thermal stability of the parent antibody (e.g., the first parent antibody, the second parent antibody). In some embodiments, the heteromultimer has a Tm that is at least 1 °C / K (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 °C / K) greater than the Tm of the Fc region of the parent antibody (e.g., the first parent antibody, the second parent antibody).

[0199] In some embodiments, the stability is thermal stability. In some embodiments, the thermal stability comprises a thermodynamic index. In some embodiments, the thermodynamic index is a Tm value.

[0200] In some embodiments, the method of making a heteromultimer as previously described, wherein the heteromultimer has a recombination efficiency of less than 30% (e.g., less than 25%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%) without the addition of an organic solvent. In some embodiments, the method of making a heteromultimer as previously described, wherein the heteromultimer has a recombination efficiency of less than 20% without the addition of an organic solvent. In some embodiments, the method of making a heteromultimer as previously described, wherein the heteromultimer has a recombination efficiency of less than 10% without the addition of an organic solvent. In some embodiments, the method of making a heteromultimer as previously described, wherein the heteromultimer has a recombination efficiency of less than 5% without the addition of an organic solvent.

[0201] In some embodiments, the method of making a heteromultimer as previously described, wherein: step a) further comprises a step of purifying the molecules comprising the first polypeptide homomer and the molecules comprising the second polypeptide homomer. In some embodiments, the purification method comprises, but is not limited to, protein A or protein G chromatography, antigen-binding based affinity chromatography, anti-idiotypic antibody based affinity chromatography, ion exchange, hydrophobic interaction chromatography, mixed mode chromatography (such as hydroxyapatite), immobilized metal affinity chromatography, thiophilic adsorption chromatography, and size exclusion chromatography (SEC), etc. In some embodiments, the purification method is affinity chromatography. In some embodiments, the purification method is protein A chromatography.

[0202] In some embodiments, the method of preparing a heteromultimer as previously described, wherein step c) is followed by a step of removing the reducing agent and the organic solvent. In some embodiments, the method of preparing a heteromultimer as previously described, the reducing agent and the organic solvent are removed by, but not limited to, dialysis, precipitation, chromatography, or filtration. In some embodiments, the method of preparing a heteromultimer as previously described, the reducing agent and the organic solvent are removed by dialysis.

[0203] The step for removing the reducing agent and the organic solvent can in principle be any method that results in or is capable of separating both without impairing the heteromultimer. Such methods include, but are not limited to, dialysis, precipitation, chromatography, or filtration. The step of removing the reducing agent and the organic solvent can be carried out as a continuous process or it can be carried out as a batch process.

[0204] In some embodiments, the method of preparing a heteromultimer as previously described, the content of heteromultimer in the product obtained in step c) exceeds 30% (e.g., exceeds 35%, exceeds 40%, exceeds 50%, exceeds 60%, exceeds 70%, exceeds 80%, exceeds 85%, exceeds 88%, exceeds 90%, exceeds 91%, exceeds 92%, exceeds 93%, exceeds 94%, exceeds 95%, exceeds 96%, exceeds 97%, exceeds 98%, or exceeds 99%). In some embodiments, the method of preparing a heteromultimer as previously described, wherein step d) further comprises a method of purifying the product obtained from step c). In some embodiments, the method of purifying comprises, but is not limited to, protein A or protein G chromatography, antigen-binding based affinity chromatography, anti-idiotypic antibody based affinity chromatography, ion exchange, hydrophobic interaction chromatography, mixed mode chromatography (such as hydroxyapatite), immobilized metal affinity chromatography, thiophilic adsorption chromatography, and size exclusion chromatography (SEC), etc. In some embodiments, the method of purifying is affinity chromatography. In some embodiments, the method of purifying is size exclusion chromatography (SEC).

[0205] In some embodiments, methods can be cited such as: a method in which a cell strain producing a molecule comprising a first polypeptide homomer and a cell strain producing a molecule comprising a second polypeptide homomer are separately cultured, the culture supernatants are purified, and the purified antibodies are used to induce a FAE (Fab arm exchange) reaction; a method in which a cell strain producing a molecule comprising a first polypeptide homomer and a cell strain producing a molecule comprising a second polypeptide homomer are separately cultured, the culture supernatants are mixed without purification, a FAE reaction is induced in the mixed culture supernatant, and then purification is performed; a method in which a cell strain producing a molecule comprising a first polypeptide homomer and a cell strain producing a molecule comprising a second polypeptide homomer are mixed and cultured, the culture supernatant is purified, and the purified antibodies are used to induce a FAE reaction; and a method in which a cell strain producing a molecule comprising a first polypeptide homomer and a cell strain producing a molecule comprising a second polypeptide homomer are mixed and cultured, a FAE reaction is induced in the culture supernatant, and then purification is performed.

[0206] In some embodiments, the present disclosure provides a method of producing a heteromultimer, comprising the steps of:

[0207] a) providing a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer;

[0208] b) mixing the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer to form a mixture; and

[0209] c) adding a reducing agent and an organic solvent to the mixture and incubating;

[0210] d) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide;

[0211] wherein the first polypeptide and the second polypeptide each comprise (e.g., each comprise one) CH3 domain, the CH3 domain each comprising one or more mutations that promote heteromerization.

[0212] In some embodiments, the present disclosure provides a method of producing a heteromultimer, the method comprising the steps of a) to d):

[0213] a) providing a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer;

[0214] b) mixing the molecule comprising the first polypeptide homomer and the molecule comprising the second polypeptide homomer to form a mixture;

[0215] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0216] d) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide,

[0217] wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer bind to different antigens or epitopes, the first and second polypeptides each comprise (e.g., each comprise one) CH3 domain, and the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439.

[0218] In some embodiments, the disclosure provides a method of making a heteromultimer, the method comprising the steps of a) to d) below:

[0219] a) a step of providing a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer;

[0220] b) mixing the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer to form a mixture;

[0221] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0222] d) obtaining a heteromultimer comprising the first and second polypeptides,

[0223] wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer bind to different antigens or epitopes, the first and second polypeptides each comprise (e.g., each comprise one) CH3 domain, and the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E;

[0224] wherein the reducing agent is selected from one or more of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, D-cysteine, and β-mercapto-ethanol, and chemical derivatives thereof, and the final concentration of the reducing agent is 5 mM to 500 mM;

[0225] wherein the organic solvent is acetonitrile, ethanol, or isopropanol, and the final volume fraction of the organic solvent is 6% to 10%.

[0226] In some embodiments, the disclosure provides a method of making a heteromultimer, the method comprising the steps of a) to d) below:

[0227] a) a step of providing a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer;

[0228] b) mixing the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer to form a mixture;

[0229] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0230] d) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide;

[0231] wherein the molecule comprising a homomultimer of the first polypeptide and the molecule comprising a homomultimer of the second polypeptide bind to different antigens or epitopes, the first polypeptide and the second polypeptide each comprise (e.g., each comprise one) CH3 domain, and the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E;

[0232] wherein the reducing agent is selected from one or more of 2-MEA, glutathione, L- cysteine, dithiothreitol, beta-mercaptoethanol, and TCEP, and the final concentration of the reducing agent is 5 mM to 200 mM;

[0233] wherein the organic solvent is acetonitrile, ethanol, or isopropanol, and the final volume fraction of the organic solvent is 6% to 10%.

[0234] In some embodiments, the disclosure provides a method of making a heteromultimer, the method comprising the steps of a) to d):

[0235] a) providing a molecule comprising a homomultimer of a first polypeptide and a molecule comprising a homomultimer of a second polypeptide;

[0236] b) mixing the molecule comprising a homomultimer of the first polypeptide and the molecule comprising a homomultimer of the second polypeptide to form a mixture;

[0237] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0238] d) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide;

[0239] wherein the molecule comprising a homomultimer of the first polypeptide and the molecule comprising a homomultimer of the second polypeptide bind to different antigens or epitopes, the first polypeptide and the second polypeptide each comprise (e.g., each comprise one) CH3 domain, and the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E;

[0240] wherein the reducing agent is 2-MEA, and the final concentration of the reducing agent is 5 mM to 200 mM;

[0241] wherein the organic solvent is acetonitrile, ethanol, or isopropanol, and the final volume fraction of the organic solvent is 6% to 10%.

[0242] In some embodiments, the disclosure provides a method of making a heteromultimer, the method comprising the steps of a) to d):

[0243] a) providing a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer;

[0244] b) mixing the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer to form a mixture;

[0245] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0246] d) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide;

[0247] wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer bind different antigens or epitopes, the first polypeptide and the second polypeptide each comprise (e.g., each comprise one) CH3 domain, and the CH3 domain of the first polypeptide comprises an amino acid mutation of D / E356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of K439E;

[0248] wherein the reducing agent is 2-MEA, and the final concentration of the reducing agent is 5 mM to 200 mM;

[0249] wherein the organic solvent is acetonitrile, ethanol, or isopropanol, and the final volume fraction of the organic solvent is 6% to 10%, preferably 6% to 8%.

[0250] In some embodiments, the disclosure provides a method of making a heteromultimer, the method comprising the steps of a) to d):

[0251] a) providing a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer;

[0252] b) mixing the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer to form a mixture;

[0253] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0254] d) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide;

[0255] wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer bind to different antigens or epitopes, the first polypeptide and the second polypeptide each comprise (e.g., each comprise one) CH3 domain, and the CH3 domain of the first polypeptide comprises an amino acid mutation of D / E356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of K439E;

[0256] wherein the reducing agent is 2-MEA, and the final concentration of the reducing agent is about 75 mM;

[0257] wherein the organic solvent is acetonitrile, ethanol, or isopropanol, and the final volume fraction of the organic solvent is about 6%, about 8%, about 10%.

[0258] In some embodiments, the disclosure provides a method of making a heteromultimer, the method comprising the steps of a) to d):

[0259] a) providing a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer;

[0260] b) mixing the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer to form a mixture;

[0261] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0262] d) obtaining a heteromultimer comprising the first polypeptide and the second polypeptide;

[0263] wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer bind to different antigens or epitopes, the first polypeptide and the second polypeptide each comprise (e.g., each comprise one) CH3 domain, and the CH3 domain of the first polypeptide comprises an amino acid mutation of D / E356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of K439E, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer in the mixture have the same molar concentration;

[0264] wherein the reducing agent is 2-MEA, and the final concentration of the reducing agent is about 75 mM;

[0265] wherein the organic solvent is acetonitrile, ethanol, or isopropanol, and the final volume fraction of the organic solvent is about 6%, about 8%, about 10%.

[0266] In some embodiments, the disclosure provides a method of making a heteromultimer, the method comprising the steps of a) to c):

[0267] a) a step of culturing separately a cell strain producing a molecule comprising a first polypeptide homomer and a cell strain producing a molecule comprising a second polypeptide homomer;

[0268] b) a step of purifying separately the culture supernatant of each cell strain to obtain a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer, and incubating together said molecule comprising a first polypeptide homomer and said molecule comprising a second polypeptide homomer in the presence of a reducing agent and an organic solvent; and

[0269] c) obtaining a heteromultimer comprising said first polypeptide and second polypeptide.

[0270] In some embodiments, the disclosure provides a method of preparing a heteromultimer, the method comprising the steps of a) to c) below:

[0271] a) a step of mixing a cell strain producing a molecule comprising a first polypeptide homomer and a cell strain producing a molecule comprising a second polypeptide homomer;

[0272] b) a step of incubating together said molecule comprising a first polypeptide homomer and said molecule comprising a second polypeptide homomer in the presence of a reducing agent and an organic solvent in the culture supernatant; and

[0273] c) obtaining a heteromultimer comprising said first polypeptide and second polypeptide.

[0274] In some embodiments, the disclosure provides a method of preparing a heteromultimer, the method comprising the steps of a) to c) below:

[0275] a) a step of culturing separately a cell strain producing a molecule comprising a first polypeptide homomer and a cell strain producing a molecule comprising a second polypeptide homomer;

[0276] b) a step of mixing the culture supernatant of each cell strain, and incubating together said molecule comprising a first polypeptide homomer and said molecule comprising a second polypeptide homomer in the presence of a reducing agent and an organic solvent; and

[0277] c) obtaining a heteromultimer comprising said first polypeptide and second polypeptide.

[0278] In another aspect, the disclosure provides a composition for preparing a heteromultimer, comprising: a molecule comprising a first polypeptide homomer, a molecule comprising a second polypeptide homomer, and an organic solvent, according to the foregoing. In some embodiments, the molecule comprising a first polypeptide homomer is a first parent antibody; and / or the molecule comprising a second polypeptide homomer is a second parent antibody. In some embodiments, the organic solvent is selected from one or more of acetonitrile, ethanol, and isopropanol.

[0279] In another aspect, the present disclosure provides a composition comprising: a heteromultimer prepared according to the methods as previously described, and an organic solvent. In some embodiments, the composition as previously described, wherein greater than 30% (e.g., greater than 35%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 85%, greater than 88%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%) is the desired heteromultimer (compared to other products in the total product, such as a half-antibody or homomultimer).

[0280] In another aspect, the present disclosure provides a heteromultimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise (e.g., each comprise one) a CH3 domain, wherein:

[0281] The CH3 domain of the first polypeptide and the CH3 domain of the second polypeptide incorporate amino acid mutations to promote formation of the heterogenized heteromultimer, wherein the amino acid mutations comprise one or more amino acid mutations selected from the group consisting of positions 347, 356, 360, 362, 368, 392, 398, 400, 411, and 439.

[0282] In some embodiments, the heteromultimer as previously described, wherein the amino acid mutations comprise one or more amino acid mutations selected from the group consisting of positions 347, 356, 360, 362, 368, 400, and 439.

[0283] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises one or more same or different amino acid mutations selected from the group consisting of positions 347, 360, 362, 368, 392, 398, 400, and 411.

[0284] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or the second polypeptide further comprises an amino acid mutation selected from the group consisting of i) to viii) below:

[0285] i) 368;

[0286] ii) 347;

[0287] iii) 347 and 360;

[0288] v) 362 and 400;

[0289] v) 392 and 398;

[0290] v) 398;

[0291] vi) 392 and 400;

[0292] vii) 400; and

[0293] viii) 400 and 411.

[0294] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, and wherein the CH3 domain of the first polypeptide and / or second polypeptide further comprises one or more same or different amino acid mutations selected from positions 347, 360, 362, 368, and 400.

[0295] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first polypeptide and / or second polypeptide further comprises an amino acid mutation selected from the group consisting of i) to iii) below:

[0296] i) 368;

[0297] ii) 347;

[0298] iii) 347 and 360; and

[0299] iv) 362 and 400.

[0300] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, and wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from any one of groups ii) to viii), and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from any one of groups ii) to viii).

[0301] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, and wherein the CH3 domain of the first polypeptide comprises an amino acid mutation selected from any one of Group ii) to viii), and the CH3 domain of the second polypeptide comprises an amino acid mutation selected from any one of Group ii) to viii).

[0302] In some embodiments, the heteromultimer as previously described, the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356 to Lys (K), Arg (R), or His (H), and / or

[0303] the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439 to Glu (E) or Asp (D).

[0304] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E.

[0305] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domain of the first polypeptide further comprises an amino acid mutation selected from any one of Group ii) to viii), and the amino acid mutation of the CH3 domain of the first polypeptide is a negatively charged amino acid, and the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from any one of Group ii) to viii), and the amino acid mutation of the CH3 domain of the second polypeptide is a positively charged amino acid.

[0306] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domain of the first polypeptide further comprises an amino acid mutation selected from any one of Group ii) to viii), and the amino acid mutation of the CH3 domain of the first polypeptide is a negatively charged amino acid, and the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from any one of Group ii) to viii), and the amino acid mutation of the CH3 domain of the second polypeptide is a positively charged amino acid.

[0307] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domain of the first polypeptide further comprises an amino acid mutation selected from any one of Group ii) to viii), and the amino acid mutation of the CH3 domain of the first polypeptide is a positively charged amino acid, and the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from any one of Group ii) to viii), and the amino acid mutation of the CH3 domain of the second polypeptide is a negatively charged amino acid.

[0308] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation of 356K, and the CH3 domain of the second polypeptide comprises an amino acid mutation of 439E, wherein the CH3 domain of the first polypeptide further comprises an amino acid mutation selected from any one of Group ii) to iv), and the amino acid mutation of the CH3 domain of the first polypeptide is a positively charged amino acid, and the CH3 domain of the second polypeptide further comprises an amino acid mutation selected from any one of Group ii) to iv), and the amino acid mutation of the CH3 domain of the second polypeptide is a negatively charged amino acid.

[0309] In some embodiments, the heteromultimer as previously described, 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).

[0310] In some embodiments, the heteromultimer as previously described, wherein the CH3 domain of the first polypeptide and / or the second polypeptide comprises:

[0311] (1) an amino acid mutation at position 347, 360, 362, 392, 398, 400, or 411 to Lys (K) or Arg (R); and / or

[0312] an amino acid mutation at position 347, 360, 362, 392, 398, 400, or 411 to Glu (E) or Asp (D); or

[0313] (2) an amino acid mutation at position 368 to Met (M).

[0314] In some embodiments, the heteromultimer as previously described, wherein:

[0315] (1) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 347, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 347, and 360; or

[0316] (2) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 347, and 360, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 347; or

[0317] (3) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 368, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 368; or

[0318] (4) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 362, and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 362, and 400.

[0319] In some embodiments, the heteromultimer as previously described, wherein:

[0320] (1) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 347, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 347, and 360; or

[0321] (2) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 347, and 360, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 347; or

[0322] (4) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 362, and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 362, and 400.

[0323] In some embodiments, the heteromultimer as previously described, wherein:

[0324] the amino acid mutation at position 356 is Lys (K), Arg (R), or His (H); and / or

[0325] the amino acid mutation at position 439 is Glu (E) or Asp (D); and / or

[0326] the amino acid mutation at position 347, 360, 362, or 400 is Lys (K) or Arg (R); and / or

[0327] the amino acid mutation at position 347, 360, 362, or 400 is Glu (E) or Asp (D);

[0328] the positions of the amino acids are determined according to the EU numbering system.

[0329] In some embodiments, the heteromultimer as previously described, wherein:

[0330] (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347D / E, and 360D / E; or

[0331] (2) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 347D / E, and 360D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 347K / R; or

[0332] (3) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368M, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368M; or

[0333] (4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362D / E, and 400D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362K / R, and 400K / R; or

[0334] (5) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362K / R, and 400K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362D / E, and 400D / E.

[0335] In some embodiments, the heteromultimer as previously described, wherein:

[0336] (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347E, and 360E; or

[0337] (2) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 347E, and 360E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 347K; or

[0338] (3) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368M, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368M; or

[0339] (4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362E, and 400E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362K, and 400K; or

[0340] (5) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362K, and 400K, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362E, and 400E.

[0341] In some embodiments, the heteromultimer as previously described, wherein:

[0342] (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347E, and 360E; or

[0343] (2) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 347E, and 360E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 347K; or

[0344] (3) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368M, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368M; or

[0345] (4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362E, and 400E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362K, and 400K.

[0346] In some embodiments, the heteromultimer as previously described, wherein:

[0347] (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347E, and 360E; or

[0348] (2) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 347E, and 360E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 347K; or

[0349] (4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362E, and 400E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362K, and 400K.

[0350] In some embodiments, the heteromultimer as previously described, wherein:

[0351] (1) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 347K, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 347E and 360E; or

[0352] (2) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K, 347E and 360E, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 347K.

[0353] In some embodiments, the heteromultimer as previously described, wherein:

[0354] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 392 and 398, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 398; or

[0355] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 398, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 392 and 398; or

[0356] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 392 and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 400; or

[0357] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 392 and 400; or

[0358] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 400 and 411, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 400 and 411; or

[0359] the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 362 and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 362 and 400.

[0360] In some embodiments, the heteromultimer as previously described, wherein the CH3 domains of the first and second polypeptides are derived from a CH3 domain of an IgG. In some embodiments, the heteromultimer as previously described, wherein the CH3 domains of the first and second polypeptides are derived from a CH3 domain of an IgGl, IgG2, IgG3, or IgG4 isotype. In some embodiments, the heteromultimer as previously described, wherein the CH3 domains of the first and second polypeptides are derived from a CH3 domain of an IgGl, IgG2, or IgG3 isotype. In some embodiments, the heteromultimer as previously described, wherein the CH3 domains of the first and second polypeptides are derived from a CH3 domain of an IgGl. In some embodiments, the heteromultimer as previously described, the CH3 domains of the first and second polypeptides are derived from a CH3 domain of a human IgGl. In some embodiments, the human IgGl has an amino acid sequence set forth in SEQ ID NO: 5, 6, or 7.

[0361] In some embodiments, the heteromultimer as previously described, wherein the amino acid mutations in the CH3 domains of the first and second polypeptides are mutations made to a CH3 domain of a native IgGl, IgG2, IgG3, or IgG4. In some embodiments, the heteromultimer as previously described, wherein the amino acid mutations in the CH3 domains of the first and second polypeptides are mutations made to a CH3 domain of a native human IgGl. In some embodiments, the heteromultimer as previously described, wherein the amino acid mutations in the CH3 domains of the first and second polypeptides are mutations made to SEQ ID NO: 5, 6, or 7. In some embodiments, the heteromultimer as previously described, wherein the amino acid sequence of the native human IgGl is set forth in SEQ ID NO: 5, 6, or 7. In some embodiments, the heteromultimer as previously described, wherein the sequence of the CH3 domain of the native human IgGl is set forth in SEQ ID NO: 32 or 33.

[0362] In some embodiments, the heteromultimer as previously described, wherein:

[0363] (1) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and Q347K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347D / E, and K360D / E; or

[0364] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q347D / E, and K360D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K / R; or

[0365] (3) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L368M; or

[0366] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362D / E, and S400D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K / R, and S400K / R; or

[0367] (5) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362K / R, and S400K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362D / E, and S400D / E.

[0368] In some embodiments, the heteromultimer as previously described, wherein:

[0369] (1) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0370] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0371] (3) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L368M; or

[0372] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K; or

[0373] (5) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362K, and S400K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362E, and S400E.

[0374] In some embodiments, the heteromultimer as previously described, wherein:

[0375] (1) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0376] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0377] (3) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L368M; or

[0378] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K.

[0379] In some embodiments, the heteromultimer as previously described, wherein:

[0380] (1) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0381] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0382] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D / E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K.

[0383] In some embodiments, the heteromultimer as previously described, wherein:

[0384] (1) the CH3 domain of the first polypeptide comprises amino acid mutations D356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0385] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0386] (3) the CH3 domain of the first polypeptide comprises amino acid mutations D356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L368M; or

[0387] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K.

[0388] In some embodiments, the heteromultimer as previously described, wherein:

[0389] (1) the CH3 domain of the first polypeptide comprises amino acid mutations D356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0390] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0391] (4) the CH3 domain of the first polypeptide comprises amino acid mutations D356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K.

[0392] In some embodiments, the heteromultimer as previously described, wherein:

[0393] (1) the CH3 domain of the first polypeptide comprises amino acid mutations D356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0394] (2) the CH3 domain of the first polypeptide comprises amino acid mutations D356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0395] (3) the CH3 domain of the first polypeptide comprises amino acid mutations D356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and L368M; or

[0396] (4) the CH3 domain of the first polypeptide comprises amino acid mutations E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K.

[0397] In some embodiments, the heteromultimer as previously described, wherein:

[0398] (1) the CH3 domain of the first polypeptide comprises amino acid mutations E356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q347E, and K360E; or

[0399] (2) the CH3 domain of the first polypeptide comprises amino acid mutations E356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E and Q347K; or

[0400] (4) the CH3 domain of the first polypeptide comprises amino acid mutations E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations K439E, Q362K, and S400K.

[0401] In some embodiments, the CH3 domain mutation sites are indicated by EU numbering.

[0402] In some embodiments, the heteromultimer as previously described is efficiently formed in the presence of a reducing agent and an organic solvent.

[0403] In some embodiments, the heteromultimer as previously described is at least 1-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, or 100-fold) more efficient at recombination in the presence of an organic solvent than in the absence of an organic solvent. In some embodiments, the heteromultimer as previously described is about 10-fold more efficient at recombination in the presence of an organic solvent than in the absence of an organic solvent.

[0404] In some embodiments, the heteromultimer as previously described has at least a 30% (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) increase in recombination efficiency in the presence of an organic solvent compared to the recombination efficiency in the absence of an organic solvent. In some embodiments, the heteromultimer as previously described, wherein the first polypeptide is an antibody heavy chain, and / or the second polypeptide is an antibody heavy chain.

[0405] In some embodiments, the heteromultimer as previously described further comprises one or more antibody light chains.

[0406] In some embodiments, the heteromultimer as previously described is a multispecific antibody or a hetero-Fc fusion protein. In some embodiments, the heteromultimer as previously described is a heterodimer. In some embodiments, the heteromultimer as previously described is a bispecific antibody or a trispecific antibody. In some embodiments, the heteromultimer as previously described is a bispecific antibody.

[0407] In some embodiments, the heteromultimer as previously described comprises a core hinge region. In some embodiments, the heteromultimer as previously described, wherein the core hinge region of the heteromultimer comprises a Cys-Pro-Pro-Cys sequence. In some embodiments, the heteromultimer as previously described, wherein the amino acids of the core hinge region form a disulfide bond.

[0408] In some embodiments, the heteromultimer as previously described, wherein there is a difference in isoelectric point between the first polypeptide and the second polypeptide. In some embodiments, the heteromultimer as previously described, wherein the amino acid mutation described herein is to impart or increase the difference in isoelectric point between the first polypeptide and the second polypeptide. In some embodiments, the heteromultimer as previously described, wherein the first polypeptide and the second polypeptide further comprise an additional amino acid mutation to impart or increase the difference in isoelectric point between the first polypeptide and the second polypeptide. In some embodiments, the heteromultimer as previously described, wherein the first polypeptide and the second polypeptide further comprise an additional amino acid mutation such that there is a difference in isoelectric point between the first polypeptide and the second polypeptide.

[0409] In another aspect, the present disclosure provides a pharmaceutical composition comprising the heteromultimer as previously described and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0410] In another aspect, the present disclosure provides an immunoconjugate comprising: a heteromultimer as previously described and a payload, wherein the payload is conjugated to the heteromultimer. In some embodiments, the payload is selected from the group consisting of an antitumor agent, an immunomodulatory agent, a biological response modifier, a lectin, a cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof.

[0411] In another aspect, the present disclosure provides a method of making an immunoconjugate comprising the steps of:

[0412] a) providing a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer;

[0413] b) mixing the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer to form a mixture; and

[0414] c) adding a reducing agent and an organic solvent to the mixture and incubating;

[0415] d) obtaining an immunoconjugate comprising the first polypeptide and the second polypeptide;

[0416] wherein the first polypeptide and the second polypeptide each comprise a CH3 domain, the CH3 domain each comprising one or more mutations that promote heteromerization formation;

[0417] wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer comprise an Fc region conjugated to a prodrug, a peptide, a drug, or a toxin.

[0418] In some embodiments, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer comprise an Fc region conjugated to a toxin.

[0419] In some embodiments, wherein the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are antibody drug conjugates.

[0420] In another aspect, the present disclosure provides a method of making an immunoconjugate comprising:

[0421] a heteromultimer obtained using any one of the methods of making a heteromultimer described herein conjugated to a toxin.

[0422] In another aspect, the present disclosure provides an isolated nucleic acid encoding a heteromultimer as previously described.

[0423] In another aspect, the present disclosure provides a vector comprising an isolated nucleic acid as previously described.

[0424] In another aspect, the present disclosure provides a host cell comprising the isolated nucleic acid as previously described.

[0425] In another aspect, the present disclosure provides a method of making the heteromultimer as previously described.

[0426] In another aspect, the present disclosure provides a method of making the heteromultimer as previously described, comprising: (a) a step of altering a nucleic acid encoding an amino acid residue that forms an inter-polypeptide interface; (b) a step of culturing a host cell having the nucleic acid to express the polypeptide; (c) a step of recovering the polypeptide from the culture of the host cell; and (d) a step of incubating each polypeptide in the presence of a reducing agent and an organic solvent to recover the desired heteromultimer.

[0427] Unless otherwise indicated, in the present disclosure, the positions of amino acids in the variable region are defined according to the Kabat numbering convention, and the positions of amino acids in the constant region are defined according to the Eu index. BRIEF DESCRIPTION OF DRAWINGS

[0428] Figure 1A represents the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E under conditions where no organic solvent was included in the reaction system.

[0429] Figure 1B represents the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E under conditions where a final volume fraction of 10% ACN was included in the reaction system.

[0430] Figure 1C represents the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E under conditions where a final volume fraction of 10% IPA was included in the reaction system.

[0431] Figure 1D represents the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E under conditions where a final volume fraction of 10% ETOH was included in the reaction system.

[0432] Figure 1E represents the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K under conditions where no organic solvent was included in the reaction system.

[0433] Figure 1F shows the HPLC-IEC profile of the recombinant reaction product Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K under conditions where the reaction mixture contained a final volume fraction of 10% ACN.

[0434] Figure 1G shows the HPLC-IEC profile of the recombinant reaction product Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K under conditions where the reaction mixture contained a final volume fraction of 10% IPA.

[0435] Figure 1H shows the HPLC-IEC profile of the recombinant reaction product Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K under conditions where the reaction mixture contained a final volume fraction of 10% ETOH.

[0436] Figure 2A shows the HPLC-IEC profile of the recombinant reaction product Ipi-D356K*CP11-K439E under conditions where the reaction mixture did not contain an organic solvent.

[0437] Figure 2B shows the HPLC-IEC profile of the recombinant reaction product Ipi-D356K*CP11-K439E under conditions where the reaction mixture contained a final volume fraction of 6% ACN.

[0438] Figure 2C shows the HPLC-IEC profile of the recombinant reaction product Ipi-D356K*CP11-K439E under conditions where the reaction mixture contained a final volume fraction of 8% ACN.

[0439] Figure 2D shows the HPLC-IEC profile of the recombinant reaction product Ipi-D356K*CP11-K439E under conditions where the reaction mixture contained a final volume fraction of 10% ACN.

[0440] Figure 2E shows the HPLC-IEC profile of the recombinant reaction product Ipi-D356K+L368M*CP11-K439E+L368M under conditions where the reaction mixture did not contain an organic solvent.

[0441] Figure 2F shows the HPLC-IEC profile of the recombinant reaction product Ipi-D356K+L368M*CP11-K439E+L368M under conditions where the reaction mixture contained a final volume fraction of 6% ACN.

[0442] Figure 2G shows the HPLC-IEC profile of the recombinant reaction product Ipi-D356K+L368M*CP11-K439E+L368M under conditions where the reaction mixture contained a final volume fraction of 8% ACN.

[0443] Figure 2H shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+L368M*CP11-K439E+L368M with 10% final volume fraction of ACN in the reaction system.

[0444] Figure 21 shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E with no organic solvent in the reaction system.

[0445] Figure 2J shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E with 6% final volume fraction of ACN in the reaction system.

[0446] Figure 2K shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E with 8% final volume fraction of ACN in the reaction system.

[0447] Figure 2L shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E with 10% final volume fraction of ACN in the reaction system.

[0448] Figure 2M shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K with no organic solvent in the reaction system.

[0449] Figure 2N shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K with 6% final volume fraction of ACN in the reaction system.

[0450] Figure 20 shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K with 8% final volume fraction of ACN in the reaction system.

[0451] Figure 2P shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K with 10% final volume fraction of ACN in the reaction system.

[0452] Figure 2Q shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q362E+S400E*CP11-K439E+Q362K+S400K under conditions where no organic solvent was included in the reaction system.

[0453] Figure 2R shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q362E+S400E*CP11-K439E+Q362K+S400K under conditions where a final volume fraction of 6% ACN was included in the reaction system.

[0454] Figure 2S shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q362E+S400E*CP11-K439E+Q362K+S400K under conditions where a final volume fraction of 8% ACN was included in the reaction system.

[0455] Figure 2T shows the HPLC-IEC profile of the recombination reaction product Ipi-D356K+Q362E+S400E*CP11-K439E+Q362K+S400K under conditions where a final volume fraction of 10% ACN was included in the reaction system. DETAILED DESCRIPTION

[0456] TERMINOLOGY

[0457] For the purposes of the present disclosure, certain technical and scientific terms are described below. Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0458] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0459] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0460] Unless the context clearly indicates otherwise, throughout the patent specification and claims, the words "comprise", "have" "include" and the like are to be construed in an inclusive sense as "including but not limited to".

[0461] The term "and / or" means include both "and" and "or". For example, the phrase "A, B, and / or C" is intended to cover each of the following aspects: 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).

[0462] The three letter code and one letter code for amino acids used herein are as described in J. Biol. Chem, 243, p 3558 (1968).

[0463] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation. Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an alpha carbon bonded to a hydrogen, a carboxyl, an amino, and an R group) as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but otherwise function in a manner similar to naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0464] The term "amino acid mutation" includes amino acid substitutions (also referred to as amino acid replacements), deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to arrive at the final construct, as long as the final construct possesses the desired properties, such as reduced or no binding to an Fc receptor. Amino acid sequence deletions and insertions include deletions and insertions at the amino- and / or carboxyl-terminus of a polypeptide chain. A particular amino acid mutation can be an amino acid substitution. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid of different structure and / or chemical characteristics. Amino acid substitutions include substitutions by non-naturally occurring amino acids or by derivatives of the 20 natural 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, and the like. Methods other than genetic engineering to alter the side chain groups of amino acids, such as chemical modifications, are also expected to be useful. Various names can be used herein to refer to the same amino acid mutation. In this context, the position + amino acid residue format can be used to indicate an amino acid residue at a particular position, e.g., 356K, indicates that the amino acid residue at position 356 is K. D356K indicates that the amino acid residue at position 356 is mutated from D to K. D / E356K indicates that the amino acid residue at position 356 is mutated from D to K or the amino acid residue at position 356 is mutated from E to K. S400D / E indicates that the amino acid residue at position 400 is mutated from S to D or the amino acid residue at position 400 is mutated from S to E. And so on. It is understood that when an amino acid sequence is defined in a claim in the position + residue format, the amino acid at that position prior to mutation does not limit the claim. In this context, "the Fc region comprises an amino acid mutation of 356K and 349S" indicates that the Fc region comprises an amino acid mutation of position 356 to lysine (K) and position 349 to serine (S).

[0465] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0466] The term "heteromultimer" refers to a protein multimer composed of a plurality of different polypeptides which can associate with each other. In more detail, the "heteromultimer" has at least a first polypeptide and a second polypeptide, where the second polypeptide is a molecule which differs from the first polypeptide by at least one amino acid residue in its amino acid sequence. Furthermore, although not particularly limited, the heteromultimer can have an antigen-binding activity for at least two different ligands, antigens (or epitopes), receptors, or substrates, etc. The heteromultimer can have other polypeptides in addition to the "heterodimer" formed by the first and second polypeptides. That is, the "heteromultimer" of the present disclosure is not limited to a heterodimer, and for example, also includes a heterotrimer, a heterotetramer, etc.

[0467] The term "homomultimer" refers to a state in which polypeptides having the same amino acid sequence are associated with each other.

[0468] In the present disclosure, the terms "multispecific antibody" and "polyspecific antibody" mean the same and refer to an antibody which can specifically bind to a plurality of different epitopes. That is, the multispecific antibody is an antibody which has specificity for at least two different epitopes, and in addition to an antibody which recognizes different antigens, also includes an antibody which recognizes different epitopes on the same antigen (for example, when the antigen is a heterologous receptor, the multispecific antibody recognizes different domains constituting the heterologous receptor; or when the antigen is a monomer, the multispecific antibody recognizes a plurality of sites of the monomeric antigen). Usually, such a molecule binds to two antigens (bispecific antibody, which means the same as "dual-specific antibody" in the present specification), but can have specificity for more than two (for example, three) antigens.

[0469] 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); and any molecule containing the aforementioned antibody structures, 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.).

[0470] "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. The natural IgG heavy chain constant region typically contains 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).

[0471] 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.

[0472] A "full length antibody" as described herein can be an antibody that specifically binds a single epitope, or can be a bispecific antibody that can bind two different epitopes simultaneously.

[0473] The antibodies used in the methods of the disclosure can be of any allotype. The allotype is not expected to have an effect on the Fab-arm exchange. Antibody allotype is associated with amino acid sequence variations at specific positions in the constant region sequence of the antibody.

[0474] A common wild-type human IgGl heavy chain constant region sequence is shown below:

[0475] > hlgGl-1

[0476] > hlgGl-2

[0477] > hlgGl-3

[0478] The term "bispecific antibody" refers to an antibody (including an antibody or an antigen-binding fragment thereof, such as a single-chain antibody) capable of specifically binding to two different antigens or at least two different antigen epitopes of the same antigen. The prior art has disclosed various structures of bispecific antibodies, which can be divided into IgG-like bispecific antibodies and antibody fragment type bispecific antibodies according to the integrity of IgG molecules; according to the number of antigen binding regions, it can be divided into divalent, trivalent, tetravalent or more valent bispecific antibodies, according to whether the structure is symmetrical or not, it can be divided into symmetrical structure bispecific antibodies and asymmetrical structure bispecific antibodies. Among them, the bispecific antibody based on antibody fragments, such as Fab fragments lacking Fc fragments, which forms a bispecific antibody by combining 2 or more Fab fragments in one molecule, has lower immunogenicity, smaller molecular weight, and higher tumor tissue penetration. The typical antibody structure of this type of bispecific antibody is F(ab)2, scFv-Fab, (scFv)2-Fab, etc.; IgG-like bispecific antibodies (e.g. with Fc fragments), which have relatively large molecular weights, Fc fragments help the purification of antibodies in the later stage, and improve their solubility, stability, Fc part may also be combined with receptor FcRn, increase the serum half-life of the antibody, typical bispecific antibody structure models are KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG 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, F(ab)4-CrossMAb, etc. (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).

[0479] The term“parent antibody” is an antibody that serves as a source of one or more antibody fragments. A parent antibody can comprise a native or wild-type sequence. A parent antibody can comprise an amino acid sequence in which one or more amino acid residues are replaced by one or more cysteine residues. A parent antibody can have pre-existing amino acid sequence modifications (such as additions, deletions, and / or substitutions) relative to other native, wild-type, or modified forms of the antibody. A parent antibody can be directed to a target antigen of interest, e.g., a biologically important polypeptide. A parent antibody can be directed to a non-polypeptide antigen (e.g., a human CCR8 antigen; e.g., patent WO2023208182A1). Exemplary parent antibodies include, but are not limited to, antibodies having affinity and selectivity for cell surface receptors and transmembrane receptors and tumor-associated antigens (TAAs).

[0480] The term“Fc region” or“fragment crystallizable region” is used to define a C-terminal region of an antibody heavy chain, including native and engineered Fc regions. In some embodiments, an Fc region comprises two subunits, which are identical or different. In some embodiments, the Fc region of a human IgG heavy chain is defined as stretching from an amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. Suitable Fc regions for use in the antibodies described herein include Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region can also vary, e.g., by deletion or addition of a C-terminal lysine (residue 447 according to EU numbering system) or deletion of a C-terminal glycine and lysine (residue 446 and 447 according to EU numbering system). Unless otherwise specified, numbering of amino acid residues in the Fc region is according to the EU numbering system, also called the EU index (Kabat EA et al., 1991. Sequences of Proteins of Immunological Interest. NIH). Although in a particular embodiment, one numbering system (e.g., EU) is used to define the amino acid residues, other numbering systems are considered to be equivalent.

[0481] The Fc region can be suitably obtained by partially digesting an IgG monoclonal antibody or the like with a proteolytic enzyme such as pepsin, and then eluting the fraction adsorbed to a protein A or protein G column. As the proteolytic enzyme, an enzyme that can digest a full-length antibody in a limited manner to produce Fab, F(ab')2, by suitably setting the reaction conditions of the enzyme such as pH, and is not particularly limited, and for example, pepsin, papain, or the like can be exemplified.

[0482] In the present disclosure, the term "Fc region" or "Fc domain" refers to an antibody region comprising at least a hinge region, a CH2 domain and a CH3 domain (see e.g. Kabat EA, US Department of Health and Human Services, NIH publication n91-3242, Edn. 5th edition 662, 680, 689 (1991).

[0483] The C-terminus of the Fc region can be the complete C-terminus ending with the amino acid residue PGK; it can also be a shortened C-terminus, e.g. 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 complete antibodies can comprise a population of antibodies in which all K447 residues and / or G446+K447 residues have been removed. In some embodiments, the composition of complete antibodies can comprise a population of antibodies in which K447 residues and / or G446+K447 residues have not been removed. In some embodiments, the composition of complete antibodies has a population of antibodies with and without K447 residues and / or G446+K447 residues.

[0484] In the present 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 IgGl antibody corresponds to amino acids 228-340 according to the EU numbering system. However, the CH2 region can also be of any other antibody isotype as described in the present disclosure.

[0485] In the present disclosure, the term "CH3 region", "CH3 domain" or "CH3 domain" is intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgGl antibody corresponds to amino acids 341-447 according to the EU numbering system. However, the CH3 region can also be of any other antibody isotype as described in the present disclosure.

[0486] The CH3 region sequence of a common wild-type human IgGl is shown below:

[0487] > hlgGl-1-CH3

[0488] > hlgGl-3-CH3

[0489] In the present disclosure, association of a polypeptide refers to the state in which, for example, a plurality of polypeptide regions interact.

[0490] In the present disclosure, "modulating association" refers to modulating the desired state of association, more specifically, to preventing the formation of undesired associations within a polypeptide.

[0491] In the present disclosure, "interface" generally refers to a binding surface (interface) at the time of binding (interaction). The amino acid residues forming the interface generally refer to one or more amino acid residues contained in the polypeptide region subjected to the binding, more preferably the amino acid residues close to and involved in the interaction at the time of binding. The interaction specifically includes the case where a hydrogen bond, an electrostatic interaction, a salt bridge, etc. are formed between the amino acid residues close to each other at the time of binding.

[0492] In the present disclosure, "amino acid residues forming the interface" specifically refer to the amino acid residues contained in the polypeptide region constituting the interface. Examples of the polypeptide region constituting the interface include a polypeptide region in an antibody, a ligand, a receptor, a substrate, etc. that bears a selective bonding within or between molecules. In the present disclosure, it can refer to the amino acid residues on the interface at which two CH3 regions interact.

[0493] The term "Fab-arm" refers to one heavy chain-light chain pair of an antibody, which can also be called a half-antibody.

[0494] The term "reducing agent" refers to an agent capable of reducing the interchain disulfide bond in the hinge region of an antibody.

[0495] Those skilled in the art will appreciate that "about" can mean within one or more than one standard deviation when used in reference to a numerical range, cutoff, or specific value. Alternatively, "about" can mean a range of up to 20% (i.e., ±20%). Since many of the numerical values used herein are determined experimentally, those skilled in the art will appreciate that such determinations can vary from experiment to experiment and generally vary from experiment to experiment. Because of this inherent variability, the values used herein are not to be taken as overly limiting. Thus, the term "about" is used to encompass variations of ±20% or less, ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from the stated value.

[0496] While the disclosure provides a range or value of an amount, one of ordinary skill in the art understands that the range or value of the amount encompasses acceptable variations of the determined particular value. The terms antibody "variable region" or "variable domain" refer to the domains of an antibody heavy or light chain that are involved in binding the antibody to an antigen. Herein, the antibody heavy chain variable region (VH) and light chain variable region (VL) each comprise four conserved framework regions (FRs) and three complementarity determining regions (CDRs). Within the variable domain, the term "complementarity determining region" or "CDR" refers to a region that is primarily responsible for binding an antigen; "framework" 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 of the VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus (also referred to as N-terminus) to carboxy-terminus (also referred to as C-terminus) in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0497] The boundaries of the CDRs can be determined by various well-known schemes, such as the "Kabat" numbering convention, the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention, and the ImMunoGenTics (IMGT) numbering convention, etc.; the correspondence between the various numbering systems is well known to those skilled in the art, and exemplary is shown in Table 1 below.

[0498] Table 1. Relationship between CDR numbering systems

[0499] Unless otherwise specified, the variable regions and CDRs in the embodiments of the disclosure are applicable to the "Kabat" numbering convention. Although the Kabat numbering convention is used to define the amino acid residues in specific embodiments, the corresponding technical solutions of other numbering systems are deemed to be equivalent technical solutions.

[0500] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds 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 antibody (sdAb, e.g., VH, VL, VHH or V HH ), single chain Fab (scFab), diabody, linear antibody, single chain antibody (e.g., scFv, sc(Fv)2); and multispecific antibody formed from antibody fragments.

[0501] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portion of the heavy and / or light chain is derived from a different, distinct source or species.

[0502] The term "humanized" antibody is an antibody that retains reactivity of a non-human antibody, while having lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterpart (i.e., the constant region as well as the framework region portion of the variable region).

[0503] The terms "human antibody," "fully human antibody," "completely human antibody" are used interchangeably herein and mean an antibody in which the variable and constant regions are of human sequence. The term encompasses antibodies that are derived from human genes but have had sequences altered, for example, to reduce possible immunogenicity, increase affinity, eliminate a cysteine or glycosylation site that can cause undesirable folding, etc. The term encompasses these antibodies that are recombinantly produced in non-human cells that can impart non-human cell characteristic glycosylation. The term also encompasses antibodies that have been produced in transgenic mice that contain some or all human immunoglobulin heavy and light chain loci. The definition of human antibody expressly excludes humanized antibodies that contain non-human antigen binding residues.

[0504] The term "affinity" refers to the overall strength of the noncovalent interactions between individual binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, binding "affinity" refers to the intrinsic binding affinity, which reflects the 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its ligand Y can generally be represented by the dissociation constant (KD). Affinity can be measured by routine methods known in the art, including those described herein.

[0505] The term "kassoc" or "ka", as used herein, refers to the association rate of a particular antibody-antigen interaction. The term "kdis" or "kd", as used herein, refers to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). KDvalues for antibodies can be determined using methods well known in the art. For example, affinity in solution can be measured using a biosensor system such as a Biacore® system measures surface plasmon resonance (e.g., Biacore), or by solution equilibrium titration (SET).

[0506] The term "surface plasmon resonance" refers to an optical phenomenon that detects alterations in protein concentrations within a biosensor matrix in real-time, for example, using the BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0507] The term "effector function" refers to those biological activities attributable to an antibody Fc region (a native sequence Fc region or an amino acid sequence mutated Fc region) and which vary with the antibody isotype. Examples of antibody effector functions include but are not limited to: Clq binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of B cell receptor (BCR) expression, and B cell activation.

[0508] The term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in their amino acid sequences except for possible naturally occurring mutations that can be present in minor amounts. In contrast, polyclonal antibody preparations typically include a multitude of different antibodies that are specific for different epitopes of the antigen, often due to immunization with a whole organism or with a protein complex, as opposed to a single epitope. "Monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

[0509] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by an antigen binding protein, including, for example, an antibody. An antigen can have one or more epitopes capable of interacting with different antigen binding proteins, e.g., antibodies.

[0510] The term "epitope" refers to a region (area or region) on an antigen to which an antibody or antigen-binding fragment thereof specifically binds. An epitope can be formed by contiguous amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), e.g., brought into spatial proximity by folding of the antigen (i.e., tertiary folding of the antigen by virtue of its protein nature). The difference between a conformational epitope and a linear epitope is that binding of an antibody to a conformational epitope is lost in the presence of a denaturing solvent. An epitope comprises 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 a particular epitope (i.e., those that bind the same epitope) can be performed using routine methods in the art, such as, but not limited to, alanine scanning, peptide mapping, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0511] The terms "capable of specifically binding," "specifically binds," or "binds" mean that an antibody is capable of binding to a certain antigen or epitope within that antigen with a higher affinity than to other antigens or epitopes. Typically, an antibody binds to an antigen or an epitope within that antigen with an equilibrium dissociation constant (KD) of about 1 x 10 -7 M or less (e.g., about 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M or less). In some embodiments, the KD of an antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of that antibody binding to a non-specific antigen (e.g., BSA, casein). KD can be measured using known methods, e.g., by surface plasmon resonance assays. However, an antibody that specifically binds to an antigen or an epitope within that antigen can have cross-reactivity to other related antigens, e.g., cross-reactivity to the corresponding antigen from other species (homologs), such as human or monkey, e.g., Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp), or Callithrix jacchus (common marmoset, marmoset).

[0512] The term "does not bind" means that an antibody is not capable of binding to a certain antigen or epitope thereof in the manner of specific binding described above. For example, an antibody does not bind to an antigen or an epitope thereof with an equilibrium dissociation constant (KD) of about 1 x 10 -6 M or greater.

[0513] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with effector cells having lytic activity, such as natural killer cells (NK), monocytes, macrophages, and neutrophils, via Fc gamma receptors (FcγRs) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies provided herein can be assessed using an in vitro assay using cells expressing an antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of a label (e.g., a radioactive substrate, a fluorescent dye, or a native intracellular protein) from the lysed cells.

[0514] ​The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism of eliminating antibody-coated target cells through internalization by phagocytic cells, such as macrophages or dendritic cells.

[0515] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism of inducing cell death in which the Fc effector domain of a target-bound antibody binds and activates complement component Clq, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0516] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties to the reference nucleotide, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0517] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid encoding a polypeptide is one that is

[0518] The term "sequence identity" refers to the extent to which the amino acid / nucleic acid of two sequences are identical at equivalent positions (percent); wherein, when the two sequences are optimally aligned, gaps are introduced if necessary, to achieve maximum percent sequence identity, and no conservative substitutions are part of the sequence identity. To determine percent sequence identity, the comparison can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0519] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, e.g., a adenoviral associated virus vector (AAV or AAV2), wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. The term "expression vector" or "expression construct" refers to a vector that can be transformed into a host cell, and which contains nucleic acid sequences that direct and / or control the expression of one or more heterologous coding regions to which they are operably linked, along with the host cell. An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, where introns are present, RNA splicing of coding regions operably linked thereto.

[0520] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. The progeny can not be completely identical to the parent cell both in genetic and protein expression makeup, but can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformant cell are included herein. Host cells include prokaryotic and eukaryotic host cells, with eukaryotic host cells including, but not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby 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 opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus sp., Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.

[0521] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0522] The term "pharmaceutical composition" denotes a mixture that comprises one or more multispecific antibodies described herein with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0523] The term "pharmaceutically acceptable carrier, diluent or excipient" refers to a component of a pharmaceutical formulation that does not itself induce the production of antibodies in recipients, that is well-tolerated in a subject, and that does not interfere with the efficacy of the active ingredient. Pharmaceutically acceptable carriers, diluents or excipients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0524] The term "subject" or "individual" includes both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein, unless otherwise indicated. In certain embodiments, the individual or subject is a human.

[0525] "Administering" or "administration," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, means the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.

[0526] The term "sample" refers to a collection (e.g., fluid, cell, or tissue) isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are biological fluids, such as blood, serum and serosal fluids, plasma, lymphatic fluid, urine, saliva, cyst fluid, lacrimal fluid, fecal matter, sputum, mucosal secretions of secretory tissues or organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal and other body cavity fluids, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, e.g., culture media (including conditioned media), lavage fluids, and the like, tissue biopsy samples, fine needle aspirates, surgically removed tissues, organ cultures, or cell cultures.

[0527] "Treatment" and "treating" (and grammatical variations thereof) refer to clinical intervention by which an individual's clinical status is modified. The desired effect of treatment includes, but is not limited to, preventing or delaying the onset of a disease or its recurrence, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, improving or lessening the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the disclosure are used to delay development of a disease or to slow the progression of a disease.

[0528] An "effective amount" is generally an amount that is sufficient to reduce the severity and / or frequency of symptoms, eliminate such symptoms and / or their underlying cause, prevent symptoms and / or their underlying cause from occurring, and / or ameliorate or improve damage caused or associated with a disease state (e.g., a lung disease). In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0529] A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptoms, especially states or symptoms associated with the disease state, or otherwise to prevent, hinder, delay, or reverse the progression of the disease state or any other undesirable symptoms associated with the disease in any way. A "prophylactically effective amount" is an amount that will have the intended prophylactic effect, e.g., 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, when administered to a subject. Complete treatment or prevention need not occur in order for a dose to be considered therapeutically or prophylactically effective. Thus, a therapeutically or prophylactically effective amount can be administered in one or more doses. "Therapeutically effective amount" and "prophylactically effective amount" can vary depending on factors such as the disease state of the individual, the age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, e.g., improved health status of the patient.

[0530] Exemplary heteromultimers (multispecific antibodies)

[0531] Multispecific antibodies of the disclosure and methods of making the same

[0532] In one aspect, the disclosure provides a method of making a multispecific antibody, comprising the steps of:

[0533] a) providing a first parent antibody and a second parent antibody;

[0534] b) mixing the first parent antibody and the second parent antibody to form a mixture; and

[0535] c) adding a reducing agent and an organic solvent to the mixture and incubating;

[0536] wherein the first parent antibody and the second parent antibody each comprise a CH3 domain, the CH3 domain each comprising one or more mutations that promote heteromerization.

[0537] In some embodiments, the method of making a multispecific antibody as previously described, further comprising d) obtaining the multispecific antibody.

[0538] In some embodiments, the method of making a multispecific antibody as previously described, wherein the CH3 domains of the first parent antibody and the second parent antibody each comprise one or more oppositely charged amino acid mutations (e.g., positively charged amino acids, negatively charged amino acids).

[0539] In some embodiments, the method of making a multispecific antibody as previously described, wherein the CH3 domains of the first and second parent antibodies each comprise one or more charged amino acid mutations (e.g., positively charged amino acids, negatively charged amino acids), wherein the amino acid in the CH3 domain of the first parent antibody and the amino acid in the same position of the CH3 domain of the second parent antibody are of opposite charge.

[0540] In some embodiments, the method of making a multispecific antibody as previously described, wherein the CH3 domain of the first parent antibody comprises one or more amino acid mutations of opposite charge, and the CH3 domain of the second parent antibody comprises one or more amino acid mutations of opposite charge (e.g., positively charged amino acids, negatively charged amino acids).

[0541] In some embodiments, the method of making a multispecific antibody as previously described, wherein the negatively charged amino acid is selected from the group consisting of glutamic acid (E) and aspartic acid (D), and the positively charged amino acid is selected from the group consisting of lysine (K), arginine (R), and histidine (H). In some embodiments, the method of making a multispecific antibody as previously described, wherein the negatively charged amino acid is selected from the group consisting of glutamic acid (E) and aspartic acid (D), and the positively charged amino acid is selected from the group consisting of lysine (K) and arginine (R).

[0542] In some embodiments, the method of making a multispecific antibody as previously described, wherein the CH3 domain of the first parent antibody comprises an amino acid mutation at position 356, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439.

[0543] In some embodiments, the method of making a multispecific antibody as previously described, wherein the CH3 domain of the first parent antibody comprises an amino acid mutation of 356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 439E, wherein the CH3 domain mutation positions are indicated by EU numbering.

[0544] In some embodiments, the method of making a multispecific antibody as previously described, wherein the CH3 domain of the first parent antibody comprises an amino acid mutation at position 356, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439, the CH3 domain of the first and / or second parent antibody further comprising one or more same or different amino acid mutations selected from the group consisting of positions 347, 360, 362, 368, and 400.

[0545] In some embodiments, the method of making a heteromultimer as previously described, wherein the CH3 domain of the first parent antibody comprises an amino acid mutation at position 356, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439, the CH3 domain of the first and / or second parent antibody further comprises one or more amino acid mutations selected from the group consisting of i) to iii) below:

[0546] i) 368;

[0547] ii) 347;

[0548] iii) 347 and 360; and

[0549] iv) 362 and 400.

[0550] In some embodiments, the method of making a multispecific antibody as previously described, wherein:

[0551] (1) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 347, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439, 347, and 360; or

[0552] (2) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356, 347, and 360, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439 and 347; or

[0553] (3) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356 and 368, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439 and 368; or

[0554] (4) the CH3 domain of the first parent antibody comprises amino acid mutations at positions 356, 362, and 400, and the CH3 domain of the second parent antibody comprises amino acid mutations at positions 439, 362, and 400;

[0555] wherein the CH3 domain mutation positions are indicated by EU numbering.

[0556] In some embodiments, the method of making a multispecific antibody as previously described, wherein:

[0557] (1) the CH3 domain of the first parent antibody comprises amino acid mutations 356K and 347K, and the CH3 domain of the second parent antibody comprises amino acid mutations 439E, 347E, and 360E; or

[0558] (2) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K, 347E, and 360E, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 347K; or

[0559] (3) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K and 368M, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E and 368M; or

[0560] (4) the CH3 domain of the first parent antibody comprises amino acid mutations of 356K, 362E, and 400E, and the CH3 domain of the second parent antibody comprises amino acid mutations of 439E, 362K, and 400K.

[0561] In some embodiments, the method of making a multispecific antibody as previously described, wherein the CH3 domains of the first parent antibody and the second parent antibody are derived from a CH3 domain of an IgG. In some embodiments, the method of making a multispecific antibody as previously described, wherein the CH3 domains of the first parent antibody and the second parent antibody are derived from a CH3 domain of an IgG1. In some embodiments, the method of making a multispecific antibody as previously described, wherein the CH3 domains of the first parent antibody and the second parent antibody are derived from a CH3 domain of a human IgG1. In some embodiments, the human IgG1 has an amino acid sequence set forth in SEQ ID NO: 5, 6, or 7. In some embodiments, the CH3 domain of the human IgG1 has an amino acid sequence set forth in SEQ ID NO: 32 or 33.

[0562] In some embodiments, the method of making a multispecific antibody as previously described, wherein:

[0563] (1) the CH3 domain of the first parent antibody comprises amino acid mutations of D / E356K and Q347K, and the CH3 domain of the second parent antibody comprises K439E, Q347E, and K360E; or

[0564] (2) the CH3 domain of the first parent antibody comprises amino acid mutations of D / E356K, Q347E, and K360E, and the CH3 domain of the second parent antibody comprises K439E and Q347K; or

[0565] (3) the CH3 domain of the first parent antibody comprises amino acid mutations of D / E356K and L368M, and the CH3 domain of the second parent antibody comprises K439E and L368M; or

[0566] (4) the CH3 domain of the first parent antibody comprises amino acid mutations D / E356K, Q362E, and S400E, and the CH3 domain of the second parent antibody comprises amino acid mutations K439E, Q362K, and S400K.

[0567] In some embodiments, the method of making a multispecific antibody as previously described, wherein the first parent antibody is a first parent monoclonal antibody, and the second parent antibody is a second parent monoclonal antibody. In some embodiments, the method of making a multispecific antibody as previously described, wherein the first parent antibody is a first parent monospecific antibody, and the second parent antibody is a second parent monospecific antibody. In some embodiments, the method of making a multispecific antibody as previously described, wherein the first parent antibody is a first parent monospecific antibody, and the second parent antibody is a second parent monospecific antibody or a second parent bispecific antibody. In some embodiments, the method of making a multispecific antibody as previously described, wherein the first parent antibody is a first parent monoclonal antibody, and the second parent antibody is a second parent monoclonal antibody or a second parent bispecific antibody.

[0568] In some embodiments, the method of making a multispecific antibody as previously described, wherein the reducing agent is selected from one or more of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, D-cysteine, and β-mercapto-ethanol and chemical derivatives thereof. In some embodiments, the method of making a multispecific antibody as previously described, wherein the reducing agent is selected from one or more of 2-MEA, glutathione, L-cysteine, dithiothreitol, β-mercaptoethanol, and TCEP. In some embodiments, the method of making a multispecific antibody as previously described, wherein the reducing agent is 2-MEA.

[0569] In some embodiments, the method of making a multispecific antibody as previously described, wherein the final concentration of the reducing agent is between 5 mM and 500 mM. In some embodiments, the method of making a multispecific antibody as previously described, wherein the final concentration of the reducing agent is between 5 mM and 200 mM. In some embodiments, the method of making a multispecific antibody as previously described, wherein the final concentration of the reducing agent is about 75 mM.

[0570] In some embodiments, the method of making a multispecific antibody as previously described, wherein the organic solvent is selected from the group consisting of nitriles and alcohols. In some embodiments, the method of making a multispecific antibody as previously described, wherein the nitrile is acetonitrile or propionitrile and the alcohol is ethanol, propanol or isopropanol. In some embodiments, the method of making a multispecific antibody as previously described, wherein the nitrile is selected from the group consisting of acetonitrile and the alcohol is selected from the group consisting of ethanol or isopropanol.

[0571] In some embodiments, the method of making a multispecific antibody as previously described, wherein the final volume fraction of the organic solvent is between 1% and 20%. In some embodiments, the method of making a multispecific antibody as previously described, wherein the final volume fraction of the organic solvent is between 4% and 10%. In some embodiments, the method of making a multispecific antibody as previously described, wherein the final volume fraction of the organic solvent is between 6% and 10%. In some embodiments, the method of making a multispecific antibody as previously described, wherein the final volume fraction of the organic solvent is between 6% and 8%. In some embodiments, the method of making a multispecific antibody as previously described, wherein the final volume fraction of the organic solvent is 6% ± 1%.

[0572] In some embodiments, the method of making a multispecific antibody as previously described, wherein the molarity of the first parent antibody and the second parent antibody in the mixture is the same.

[0573] In some embodiments, the method of making a multispecific antibody as previously described, wherein the incubation temperature of the mixture is between 15°C and 40°C. In some embodiments, the method of making a multispecific antibody as previously described, wherein the incubation temperature of the mixture is between 20°C and 40°C. In some embodiments, the method of making a multispecific antibody as previously described, wherein the incubation temperature of the mixture is about 37°C.

[0574] In some embodiments, the method of making a multispecific antibody as previously described, wherein the incubation time of the first parent antibody and the second parent antibody is between 0.5 hours and 3 hours. In some embodiments, the method of making a multispecific antibody as previously described, wherein the incubation time of the first parent antibody and the second parent antibody is between 2 hours and 3 hours. In some embodiments, the method of making a multispecific antibody as previously described, wherein the incubation time of the first parent antibody and the second parent antibody is about 2.5 hours.

[0575] In some embodiments, the method of producing a multispecific antibody as previously described, step c) is further followed by a step of removing the reducing agent and the organic solvent. In some embodiments, the reducing agent and the organic solvent are removed by, but not limited to, dialysis, precipitation, chromatography, or filtration. In some embodiments, the reducing agent and the organic solvent are removed by dialysis.

[0576] In some embodiments, the method of producing a multispecific antibody as previously described, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0577] In some embodiments, the method of producing a multispecific antibody as previously described, wherein more than 30% (e.g., more than 35%, more than 40%, more than 50%, more than 60%, more than 70%, 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 multispecific antibody.

[0578] In some embodiments, the method of producing a multispecific antibody as previously described, wherein the multispecific antibody has a recombination efficiency of less than 30% (e.g., less than 25%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%) under conditions without the organic solvent.

[0579] In some embodiments, the present disclosure provides a method of producing a multispecific antibody, the method comprising the steps of a) to d):

[0580] a) providing a step of providing a first parent antibody and a second parent antibody;

[0581] b) mixing the first parent antibody and the second parent antibody to form a mixture;

[0582] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0583] d) obtaining a step of obtaining a multispecific antibody,

[0584] wherein the first parent antibody and the second parent antibody bind to different antigens or epitopes, the CH3 domain of the first parent antibody comprises an amino acid mutation at position 356, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439.

[0585] In some embodiments, the present disclosure provides a method of preparing a multispecific antibody, the method comprising the steps of a) to d) below:

[0586] a) a step of providing a first parent antibody and a second parent antibody;

[0587] b) mixing the first parent antibody and the second parent antibody to form a mixture;

[0588] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0589] d) a step of obtaining a multispecific antibody,

[0590] wherein the first parent antibody and the second parent antibody bind to different antigens or epitopes, the CH3 domain of the first parent antibody comprises an amino acid mutation at position 356, and the CH3 domain of the second parent antibody comprises an amino acid mutation at position 439;

[0591] wherein the reducing agent is selected from one or more of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, D-cysteine, and β-mercapto-ethanol and chemical derivatives thereof, the final concentration of the reducing agent is 5 mM to 500 mM;

[0592] wherein the organic solvent is acetonitrile, ethanol, or isopropanol, the final volume fraction of the organic solvent is 4% to 10%.

[0593] In some embodiments, the present disclosure provides a method of preparing a multispecific antibody, the method comprising the steps of a) to d) below:

[0594] a) a step of providing a first parent antibody and a second parent antibody;

[0595] b) mixing the first parent antibody and the second parent antibody to form a mixture;

[0596] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0597] d) a step of obtaining a multispecific antibody,

[0598] wherein the first parent antibody and the second parent antibody bind to different antigens or epitopes, the CH3 domain of the first parent antibody comprises an amino acid mutation of 356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 439E;

[0599] wherein the reducing agent is selected from one or more of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, D-cysteine, and b-mercapto-ethanol and chemical derivatives thereof, at a final concentration of 5 mM to 500 mM;

[0600] wherein the organic solvent is acetonitrile, ethanol, or isopropanol, at a final volume fraction of 6% to 10%.

[0601] In some embodiments, the present disclosure provides a method of preparing a multispecific antibody, the method comprising the steps of a) to d) below:

[0602] a) a step of providing a first parent antibody and a second parent antibody;

[0603] b) a step of mixing the first parent antibody and the second parent antibody to form a mixture;

[0604] c) a step of adding a reducing agent and an organic solvent to the mixture and incubating; and

[0605] d) a step of obtaining a multispecific antibody,

[0606] wherein the first parent antibody and the second parent antibody bind to different antigens or epitopes, the CH3 domain of the first parent antibody comprises an amino acid mutation of 356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 439E;

[0607] wherein the reducing agent is selected from one or more of 2-MEA, glutathione, L-cysteine, dithiothreitol, b-mercaptoethanol, and TCEP, at a final concentration of 5 mM to 200 mM;

[0608] wherein the organic solvent is acetonitrile, ethanol, or isopropanol, at a final volume fraction of 6% to 10%.

[0609] In some embodiments, the present disclosure provides a method of preparing a multispecific antibody, the method comprising the steps of a) to d) below:

[0610] a) a step of providing a first parent antibody and a second parent antibody;

[0611] b) a step of mixing the first parent antibody and the second parent antibody to form a mixture;

[0612] c) a step of adding a reducing agent and an organic solvent to the mixture and incubating; and

[0613] d) a step of obtaining a multispecific antibody,

[0614] wherein the first parent antibody and the second parent antibody bind to different antigens or epitopes, the CH3 domain of the first parent antibody comprises an amino acid mutation of 356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of 439E;

[0615] wherein the reducing agent is 2-MEA, and the final concentration of the reducing agent is 5mM to 200mM;

[0616] wherein the organic solvent is acetonitrile, ethanol or isopropanol, and the final volume fraction of the organic solvent is 6%-to 10%.

[0617] In some embodiments, the present disclosure provides a method of preparing a multispecific antibody, the method comprising the steps of a) to d) below:

[0618] a) a step of providing a first parent antibody and a second parent antibody;

[0619] b) mixing the first parent antibody and the second parent antibody to form a mixture;

[0620] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0621] d) a step of obtaining a multispecific antibody,

[0622] wherein the first parent antibody and the second parent antibody bind to different antigens or epitopes, the CH3 domain of the first parent antibody comprises an amino acid mutation of D / E356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of K439E;

[0623] wherein the reducing agent is 2-MEA, and the final concentration of the reducing agent is 5mM to 200mM;

[0624] wherein the organic solvent is acetonitrile, ethanol or isopropanol, and the final volume fraction of the organic solvent is 6%-to 10%, preferably 6% to 8%.

[0625] In some embodiments, the present disclosure provides a method of preparing a multispecific antibody, the method comprising the steps of a) to d) below:

[0626] a) a step of providing a first parent antibody and a second parent antibody;

[0627] b) mixing the first parent antibody and the second parent antibody to form a mixture;

[0628] c) adding a reducing agent and an organic solvent to the mixture and incubating; and

[0629] d) a step of obtaining a multispecific antibody,

[0630] wherein the first parent antibody and the second parent antibody bind to different antigens or epitopes, the CH3 domain of the first parent antibody comprises an amino acid mutation of D / E356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of K439E;

[0631] wherein the reducing agent is 2-MEA, and the final concentration of the reducing agent is about 75 mM;

[0632] wherein the organic solvent is acetonitrile, ethanol, or isopropanol, and the final volume fraction of the organic solvent is about 6%, about 8%, about 10%.

[0633] In some embodiments, the present disclosure provides a method of preparing a multispecific antibody, comprising the steps of a) to d) below:

[0634] a) a step of providing a first parent antibody and a second parent antibody;

[0635] b) a step of mixing the first parent antibody and the second parent antibody together to form a mixture;

[0636] c) a step of adding a reducing agent and an organic solvent to the mixture and incubating; and

[0637] d) a step of obtaining a multispecific antibody,

[0638] wherein the first parent antibody and the second parent antibody bind to different antigens or epitopes, the CH3 domain of the first parent antibody comprises an amino acid mutation of D / E356K, and the CH3 domain of the second parent antibody comprises an amino acid mutation of K439E, wherein the molar concentration of the first parent antibody and the second parent antibody in the mixture is the same;

[0639] wherein the reducing agent is 2-MEA, and the final concentration of the reducing agent is about 75 mM;

[0640] wherein the organic solvent is acetonitrile, ethanol, or isopropanol, and the final volume fraction of the organic solvent is about 6%, about 8%, about 10%.

[0641] In some embodiments, the present disclosure provides a method of preparing a multispecific antibody, comprising the steps of a) to c) below:

[0642] a) a step of culturing a cell strain producing a first parent antibody and a cell strain producing a second parent antibody, respectively;

[0643] b) a step of purifying the culture supernatant of each cell strain to obtain the first parent antibody and the second parent antibody, respectively, and incubating the first parent antibody and the second parent antibody together in the presence of a reducing agent and an organic solvent; and

[0644] c) obtaining the multispecific antibody.

[0645] In some embodiments, the present disclosure provides a method of producing a multispecific antibody, the method comprising the steps of a) to c):

[0646] a) a step of mixing a cell line producing a first parent antibody with a cell line producing a second parent antibody;

[0647] b) a step of incubating the first parent antibody and the second parent antibody together in the presence of a reducing agent and an organic solvent in the culture supernatant; and

[0648] c) obtaining the multispecific antibody.

[0649] In some embodiments, the present disclosure provides a method of producing a multispecific antibody, the method comprising the steps of a) to c):

[0650] a) a step of culturing separately a cell line producing a first and a second parent antibody;

[0651] b) a step of mixing the culture supernatant of each cell line, incubating the first parent antibody and the second parent antibody together in the presence of a reducing agent and an organic solvent; and

[0652] c) obtaining the multispecific antibody.

[0653] In another aspect, the present disclosure provides a multispecific antibody produced according to the method as described previously.

[0654] In another aspect, the present disclosure provides a multispecific antibody comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise (e.g., each comprises one) CH3 domain, wherein:

[0655] the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first and / or second polypeptide further comprises one or more same or different amino acid mutations selected from positions 347, 360, 362, 368, and 400;

[0656] the CH3 domain mutation positions are indicated by EU numbering.

[0657] In some embodiments, the multispecific antibody as described previously, wherein the CH3 domain of the first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of the second polypeptide comprises an amino acid mutation at position 439, wherein the CH3 domain of the first and / or second polypeptide further comprises an amino acid mutation selected from the group consisting of i) to iii) below:

[0658] i) 368;

[0659] ii) 347;

[0660] iii) 347 and 360; and

[0661] iv) 362 and 400.

[0662] In some embodiments, the multispecific antibody as previously described, wherein:

[0663] (1) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 347, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 347, and 360; or

[0664] (2) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 347, and 360, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 347; or

[0665] (3) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 368, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 368; or

[0666] (4) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 362, and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 362, and 400.

[0667] In some embodiments, the multispecific antibody as previously described, wherein:

[0668] (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347E, and 360E; or

[0669] (2) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 347E, and 360E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 347K; or

[0670] (3) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368M, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368M; or

[0671] (4) the CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E, Q362K, and S400K.

[0672] In some embodiments, the multispecific antibody as previously described, wherein the CH3 domains of the first and second polypeptides are derived from a CH3 domain of an IgG. In some embodiments, the multispecific antibody as previously described, wherein the CH3 domains of the first and second polypeptides are derived from a CH3 domain of an IgG1. In some embodiments, the multispecific antibody as previously described, wherein the CH3 domains of the first and second polypeptides are derived from a CH3 domain of a human IgG1. In some embodiments, the human IgG1 has an amino acid sequence set forth in SEQ ID NO: 5, 6, or 7. In some embodiments, the CH3 domain of the human IgG1 has an amino acid sequence set forth in SEQ ID NO: 32 or 33.

[0673] In some embodiments, the multispecific antibody as previously described, wherein:

[0674] (1) the CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and Q347K, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E, Q347E, and K360E; or

[0675] (2) the CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K, Q347E, and K360E, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and Q347K; or

[0676] (3) the CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K and L368M, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E and L368M; or

[0677] (4) the CH3 domain of the first polypeptide comprises amino acid mutations of D / E356K, Q362E, and S400E, and the CH3 domain of the second polypeptide comprises amino acid mutations of K439E, Q362K, and S400K.

[0678] In some embodiments, the multispecific antibody as previously described is efficiently formed in the presence of a reducing agent and an organic solvent.

[0679] In some embodiments, the multispecific antibody as described above exhibits at least a 1-fold increase in recombination efficiency in the presence of an organic solvent compared to the absence of an organic solvent (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, or 100-fold). In some embodiments, the multispecific antibody as described above exhibits approximately a 10-fold increase in recombination efficiency in the presence of an organic solvent compared to the absence of an organic solvent.

[0680] In some embodiments, the multispecific antibody as described above exhibits a recombination efficiency at least 30% higher in the presence of an organic solvent than in the absence of an organic solvent (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%). In some embodiments, the multispecific antibody as described above, wherein the first polypeptide is an antibody heavy chain, and / or the second polypeptide is an antibody heavy chain.

[0681] In some implementations, the multispecific antibody, as described above, also comprises one or more antibody light chains.

[0682] In some implementations, the multispecific antibody as described above is a bispecific antibody or a trispecific antibody.

[0683] In another aspect, this disclosure provides a pharmaceutical composition comprising the multispecific antibody as described above and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0684] In another aspect, this disclosure provides an immunoconjugate comprising: a multispecific antibody as described above and a payload, wherein the payload is conjugated to the multispecific antibody. 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.

[0685] In another aspect, this disclosure provides an isolated nucleic acid that encodes a multispecific antibody as described above.

[0686] In another aspect, this disclosure provides a host cell containing the isolated nucleic acids as described above.

[0687] In another aspect, this disclosure provides a method for preparing the multispecific antibodies as described above.

[0688] In another aspect, the present disclosure provides a method of preparing the multispecific antibody as described above, comprising: (a) a step of altering a nucleic acid encoding an amino acid residue forming the inter-polypeptide interface; (b) a step of culturing a host cell having the nucleic acid to express the polypeptide; (c) a step of recovering the polypeptide from the culture of the host cell; and (d) a step of incubating each polypeptide in the presence of a reducing agent and an organic solvent to recover the desired multispecific antibody.

[0689] Combinations with isoelectric point alteration techniques and the like

[0690] As a further preferred aspect of the present disclosure, by introducing an amino acid variation that alters the isoelectric point (pI value) of a polypeptide into the polypeptides of the present disclosure, a polypeptide multimer having the target 1st to 4th polypeptides can be purified or prepared with higher purity and efficiency (WO2007114325, US20130171095). As the amino acid variation introduced for facilitating the association of polypeptides, a method for hetero-associating a polypeptide comprising 2 heavy chain constant regions by altering the CH3 domain of the heavy chain constant region, as described in 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, and the like; and a method for facilitating the association of a specific combination of heavy and light chains, as described in WO2009080251, WO2009080252, WO2009080253, and the like, and the like, can also be used.

[0691] Combinations with other constant region and / or variable region alteration techniques

[0692] As a non-limiting embodiment of the present disclosure, a combination with a constant region alteration technique aimed at enhancing the binding to FcγR (WO2013047752) can be cited.

[0693] As a combination of the present disclosure with other constant region alteration techniques, a combination with a technique for controlling and complement binding can be exemplified. As a complement, any of the complement components can be used as long as it is a polypeptide that forms a complement cascade, but as a preferred complement, a complement component of Clq, Clr, or Cls involved in opsonin binding can be appropriately exemplified. An Fc region having a higher complement binding activity than that of a native Fc region can be produced by altering the amino acids of the native Fc region. The native Fc region described herein refers to an Fc region represented by human IgGl, IgG2, IgG3, or IgG4. Whether or not the Fc region has a higher complement binding activity than that of the native Fc region can be appropriately performed using known immunological methods such as FACS, ELISA, and the like. The "alteration of amino acids" or "amino acid alteration" of the Fc region includes an alteration to an amino acid sequence different from the amino acid sequence of the starting Fc region. Any of the Fc regions can be used as a starting domain as long as the modified alteration binds to a complement in the neutral pH range. Furthermore, an Fc region to which a further alteration has been added can also be appropriately used as the Fc region of the present disclosure, as a starting Fc region to which an alteration has been added. The starting Fc region refers to a polypeptide itself, a composition including the starting Fc region, or an amino acid sequence encoding the starting Fc region. In the starting Fc region, the Fc region of a known IgG antibody produced by recombination outlined in the items of the antibody can be included. The origin of the starting Fc region is not particularly limited, and can be obtained from any organism of a non-human animal or a human. As the any organism, an organism selected from the group consisting of a mouse, a rat, a guinea pig, a hamster, a gerbil, a cat, a rabbit, a dog, a goat, a sheep, a cow, a horse, a camel, and a non-human primate can be appropriately exemplified. In another aspect, the starting Fc region can also be obtained from a cynomolgus monkey, a marmoset, a macaque, a chimpanzee, or a human. The starting Fc region can be obtained from human IgGl, but is not limited to a specific type of IgG. This means that the Fc region of human IgGl, IgG2, IgG3, or IgG4 can be appropriately used as the starting Fc region. Also, this means that in the present disclosure, the Fc region of any type or subclass of IgG from any of the organisms described above can be appropriately used as the starting Fc region. Examples of naturally occurring variants or engineered models of IgG are described in known documents (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.

[0694] The amino acid to be changed can be changed at any position as long as it has a binding activity to complement or improves the binding activity to complement. When the antibody comprises the Fc region of human IgGl as the human Fc region, it is preferable to comprise a change that produces an effect of increasing the binding activity to complement stronger than the starting Fc region of human IgGl. As the amino acid for changing the binding activity to complement, for example, the amino acid of the Fc region in which the binding activity to Clq is changed 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, and the like can be exemplified.

[0695] As such an amino acid that can be changed to enhance the binding activity to Clq, for example, one or more amino acids selected from the group consisting of positions 231 to 238 and positions 318 to 337 represented by EU numbering can be exemplified. As an example of the amino acid without limitation, one or more amino acids selected from the group consisting of positions 235, 237, 318, 320, 322, 324, 327, 331, and 333 can be exemplified. By changing these amino acids, the Fc region of IgG-type immunoglobulin can be enhanced in the binding to complement.

[0696] As other combinations of the present disclosure with other constant region alteration techniques, combinations of antibody alteration techniques such as Fc technology for enhancing binding to FcRn at acidic pH (WO2002060919, WO2004035752, WO2000042072), Fc technology for enhancing binding to FcRn at neutral pH (WO2011122011, WO2012133782), Fcγ receptor selective binding enhancement technology (WO2012115241, WO2013125667), Fcγ receptor selective binding enhancement technology (ADCC activity enhancement technology) (WO2013002362), technology for reducing binding activity to rheumatoid factor (WO2013046704), and the like, combinations of Fc mutation for modulating effector functions (Liu R, Oldham R J, Teal E, et al. Fc-engineering for modulated effector functions - improving antibodies for cancer treatment [J]. Antibodies, 2020, 9(4):64.) can be mentioned.

[0697] Alteration of nucleic acid

[0698] In another embodiment of the production method of the present disclosure, the present disclosure provides a production method of a heteromultimer, which is a production method of a heteromultimer having variation in an amino acid residue forming an inter-polypeptide interface (e.g., one or more amino acid residues selected from the group consisting of positions 347, 356, 360, 362, 368, 392, 398, 400, 411, and 439, according to EU numbering) to control dissociation and / or association between polypeptides, the production method comprising the steps of: (a) a step of altering a nucleic acid encoding an amino acid residue forming the inter-polypeptide interface or the like according to the original nucleic acid to control dissociation and association between polypeptides; (b) a step of culturing a host cell having the nucleic acid to express the polypeptide; (c) a step of recovering the polypeptide from the culture of the host cell; and (d) a step of incubating each polypeptide in the presence of a reducing agent and an organic solvent to recover the desired heteromultimer.

[0699] A method comprising a step of altering a nucleic acid encoding an amino acid residue forming an inter-polypeptide interface according to the original nucleic acid to suppress association between polypeptides using the above-mentioned dissociation and / or association control method of the present disclosure is also one of the preferred embodiments of the above-mentioned production method of the present disclosure.

[0700] In the above-described method of the present disclosure, "altering a nucleic acid" means changing a nucleic acid to correspond to an amino acid residue introduced by "alteration" in the present disclosure. More specifically, it means changing a nucleic acid encoding an original (before alteration) amino acid residue to a nucleic acid encoding an amino acid residue introduced by alteration. Generally, it means a genetic manipulation or mutagenesis treatment of at least one base insertion, deletion, or substitution to the original nucleic acid to form a codon encoding a target amino acid residue. That is, a codon encoding an original amino acid residue is replaced with a codon encoding an amino acid residue introduced by alteration. Such a change in nucleic acid can be appropriately performed using techniques known to those skilled in the art, such as site-specific mutagenesis, PCR mutagenesis, and the like.

[0701] The nucleic acid in the present disclosure is usually carried (inserted) into an appropriate vector and introduced into a host cell. As the vector, there is no particular limitation as long as the inserted nucleic acid is stably maintained.

[0702] The host cell is not particularly limited, and various host cells can be used according to the purpose.

[0703] In order to allow a polypeptide expressed in a host cell to be secreted into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, an appropriate secretion signal can be combined with the target polypeptide. The signal can be an endogenous signal or a heterologous signal to the target polypeptide.

[0704] Regarding the recovery of the polypeptide in the above-described production method, when the polypeptide of the present disclosure is secreted into the culture medium, the culture medium is recovered. When the polypeptide of the present disclosure is produced in a cell, the cell is first lysed, and then the polypeptide is recovered.

[0705] When the polypeptide of the present disclosure is recovered from a recombinant cell culture and purified, known methods involving ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography can be used.

[0706] Recombinant methods

[0707] Heteromultimers can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the heteromultimer are provided.

[0708] In one embodiment, the disclosure provides an isolated nucleic acid encoding a heteromultimer as previously described. Such a nucleic acid can independently encode any of the polypeptide chains previously described. In another aspect, the disclosure provides one or more vectors (e.g., expression vectors) comprising such a nucleic acid. In another aspect, the disclosure provides a host cell comprising such a nucleic acid. In one embodiment, a method of making a heteromultimer is provided, wherein the method comprises, culturing a host cell comprising a nucleic acid encoding the heteromultimer, as provided above, under conditions suitable for expression, and optionally recovering the protein from the host cell (or host cell culture medium).

[0709] For recombinant production of a heteromultimer, nucleic acids encoding the proteins are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures, or produced by recombinant methods or by chemical synthesis.

[0710] Suitable host cells for cloning or expression of a vector encoding a heteromultimer include prokaryotic or eukaryotic cells described herein. For example, production in bacteria is possible, particularly when glycosylation and Fc effector functions are not needed. Upon expression, the protein can be isolated from bacterial cell paste in a soluble fraction, and can be further purified.

[0711] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains. Suitable host cells for the expression of antibodies can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells; plant cell cultures also can be utilized as hosts, e.g., US5959177, US6040498, US6420548, US7125978, and US6417429; vertebrate cells can also be used as hosts, e.g., mammalian cell lines adapted to grow in suspension, e.g., the CHO cell line; other examples of appropriate mammalian host cell lines are monkey kidney CVl line (COS-7) transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells); monkey kidney cells (CVl); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (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, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for production of antibodies, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0712] assays

[0713] The heteromultimers provided herein can be identified, screened, or characterized for their physical / chemical characteristics and / or biological activities by a variety of assays known in the art. In one aspect, the activities of the heteromultimers of the disclosure are tested, e.g., by known methods such as flow cytometry, ELISA, Western blotting, HPLC, SEC, IEC, mass spectrometry, and the like.

[0714] Methods of treatment and routes of administration

[0715] Any of the heteromultimers provided herein can be used in a method of treatment. In yet another aspect, the disclosure provides use of a heteromultimer in the manufacture or preparation of a medicament. In some embodiments, in one such embodiment, the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents). A “subject” according to any of the above embodiments can be a human.

[0716] In yet another aspect, a pharmaceutical composition comprising the heteromultimer is provided, e.g., for any of the above pharmaceutical uses or methods of treatment. In one embodiment, the pharmaceutical composition comprises any of the heteromultimers provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0717] The heteromultimers of the disclosure (and any additional therapeutic agents) can be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and, if local treatment is required, intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short term or long term. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0718] The heteromultimers of the disclosure will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The heteromultimers can be formulated with or without one or more agents currently used in prevention or treatment of the disorder in question. The effective amount of such other agents depends on the amount of heteromultimer present in the composition, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages are used, or other dosages are used, in conjunction with the dosages and routes of administration of the heteromultimers herein described.

[0719] For prevention or treatment of disease, the appropriate dosage of the heteromultimers of the disclosure (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 treatment molecule, the severity and course of the disease, whether the agent is for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician. The treatment molecule is suitably administered to the patient at one time or over a series of treatments.

[0720] Articles of manufacture

[0721] 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 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 a 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.

[0722] Although the antibodies used in the examples target specific antigens, those skilled in the art will understand, based on the teachings of this disclosure, that the technical effect is achieved not by a specific CDR sequence, nor by a specific antigen sequence, but by a mutation in an amino acid of the CH3 domain (e.g., one or more amino acids selected from positions 347, 356, 360, 362, 368, 392, 398, 400, 411, and 439) to promote the formation of heteropolymers.

[0723] Example

[0724] The following examples further describe the present disclosure and are not to be construed as limiting the scope of the present disclosure. The examples of the present disclosure do not include detailed descriptions of conventional methods, experimental procedures, experimental results not specifically mentioned, which are typically performed according to conventional methods, as such methods are well known to those skilled in the art and are described in many publications, such as “Molecular Cloning” published by Cold Spring Harbor Laboratory (Green M R, Sambrook J. Molecular cloning. A Laboratory Manual 4th, 2012.) or “Antibody engineering: methods and protocols” of Springer protocols (Antibody engineering: methods and protocols. Humana Press, 2018.); or according to the conditions recommended by the manufacturer of the raw materials or commercial suppliers; reagent materials not specifically mentioned for their source, are obtained commercially.

[0725] Example 1.1: Sources and preparation of anti-CTLA4 monoclonal antibody Ipilimumab and anti-CCR8 monoclonal antibody CP11H2

[0726] The Ipilimumab heavy and light chain amino acid sequences are from patent WO2016059602A2; the CP11H2 heavy and light chain amino acid sequences are from patent WO2023208182A1.

[0727] > Ipilimumab heavy chain amino acid sequence

[0728] > Ipilimumab light chain amino acid sequence

[0729] > CP11H2 heavy chain amino acid sequence

[0730] > CP11H2 light chain amino acid sequence

[0731] Note: the single underlined region represents the antibody variable region.

[0732] DNA encoding the above variable regions were synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd. The heavy chain variable region of Ipilimumab (Ipilimumab-VH) and the light chain variable region of Ipilimumab (Ipilimumab-VL) were connected with the DNA of human IgG1 heavy chain constant region and 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 heavy chain variable region of CP11H2 (CP11H2-VH) and the light chain variable region of CP11H2 (CP11H2-VL) were connected with the DNA of human IgG1 heavy chain constant region and 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 above heavy chain and light chain genes were cloned into the expression vector pcDNA3.4, respectively, and the expression vectors of the heavy chain and light chain of each antibody were transfected into Expi293F cells (Thermo Fisher Scientific, Cat No: A14527) at the same time to express the antibody using PEI (Polyethylenimine, polyethyleneimine). TM The 293 cells were cultured in serum-free medium for about 5 days, and the cell supernatant was collected and the antibody was purified by Protein A affinity chromatography.

[0733] The purification steps are described as follows: high-speed centrifugation was used to remove impurities in the cell culture supernatant. First, the MabSelect Sure (Cytiva, Cat No: 17543801) affinity chromatography column was washed with 0.2M NaOH, then rinsed with pure water and equilibrated with PBS. The supernatant was passed through the affinity column, and the affinity column was washed with PBS until the A280 decreased to the baseline. The target protein was eluted with 0.1M acetic acid buffer at pH 3.5, and the antibody solution was neutralized with 1M Tris-HCl (pH 8.0). The antibody solution was appropriately concentrated by ultrafiltration, and then further purified by gel chromatography column HiLoad Superdex 200 (Cytiva, Cat No: 28989335). The antibody concentration was determined by ultraviolet spectrophotometry, filtered to remove bacteria, and stored in the refrigerator (4°C). This method is used to purify related monoclonal antibodies, and this method can also be used to purify other antibodies or recombinant proteins in the present disclosure.

[0734] Example 1.2: Preparation of Ipilimumab and CP11H2 monoclonal mutants

[0735] Site-directed mutagenesis was used to introduce mutations in the coding region of the CH3 domain of the heavy chain of Ipilimumab and CP11H2 to mutate the amino acid residue at a specific position to a specific amino acid, using the Eu numbering scheme to identify the position of the amino acid residue. Ipilimumab and CP11H2 with the mutations were prepared according to the expression and purification method described in Example 1.1. The resulting antibody samples were filtered to remove bacteria using a 0.2-μm filter and the concentration of the antibodies was determined using a NanoDrop microspectrophotometer. The naming rule of the antibody mutants is as follows, for example, Ipilimumab-D356K indicates that the D at position 356 of the heavy chain of Ipilimumab is mutated to K, and the rest is inferred by analogy, as shown in Table 2. Ipilimumab and CP11H2 are abbreviated as Ipi and CP11, respectively, in the recombination reaction.

[0736] Table 2. Parent monoclonal mutants and corresponding recombination reactions Note: * indicates that the recombination reaction is performed between two monoclonal antibodies.

[0737] Example 1.3: Recombination reaction method of anti-CTLA4 / CCR8 bispecific antibody

[0738] Prepare a 2-MEA stock solution by weighing 1700 mg 2-MEA (Sigma-Aldrich, Cat No: 30078 / 100G), dissolving in 20 mL PBS, and adjusting the pH to 7.4 with 1M NaOH. The 2-MEA stock solution has a concentration of about 750 mM. Dilute three organic solvents, acetonitrile (ACN, purchased from Sinopharm), ethanol (ETOH, purchased from Sinopharm), and isopropanol (IPA, purchased from Sinopharm) to a 20% stock solution in PBS. Adjust the concentrations of the Ipilimumab and CP11H2 mutants prepared in the above examples to the same molar concentration (3.45 mM-690 mM; corresponding mass concentration range: 0.5-100 mg / mL) with PBS. Mix equal volumes of the Ipilimumab mutant and the corresponding CP11H2 mutant, then mix an appropriate amount of the 2-MEA stock solution with the organic solvent (ACN, ETOH, or IPA) and add it to the antibody mixture, mix well to make the final concentration of 2-MEA 75 mM, and the final volume fraction of the three organic solvents 6%, 8%, and 10%, respectively. Place the mixed solution (containing Ipilimumab mutant, CP11H2 mutant, 2-MEA, and different volume fractions of organic solvent) in a 37°C water bath for 2.5 h. Transfer the mixed solution to Slide-A-Lyzer TM G3 Dialysis Cassettes (Thermo Fisher Scientific, Cat No: A52971) and dialyze in 5 L of PBS. After 5 hours, replace the PBS and place it in the refrigerator (4°C) for thorough dialysis. After 18 hours of dialysis, collect the sample and re-determine the antibody concentration. Use HPLC-SEC and IEC to determine the physicochemical properties of the parent monoclonal antibody and the corresponding recombinant reaction product.

[0739] Example 1.4: Recombination reaction efficiency of anti-CTLA4 / CCR8 bispecific antibody

[0740] The physicochemical property analysis methods used in this example are described as follows.

[0741] 1. High-performance liquid chromatography-size exclusion chromatography (HPLC-SEC)

[0742] Antibodies are high molecular weight proteins with highly complex secondary and tertiary structures. Antibodies are heterogeneous in biochemical and biophysical properties due to changes such as post-translational modifications, aggregation, and degradation. Variants, aggregates, and degradation fragments are often observed when bispecific antibodies are analyzed by separation techniques, and their presence can compromise safety and efficacy. Aggregates, degradation fragments, and incompletely assembled molecules are prone to occur during the production and storage of antibodies. The present disclosure uses HPLC-SEC to detect the content of the above impurities in the sample. The molecular weight of the aggregate is greater than that of the monomer, and the retention time of the corresponding spectrum peak is shorter; the molecular weight of the degradation fragment or the incompletely assembled molecule is smaller than that of the monomer, and the retention time of the corresponding spectrum peak is longer.

[0743] The chromatograph used in HPLC-SEC is Waters e2695-2489. The mobile phase is prepared as follows: weigh 3.06 g of NaH2PO4, 3.48 g of Na2HPO4, and 17.53 g of NaCl, dissolve with purified water, continue to add purified water to a total volume of 1 L, and adjust the pH to 6.8 with 2M NaOH solution. The column is Waters XBridge BEH 200A SEC, with a size of 7.8 x 300 mm 3.5 μm. The injection amount is set to 30 μg, the flow rate is 0.5 mL / min, the elution time is 30 minutes, the column temperature is 30°C, the sample chamber temperature is 15°C, and the detection wavelength is 280 nm.

[0744] 2. High-performance liquid chromatography-ion exchange chromatography (HPLC-IEC)

[0745] Many post-translational modifications of proteins (e.g. N-glycosylation, modification of C-terminal lysine residues, N-terminal glutamine or glutamic acid cyclization, asparagine deamidation, aspartate isomerization, and oxidation of amino acid residues, etc.) directly or indirectly cause changes in the surface charge of antibodies, leading to the generation of charge heterogeneity. Charge variants can be separated and analyzed based on the charge they carry, and commonly used analytical methods are cation exchange chromatography (CEX) and anion exchange chromatography (AEX). When analyzed by chromatography-based methods, acidic species and basic species are defined based on their retention times relative to the main peak. Acidic species are variants that elute earlier than the main peak on CEX or later than the main peak on AEX, while basic species are variants that elute later than the main peak on CEX or earlier than the main peak on AEX. The spectral peaks corresponding to acidic species and basic species are referred to as acidic peaks and basic peaks, respectively. Charge variants are prone to be generated during the production and storage of antibodies. The charge heterogeneity of samples is analyzed using HPLC-IEC herein.

[0746] The chromatograph used for HPLC-IEC is Agilent 1260 Infinity II DAD BIO-LC. The mobile phase A is 20 mM MES pH 6.0 (weigh 3.9048 g of MES, molecular weight 195.24 g / mol, dissolved with purified water, continue to add purified water to a total volume of 1 L, adjust the pH to 6.0 with 10 M NaOH), and the mobile phase B is 20 mM MES + 500 mM NaCl pH 6.0 (weigh 3.9048 g of MES and 29.22 g of NaCl, dissolved with purified water, continue to add purified water to a total volume of 1 L, adjust the pH to 6.0 with 10 M NaOH), the ratio of the mixture of the two mobile phases changes with time according to the pre-set program, the flow rate is 0.4 mL / min. The chromatographic column is YMC BioPro SP-F, specification 4.6 x 100 mm 5 μm. The injection amount is set to 30 μg, the column temperature is 30 °C, the sample chamber temperature is 15 °C, and the detection wavelength is 280 nm.

[0747] 3. Mass Spectrometry

[0748] The molecular weight of the recombinant reaction product is analyzed by mass spectrometry. If the molecular weight of the recombinant reaction product is exactly half the sum of the molecular weights of the two parent monoclonal mutant molecules, it is considered that the recombinant product is a bispecific antibody. The method for detecting the molecular weight of the antibody is described as follows.

[0749] The sample treatment method for determining the deglycosyl intact molecular weight is as follows: 30 μg of the expressed antibody was lyophilized and 10 μL of 8M Guanidine-HCl was added, and denaturation was performed at 70°C for 10 minutes. 90 μL of distilled water was added, and 30 μL was added to 0.8 μL of PNGase F, and incubation was performed in a 37°C water bath for 2 hours. 0.5 μg of the sample was taken for determination of the deglycosyl intact molecular weight. The sample treatment method for determining the deglycosyl reduced molecular weight is as follows: 30 μg of the expressed antibody was lyophilized and 10 μL of 8M Guanidine-HCl was added, and denaturation was performed at 70°C for 10 minutes. 90 μL of distilled water was added, and 30 μL was added to 0.8 μL of PNGase F, and incubation was performed in a 37°C water bath for 2 hours. 2 μL of 0.025M DTT was added, and reduction was performed at 70°C for 10 minutes. 0.5 μg was taken for determination of the deglycosyl reduced molecular weight.

[0750] The chromatographic separation conditions are described as follows. The chromatographic column is Poroshell 300SB-C8, with a size of 5 μm 2.1*75 mm. The mobile phase A is 0.1% HCOOH / H2O, and the mobile phase B is 0.1% HCOOH / ACN. The column temperature is 75°C. The mass spectrometry detection conditions are described as follows: the mass spectrometer is Agilent 6530 Q-TOF LC-MS, and the ionization mode is electrospray ionization (ESI). The acquisition mode is 1GHz (mass range 500-5000 m / z). The dry gas temperature is set to 325°C, the dry gas flow is 10 L / min, the atomizer is 40 psi, the sheath gas temperature is 350°C, the sheath gas flow is 12 L / min, the spray voltage is 500V, the capillary voltage is 3500V, the fragmentation voltage is 200V, the Skimmer voltage is 65V, and the Rf voltage is 7500V.

[0751] Here, the effects of different organic solvents on the recombination reaction efficiency were explored in advance. Three different organic solvents were added to the reaction system of the mutant combination Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E or Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K at a volume fraction of 10%, and the recombination reaction was carried out according to the method described in Example 1.3 above. The recombination reaction product (containing bispecific antibodies) of the mutant combination was analyzed by HPLC-SEC and HPLC-IEC, and the measured purity (here, the SEC purity is the main peak area ratio, and the IEC purity is the sum of the main peak, acidic peak and basic peak, etc. Main peak area ratio) data is summarized in Table 3. The parent monoclonal mutant and the corresponding recombination reaction product in Table 3 were analyzed by superimposing the HPLC-IEC spectrum, and the related spectrum is shown in FIGS. 1A to 1H.

[0752] Table 3. SEC and IEC purity of recombination reaction products of specific parental mutant combinations and under corresponding conditions Note: #NA means no organic solvent is added; # (10%) means a volume fraction of 10% of organic solvent is added.

[0753] The recombination reaction efficiency formula is: 100% - (percentage of peak area corresponding to parental monoclonal antibody A + percentage of peak area corresponding to parental monoclonal antibody B). The monoclonal antibodies used in the recombination reaction of the present disclosure are not fine-purified, and there are aggregates and a small amount of impurities.

[0754] Table 4. Recombination reaction efficiency of combinations in Table 3 calculated by residual amount of parental monoclonal mutants Note: #NA means no organic solvent is added; # (10%) means a volume fraction of 10% of organic solvent is added.

[0755] The results of FIG. 1A, FIG. 1E, Table 3 and Table 4 show that in the absence of organic solvents, the recombination reaction efficiencies of Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E and Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K are only 14.3% and 17.9%, respectively, and there are obvious residual peaks corresponding to parental monoclonal antibodies that do not participate in the recombination reaction in the HPLC-IEC spectrum of the recombination reaction product, indicating that the two combinations are difficult to produce bispecific antibodies in the absence of organic solvents. The results of FIG. 1B, FIG. 1F, Table 3 and Table 4 show that under the action of 10% ACN, both of the two different mutant combinations Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E and Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K can effectively undergo recombination reaction to generate bispecific antibodies, and the recombination efficiency reaches 100%. In addition, under the action of 10% IPA or ETOH, the recombination reaction efficiency of the above two mutant combinations can be increased from less than 20% to 79.9%-93.9% (FIG. 1C, FIG. 1D, FIG. 1G and FIG. 1H). The experimental data shown in this example show that three kinds of organic solvents at a volume fraction of 10% can greatly improve the efficiency of recombination reaction to produce bispecific antibodies. Among them, ACN can more effectively improve the recombination reaction efficiency than the other two organic solvents, thereby improving the yield of bispecific antibodies.

[0756] Here, ACN was selected as the research object, and the influence of organic solvents on the recombination reaction efficiency was further explored. Different volume fractions (6%, 8%, and 10%) of ACN were added to the reaction system of the mutant combination in Table 2, and the recombination reaction was carried out according to the method described in Example 1.3 above. The recombination reaction products (containing bispecific antibodies) of the mutant combination in Table 2 were analyzed by HPLC-SEC and HPLC-IEC, and the measured purity (here, the SEC purity is the main peak area ratio, and the IEC purity is the sum of the main peak, acidic peak, and basic peak, etc. Main peak area ratio of spectrum peak area ratio) data is summarized in Table 5. The parent monoclonal mutant and the corresponding recombination reaction product in Table 5 were analyzed by superimposing the HPLC-IEC spectrum, and the related spectrum is shown in FIGS. 2A to 2T.

[0757] Table 5. SEC and IEC purity of recombination reaction products of various parent mutant combinations and under corresponding conditions Note: #NA indicates that no organic solvent is added; #ACN(%) indicates that different proportions of volume fractions of ACN are added.

[0758] Table 6. Recombination reaction efficiency of the combination in Table 5 calculated by the residual amount of the parent monoclonal antibody Note: #NA indicates that no organic solvent is added; #ACN(%) indicates that different proportions of volume fractions of ACN are added.

[0759] The above results show (Figures 2A-2T, Table 5 and Table 6) that the recombination reaction efficiency of Ipi-D356K*CP11-K439E, Ipi-D356K+L368M*CP11-K439E+L368M, Ipi-D356K+Q347K*CP11-K439E+Q347E+K360E, Ipi-D356K+Q347E+K360E*CP11-K439E+Q347K and Ipi-D356K+Q362E+S400E*CP11-K439E+Q362K+S400K is only 3.3%, 17.4%, 14.3%, 17.9% and 16.9%, respectively, in the absence of organic solvent, which indicates that these five mutant combinations are difficult to recombine to form complete bispecific antibodies in the absence of organic solvent. The results of Figures 2A-2D of the present disclosure show that only the reaction combination of introducing D356K and K439E mutations in Ipilimumab and CP11H2 parent monoclonal antibodies, respectively, the recombination reaction efficiency can be increased from 3.3% to 36.1%, 66.8% and 85.1% by adding different volume fractions of ACN (6%, 8% and 10%). The results of Figures 2E-2T show that the remaining four pairs of mutant combinations that additionally introduce engineered sites on the basis of Ipi-D356K*CP11-K439E can greatly increase the recombination reaction efficiency under the reaction conditions of adding ACN. Among them, 8% and 10% volume fraction of ACN can increase the recombination reaction efficiency to more than 95%, and the highest can reach 100%.

[0760] 3. The method and conditions for detecting the molecular weight of the deglycosylated antibody by mass spectrometry (MS) are as previously described.

[0761] Table 7. Mass spectrometry analysis results of preferred monoclonal mutant combinations and corresponding recombination reaction products Note: The theoretical heavy chain, light chain and complete molecular weights in this table are theoretical values calculated by software after considering post-translational modifications and redox states of the protein.

[0762] The results of Table 7 show that under the conditions of 8% and 10% ACN, the theoretical heavy chain, light chain and complete molecular weights of the bispecific antibodies produced by different mutant combinations are very close to the actual heavy chain, light chain and complete molecular weights measured by mass spectrometry. The above results show that a certain volume fraction of ACN can promote the effective recombination reaction of originally inert mutant combinations and produce structurally correct bispecific antibodies. There are no obvious spectral peaks corresponding to the parent monoclonal mutant in the mass spectrum of the recombination reaction product.

[0763] Example 2.1: Sources and preparation of anti-HER2 monoclonal antibodies Trastuzumab and Pertuzumab

[0764] The heavy and light chain amino acid sequences of Trastuzumab were derived from patent WO2014096051A1, and the heavy and light chain amino acid sequences of Pertuzumab were derived from patent WO2006033700A2.

[0765] The heavy chain amino acid sequence of Trastuzumab is as follows (SEQ ID NO: 8):

[0766] The light chain amino acid sequence of Trastuzumab is as follows (SEQ ID NO: 9):

[0767] The heavy chain amino acid sequence of Pertuzumab is as follows (SEQ ID NO: 10):

[0768] The light chain amino acid sequence of Pertuzumab is as follows (SEQ ID NO: 11):

[0769] Note: The single underlined region represents the antibody variable region.

[0770] The DNA encoding the above variable regions was obtained by gene synthesis. The Trastuzumab heavy chain variable region (Trastuzumab-VH) and the light chain variable region (Trastuzumab-VL) were connected to human IgG1 heavy chain constant region and human Kappa light chain constant region DNA, respectively, to construct the full-length Trastuzumab heavy chain and light chain genes, designated as Trastuzumab-HC and Trastuzumab-LC, respectively. The heavy chain variable region of Pertuzumab (Pertuzumab-VH) and the light chain variable region of Pertuzumab (Pertuzumab-VL) were connected to human IgG1 heavy chain constant region and human Kappa light chain constant region DNA, respectively, to construct the full-length Pertuzumab heavy chain and light chain genes, designated as Pertuzumab-HC and Pertuzumab-LC, respectively. The above heavy and light chain genes were cloned into the expression vector pcDNA3.4, and the expression vectors of the heavy and light chains of each antibody were transfected into Expi293F cells at the same time using PEI (Polyethylenimine, polyethyleneimine). TMThe cells (Thermo Fisher Scientific, Cat. No. A14527) were used to express antibodies. The 293-F cells were cultured in serum-free medium for about 5 days, and the cell supernatant was collected and used to purify the antibodies by Protein A affinity chromatography.

[0771] The purification steps are described as follows: high-speed centrifugation was used to remove impurities in the cell culture supernatant. First, the MabSelect Sure (Cytiva, Cat. No. 17543801) affinity chromatography column was washed with 0.2M NaOH, then rinsed with pure water and equilibrated with PBS. The supernatant was then passed through the affinity column, and the affinity column was washed with PBS until the A280 dropped to the baseline. The target protein was eluted with 0.1M acetic acid buffer at pH 3.5, and the antibody solution was neutralized with 1M Tris-HCl (pH 8.0). The antibody solution was appropriately concentrated by ultrafiltration, and then further purified by gel chromatography column HiLoad Superdex 200 (Cytiva, Cat. No. 28989335). The antibody concentration was determined by ultraviolet spectrophotometry, filtered to remove bacteria, and stored in the refrigerator (4°C). This method was used to purify related monoclonal antibodies, and this method can also be used to purify other antibodies or recombinant proteins in the present disclosure.

[0772] Example 2.2: Preparation of Trastuzumab and Pertuzumab monoclonal mutants

[0773] Site-directed mutagenesis was used to introduce genetic mutations in the coding region of the Trastuzumab and Pertuzumab heavy chain CH3 domain, mutating the amino acid residues at specific positions to specific amino acids, using the Eu numbering scheme to identify the positions of the amino acid residues. Trastuzumab and Pertuzumab with mutations were prepared according to the expression and purification method described in Example 2.1. The resulting antibody samples were filtered to remove bacteria using a 0.2-μm filter, and the concentration of the antibodies was determined using a NanoDrop microspectrophotometer. The naming rules for antibody mutants are as follows, for example, Trastuzumab-E356K indicates that the E at position 356 of the Trastuzumab heavy chain is mutated to K, and the rest is similar, as shown in Table 8. Trastuzumab and Pertuzumab are abbreviated as Tra and Per, respectively, in the recombinant reaction.

[0774] Table 8. Parental monoclonal mutants and corresponding recombinant reactions Note: * indicates that two parental monoclonal mutants were subjected to a recombinant reaction under certain conditions.

[0775] Example 2.3: Preparation method of anti-HER2 bispecific antibody

[0776] Trastuzumab and Pertuzumab mutants prepared in the above examples were adjusted to equal molar concentration (3.45 μΜ-690 μΜ; corresponding mass concentration range 0.5-100 mg / mL) with PBS respectively, and equal volume of Trastuzumab mutant and corresponding Pertuzumab mutant were mixed, then appropriate amount of 2-MEA stock solution was mixed with organic solvent ACN and added to the above antibody mixture, mixed thoroughly, so that the final concentration of 2-MEA was 75 mM, and the final volume fraction of organic solvent ACN was 6%. The mixed solution (containing Trastuzumab mutant, Pertuzumab mutant, 2-MEA and ACN) was placed in a 37°C water bath for 2.5 h. The mixed solution was transferred to Slide-A-Lyzer TM G3 Dialysis Cassettes (Thermo Fisher Scientific, Cat: A52971) and dialyzed in 5 L of PBS, and the PBS was replaced after 5 h and placed in a refrigerator (4°C) for thorough dialysis. The sample was collected after 18 h of dialysis, and the antibody concentration was re-determined.

[0777] Example 2.4: Recombination efficiency of anti-HER2 bispecific antibody

[0778] In this example, HPLC-SEC and IEC were used to determine the physicochemical properties of the parent monoclonal antibody and the above recombination reaction product. The physicochemical analysis methods such as HPLC-SEC, HPLC-IEC and mass spectrometry used here are as described in the previous examples.

[0779] Since IEC cannot effectively separate and analyze various components in a specific recombination reaction product, High-Performance Liquid Chromatography-Reversed-Phase (HPLC-RP) was used to analyze the specific recombination reaction product, and the recombination reaction efficiency was calculated according to the corresponding results. The experimental method of HPLC-RP is described as follows: the used chromatograph is Agilent 1290 UPLC DAD; the mobile phase is prepared as follows: mobile phase A is 0.1% aqueous perchloric acid, mobile phase B is acetonitrile, gradient elution B%: 0-15 min (20%-45%); 25 min (85%); 25.1-30 min (20%); injection amount: 3 μg; the chromatographic column is Agilent AdvanceBio RP-mAb Diphenyl, 2.1 x 100 mm 3.5 μM; flow rate 0.3 mL / min, elution time 30 min; column temperature 60°C, sample chamber temperature 15°C; detection wavelength 280 nm.

[0780] The effect of organic solvent ACN on the recombination reaction efficiency of various mutant combinations was tested. 6% volume fraction of ACN was added to the reaction system of the mutant combinations in Table 9, and the recombination reaction was carried out according to the method described in Example 2.3 above. The recombination reaction products (containing bispecific antibodies) of the mutant combinations in Table 9 were analyzed by HPLC-SEC, HPLC-IEC and HPLC-RP, and the measured purity (here, SEC purity is the main peak area ratio, IEC purity is the sum of the main peak, acidic peak and basic peak area ratio of the main spectrum peak of the recombination reaction product / bispecific antibody, and RP purity is the main peak area ratio) data is summarized in Table 9.

[0781] Table 9. SEC and IEC purity of parent monoclonal mutant combinations and recombination reaction products Note: #NA indicates no addition of organic solvent; #ACN (6%) indicates the addition of ACN with a final volume fraction of 6%.

[0782] The recombination reaction efficiency calculation formula is: 100% - (parent monoclonal antibody A corresponding spectrum peak area percentage + parent monoclonal antibody B corresponding spectrum peak area percentage).

[0783] Table 10. Recombination reaction efficiency of combinations in Table 9 calculated by the residual amount of parent monoclonal mutants Note: #NA indicates no addition of organic solvent. #ACN (6%) indicates the addition of ACN with a final volume fraction of 6%.

[0784] The above results (Tables 9 and 10) show that the recombination reaction efficiencies of Tra-E356K*Per-K439E, Tra-E356K+L368M*Per-K439E+L368M, Tra-E356K+Q347K*Per-K439E+Q347E+K360E, Tra-E356K+Q347E+K360E*Per-K439E+Q347K and Tra-E356K+Q362E+S400E*Per-K439E+Q362K+S400K are only 1.9%, 19.5%, 19.1%, 13.0% and 10.4%, respectively, in the absence of organic solvent, which indicates that these five combinations of mutants are difficult to recombine to form bispecific antibodies in the absence of organic solvent. The above results (Group 1 and 2 in Table 10) show that the recombination reaction efficiency can be increased from 1.9% to 37.4% by adding 6% ACN by volume to the reaction system of Tra-E356K*Per-K439E. The results of Groups 3 to 10 in Table 10 show that the recombination reaction efficiencies of the four combinations of mutants, including Tra-E356K+L368M*Per-K439E+L368M, Tra-E356K+Q347K*Per-K439E+Q347E+K360E, Tra-E356K+Q347E+K360E*Per-K439E+Q347K and Tra-E356K+Q362E+S400E*Per-K439E+Q362K+S400K, which are based on Tra-E356K and Per-K439E and introduce additional engineered mutations, can be greatly increased to 94% or even more by adding 6% ACN to the corresponding reaction system.

[0785] Here, the molecular weight of the recombination reaction product / bispecific antibody is determined by mass spectrometry, and it is determined that the recombination reaction product is the expected bispecific antibody. The method and conditions for determining the molecular weight of the deglycosylated antibody by mass spectrometry are as described above.

[0786] Table 11. Mass spectrometry analysis results of parent monoclonal mutant combinations and corresponding recombination reaction products Note: The theoretical heavy chain, light chain and complete molecular weights in the table are theoretical values calculated by software after considering post-translational modifications and redox states of the protein.

[0787] The results in Table 11 show that the theoretical heavy chain, light chain, and intact molecular weight of the bispecific antibody produced by recombination of different mutant combinations in the presence of 6% ACN are very close to the actual heavy chain, light chain, and intact molecular weight measured by mass spectrometry, which is consistent with the theoretical expectation. The above results show that a certain volume fraction of ACN can promote the effective recombination reaction of originally inert mutant combinations and produce structurally correct bispecific antibodies.

[0788] Example 3.1: Sources and humanization of mouse anti-human CD3 monoclonal antibody

[0789] The heavy chain and light chain variable region amino acid sequences of the mouse anti-human CD3 monoclonal antibody (anti-CD3) prepared by hybridoma technology are from US2009 / 0252683A1. The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of anti-CD3 were analyzed, and the framework region (FR) and complementarity determining region (CDR) of each VH and VL were determined according to the Kabat numbering scheme. The CDR sequences of the heavy chain and light chain variable regions of anti-CD3 are shown in Table 12 below.

[0790] > Anti-CD3-VH amino acid sequence (SEQ ID NO: 12):

[0791] > Anti-CD3-VL amino acid sequence (SEQ ID NO: 13):

[0792] Note: The italicized part is the CDR determined according to the Kabat coding scheme.

[0793] The humanization process of the murine anti-CD3 antibody is as follows. Using the IgBlast tool, anti-CD3-VH and anti-CD3-VL were compared with the NCBI human antibody variable region germline gene database, and through homology analysis, IGHV3-23*05 and IGLV7-43*01 were selected as the templates for humanization of anti-CD3-VH and anti-CD3-VL, respectively. The CDRs of anti-CD3-VH and anti-CD3-VL were transplanted into the corresponding human templates, and WGQGTTVTVSS and FGGGTKLTVL were selected as the FR4 of VH and VL, respectively, to form CDR-grafted VH and VL with the primary structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0794] In this case, the amino acid residues of the CDR-grafted VH and VL are back-mutated or otherwise mutated.

[0795] The amino acid residues at positions 73, 94, 96 and 100 of the CDR-grafted VH are mutated, specifically the combination of mutations N73D, K94R, G96E and N100Q, to obtain a humanized VH, designated as anti-CD3-Hu-VH. The amino acid residues at positions 24, 33, 36, 46, 49, 57, 58 and 94 of the CDR-grafted VL are mutated, specifically the combination of mutations R24A, A33P, F36V, A46G, Y49G, W57G, T58V and N94E, to obtain a humanized VL, designated as anti-CD3-Hu-VL.

[0796] Table 12. CDR sequences of anti-CD3-Hu antibody

[0797] > Anti-CD3-Hu-VH amino acid sequence (SEQ ID NO: 20):

[0798] > Anti-CD3-Hu-VL amino acid sequence (SEQ ID NO: 21):

[0799] Note: italicized portions are CDRs determined according to the Kabat numbering scheme, underlined and bolded portions represent back-mutations or other mutations.

[0800] DNA encoding the above humanized heavy and light chain variable regions are synthesized. The anti-CD3-Hu-VH is recombined with a gene encoding a human IgGl heavy chain constant region (SEQ ID NO: 22) containing AAA mutations (L234A+L235A+G237A, silent Fc-mediated effector function) to obtain a full-length humanized heavy chain gene, designated as anti-CD3-Hu-HC (SEQ ID NO: 24); the anti-CD3-Hu-VL is recombined with a gene encoding a human Lambda light chain constant region (SEQ ID NO: 23) to obtain a full-length humanized light chain gene, designated as anti-CD3-Hu-LC (SEQ ID NO: 25).

[0801] > Silenced human IgGl heavy chain constant region amino acid sequence (SEQ ID NO: 22):

[0802] > Human Lambda light chain constant region amino acid sequence (SEQ ID NO: 23):

[0803] Anti-CD3-Hu-HC amino acid sequence (SEQ ID NO: 24):

[0804] Anti-CD3-Hu-LC amino acid sequence (SEQ ID NO: 25):

[0805] Note: underlined part is variable region, italic part is CDR determined according to Kabat coding scheme.

[0806] The anti-CD3-Hu-HC and anti-CD3-Hu-LC genes were constructed into pcDNA3.4 expression vectors respectively, and the obtained heavy chain and light chain expression vectors were simultaneously transfected into Expi293F cells (Thermo Fisher Scientific, Cat. No. A14527) by PEI transfection method to express the antibody. The 293 cells were cultured in serum-free medium for 5 days, and the cell supernatant was collected. The antibody was purified by Protein A affinity chromatography, and the obtained antibody was named anti-CD3-Hu. TM

[0807] Example 3.2: Sources of anti-CEA and anti-MUC1 antibodies and preparation of corresponding bispecific antibodies

[0808] The heavy chain and light chain amino acid sequences of the humanized anti-CEA monoclonal antibody (Hu67-14) are from WO2021121204A1. The heavy chain and light chain amino acid sequences of the humanized anti-MUC1 monoclonal antibody (M6H2L2) are from WO2024213106A1.

[0809] Hu67-14 heavy chain amino acid sequence (SEQ ID NO: 26):

[0810] Hu67-14 light chain amino acid sequence (SEQ ID NO: 27):

[0811] M6H2L2 heavy chain amino acid sequence (SEQ ID NO: 28):

[0812] M6H2L2 light chain amino acid sequence (SEQ ID NO: 29):

[0813] Note: underlined part is variable region, italic part is CDR determined according to Kabat coding scheme. ​

[0814] BLAST (Basic Local Alignment Search Tool) analysis revealed that the light chain variable region amino acid sequences of Hu67-14 and M6H2L2 were highly similar (Identities: 82%; Positives: 94%). Comparison of the light chain CDRs of the two antibodies revealed high similarity between the corresponding CDRs. Here, the light chain of Hu67-14 was chosen as the potential common light chain to construct a symmetric tetravalent bispecific antibody. The method of constructing the anti-CEA*MUC1 bispecific antibody is described as follows. The VH and CH1 domains of M6H2L2 were linked to the heavy chain of Hu67-14 via three GGGGS linkers, resulting in a long heavy chain. Q105E mutations were introduced into the two VHs of the long heavy chain to lower the isoelectric point of the potential bispecific antibody. AAA mutations (L234A+L235A+G237A) were introduced into the Fc domain of the long heavy chain to silence the Fc-mediated effector functions. The long heavy chain produced after the foregoing modifications was named anti-CEA*MUC1-HC (SEQ ID NO: 30). K42E mutations were introduced into the light chain of Hu67-14 to further lower the isoelectric point of the potential bispecific antibody, and the common light chain thus produced was named anti-CEA*MUC1-LC (SEQ ID NO: 31).

[0815] > Anti-CEA*MUC1-HC amino acid sequence is as follows (SEQ ID NO: 30):

[0816] > Anti-CEA*MUC1-LC amino acid sequence is as follows (SEQ ID NO: 31):

[0817] The anti-CEA*MUC1-HC and anti-CEA*MUC1-LC genes were constructed into pcDNA3.4 expression vectors, respectively, and the resulting heavy chain and light chain expression vectors were simultaneously transfected into Expi293F cells (Thermo Fisher Scientific, Cat. No. A14527) using PEI transfection to express the antibodies. The 293 cells were cultured in serum-free medium for 5 days, and the cell supernatant was collected. The antibodies were purified using Protein A affinity chromatography, and the resulting antibodies were named anti-CEA*MUC1. TM

[0818] Example 3.3: Preparation of Anti-CD3-Hu and Anti-CEA*MUC1 Mutants ​

[0819] Site-directed mutagenesis was used to introduce mutations in the heavy chain CH3 domain of anti-CD3-Hu monoclonal antibody and anti-CEA*MUC1 bispecific antibody. The positions of the amino acid residues were identified using Eu numbering scheme. The anti-CD3-Hu monoclonal antibody and anti-CEA*MUC1 bispecific antibody with mutations were prepared according to the expression and purification methods described in the previous examples. The resulting antibody samples were filtered to remove bacteria using 0.2-μm filter heads, and the concentrations of the antibodies were determined using a NanoDrop micro-volume spectrophotometer. The antibody mutants and corresponding recombination reactions are shown in Table 13. The naming rule of the antibody mutants is as follows, for example, E356K means that the E at position 356 is mutated to K, and the rest is similar. Anti-CD3-Hu and anti-CEA*MUC1 are abbreviated as C and CM, respectively, in the recombination reactions.

[0820] Table 13. Parental antibodies and corresponding recombination reactions Note: * indicates that two parental antibodies were subjected to recombination reactions under certain conditions.

[0821] Example 3.4: Preparation method of anti-CD3*CEA*MUC1 trispecific antibody

[0822] A 1700 mg of 2-MEA (Sigma-Aldrich, item number / specification: 30078 / 100G) was dissolved in 20 mL of PBS, and the pH was adjusted to 7.4 using 1M NaOH to prepare a 2-MEA stock solution, and the concentration of 2-MEA in this solution was about 750 mM. An organic solvent acetonitrile (abbreviated as ACN, purchased from Sinopharm Group) was diluted with PBS to prepare a stock solution with a volume fraction of 20%. The concentrations of the anti-CD3-Hu and anti-CEA*MUC1 mutants prepared in the above examples were adjusted to equal molar concentrations (3.45 μM-690 μM) using PBS, and equal volumes of the anti-CD3-Hu mutant and the corresponding anti-CEA*MUC1 mutant were mixed, and then an appropriate amount of the 2-MEA stock solution was mixed with the organic solvent ACN and added to the above antibody mixture, and the mixture was thoroughly mixed to obtain a final concentration of 75 mM of 2-MEA and a final volume fraction of 6% of the organic solvent ACN. The mixed solution (containing anti-CD3-Hu mutant, anti-CEA*MUC1 mutant, 2-MEA, and organic solvent) was placed in a 37°C water bath for 2.5 h. The mixed solution was transferred to a Slide-A-Lyzer dialysis caddy (Thermo Scientific, item number: 6175000) with a molecular weight cut-off of 3500 Da, and dialyzed against 2 L of PBS for 2 h. The dialyzed solution was filtered to remove bacteria using a 0.2-μm filter head, and the concentration of the antibody was determined using a NanoDrop micro-volume spectrophotometer. The resulting anti-CD3*CEA*MUC1 trispecific antibody was stored at -80°C. TMG3 Dialysis Cassettes (Thermo Fisher Scientific, Cat. No. A52971) were used for dialysis in 5 L of PBS, and the PBS was replaced after 5 hours and the dialysis was performed in a cold room (4°C) for a sufficient time. The sample was collected after 18 hours of dialysis, and the antibody concentration was re-determined.

[0823] Example 3.5: Recombination reaction efficiency of anti-CD3*CEA*MUC1 trispecific antibodies

[0824] This example determined the physicochemical properties of the parent antibodies and the recombination reaction products described above using HPLC-SEC, IEC, and HPLC-RP. The physicochemical analysis methods used here, such as HPLC-SEC, HPLC-IEC, HPLC-RP, and mass spectrometry, were as described in the previous examples.

[0825] The effect of the organic solvent ACN on the recombination reaction efficiency of various mutant combinations was tested. 6% ACN was added to the reaction system of the mutant combinations in Table 13, and the recombination reaction was performed according to the method described in Example 3.4 above. The recombination reaction products (containing trispecific antibodies) of the mutant combinations in Table 13 were analyzed using HPLC-SEC, HPLC-IEC, and HPLC-RP, and the measured purity (here, the SEC purity is the main peak area ratio, the IEC purity is the sum of the main peak, acidic peak, and basic peak area ratios of the main spectrum peaks of the recombination reaction product / trispecific antibody, and the RP purity is the main peak area ratio) data are summarized in Table 14.

[0826] Table 14. SEC and IEC purity of various parent antibody mutant combinations and recombination reaction products under corresponding conditions Note: #NA indicates no addition of organic solvent; #ACN (6%) indicates the addition of ACN with a final volume fraction of 6%.

[0827] The recombination reaction efficiency calculation formula is: 100% - (parent antibody A corresponding spectrum peak area percentage + parent antibody B corresponding spectrum peak area percentage).

[0828] Table 15. Recombination reaction efficiency of combinations in Table 14 calculated by the residual amount of parent antibody mutants Note: #NA indicates no addition of organic solvent; #ACN (6%) indicates the addition of ACN with a final volume fraction of 6%.

[0829] The results above (Tables 14 and 15) show that, in the absence of organic solvents, the recombination efficiencies of C-E356K*CM-K439E, C-E356K+L368M*CM-K439E+L368M, C-E356K+Q347K*CM-K439E+Q347E+K360E, C-E356K+Q347E+K360E*CM-K439E+Q347K, and C-E356K+Q362E+S400E*CM-K439E+Q362K+S400K are only 1.1%, 23.1%, 22.8%, 13.6%, and 13.0%, respectively. This indicates that these five mutant combinations are difficult to recombine to form trispecific antibodies in the absence of organic solvents. The results disclosed above (Groups 1 and 2 in Table 15) show that adding 6% volume fraction of ACN to the C-E356K*CM-K439E reaction system can increase the recombination reaction efficiency from 1.1% to 51.3%. The results in groups 3 to 10 of Table 15 show that the combination of four mutant pairs generated by introducing additional engineered mutations on the basis of C-E356K and CM-K439E, including C-E356K+L368M*CM-K439E+L368M, C-E356K+Q347K*CM-K439E+Q347E+K360E, C-E356K+Q347E+K360E*CM-K439E+Q347K and C-E356K+Q362E+S400E*CM-K439E+Q362K+S400K, can significantly improve the recombination reaction efficiency to 95% or even higher when 6% ACN is added to the corresponding reaction system.

[0830] Here, the molecular weight of the recombinant reaction product / trispecific antibody was determined by mass spectrometry to confirm that the recombinant reaction product was the expected trispecific antibody. The method and conditions for determining the molecular weight of the deglycosylated antibody by mass spectrometry are as described above.

[0831] Table 16. Mass spectrometry analysis results of maternal antibody mutant combinations and corresponding recombinant reaction products Note: The theoretical heavy chain, light chain, and complete molecular weight in this table are theoretical values ​​calculated by software after taking into account the protein's post-translational modifications and redox state.

[0832] Table 16 shows that, in the presence of 6% ACN, the theoretical heavy chain, light chain, and intact molecular weights of the trispecific antibodies produced by different mutant combinations are very close to the actual heavy chain, light chain, and intact molecular weights measured by mass spectrometry. This indicates that the structure of the trispecific antibodies conforms to theoretical expectations. These results demonstrate that a certain volume fraction of ACN can effectively induce recombination in originally inert mutant combinations and produce structurally correct trispecific antibodies.

[0833] While the foregoing application has been described in some detail for purposes of clarity and the specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be practiced within the scope of the application. Accordingly, the disclosure is not to be limited to the exact details shown and described. The disclosure expressly encompasses all alternatives falling within the scope of the claims.

Claims

1. A method of making a heteromultimer, comprising the steps of: a) providing a molecule comprising a first polypeptide homomer and a molecule comprising a second polypeptide homomer; b) mixing said molecule comprising a first polypeptide homomer and said molecule comprising a second polypeptide homomer to form a mixture; and c) adding a reducing agent and an organic solvent to the mixture of step b) and incubating; d) obtaining a heteromultimer comprising said first polypeptide and second polypeptide; wherein said first polypeptide and second polypeptide each comprise a CH3 domain, said CH3 domain each comprising one or more mutations that promote heteromerization.

2. The method of making a heteromultimer according to claim 1, wherein: the CH3 domains of said first polypeptide and second polypeptide are independently selected from the group consisting of CH3 domains of IgGl, IgG2, IgG3 and IgG4; preferably a CH3 domain of human IgGl.

3. The method of making a heteromultimer according to claim 1 or 2, wherein the CH3 domain of said first polypeptide comprises an amino acid mutation at position 356, and the CH3 domain of said second polypeptide comprises an amino acid mutation at position 439, said positions of amino acids being determined according to the EU numbering system.

4. The method of making a heteromultimer according to claim 3, wherein: the CH3 domain of said first polypeptide comprises an amino acid mutation at position 356 to Lys (K), Arg (R) or His (H), and the CH3 domain of said second polypeptide comprises an amino acid mutation at position 439 to Glu (E) or Asp (D); preferably, the CH3 domain of said first polypeptide comprises an amino acid mutation 356K, and the CH3 domain of said second polypeptide comprises an amino acid mutation 439E; said positions of amino acids being determined according to the EU numbering system.

5. The method of making a heteromultimer according to claim 3 or 4, wherein the CH3 domain of said first polypeptide and / or second polypeptide comprises one or more amino acid mutations selected from the group consisting of positions 347, 360, 362, 368, 392, 398, 400 and 411, said positions of amino acids being determined according to the EU numbering system.

6. The method of making a heteromultimer according to claim 5, wherein: one or more amino acid mutations in the CH3 domain of said first polypeptide are to a charged amino acid, one or more amino acid mutations in the CH3 domain of said second polypeptide are to a charged amino acid, and the amino acid at the same position in the CH3 domain of said first polypeptide and the CH3 domain of said second polypeptide are oppositely charged, said same position being selected from the group consisting of amino acids at positions 347, 360, 362, 392, 398, 400 and 411, said positions of amino acids being determined according to the EU numbering system; preferably, said charged amino acid is a positively charged amino acid or a negatively charged amino acid, wherein: the positively charged amino acid is selected from the group consisting of lysine (K), arginine (R) and histidine (H); the negatively charged amino acid is selected from the group consisting of glutamic acid (E) and aspartic acid (D).

7. The method of making a heteromultimer according to claim 5, wherein the CH3 domain of the first and / or second polypeptide further comprises at least one amino acid mutation independently selected from the group consisting of i) to viii) below: ⅰ)368； ⅱ)347； ii) 347 and 360; iii) 362 and 400; iv) 392 and 398; ⅵ)398； vi) 392 and 400; vii) 400; and viii) 400 and 411 ; preferably, the CH3 domain of the first and / or second polypeptide further comprises an amino acid mutation independently selected from the group consisting of i) to iii) below: ⅰ)368； ⅱ)347； ii) 347 and 360; and iii) 362 and 400; the positions of the amino acids are determined according to the EU numbering system.

8. The method of making a heteromultimer according to any one of claims 1 to 7, wherein: (1) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 347, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 347 and 360; or (2) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 347 and 360, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 347; or (3) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 368, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 368; or (4) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 362 and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 362 and 400; the positions of the amino acids are determined according to the EU numbering system.

9. The method of making a heteromultimer according to claim 8, wherein: (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347D / E and 360D / E; or (2) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 347D / E and 360D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 347K / R; or (3) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 368M, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 368M; or (4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362D / E and 400D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362K / R and 400K / R; or (5) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362K / R and 400K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362D / E and 400D / E; preferably, (1) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 347K, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 347E and 360E; or (2) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K, 347E and 360E, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 347K; or (3) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K and 368M, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E and 368M; or (4) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K, 362E and 400E, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 362K and 400K; or (5) the CH3 domain of the first polypeptide comprises amino acid mutations of 356K, 362K and 400K, and the CH3 domain of the second polypeptide comprises amino acid mutations of 439E, 362E and 400E; The positions of the amino acids are determined according to the EU numbering system.

10. The method for preparing a heteromultimer according to any one of claims 1 to 9, wherein the reducing agent is selected from one or more of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, D-cysteine and β-mercaptoethanol and chemical derivatives thereof; Preferably, the reducing agent is selected from one or more of 2-MEA, glutathione, L-cysteine, dithiothreitol, β-mercaptoethanol and TCEP; More preferably, the reducing agent is 2-MEA.

11. The method for preparing a heteromultimer according to any one of claims 1 to 10, wherein the organic solvent is selected from polar organic solvents; Preferably, the organic solvent is selected from one or more of nitriles, alcohols, ketones, ethers, amines, amides, dimethyl sulfoxide, carbon disulfide, 1,4-dioxane and pyridine; Further preferably, the nitrile is selected from acetonitrile, propionitrile and succinonitrile, the alcohol is selected from ethanol, propanol, isopropanol and ethylene glycol, the ether is selected from tetrahydrofuran, diethyl ether and ethylene glycol dimethyl ether, the ketone is selected from acetone and butanone, and the amide is selected from dimethylformamide and dimethylacetamide; More preferably, the organic solvent is selected from one or more of acetonitrile, ethanol and isopropanol.

12. The method for preparing a heteromultimer according to any one of claims 1 to 11, wherein the final volume fraction of the organic solvent is 1% to 20%; Preferably, the final volume fraction of the organic solvent is 4% to 10%; More preferably, the final volume fraction of the organic solvent is 6% to 10%.

13. The method for preparing a heteromultimer according to any one of claims 1 to 12, further comprising a step of removing the reducing agent and the organic solvent after step c).

14. The method of making a heteromultimer according to any one of claims 1 to 13, wherein the heteromultimer is a multispecific antibody or a hetero-Fc fusion protein, preferably a bispecific antibody or a trispecific antibody.

15. The method of making a heteromultimer according to any one of claims 1 to 14, wherein: the molecule comprising a first polypeptide homomer is a first parent antibody; and / or the molecule comprising a second polypeptide homomer is a second parent antibody; preferably, the molecule comprising a first polypeptide homomer and the molecule comprising a second polypeptide homomer are independently selected from a monoclonal antibody, a monospecific antibody, or a bispecific antibody.

16. A heteromultimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprise a CH3 domain, wherein: (1) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356 and 347, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 347, and 360; or (2) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 347, and 360, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439 and 347; or (3) the CH3 domain of the first polypeptide comprises amino acid mutations at positions 356, 362, and 400, and the CH3 domain of the second polypeptide comprises amino acid mutations at positions 439, 362, and 400; the positions of the amino acids are determined according to the EU numbering system.

17. The heteromultimer according to claim 16, wherein: the amino acid mutation at position 356 is Lys (K), Arg (R), or His (H); and / or the amino acid mutation at position 439 is Glu (E) or Asp (D); and / or the amino acid mutation at position 347, 360, 362, or 400 is Lys (K) or Arg (R); and / or the amino acid mutation at position 347, 360, 362, or 400 is Glu (E) or Asp (D); the positions of the amino acids are determined according to the EU numbering system.

18. The heteromultimer according to claim 16 or 17, wherein: (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347D / E, and 360D / E; or (2) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 347D / E, and 360D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 347K / R; or (3) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362D / E, and 400D / E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362K / R, and 400K / R; or (4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362K / R and 400K / R, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362D / E and 400D / E; Preferably, (1) the CH3 domain of the first polypeptide comprises amino acid mutations 356K and 347K, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 347E and 360E; or (2) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 347E and 360E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E and 347K; or (3) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362E and 400E, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362K and 400K; or (4) the CH3 domain of the first polypeptide comprises amino acid mutations 356K, 362K and 400K, and the CH3 domain of the second polypeptide comprises amino acid mutations 439E, 362E and 400E; The positions of the amino acids are determined according to the EU numbering system.

19. The heteromultimer according to any one of claims 16 to 18, wherein: the CH3 domains of the first and second polypeptides are independently selected from the CH3 domains of IgGl, IgG2, IgG3 and IgG4; preferably the CH3 domain of human IgGl.

20. The heteromultimer according to any one of claims 16 to 19, wherein the heteromultimer is a multispecific antibody or a hetero-Fc fusion protein, preferably a bispecific antibody or a trispecific antibody.

21. A pharmaceutical composition comprising the heteromultimer according to any one of claims 16 to 20 and one or more pharmaceutically acceptable carriers, diluents or excipients.

22. An isolated nucleic acid encoding the heteromultimer according to any one of claims 16 to 20.

23. A host cell comprising the isolated nucleic acid of claim 22.

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