Selective heterodimeric Fc variant for Fc gamma RIIB

Abstract

This invention provides a heterodimeric Fc variant that is selective for FcγRIIb. [Solution] The present invention provides a heterodimeric Fc variant comprising one or more asymmetric amino acid mutations in the CH2 domain, which exhibits increased binding selectivity to FcγRIIb compared to the parent Fc region; a polypeptide comprising this heterodimeric Fc variant; and a polynucleotide encoding this heterodimeric Fc variant. The one or more asymmetric mutations include loop substitution in the CH2 domain, a mutation at position 236 in the CH2 domain, or a combination of loop substitution in the CH2 domain and a mutation at position 236 in the CH2 domain.

Description

[Technical Field]

[0001] field This disclosure relates to the field of Fc variants, and more particularly to heterodimeric Fc variants that are selective for FcγRIIb. [Background technology]

[0002] background The interaction between the Fc domain of an antibody and members of the cellular Fcγ receptor (FcγR) family is deeply related to the strength of the immune response. In the development of therapeutic drugs, there is particular interest in two members of the FcγR family: FcγRIIa, which upregulates immune activity when bound to antibody Fc, and FcγRIIb, which downregulates immune activity when bound to antibody Fc. FcγRIIb is the only inhibitory IgG receptor and downregulates immune activity by suppressing the activation of B lymphocytes, monocytes, mast cells, and basophils induced by activating receptors.

[0003] Fc engineering has been employed to modulate the ability of antibodies to interact with FcγR (Carter, 2006, Nat Rev Immunol., 6:343-357 (Non-patent Literature 1); Presta, 2008, Curr Opin Immunol., 20:460-470 (Non-patent Literature 2)). Fc engineering to increase the affinity and selectivity of the Fc region to FcγRIIb is described (Chu, et al., 2008, Mol Immunol., 45:3926-3933 (Non-Patent Literature 3); Mimoto et al., 2013, Protein Eng. Des. Sel., 26:589-598 (Non-Patent Literature 4); U.S. Patent No. 9,540,451 (Patent Literature 1); U.S. Patent No. 9,902,773 (Patent Literature 2) and U.S. Patent No. 9,914,778 (Patent Literature 3); U.S. Patent Application Publication No.: US2009 / 0042291 (Patent Literature 4); U.S. Patent No. 2015 / 0299296 (Patent Literature 5); U.S. Patent No. 2016 / 0039912 (Patent Literature 6) and U.S. Patent No. 2016 / 0046693 (Patent Literature 7)).

[0004] Fc engineering approaches, including inserting additional amino acids into the Fc region to modify the FcγR or FcRn bond, have also been described (U.S. Patent No. 9,890,216 (Patent Document 8); U.S. Patent Application Publication Nos.: US2008 / 0227958 (Patent Document 9) and US2014 / 0356358 (Patent Document 10)).

[0005] This background information is provided for the purpose of making known any information that the applicant considers to be potentially relevant to this disclosure. None of the aforementioned information is intended to be, nor should it be interpreted as, an admission that it constitutes prior art with respect to the claimed invention. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent No. 9,540,451 [Patent Document 2] U.S. Patent No. 9,902,773 [Patent Document 3] U.S. Patent No. 9,914,778 [Patent Document 4] US2009 / 0042291 [Patent Document 5] US2015 / 0299296 [Patent Document 6] US2016 / 0039912 [Patent Document 7] US2016 / 0046693 [Patent Document 8] U.S. Patent No. 9,890,216 [Patent Document 9] US2008 / 0227958 [Patent Document 10] US2014 / 0356358 [Non-patent literature]

[0007] [Non-Patent Document 1] Carter,2006,Nat Rev Immunol.,6:343-357 [Non-Patent Document 2] Presta,2008,Curr Opin Immunol.,20:460-470 [Non-Patent Document 3] Chu,et al.,2008,Mol Immunol.,45:3926-3933 [Non-Patent Document 4] Mimoto et al.,2013,Protein Eng.Des.Sel.,26:589-598 [Overview of the project]

[0008] overview A heterodimeric Fc variant selective to FcγRIIb is described herein. In one embodiment, the disclosure relates to a heterodimeric Fc variant comprising a first Fc polypeptide and a second Fc polypeptide, wherein the heterodimeric Fc variant exhibits increased selectivity for binding to FcγRIIb compared to a parent Fc region, wherein one of the Fc polypeptides comprises a substitution of all or part of the native loop in the CH2 domain of the Fc polypeptide with an alternative amino acid sequence such that the length of the native loop is extended, and at least one of the amino acid residues of the alternative amino acid sequence is within a heavy atom distance of 3 Å from a target amino acid residue in FcγRIIb when the heterodimeric Fc variant is bound to FcγRIIb, wherein the heterodimeric Fc variant is a variant of immunoglobulin G (IgG)Fc.

[0009] In another embodiment, the present disclosure relates to a heterodimer Fc variant comprising a first Fc polypeptide and a second Fc polypeptide, wherein one of the Fc polypeptides comprises a substitution of amino acids 325-331 with a polypeptide of 8-15 amino acid length, wherein the heterodimer Fc variant has increased selectivity for binding to FcγRIIb compared to the parent Fc region, the heterodimer Fc variant is a variant of immunoglobulin G (IgG)Fc, and the amino acid numbering follows the EU index.

[0010] In another embodiment, the present disclosure relates to a method for preparing a heterodimeric Fc variant comprising a first Fc polypeptide and a second Fc polypeptide having increased selectivity for a target receptor compared to a parent Fc region, the method comprising: (a) using an in silico model of a parent Fc region complexed with a target receptor, (i) inserting a sequence of one or more amino acid residues into one of the native loops of the Fc polypeptide to provide a variant candidate, such that the length of the native loop is extended; (ii) determining the distance of at least one amino acid residue of the insertion sequence from a target amino acid residue in the receptor; (iii) selecting the variant candidate as a heterodimeric Fc variant if at least one amino acid residue of the insertion sequence is within a heavy atom distance of 3 Å to the target amino acid residue in the receptor; (b) preparing a nucleic acid encoding the heterodimeric Fc variant; and (c) expressing the nucleic acid in a host cell to provide the heterodimeric Fc variant, wherein the target receptor is FcγRIIb.

[0011] In another embodiment, the present disclosure relates to a heterodimer Fc variant comprising a first Fc polypeptide and a second Fc polypeptide, wherein the heterodimer Fc variant has increased selectivity for binding to FcγRIIb compared to the parent Fc region, and comprises an asymmetric mutation at position 236, where one of the Fc polypeptides comprises the mutation G236N or G236D, and the heterodimer Fc variant is a variant of immunoglobulin G (IgG)Fc, and the amino acid numbering follows the EU index.

[0012] In another embodiment, the disclosure relates to polypeptides comprising a heterodimeric Fc variant disclosed herein and one or more protein moieties fused to or covalently bonded to the heterodimeric Fc variant.

[0013] In another embodiment, the present disclosure relates to a pharmaceutical composition comprising a heterodimer Fc variant disclosed herein, or a polypeptide comprising a heterodimer variant and one or more protein moieties, and a pharmaceutically acceptable carrier or diluent.

[0014] In another embodiment, the present disclosure relates to polypeptides for therapeutic use, comprising a heterodimeric Fc variant disclosed herein and one or more protein moieties fused to or covalently bonded to the heterodimeric Fc variant.

[0015] In another embodiment, the disclosure relates to a polypeptide for use in the treatment of cancer, comprising a heterodimeric Fc variant disclosed herein and one or more protein moieties fused to or covalently bound to the heterodimeric Fc variant, wherein at least one of the protein moieties is an antigen-binding domain that binds to a tumor-associated antigen or a tumor-specific antigen.

[0016] In another embodiment, the present disclosure relates to a method of treatment comprising administering to a patient in need a polypeptide comprising a heterodimeric Fc variant and one or more protein moieties fused to or covalently bonded to the heterodimeric Fc variant.

[0017] In another embodiment, the present disclosure relates to a method for treating cancer, comprising administering to a patient in need thereof a polypeptide comprising a heterodimeric Fc variant and one or more protein moieties fused to or covalently bound to the heterodimeric Fc variant, wherein at least one of the protein moieties is an antigen-binding domain that binds to a tumor-associated antigen or a tumor-specific antigen.

[0018] In another embodiment, the disclosure relates to nucleic acids encoding heterodimeric Fc variants disclosed herein, or polypeptides comprising a heterodimeric Fc variant and one or more protein moieties fused to or covalently bonded to the heterodimeric Fc variant. In another embodiment, the disclosure relates to host cells comprising nucleic acids.

[0019] In another embodiment, the present disclosure relates to a method for preparing a heterodimeric Fc variant disclosed herein, or a polypeptide comprising a heterodimeric Fc variant and one or more protein moieties fused to or covalently bound to the heterodimeric Fc variant, the method comprising expressing a nucleic acid encoding the heterodimeric Fc variant or polypeptide in a host cell. [Brief explanation of the drawing]

[0020] [Figure 1] The steps taken to generate a selective variant for FcγRIIb are outlined below. LVG1 = Read Variant Generation 1; LVG2 = Read Variant Generation 2. [Figure 2] Two approaches were taken to introduce FcγRIIb selectivity into the Fc region: (A) introduction of an asymmetric point mutation, and (B) asymmetric substitution of loop 3. [Figure 3] This shows a cartoon representation of the in silico model constructed for IgG1 Fc bound to FcγRIIb. [Figure 4]The sequence alignment of IgG1 and IgG4 is shown, illustrating the differences in the positions of the lower hinge and CH2 domain at 234, 268, 274, 296, 327, and 331. [Figure 5] This paper compares the crystal structures of the Fc / FcγR complex 1E4K and 1T83, and shows two binding modes in which FcγR is thought to be able to bind to the Fc region. [Figure 6] A schematic diagram of the method used to determine the contribution of a given mutation in each Fc chain to FcγR binding is shown. Mutation G236A was used as an exemplary mutation, and E269K was used as a polarity driver. This blocks binding to FcγR only in the binding mode closest to receptor position L135 (and R134). This binding mode is indicated by an "x" in Figure 6. [Figure 7] This shows the generalized loop "template" portion. The loop template consists of N-side and C-side β-strand regions and an unstructured loop region (shown in dark gray) which are extensions of the existing β-strands of the CH2 domain (shown in light gray). The template was grafted into the CH2 domain by aligning the template's anchor residues with residues B / 324 and B / 332 of the CH2 domain. The anchor residues are not grafted with the rest of the template. [Figure 8] This shows the length distribution of loop templates identified in the initial search of the Protein Data Bank (PDB). [Figure 9] A schematic diagram of the human IgG1 Fc / FcγRIII complex structure, available in the Protein Data Bank (PDB) under ID 1E4K, is shown (chain A (green) is characterized by hotspot P329, and chain B (cyan) is characterized by hotspot D270). [Figure 10A]This section outlines the improved affinity of FcγRIIb to the wild type (WT) for variants generated by optimizing strategy 1 of lead variant v19544. Positions 325–331B are located within the insertion loop sequence and are indicated separately with asterisks herein (i.e., 325*, 326*, etc.). The inset shows a positional heatmap illustrating the approximate placement of Fc positions 329 and 330 (329* and 330*) relative to FcγRIIb position S135. [Figure 10B] This section outlines the improvement in FcγRIIb selectivity over wild-type (WT) for variants generated by optimizing strategy 1 for read variant v19544. Positions 325–331B are located within the insertion loop sequence and are indicated separately with asterisks herein (i.e., 325*, 326*, etc.). The inset shows a positional heatmap illustrating the approximate placement of Fc positions 329 and 330 (329* and 330*) relative to FcγRIIb position S135. [Figure 11A] This paper outlines the improvement in FcγRIIb affinity to the wild type (WT) for variants generated by optimizing strategy 2 for lead variant v19585. [Figure 11B] This paper outlines the improvement in FcγRIIb selectivity compared to the wild type (WT) for variants generated by optimizing strategy 2 for lead variant v19585. [Figure 12A] This section outlines the improvement in affinity of FcγRIIb to wild-type (WT) for variants generated by Strategy 3 (combinations of read variant v19544 including various loop substitutions). [Figure 12B] This paper outlines the improvement in FcγRIIb selectivity over wild-type (WT) for variants generated by Strategy 3 (combinations of read variant v19544 including various loop substitutions). [Figure 13A] This section outlines the improved affinity of FcγRIIb to wild-type (WT) for variants generated by Strategy 4 (combinations of read variant v19544 including longer loop substitutions). [Figure 13B] This section outlines the improved selectivity of FcγRIIb over wild-type (WT) for variants generated by Strategy 4 (combinations of read variant v19544 including longer loop substitutions). [Figure 14] The following plots summarize the binding and selectivity of FcγRIIb, C1q binding, changes in FcγRIIb binding and aggregation tendency with pH, ​​and changes in Tm for variants v32210, v32226, v32295, v32230, v32227, v32274, and v32284. [Figure 15] Variants v22096, v26370, v26774, v27092, v31186, v31188, v31191, v31192, v31213, v32210, v32211, v32212, v32226, v32227, v32230, v32231, v32242, v32274, v32282, v32284, The correlation between CDC activity and C1q binding is shown using Spearman's rank test (R=0.94, p<1e-12) for anti-CD40 antibodies, including v32287, v32288, v32292, v32293, v32294, v32295, and v32296, as well as controls (wild-type, negative, v12, and SELF). [Figure 16] This shows serum human C5 antigen levels in human FcγR2b transgenic mice after administration of 1 mg / kg of anti-C5 antibodies with different affinities to human FcγRIIb. The treatment groups consisted of n=5 (Neg, v31188 and v32227), n=4 (v21653 (WT) and v32284), and n=2 (no antibody group). The values ​​shown are mean ± SEM. [Figure 17]This shows serum antibody concentrations in human FcγR2b transgenic mice after administration of 1 mg / kg of anti-C5 antibodies with different affinities to human FcγRIIb. The treatment groups consisted of n=5 (Neg, v31188, and v32227) and n=4 (v21653 (WT) and v32284). Results from a single animal in each group (v32227 and v32284) were omitted because the profile was similar to SC / IP rather than intravenous administration. Values ​​shown are mean ± SEM. [Modes for carrying out the invention]

[0021] Detailed explanation Described herein are heterodimeric Fc variants comprising one or more asymmetric amino acid mutations in the CH2 domain, which increase the selectivity for binding to FcγRIIb compared to the parental Fc region. In some embodiments, the heterodimeric Fc variants described herein have increased selectivity for binding to FcγRIIb and increased binding affinity to FcγRIIb compared to the parental Fc region. The “parental Fc region” is an Fc region that is identical to that of the heterodimeric Fc variant except that it lacks one or more amino acid mutations in the CH2 domain that increase the binding selectivity and / or affinity to FcγRIIb. The one or more asymmetric mutations include loop substitution in the CH2 domain, a mutation at position 236 in the CH2 domain, or a combination of loop substitution in the CH2 domain and a mutation at position 236 in the CH2 domain.

[0022] Certain embodiments of this disclosure relate to polypeptides comprising heterodimeric Fc variants described herein. Examples of such polypeptides include, but are not limited to, antibodies, antibody fragments, and Fc fusion proteins. Polypeptides comprising heterodimeric Fc variants may find use as therapeutic agents, diagnostic agents, or research tools.

[0023] Certain embodiments of this disclosure relate to polynucleotides encoding heterodimeric Fc variants and polynucleotides encoding polypeptides containing heterodimeric Fc variants, as well as host cells containing said polynucleotides, and methods for preparing heterodimeric Fc variants or polypeptides containing heterodimeric Fc variants using said polynucleotides and host cells.

[0024] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art.

[0025] As used herein, the term “about” refers to a variation of approximately + / - 10% from a given value. Such variation is understood to always be included in any given value provided herein, whether or not it is specifically mentioned, unless otherwise specified.

[0026] The use of the words "a" or "an," as used herein in conjunction with the term "including," may mean "one," but also coincides with the meanings of "one or more," "at least one," and "one or more than one."

[0027] Where used herein, the terms “comprising,” “having,” “including,” and “containing,” and their grammatical variations, are inclusive or open-ended and do not preclude additional unlisted elements and / or method steps. Where used herein in relation to an Fc variant, composition, use, or method, the term “essentially consisting of” indicates that additional elements and / or method steps may exist, but these additions do not substantially affect the manner in which the listed Fc variant, composition, method, or use functions. Where used herein in relation to an Fc variant, composition, use, or method, the term “consisting of” excludes the existence of additional elements and / or method steps. An Fc variant, composition, use, or method described herein as containing certain elements and / or steps may also, in certain embodiments, essentially consist of those elements and / or steps, and in other embodiments, whether specifically mentioned or not.

[0028] As used herein to describe amino acid sequences, the term "derived from" means that the amino acid sequence in question is substantially identical to the reference amino acid sequence from which it is derived.

[0029] As used herein in relation to amino acid sequences, “substantially identical” means that, when optimally aligned (e.g., using the methods described below), the amino acid sequence shares at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with its reference amino acid sequence. The percentage of identity between two amino acid sequences can be determined by various methods known in the art, e.g., commonly available computer software, such as Smith Waterman Alignment (Smith & Waterman, 1981, J Mol Biol 147:195-7); “BestFit” (Smith & Waterman, 1981, Advances in Applied Mathematics, 482-489); BLAST (Basic Local Alignment Search Tool; (Altschul, et al., 1990, J Mol Biol)). Biol, 215:403-10) and its variations and updates; can be determined using ALIGN, ALIGN-2, CLUSTAL or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including the algorithms necessary to achieve the maximum alignment over the lengths of the sequences being compared. Generally, for peptides, the length of the comparison sequence is at least 10 amino acids, but those skilled in the art will understand that the actual length depends on the full length of the sequences being compared. In certain embodiments, the length of the comparison sequence may be the full length of the peptide sequence or polypeptide sequence.

[0030] When used herein in relation to a substance, the term "isolated" means that the substance has been removed from its original environment (e.g., its natural environment if it exists in nature). For example, a naturally occurring polynucleotide or polypeptide present in an animal organism is not isolated, but the same polynucleotide or polypeptide separated from some or all of the substances coexisting in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, but such a vector or composition is isolated in that it is not part of its natural environment.

[0031] The terms “Fc region” and “Fc” are used without distinction herein and refer to the C-terminal region of an immunoglobulin heavy chain. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region sequence of a human IgG heavy chain is typically defined as extending, for example, from position 239 to the C-terminus. The “Fc polypeptide” of dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., the polypeptide containing the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-assembly. The Fc region typically contains the CH2 domain and the CH3 domain. In certain embodiments, the Fc region may also be considered to encompass a hinge region.

[0032] The "CH2 domain" of the human IgG Fc region is typically defined as extending from position 239 to position 340. The "CH3 domain" is typically defined as including the C-terminus to the CH2 domain of the Fc region, i.e., amino acid residues from position 341 to position 447. The "hinge region" of human IgG1 is generally defined as extending from position 216 to position 238 (Burton, 1985, Molec. Immunol., 22:161-206). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by matching the first and last cysteine ​​residues that form the inter-heavy chain disulfide bond.

[0033] Unless otherwise specified herein, the numbering of amino acid residues in the Fc region follows the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0034] It should be understood that the positive enumeration of certain features in one embodiment may serve as a basis for excluding those features in an alternative embodiment. In particular, when a list of options is presented for a given embodiment or claim, it should be understood that one or more options may be removed from the list, and the shortened list may form an alternative embodiment, whether or not such alternative embodiments are specifically mentioned.

[0035] Any embodiment discussed herein is intended to be performed with respect to the Fc variants, methods, uses, or compositions disclosed herein, and vice versa.

[0036] Heterodimer Fc variant The heterodimeric Fc variants of this disclosure contain one or more asymmetric amino acid mutations in the CH2 domain and exhibit increased selectivity for binding to FcγRIIb compared to the parental Fc region. In some embodiments, the heterodimeric Fc variants also exhibit increased binding affinity to FcγRIIb compared to the parental Fc region.

[0037] Increased selectivity for binding to FcγRIIb is also referred to herein as “increased selectivity for FcγRIIb,” and means that the heterodimeric Fc variant exhibits greater binding affinity to FcγRIIb than to other Fcγ receptors, particularly FcγRIIaR, compared to the parent Fc region. In certain embodiments, the increased selectivity of the heterodimeric Fc region for FcγRIIb is defined relative to the binding affinity for FcγRIIaR. In certain embodiments described herein, the increased selectivity of the heterodimeric Fc variant for FcγRIIb relative to FcγRIIaR may be expressed as an increase in the ratio of the parent Fc region's selectivity for FcγRIIb. For example, in some embodiments, the heterodimeric Fc variant may have at least 1.5 times, or at least 2 times, increased selectivity for FcγRIIb compared to the parent Fc region.

[0038] An increase in FcγRIIb selectivity may or may not be accompanied by an increase in FcγRIIb affinity compared to the parent Fc region. Therefore, in certain embodiments, a heterodimeric Fc variant may have increased selectivity for FcγRIIb compared to the parent Fc region, for example, at least 1.5 times the FcγRIIb selectivity compared to the parent Fc region, but without an increase in FcγRIIb affinity. In certain embodiments, a heterodimeric Fc variant may have increased selectivity for FcγRIIb compared to the parent Fc region, for example, at least 1.5 times the FcγRIIb selectivity compared to the parent Fc region, but with decreased FcγRIIb affinity compared to the parent Fc region.

[0039] In certain embodiments, the heterodimeric Fc variant may have increased selectivity for FcγRIIb compared to the parent Fc region, for example, at least 1.5 times increased selectivity for FcγRIIb compared to the parent Fc region, and may also have substantially the same FcγRIIb affinity compared to the parent Fc region.

[0040] An increase in binding affinity to FcγRIIb is also referred to herein as "increased affinity to FcγRIIb," and means that the heterodimeric Fc variant has increased binding affinity to FcγRIIb compared to the binding affinity of the parent Fc to FcγRIIb. In certain embodiments described herein, the increase in affinity of the heterodimeric Fc variant to FcγRIIb may be expressed as an increase in the ratio of the affinity of the parent Fc region to FcγRIIb. For example, in some embodiments, the heterodimeric Fc variant may have an affinity for FcγRIIb that is at least 10 times higher than that of the parent Fc region.

[0041] A heterodimer Fc variant comprises two heavy-chain constant-domain polypeptides, referred to herein as the first Fc polypeptide and the second Fc polypeptide. The designations "first" and "second" with respect to the Fc polypeptides are for convenience only, and it is understood that the two Fc polypeptides are interchangeable, provided that the Fc variant comprises one first Fc polypeptide and one second Fc polypeptide.

[0042] In this disclosure, “asymmetric” amino acid mutation means that one Fc polypeptide contains an amino acid mutation at a specific position, while the other Fc polypeptide either does not contain an amino acid mutation at the corresponding position, or contains a different amino acid mutation at the corresponding position. The first and second Fc polypeptides of the heterodimeric Fc variant may contain one or more asymmetric amino acid mutations. The amino acid mutation may be an amino acid substitution, insertion or deletion, or a substitution of one or more amino acid sequences with alternative sequences. The alternative sequence may be the same length as the sequence being substituted (i.e., containing the same number of amino acids) or it may be longer than the sequence being substituted (i.e., containing additional amino acids). In certain embodiments, one or more asymmetric amino acid mutations in the heterodimeric Fc variant include one or more amino acid substitutions. In some embodiments, one or more asymmetric amino acid mutations in the heterodimeric Fc variant include an asymmetric loop substitution in which a loop sequence in the CH2 domain of one Fc polypeptide is replaced with a different polypeptide loop sequence. In some embodiments, one or more asymmetric amino acid mutations in a heterodimer Fc variant include one or more amino acid substitutions and asymmetric loop substitutions in which a loop sequence in the CH2 domain of one Fc polypeptide is replaced with a different polypeptide loop sequence.

[0043] In certain embodiments, one or more asymmetric amino acid mutations in a heterodimer Fc variant include an asymmetric loop substitution in the CH2 domain, a mutation at position 236, or a combination of an asymmetric loop substitution in the CH2 domain and a mutation at position 236. If the heterodimer Fc variant includes an asymmetric loop substitution in the CH2 domain and a mutation at position 236, the mutation at position 236 may be symmetric or asymmetric. In some embodiments, the heterodimer Fc variant includes an asymmetric loop substitution in the CH2 domain and a symmetric mutation at position 236. In some embodiments, the heterodimer Fc variant includes an asymmetric loop substitution in the CH2 domain and an asymmetric mutation at position 236.

[0044] In certain embodiments, the heterodimer Fc variant includes an asymmetric loop substitution in the CH2 domain. In some embodiments, the heterodimer Fc variant includes an asymmetric loop substitution in the CH2 domain and one or more additional amino acid mutations in the CH2 domain. The one or more additional amino acid mutations may be asymmetric or symmetric mutations.

[0045] In certain embodiments, the heterodimer Fc variant includes an asymmetric mutation at position 236. In some embodiments, the heterodimer Fc variant includes an asymmetric mutation at position 236 and one or more additional amino acid mutations in the CH2 domain. The one or more additional amino acid mutations may be asymmetric or symmetric mutations.

[0046] Examples of heterodimeric Fc variants include, but are not limited to, heterodimeric Fc variants containing amino acid mutations listed in any one of the variants shown in Tables 5A, 5B, 5C, 13.1, 6.22, 6.23, 6.24, 6.25, 6.26, and 6.27. Additional heterodimeric Fc variants are listed below.

[0047] In certain embodiments, the heterodimer Fc variant includes an amino acid mutation described in any one of the variants shown in Tables 5A, 5B, 5C, 13.1, 6.22, 6.23, and 6.24. In some embodiments, the heterodimer Fc variant includes an amino acid mutation described in any one of the variants shown in Tables 5A, 5B, 5C, and 13.1.

[0048] When a heterodimeric Fc variant contains more than one amino acid mutation, each individual mutation in the heterodimeric Fc variant may result in increased selectivity of the heterodimeric Fc variant for FcγRIIb, increased affinity of the heterodimeric Fc variant for FcγRIIb, or increased selectivity and affinity of the heterodimeric Fc variant for FcγRIIb. However, when amino acid mutations are combined, a heterodimeric Fc variant may result in increased selectivity for FcγRIIb and, optionally, increased affinity for FcγRIIb. Therefore, in a particular embodiment, the amino acid mutations in a heterodimeric Fc variant may include one or more amino acid mutations that result in increased selectivity of the heterodimeric Fc variant for FcγRIIb, and, optionally, one or more different amino acid mutations that result in increased affinity for FcγRIIb. In some embodiments, one or more amino acid mutations in the heterodimer Fc result in increased selectivity of the heterodimer Fc variant to FcγRIIb and increased affinity for FcγRIIb.

[0049] If the heterodimer Fc variant described herein contains more than one amino acid mutation that increases selectivity and / or affinity for FcγRIIb, the heterodimer Fc variant may contain a total of up to 20 such amino acid mutations, where an asymmetric loop insertion is considered one amino acid mutation. In certain embodiments, the heterodimer Fc variant contains 1 to 20 amino acid mutations, where an asymmetric loop insertion is considered one amino acid mutation. In certain embodiments, the heterodimer Fc variant contains 1 to 18 amino acid mutations, 1 to 16 amino acid mutations, or 1 to 15 amino acid mutations, where an asymmetric loop insertion is considered one amino acid mutation.

[0050] In certain embodiments, the heterodimer Fc variant is a variant of immunoglobulin G (IgG) Fc. In some embodiments, the heterodimer Fc variant is a variant of human IgG Fc. In some embodiments, the heterodimer Fc variant is a variant of IgG1 Fc. In some embodiments, the heterodimer Fc variant is a variant of human IgG1 Fc. The amino acid sequence of native human IgG1 Fc from positions 231 to 447 is provided in Table 1 (SEQ ID NO: 1).

[0051] [Table 1]

[0052] A heterodimeric Fc variant containing an asymmetric loop substitution in the CH2 domain. Certain embodiments of this disclosure relate to heterodimeric Fc variants that exhibit increased selectivity for FcγRIIb compared to a parent Fc region, wherein one of the Fc polypeptides of the heterodimeric Fc variant includes substitution of all or part of the native loop in the CH2 domain of the Fc polypeptide with an alternative amino acid sequence such that the length of the native loop is extended and the affinity of the heterodimeric variant for FcγRIIb is increased. Some embodiments relate to methods for designing such heterodimeric Fc variants.

[0053] Accordingly, certain embodiments of the present disclosure relate to a method for designing a heterodimeric Fc variant with increased selectivity for a target receptor compared to a parent Fc region, comprising: (i) providing a variant candidate by replacing all or part of the native loop sequence in one CH2 domain of the Fc polypeptide of the Fc variant with an alternative amino acid sequence such that the length of the native loop is extended in an in silico model of the parent Fc region complexed with a target receptor; (ii) determining the distance of at least one amino acid residue of the alternative amino acid sequence from a target amino acid residue in the receptor; and (iii) selecting the variant candidate as a heterodimeric Fc variant if at least one amino acid residue of the alternative amino acid sequence is within a heavy atom distance of 3 Å from the target amino acid residue in the receptor. In certain embodiments, the target receptor is FcγRIIb.

[0054] In some embodiments, the method further comprises preparing a nucleic acid encoding a heterodimeric Fc variant and expressing the nucleic acid in a host cell to provide the heterodimeric Fc variant.

[0055] Certain embodiments of the present disclosure relate to a heterodimeric Fc variant with increased selectivity for FcγRIIb compared to a parent Fc region, wherein one of the Fc polypeptides of the heterodimeric Fc variant includes substitution of all or part of a native loop in the CH2 domain of the Fc polypeptide with an alternative amino acid sequence such that the length of the loop is extended and the interaction between the Fc polypeptide and the receptor is increased. For example, the substituted loop may modify the interaction between one or more other loops in the Fc polypeptide and the receptor so as to improve the binding of the Fc polypeptide to the receptor, or at least one of the residues of the substituted loop may be brought closer to a target amino acid in the receptor so as to increase the interaction between the Fc polypeptide and the receptor. In certain embodiments, at least one of the amino acid residues of the substituted loop is within a heavy atom distance of 3 Å from a target amino acid residue in the receptor when the heterodimeric Fc variant is bound to the receptor. In certain embodiments, the target amino acid residue in the receptor is Ser135.

[0056] In some embodiments, the substituted loop sequence is a polypeptide with a length of 7-15 amino acids or 8-15 amino acids. In some embodiments, the native loop contains amino acids 325-331 of the Fc polypeptide.

[0057] The terms “substitution loop,” “substitution loop sequence,” and “loop substitution” are used interchangeably herein with respect to sequences used to replace all or part of a selected native loop in the CH2 domain of a heterodimeric Fc polypeptide. Similarly, the terms “polypeptide” and “polypeptide loop” are used interchangeably when describing substitution loop sequences.

[0058] As described herein, the loop at positions 325–331 in one CH2 domain of the IgG Fc polypeptide does not directly participate in FcγR binding because the residues contained in this loop are typically far from position 135 on FcγR (see Figure 2B). The loop at positions 325–331 in the IgG1 CH2 domain is sometimes referred to as the “FG loop” or “loop 3”. As also described herein, selective binding of Fc to the receptor is improved by replacing one FG loop of the Fc polypeptide with a polypeptide loop engineered to interact with FcγRIIb near residue 135. In certain embodiments, the heterodimeric Fc variants of this disclosure include an asymmetric substitution of the FG loop and, optionally, one or more additional amino acid mutations in the CH2 domain and include increased selectivity for FcγRIIb compared to the parental Fc. In some embodiments, the heterodimeric Fc variant comprises an asymmetric substitution of the FG loop and, optionally, one or more additional amino acid mutations in the CH2 domain, resulting in increased selectivity and affinity for FcγRIIb compared to the parental Fc. The one or more additional amino acid mutations may be asymmetric or symmetric. In certain embodiments, the one or more additional amino acid mutations comprise a mutation at position 236 in one or both of the Fc polypeptides.

[0059] Asymmetric Loop Substitution In certain embodiments, the heterodimer Fc variant includes an asymmetric loop substitution in the CH2 domain, resulting in increased selectivity for FcγRIIb compared to the parent Fc. In some embodiments, the asymmetric loop substitution in the heterodimer Fc variant includes substitution of the native loop at positions 325-331 in one Fc polypeptide with a polypeptide loop of 7-15 amino acids, e.g., 7-12 amino acids. In some embodiments, the asymmetric loop substitution in the heterodimer Fc variant includes substitution of the native loop at positions 325-331 in one Fc polypeptide with a longer polypeptide loop, e.g., 8-15 amino acids, 8-14 amino acids, or 8-12 amino acids. In some embodiments, the asymmetric loop substitution in the heterodimer Fc variant includes substitution of the native loop at positions 325-331 in one Fc polypeptide with a polypeptide loop of 9-15 amino acids, 9-14 amino acids, 10-15 amino acids, or 10-14 amino acids.

[0060] In some embodiments, the polypeptide loop replacing the native loop in the Fc variant is derived from the sequence of the loop-forming segment of a second protein. Identifying suitable loop-forming segments of known proteins can be achieved using methods such as those described herein (see Example 2). For example, candidate loop sequences can be identified by analyzing structures of known proteins, such as those available from the Protein Data Bank (PDB) (Berman, et al., 2000, Nucl. Acids Res., 28:235-242). The PDB can be accessed, for example, from a website managed by the Research Collaboratory for Structural Bioinformatics (RCSB). To facilitate the identification of candidate loop sequences, the protein structures selected for analysis may be limited to those having a specific level of resolution, e.g., crystal structures with a resolution of 2.5 Å or higher.

[0061] Candidate loop sequences ("templates") are typically loop sequences anchored to the parent protein by β-strands. A general structure of a suitable loop sequence is shown in Figure 7. In this general structure, the loop template consists of an unstructured loop region and N-terminal and C-terminal β-strand regions, which can function to extend existing β-strands present in the Fc CH2 domain. The anchor residues of the template enable alignment with amino acids located at positions 324 and 332 in the CH2 domain, but the anchor residues do not form part of the template.

[0062] Once loop sequence candidates have been identified, a secondary structure may be assigned to the amino acids of the selected PDB protein structure using one or a combination of various algorithms known in the art, such as STRIDE (Frishman & Argos, 1995, Proteins Struct.Funct.Bioinf., 23:566-579), DSSP (Kabsch & Sander, 1983, Biopolymers, 22:2577-2637), DEFINE (Richards & Kundrot, 1988, Proteins, 3:71-84), ScrewFit (Calligari & Kneller, 2012, Acta Crystallographica Section D.68:1690-3), or SST (Konagurthuet al., 2012, Bioinformatics, 28:i97-i105).

[0063] In some embodiments, polypeptide loop candidates can be identified from PDB protein structures using the following selection criteria: i) The loop sequence is fixed to the parent protein by the beta strand; ii) The loop sequence contains one or more beta-strand amino acids at both the N-terminus and the C-terminus of the loop; iii) One or more beta-strand amino acids at the C-terminus of the polypeptide loop do not form hydrogen bonds with any amino acids in the parent protein except for the beta-strand amino acid at the N-terminus of the polypeptide loop; and iv) The mean squared deviation of the skeletal heavy atoms of one or more beta-strand amino acids at the N-terminus and C-terminus of the polypeptide loop, relative to one or more amino acids that terminate at site 324 (if N-terminus) of the CH2 domain and begin at site 332 (if C-terminus), is <0:85 Å.

[0064] In some embodiments, the following additional criteria may be used to identify polypeptide loop candidates: v) The loop sequence contains at least one hydrogen bond between the beta-strand amino acids at both ends of the polypeptide loop.

[0065] Once polypeptide loop candidates are identified, they can be further analyzed to select a suitable template for use in replacing native loops in the Fc variant.

[0066] In certain embodiments, polypeptide loop candidates may be grafted in silico onto Fc / FcγRIIb complexes for further analysis. In some embodiments, in silico grafting may include the following steps: i) Remove residues 325-331 (including endpoints) from the Fc / FcγRIIb complex; ii) Introducing the template skeleton into the Fc / FcγRIIb complex by aligning the heavy atoms of the template anchor skeleton with residues 324 and 332 of the Fc / FcγRIIb complex; and iii) Minimize the coordinates of the skeletal atoms of residues 323, 324, 332, 333, and the first two and last two residues of the template.

[0067] Step iii) above can be achieved using conventional software, for example, the AMBER99SB force field (Hornak, et al., 2006, Proteins Struc.Funct.Bioinf., 65:712) and minimization by the conjugate gradient method.

[0068] Subsequently, grafted polypeptide loop candidates may be further screened by applying filters to identify templates having a length and orientation in which one or more template residues can interact with FcγRIIb at or near position 135 of FcγR. For example, a crude contactability filter may be applied to the grafted polypeptide loop candidates. In the examples provided herein, the following crude contactability filters were developed. These may be used for this purpose:

number

number

[0069] When applying the above rough contact possibility filter, a minimum rough contact number of 5 to 10 may be used. For example, a minimum rough contact number of 6, 7, or 8 may be used.

[0070] Subsequently, polypeptide loop candidates that have passed through the coarse contact filter can undergo structural optimization. This step includes side chain repacking accompanied by backbone relaxation. The side chain repacking procedure employed in the examples provided herein is a modified version of the ICM algorithm using a fine rotamer library (see Xiang & Honig, 2001, J. Mol. Biol., 311:421), and backbone coordinates are relaxed by 5000 steps of the backrub algorithm (see Betancourt, 2005, J. Chem. Phys., 123:174905; Smith & Kortemme, 2008, J. Mol. Biol., 380:742). For repacking, the sequence of the polypeptide loop candidate is set to the wild-type sequence found in the PDB structure from which the polypeptide loop sequence was obtained.

[0071] The above steps can be performed using, for example, the AMBER99SB force field (Hornak, et al., 2006, Proteins Struc. Funct. Bioinf., 65:712), the GB / OBC implicit solvent model (Onufriev, et al., 2004, Proteins Struc. Funct. Bioinf., 55:383), and a pairwise hydrophobic potential (Jacobsen, et al., 2004, Proteins Struc. Funct. Bioinf., 55:351).

[0072] After repacking and backbone optimization, the grafted polypeptide loop candidates can be checked for interatomic collisions. In certain embodiments, atoms i and j satisfy σ i +σ j -d ij > 0.4, they are considered to collide, wherein σ i is the van der Waals radius of atom i defined in the AMBER99SB force field, and d ij is the distance between atom i and atom j. Polypeptide loop candidates that do not exhibit interatomic collisions after repacking are selected for further analysis and can be re-evaluated using a coarse contact score. Any residue on the polypeptide loop and C on receptor residue 135 βMinimum C between atoms β -C β The distance was also calculated.

[0073] Next, the Pareto optimal template is determined by anchor skeleton heavy atom RMSD, rough contact score, and minimum C β -C β The models were identified based on distance. The Pareto optimal consensus (POC) method (Li, et al., 2010, BMC Struc. Biol., 10:22) is a consensus model ranking approach that integrates multiple knowledge or physics-based scoring functions. The procedure for identifying the best-in-class model in a set of models includes: 1) identifying the model that is Pareto optimally front for the set of scoring functions, and 2) ranking them based on their fuzzy superiority relative to the remaining models.

[0074] For polypeptide loop candidates, identify the loops on the first three Pareto-optimal fronts and calculate pairwise sequence similarity for all polypeptide loop candidates of common length within the optimal set.

[0075] As the next step, we will test the stability of the template conformation in the Fc / FcγRIIb complex using a simulated annealing approach based on the dynamics of simple anemia molecules. This step is performed to account for conformational changes of the polypeptide loop candidate in a novel Fc / FcγR complex environment that is expected to differ from the loop's native environment.

[0076] In the molecular dynamics-based simulated annealing approach, arginine residues are first placed at each site on the polypeptide loop candidate, and the residues are rotated using all rotational isomers of the Dunbrack rotational isomer library (Dunbrack & Karplus, 1993, J. Mol. Biol., 230:543). The migration region is defined by enumerating all Fc / FcγR residues containing heavy atoms less than 4.0 Å from the heavy atom of the test arginine in any rotational isomer configuration. All residues thus identified are combined to obtain a "migration zone." All residues not included in the migration zone remain fixed, but residues within this zone are unrestricted. Once the migration zone of the polypeptide loop candidate is defined, the loop is executed using a simulated annealing protocol with, for example, the OpenMM molecular dynamics package (Eastman, et al., 2013, J. Chem. Theory Comput., 9:461), and the AMBER99SB force field and GB / OBC anal solvent model.

[0077] An exemplary annealing protocol includes the following steps: 1. Perform a short-time (2 nanoseconds) high-temperature simulation at 500K. 2. The latter conformations of the trajectories generated in step 1 are clustered into 10 clusters using the k-means algorithm. 3. Starting from the conformation identified in Step 2, perform 10 separate annealing simulations. The sample temperature schedule includes a step of geometric cooling from 500K to 450K in 1.0 nanosecond, followed by a step of linear cooling from 450K to 300K in 19 nanoseconds. 4. Extract the low-temperature components (300K–302K) from each of the 10 annealing trajectories for subsequent analysis. In total, 10 annealing operations generate 3-nanosecond trajectory data for each polypeptide loop candidate.

[0078] Next, the ensemble trajectories generated in step 4 of the annealing procedure are clustered. Clustering is performed on the template backbone heavy atoms, for example, using the SPICKER clustering method (Zhang & Skolnick, 2004, J. Comput. Chem., 25:865). Since most of the Fc / FcγR structure was fixed during the annealing simulation, changes in the template conformation are attributed to both internal deformation of the template and relaxation of the anchor β-strand. Only the primary clusters returned by the SPICKER algorithm are considered for subsequent analysis.

[0079] By configuration, primary clusters contain 60% to 70% of all frames of the ensemble trajectories generated in step 4 of the annealing procedure. Using the primary clusters, the following quantities are calculated: 1. The average coarse contact number between a polypeptide loop candidate and residue 135 on the FcγRIIb receptor. 2. The average root mean square fluctuation (RMSF) of the template (calculated based on the template backbone heavy atoms). 3. The average backbone heavy atom root mean square deviation (RMSD) (calculated for the grafted structure of the polypeptide loop candidate).

[0080] The coarse contact score provides an indicator of whether a low-temperature structure generated by the annealing process is configured to be positioned to interact with residue 135 of FcγRIIb.

[0081] RMSF serves as an indicator of consistency between and within annealing runs. A low RMSF value indicates that the polypeptide loop candidate shows structural consistency throughout the annealing runs, demonstrating that the runs have sufficiently converged. A low RMSF value also indicates that the polypeptide loop candidate does not have excessively high flexibility. Therefore, polypeptide loop candidates with low RMSF are preferable for subsequent selection rounds.

[0082] A low skeletal RMSD relative to the grafted structure indicates that the polypeptide loop candidate does not deviate significantly from the wild-type conformation found in the native PDB structure. Therefore, polypeptide loop candidates exhibiting a low skeletal RMSD relative to the grafted conformation are also preferred.

[0083] The above set of metrics can be used to select a set of polypeptide loop candidates for experimental screening. In certain embodiments, the above set of metrics can be used to select polypeptide loop candidates using the following values: (a) crude contact number greater than 5 and reference RMSD less than 3.0 Å, or (b) crude contact number greater than 5 and RMSF less than 3.0 Å. In some embodiments, the above set of metrics can be used to select polypeptide loop candidates using the following values: (a) crude contact number greater than 3 and reference RMSD less than 1.5 Å, or (b) crude contact number greater than 3 and RMSF less than 1.5 Å.

[0084] Polypeptide loop candidates selected by the above approach can be experimentally tested by engineering the test antibody, by replacing residues 325-331 in one of the Fc polypeptides of the test antibody with the loop sequence candidate using standard molecular biology techniques, and then testing the FcγR binding of the resulting variant antibody using a standard protocol such as that described herein. If necessary or desired, one or more amino acid substitutions may be made in the loop sequence to increase the selectivity or affinity of the variant antibody to FcγRIIb, as described in the examples provided herein.

[0085] Table 2 shows examples of polypeptide loop candidates identified using the approach outlined above.

[0086] [Table 2]

[0087] In certain embodiments, the substitution loop contained in the heterodimer Fc variant is a polypeptide loop comprising an amino acid sequence substantially identical to the sequence described in any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the polypeptide loop comprises an amino acid sequence that is a variant of the sequence described in any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, where the variant comprises 1, 2, 3, 4, or 5 amino acid mutations. In some embodiments, the variant comprises 1, 2, 3, or 4 amino acid mutations. In some embodiments, the polypeptide loop comprises an amino acid sequence described in any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0088] In certain embodiments, the substitution loop contained in the heterodimer Fc variant is a polypeptide loop comprising an amino acid sequence substantially identical to the sequence described in any one of SEQ ID NOs: 6, 8, 9, 12, or 14. In some embodiments, the polypeptide loop comprises an amino acid sequence that is a variant of the sequence described in any one of SEQ ID NOs: 6, 8, 9, 12, or 14, where the variant comprises 1, 2, 3, 4, or 5 amino acid mutations. In some embodiments, the variant comprises 1, 2, 3, or 4 amino acid mutations. In some embodiments, the polypeptide loop comprises an amino acid sequence described in any one of SEQ ID NOs: 6, 8, 9, 12, or 14.

[0089] In a particular embodiment, the substitution loop contained in the heterodimer Fc variant is a polypeptide loop comprising an amino acid sequence described in any one of the following formulas: (I), (Ia), (Ib), (II), (III), (IV), (V), or (VI), where (I), (Ia), and (Ib) are derived from the sequence described in SEQ ID NO: 6, (II) and (III) are derived from the sequence described in SEQ ID NO: 8, (IV) and (V) are derived from the sequence described in SEQ ID NO: 12, and (VI) is derived from the sequence described in SEQ ID NO: 14.

[0090] Equation (I): X 1 X 2 WX 3 X 4 X 5 GX 6 X 7 T(I) During the ceremony, X 1 is A, D, N, or S, X 2 is A, D, E, F, H, I, L, N, Q, S, T, V, W, or Y, X 3 is A, D, E, F, H, I, N, Q, S, T, V, W, or Y, X 4 is D, E, G, I, L, P, or Q, X 5 is A, D, E, G, H, K, N, R, S, T, or Y, X 6 is A, D, E, F, H, P, W, or Y, X 7 These are A, D, E, F, G, H, K, L, N, Q, or R.

[0091] In some embodiments, in general formula (I), X 1 It is either A or S.

[0092] In some embodiments, in general formula (I), X 2is A, D, E, F, H, I, L, N, Q, T, V, or W. In some embodiments, in general formula (I), X 2 It is either H or T.

[0093] In some embodiments, in formula (I), X 3 is A, F, H, I, S, T, V, W, or Y. In some embodiments, in formula (I), X 3 is D, E, F, H, N, Q, S, T, or Y. In some embodiments, in formula (I), X 3 is F, H, S, T, or Y. In some embodiments, in formula (I), X 3 is E, F, H, Q, S, or T. In some embodiments, in formula (I), X 3 is F, H, S, or T. In some embodiments, in general formula (I), X 3 is E, F, or S. In some embodiments, in general formula (I), X 3 It is either F or S.

[0094] In some embodiments, in formula (I), X 4 is D, G, I, or L. In some embodiments, in formula (I), X 4 It is either D or G.

[0095] In some embodiments, in formula (I), X 5 is A, D, E, G, H, K, or R. In some embodiments, in formula (I), X 5 It is G.

[0096] In some embodiments, in formula (I), X 6 is F, W, or Y. In some embodiments, in formula (I), X 6 Y is Y.

[0097] In some embodiments, in formula (I), X 7 is A, D, E, G, H, K, L, N, Q, or R. In some embodiments, in formula (I), X7 is A, F, H, K, L or N. In some embodiments, in formula (I), X 7 is A, H, K, L or N. In some embodiments, in formula (I), X 7 is A or N.

[0098] In a specific embodiment, in formula (I), X 1 is A, D, N or S, and X 2 is A, D, E, F, H, I, L, N, Q, S, T, V, W, or Y, and X 3 is A, F, H, I, S, T, V, W or Y, and X 4 is D, E, G, I, L, P or Q, and X 5 is A, D, E, G, H, K, N, R, S, T or Y, and X 6 is A, D, E, F, H, P, W or Y, and X 7 is A, D, E, G, H, K, L, N, Q or R.

[0099] In a specific embodiment, in formula (I), X 1 is A or S, and X 2 is A, D, E, F, H, I, L, N, Q, T, V or W, and X 3 is F, H, S, T or Y, and X 4 is D, G, I or L, and X 5 is G, and X 6 is F, W or Y, and X 7 is A, F, H, K, L or N.

[0100] Other combinations of the embodiments described above for formula (I) are also contemplated, each combination forming a separate embodiment for the purposes of this disclosure.

[0101] Equation (Ia): X 1 X 2 WX 3 X 4 X 5 GYX 6 T(Ia) During the ceremony, X 1 is A, D, N, or S, X 2 is A, D, E, F, H, I, L, N, Q, S, T, V, W, or Y, X 3 is A, D, E, F, H, I, N, Q, S, T, V, W, or Y, X 4 is D, E, G, I, L, P, or Q, X 5 is A, D, E, G, H, K, N, R, S, T, or Y, X 6 These are A, D, E, F, G, H, K, L, N, Q, or R.

[0102] In some embodiments, in general formula (Ia), X 1 It is either A or S.

[0103] In some embodiments, in general formula (Ia), X 2 is A, D, E, F, H, I, L, N, Q, T, V, or W. In some embodiments, in general formula (Ia), X 2 It is either H or T.

[0104] In some embodiments, in formula (Ia), X 3 is A, F, H, I, S, T, V, W, or Y. In some embodiments, in formula (Ia), X 3 is D, E, F, H, N, Q, S, T, or Y. In some embodiments, in formula (Ia), X 3is F, H, S, T or Y. In some embodiments, in formula (Ia), X 3 is E, F, H, Q, S or T. In some embodiments, in formula (Ia), X 3 is F, H, S or T. In some embodiments, in general formula (I), X 3 is E, F or S. In some embodiments, in general formula (Ia), X 3 is F or S.

[0105] In some embodiments, in formula (Ia), X 4 is D, G, I or L. In some embodiments, in formula (Ia), X 4 is D or G.

[0106] In some embodiments, in formula (Ia), X 5 is A, D, E, G, H, K or R. In some embodiments, in formula (Ia), X 5 is G.

[0107] In some embodiments, in formula (Ia), X 6 is A, D, E, G, H, K, L, N, Q or R. In some embodiments, in formula (Ia), X 6 is A, F, H, K, L or N. In some embodiments, in formula (Ia), X 6 is A, H, K, L or N. In some embodiments, in formula (Ia), X 6 is A or N.

[0108] Combinations of any of the foregoing embodiments described for formula (Ia) are also contemplated, and each combination forms a separate embodiment for the purposes of the present disclosure.

[0109] Formula (Ib): X 1 X 2 WX 3 X 4 GGYX 5 T(Ib) During the ceremony, X 1 is A or S, X 2 is A, D, E, F, H, I, L, N, Q, T, V, or W. X 3 is D, E, F, H, N, Q, S, T, or Y, X 4 is D, G, I, or L, X 5 It is A, F, H, K, L, or N.

[0110] In some embodiments, in formula (Ib), X 2 It is either H or T.

[0111] In some embodiments, in formula (Ib), X 3 is F, H, S, or Y. In some embodiments, in formula (Ib), X 3 is E, F, H, Q, S, or T. In some embodiments, in formula (Ib), X 3 is F, H, or S. In some embodiments, in formula (Ib), X 3 is E, F, or S. In some embodiments, in formula (Ib), X 3 It is either F or S.

[0112] In some embodiments, in formula (Ib), X 4 It is either D or G.

[0113] In some embodiments, in formula (Ib), X 5 is A, F, H, K, or L. In some embodiments, in formula (Ib), X 5 is A or N. In some embodiments, in formula (Ib), X 5 A is the answer.

[0114] Any combination of the aforementioned embodiments described for formula (Ib) is also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0115] Formula (II): X 1 LDX 2 X 3 GKGX 4 V(II) During the ceremony, X 1 is F or G, X 2 is E, H, Q, or T, X 3 is E, N, R, S, or T, X 4 It is A, Y, or V.

[0116] In some embodiments, in formula (II), X 2 It is E.

[0117] In some embodiments, in formula (II), X 3 is E, N, R, or S. In some embodiments, in formula (II), X 3 It is either E or N.

[0118] Any combination of the above embodiments described for formula (II) is also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0119] Formula (III): X 1 TDEX 2 GKGX 3 T(III) During the ceremony, X 1 is F or G, X 2 is either E or N, X 3 It is either A or V.

[0120] Formula (IV): X 1 FX 2 X 3 X 4 X 5 GEVV(IV) During the ceremony, X 1 is A or D, X 2 is either D or N, X 3 is D, E, H, N, P, Q, S, or T, X 4 is D, E, N, S, or T, X 5 It is either D or Q.

[0121] In some embodiments, in formula (IV), X 1 It is D.

[0122] In some embodiments, in formula (IV), X 2 It is D.

[0123] In some embodiments, in formula (IV), X 3 It is E, H, N, S, or T.

[0124] In some embodiments, in formula (IV), X 4 It is D, N, S, or T.

[0125] Any combination of the above embodiments described for formula (IV) is also conceivable, and each combination forms a separate embodiment for the purposes of this disclosure.

[0126] Formula (V): X 1 TDX 2 X 3 X 4 GEVT(V) During the ceremony, X 1 is either A or D, X 2 is D, P, or Q, X 3 is D, E, or N, X 4 It is either D or Q.

[0127] Equation (VI): LTDX 1 X 2 GX 3 PX 4 R(VI) During the ceremony, X 1 is E or H, X 2 is D, E, or N, X 3 is R or S, X 4 is I, Q, or Y.

[0128] In some embodiments, in formula (VI), X 1 It is E.

[0129] In some embodiments, in formula (VI), X 4 It is either I or Y.

[0130] Any combination of the embodiments described above for formula (VI) is also contemplated, and each combination forms a separate embodiment for the purposes of this disclosure.

[0131] In a particular embodiment, the substitution loop contained in the heterodimer Fc variant is a polypeptide loop comprising the amino acid sequence described in one of the sequences (SEQ ID NOs: 4-172) shown in Tables 3A and 3B. Since the polypeptide loop replaces residues 325-331 in the parent Fc sequence, the following numbering system is used in Tables 3A and 3B and throughout the description: The residue immediately following position 324 in Fc is 325 * The remaining residues of the polypeptide loop are specified as follows: 326 in order. * From 331 * It is numbered as follows: 331 in the polypeptide loop * Any additional residues after this are specified by letters, i.e., 331 * A, 331 * B, 331 * Let's call it C, for example.

[0132] In some embodiments, the substitution loop contained in the heterodimer Fc variant is a polypeptide loop containing any one of the amino acid sequences described in SEQ ID NOs. 4-90 (see Table 3A). In some embodiments, the polypeptide loop is SEQ ID NOs. 6, 8, 9, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, The amino acid sequence comprises any one of the amino acids listed in 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 (Table 3A). In certain embodiments, the heterodimer Fc variant further comprises mutation I332L.

[0133] In a particular embodiment, the substitution loop contained in the heterodimer Fc variant is a polypeptide loop comprising the amino acid sequence described in any one of SEQ ID NOs: 6, 8, 47, 68, or 73. In a particular embodiment, the heterodimer Fc variant further comprises mutation I332L.

[0134] [Table 3A] TIFF2026143609000007.tif176165TIFF2026143609000008.tif173165TIFF2026143609 000009.tif198165TIFF2026143609000010.tif196165TIFF2026143609000011.tif18916 5TIFF2026143609000012.tif185165TIFF2026143609000013.tif191165TIFF2026143609 000014.tif182165TIFF2026143609000015.tif196165TIFF2026143609000016.tif57165

[0135] [Table 3B] TIFF2026143609000018.tif196165TIFF2026143609000019.tif189165TIFF20261436090 00020.tif200165TIFF2026143609000021.tif194165TIFF2026143609000022.tif192165 TIFF2026143609000023.tif180165TIFF2026143609000024.tif195165TIFF20261436090 00025.tif178165TIFF2026143609000026.tif178165TIFF2026143609000027.tif122165

[0136] Additional CH2 domain mutations In certain embodiments, the heterodimer Fc variant comprises an asymmetric loop substitution and one or more additional mutations in the CH2 domain as described in any one of the embodiments above. The one or more additional mutations in the CH2 domain may be symmetric or asymmetric and may increase the selectivity of the heterodimer Fc variant to FcγRIIb, or increase the affinity of the heterodimer Fc variant to FcγRIIb, or increase both the selectivity and affinity of the heterodimer Fc variant to FcγRIIb. In some embodiments, the heterodimer Fc variant comprises an asymmetric loop substitution and one or more additional asymmetric mutations in the CH2 domain as described in any one of the embodiments above.

[0137] In certain embodiments, the heterodimer Fc variant contains 1 to 20 amino acid mutations in the CH2 domain, one of which is an asymmetric loop substitution. In some embodiments, the heterodimer Fc variant contains an asymmetric loop substitution and 1 to 15 additional amino acid mutations in the CH2 domain. In some embodiments, the heterodimer Fc variant contains an asymmetric loop substitution and 1 to 12 additional amino acid mutations in the CH2 domain, for example, 1 to 11 additional amino acid mutations in the CH2 domain, 1 to 10 additional amino acid mutations, 1 to 9 additional amino acid mutations, or 1 to 8 additional amino acid mutations.

[0138] References to “asymmetric loop substitution” or “loop substitution” in the embodiments described above and below, combined with one or more additional amino acid mutations in the CH2 domain, are intended to encompass the asymmetric loop substitutions described in any one of the embodiments detailed above, and each combination forms an embodiment of the present disclosure, just as each combination is described individually.

[0139] In certain embodiments, the heterodimer Fc variant comprises an asymmetric loop substitution and a mutation at position 236 in the CH2 domain. The mutation at position 236 may be a symmetric or asymmetric mutation. In certain embodiments, the heterodimer Fc variant comprises an asymmetric loop substitution, a mutation at position 236, and one or more additional mutations in the CH2 domain.

[0140] In some embodiments, the heterodimeric Fc variant includes a loop substitution in one Fc polypeptide and a mutation at position 236 in the same Fc polypeptide. In some embodiments, the heterodimeric Fc variant includes a loop substitution in one Fc polypeptide and a mutation at position 236 in the other Fc polypeptide. In some embodiments, the heterodimeric Fc variant includes a loop substitution in one Fc polypeptide and a mutation at position 236 in both Fc polypeptides. In some embodiments, the heterodimeric Fc variant includes a loop substitution in one Fc polypeptide and a mutation at position 236 in both Fc polypeptides, where the mutation at position 236 is symmetric (i.e., the mutation at position 236 is the same in both Fc polypeptides). In some embodiments, the heterodimeric Fc variant comprises a loop substitution in one Fc polypeptide and a mutation at position 236 in both Fc polypeptides, where the mutation at position 236 is asymmetric (i.e., the mutation at position 236 is different in each Fc polypeptide, or one Fc polypeptide contains the mutation at position 236 and the other Fc polypeptide does not).

[0141] In certain embodiments, the heterodimer Fc variant comprises a loop substitution in one Fc polypeptide and a mutation at position 236 in the same Fc polypeptide, selected from G236D, G236E, G236K, G236N, and G236T. In some embodiments, the heterodimer Fc variant comprises a loop substitution in one Fc polypeptide and a mutation at position 236 in the same Fc polypeptide, selected from G236D and G236N.

[0142] In certain embodiments, the heterodimer Fc variant comprises a loop substitution in one Fc polypeptide and a mutation at position 236 in the other Fc polypeptide, selected from G236A, G236D, G236E, G236F, G236H, G236I, G236L, G236N, G236P, G236Q, G236S, G236T, G236V, G236W, and G236Y. In some embodiments, the heterodimer Fc variant comprises a loop substitution in one Fc polypeptide and a mutation at position 236 in the other Fc polypeptide, selected from G236D, G236K, and G236N.

[0143] In certain embodiments, the heterodimeric Fc variant includes a loop substitution in one Fc polypeptide and a mutation at position 236 in both Fc polypeptides. In some embodiments, the first Fc polypeptide of the heterodimeric Fc variant includes a mutation at position 236 selected from G236A, G236D, G236E, G236F, G236H, G236I, G236L, G236N, G236P, G236Q, G236S, G236T, G236V, G236W, and G236Y, and the second Fc polypeptide of the heterodimeric Fc variant includes a loop substitution and a mutation at position 236 selected from G236D, G236E, G236K, G236N, and G236T. In some embodiments, the first Fc polypeptide of the heterodimer Fc variant includes a mutation at position 236 selected from G236A, G236D, G236E, G236F, G236H, G236I, G236L, G236N, G236P, G236Q, G236S, G236T, G236V, G236W, and G236Y, and the second Fc polypeptide of the heterodimer Fc variant includes a loop substitution and the mutation G236D. In some embodiments, the first Fc polypeptide of the heterodimer Fc variant includes the mutation G236N, and the second Fc polypeptide of the heterodimer Fc variant includes a loop substitution and a mutation at position 236 selected from G236D, G236E, G236K, G236N, and G236T.

[0144] In some embodiments, the heterodimer Fc variant comprises a loop substitution in one Fc polypeptide and a mutation at position 236 in both Fc polypeptides, where the mutation at position 236 is symmetric and selected from G236D, G236N, and G236K.

[0145] In some embodiments, the heterodimeric Fc variant includes a loop substitution in one of the Fc polypeptides and an asymmetric mutation at position 236. In some embodiments, the first Fc polypeptide of the heterodimer Fc variant comprises a mutation at position 236 selected from G236A, G236D, G236E, G236F, G236H, G236I, G236L, G236N, G236P, G236Q, G236S, G236T, G236V, G236W, and G236Y, and the second Fc polypeptide of the heterodimer Fc variant comprises a loop substitution and a mutation at position 236 selected from G236D, G236E, G236K, G236N, and G236T, wherein the mutation at position 236 is asymmetric (i.e., the mutation at position 236 in the first Fc polypeptide is different from the mutation at position 236 in the second Fc polypeptide).

[0146] In some embodiments, the first Fc polypeptide of the heterodimer Fc variant includes a mutation at position 236 selected from G236A, G236E, G236F, G236H, G236I, G236L, G236N, G236P, G236Q, G236S, G236T, G236V, G236W, and G236Y, and the second Fc polypeptide of the heterodimer Fc variant includes a loop substitution and the mutation G236D. In some embodiments, the first Fc polypeptide of the heterodimer Fc variant includes the mutation G236N, and the second Fc polypeptide of the heterodimer Fc variant includes a loop substitution and a mutation at position 236 selected from G236D, G236E, G236K, and G236T.

[0147] In some embodiments, the first Fc polypeptide of the heterodimer Fc variant comprises a mutation at position 236 selected from G236D, G236K, and G236N, and the second Fc polypeptide of the heterodimer Fc variant comprises a loop substitution and a mutation at position 236 selected from G236D and G236N, where the mutation at position 236 is asymmetric. In some embodiments, the first Fc polypeptide of the heterodimer Fc variant comprises the mutation G236N, and the second Fc polypeptide of the heterodimer Fc variant comprises a loop substitution and the mutation G236D.

[0148] In a particular embodiment, the heterodimer Fc variant comprises a loop substitution in one of the Fc polypeptides, optionally comprising a mutation at position 236 in one or both of the Fc polypeptides as described in any one of the embodiments above, and further comprising one or more “binding enhancers”.

[0149] A “binding enhancer” is an amino acid mutation known in the art or identified herein that increases the affinity of Fc to FcγRIIb. Examples include, but are not limited to, L234F, L234W, L234D, L235F, L235W, G237F, G237A, G237L, S239D, S239E, V266I, V266L, S267A, S267E, S267I, S267Q, S267V, H268D, Y300E, K326D, K326E, K326N, I332L, and I332E.

[0150] In a particular embodiment, the heterodimer Fc variant includes one or more binding enhancers selected from L234F, L234W, L234D, L235F, L235W, G237F, G237A, G237L, S239D, S239E, V266I, V266L, S267A, S267E, S267I, S267Q, S267V, H268D, Y300E, K326D, K326E, K326N, I332L, and I332E. In some embodiments, the heterodimer Fc variant includes one or more binding enhancers selected from S239D, S239E, V266I, V266L, S267A, S267E, S267I, S267Q, S267V, H268D, Y300E, K326D, and I332E.

[0151] In a particular embodiment, the heterodimer Fc variant comprises a loop substitution in one of the Fc polypeptides, optionally comprising a mutation at position 236 in one or both Fc polypeptides as described in any one of the embodiments above, and further comprising one or more binding enhancers selected from S239D, S239E, V266I, V266L, S267A, S267E, S267I, S267Q, S267V, H268D, Y300E, K326D, and I332E. In some embodiments, the heterodimer Fc variant comprises a loop substitution in one of the Fc polypeptides, optionally comprising a mutation at position 236 in one or both Fc polypeptides as described in any one of the embodiments above, and further comprising one or more binding enhancers selected from S239D, S239E, V266I, V266L, S267A, S267I, S267V, S267Q, and H268D.

[0152] In a particular embodiment, the heterodimer Fc variant comprises a loop substitution in one of the Fc polypeptides, a mutation at position 236 in both Fc polypeptides as described in any one of the embodiments above, and further comprises one or more binding enhancers selected from S239D, S239E, V266I, V266L, S267A, S267E, S267I, S267Q, S267V, H268D, Y300E, K326D, and I332E, where one or more binding enhancers are located on the same Fc polypeptide as the loop substitution. In some embodiments, the heterodimer Fc variant comprises a loop substitution in one of the Fc polypeptides, a mutation at position 236 in both Fc polypeptides as described in any one of the embodiments above, and further comprises one or more binding enhancers selected from S239D, S239E, V266I, V266L, S267A, S267I, S267V, S267Q and H268D, where one or more binding enhancers are located on the same Fc polypeptide as the loop substitution.

[0153] In a particular embodiment, the heterodimer Fc variant comprises a loop substitution in one of the Fc polypeptides, an asymmetric mutation at position 236 in both Fc polypeptides as described in any one of the embodiments above, and further comprises one or more binding enhancers selected from S239D, S239E, V266I, V266L, S267A, S267E, S267I, S267Q, S267V, H268D, Y300E, K326D, and I332E, where one or more binding enhancers are located on the same Fc polypeptide as the loop substitution. In some embodiments, the heterodimer Fc variant comprises a loop substitution in one of the Fc polypeptides, an asymmetric mutation at position 236 in both Fc polypeptides as described in any one of the embodiments above, and further comprises one or more binding enhancers selected from S239D, S239E, V266I, V266L, S267A, S267I, S267V, S267Q and H268D, where one or more binding enhancers are located on the same Fc polypeptide as the loop substitution.

[0154] In some embodiments, the first Fc polypeptide of the heterodimer Fc variant comprises a mutation at position 236 selected from G236A, G236D, G236E, G236F, G236H, G236I, G236L, G236N, G236P, G236Q, G236S, G236T, G236V, G236W, and G236Y, and the second Fc polypeptide of the heterodimer Fc variant comprises a loop substitution, a mutation at position 236 selected from G236D, G236E, G236K, G236N, and G236T, and one or more binding enhancers selected from S239D, S239E, V266I, V266L, S267A, S267I, S267V, S267Q, and H268D. In some embodiments, the first Fc polypeptide of the heterodimer Fc variant comprises a mutation at position 236 selected from G236A, G236D, G236E, G236F, G236H, G236I, G236L, G236N, G236P, G236Q, G236S, G236T, G236V, G236W, and G236Y, and the second Fc polypeptide of the heterodimer Fc variant comprises a loop substitution, the mutation G236D, and one or more binding enhancers selected from S239D, S239E, V266I, V266L, S267A, S267I, S267V, S267Q, and H268D. In some embodiments, the first Fc polypeptide of the heterodimer Fc variant comprises the mutation G236N, and the second Fc polypeptide of the heterodimer Fc variant comprises a loop substitution, a mutation at position 236 selected from G236D, G236E, G236K, G236N, and G236T, and one or more binding enhancers selected from S239D, S239E, V266I, V266L, S267A, S267I, S267V, S267Q, and H268D.

[0155] In certain embodiments, the binding enhancers included in the heterodimer Fc variant include (i) mutation S239D or S239E, and / or (ii) mutation H268D. In some embodiments, the binding enhancers included in the heterodimer Fc variant include (i) mutation S239D or S239E, and / or (ii) mutation H268D, and / or (iii) mutations S267A, S267I, or S267V. In some embodiments, the binding enhancers included in the heterodimer Fc variant include mutations S239D and H268D. In some embodiments, the binding enhancers included in the heterodimer Fc variant include mutations S239D, H268D, and S267V. In some embodiments, the binding enhancers include mutations S239D, H268D, and S267A.

[0156] In a particular embodiment, the heterodimer Fc variant comprises (a) a mutation at position 236 of one or both of the first and second Fc polypeptides as described in any one of the embodiments above; (b) a loop substitution in the second Fc polypeptide; (c) one or more “binding enhancers” in the second Fc polypeptide as described in any one of the embodiments above; (d) an optional additional CH2 mutation at one or more of the positions 234, 235, 237, and 239 in the first Fc polypeptide; and (e) an optional additional CH2 mutation at one or more of the positions 234, 235, 237, 240, 263, 264, 266, 269, 271, 273, 323, and 332 in the second Fc polypeptide.

[0157] In some embodiments, additional CH2 mutations at one or more positions 234, 235, 237, and 239 in the first Fc polypeptide of the heterodimeric Fc variant are selected from the following: (i) The mutation at position 234 is selected from L234A, L234D, L234E, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235I, L235N, L235P, L235Q, L235S, L235T, L235V, L235W, and L235Y. (iii) The mutation at position 237 is selected from G237A, G237D, G237F, G237H, G237L, G237N, G237P, G237S, G237V, G237W and G237Y, (iv) The mutation at position 239 is selected from S239A, S239D, S239E, S239F, S239G, S239H, S239I, S239L, S239N, S239Q, S239R, S239T, S239V, S239W, and S239Y.

[0158] In some embodiments, additional CH2 mutations at one or more positions 234, 235, 237, and 239 in the first Fc polypeptide of the heterodimeric Fc variant are selected from the following: (i) The mutation at position 234 is selected from L234D, L234F, L234Q, L234T, and L234W. (ii) The mutation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235R, L235W, and L235Y. (iii) The mutation at position 237 is selected from G237A, G237D, G237L, and G237N. (iv) The mutation at position 239 is selected from S239A, S239G, S239H, S239T, and S239Y.

[0159] In some embodiments, the first Fc polypeptide of the heterodimeric Fc polypeptide comprises an additional CH2 mutation selected from L234D and L235F.

[0160] In some embodiments, additional CH2 mutations at one or more positions 234, 235, 237, 240, 263, 264, 266, 269, 271, 273, 323, and 332 in the second Fc polypeptide of the heterodimer Fc variant are selected from the following: (i) The mutation at position 234 is selected from L234A, L234E, L234F, L234G, L234H, L234I, L234K, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 235 is selected from L235A, L235D, L235F, L235G, L235N, L235S, L235W, and L235Y. (iii) The mutation at position 237 is selected from G237F, G237I, G237K, G237L, G237Q, G237T, G237V, and G237Y. (iv) The mutation at position 240 was selected from V240I and V240L, (v) The mutation at position 263 is V263T, (vi) The mutation at position 264 is V264T, (vii) The mutation at position 266 is V266I, (viii) The mutation at position 269 is E269Q, (ix) The mutation at position 271 is P271D, The mutation at position (x)273 was selected from V273A and V273I. (xi) The mutation at position 323 was selected from V323A and V323I. (xii) The mutation at position 332 is selected from I332F and I332L.

[0161] In some embodiments, the second Fc polypeptide of the heterodimer Fc variant comprises an additional CH2 mutation at one or more of positions 271, 323, and 332, selected from (i) mutation P271D, (ii) mutation V323A, and (iii) a mutation at position 332 selected from I332F and I332L.

[0162] In certain embodiments, the heterodimer Fc variant includes amino acid mutations listed in Tables 5A, 5B, and 5C for one of the variants listed under “loop substitution + symmetric 236 mutation,” “strategy 1 / 3,” or “strategy 1 / 3 + strategy 2 combination.” In certain embodiments, the heterodimer Fc variant includes amino acid mutations listed in one of the variants shown in Tables 6.22, 6.24, 6.25, and 6.27. In certain embodiments, the heterodimer Fc variant includes amino acid mutations listed in one of the variants shown in Tables 6.22 and 6.24.

[0163] In certain embodiments, the heterodimer Fc variant includes one amino acid mutation from any of the variants shown in Tables 6.17, 6.19, and 6.20, having a "IIb selectivity ratio relative to control" value ≥ 0.5 and a "IIb ratio relative to control" value ≥ 0.5 ("Criterion B"). In some embodiments, the heterodimer Fc variant includes one amino acid mutation from any of the variants shown in Tables 6.17, 6.19, and 6.20, having a "IIb selectivity ratio relative to control" value ≥ 1.0 and a "IIb ratio relative to control" value ≥ 0.3 ("Criterion C"). In some embodiments, the heterodimer Fc variant includes one amino acid mutation from any of the variants shown in Tables 6.17, 6.19, and 6.20, having a "IIb selectivity ratio relative to control" value ≥ 1.0 and a "IIb ratio relative to control" value ≥ 0.5 ("Criterion D"). In some embodiments, the heterodimer Fc variant includes one amino acid mutation from any of the variants shown in Tables 6.17, 6.19, and 6.20, having a "ratio of IIb selectivity to control" value ≥ 1.5 and a "ratio of IIb to control" value ≥ 0.3 ("reference A").

[0164] A heterodimer Fc variant containing an asymmetric mutation at position 236. As described herein, it has been shown that incorporating an asymmetric mutation at position 236 in the CH2 domain of Fc increases selectivity for FcγRIIb. Accordingly, certain embodiments of this disclosure relate to a heterodimeric Fc variant that includes an asymmetric mutation at position 236 and exhibits increased selectivity for FcγRIIb compared to parental Fc. The asymmetric mutation at position 236 may include an amino acid mutation at position 236 of one Fc polypeptide and no mutation at position 236 of the other Fc polypeptide, or it may include a mutation at position 236 of one Fc polypeptide and a different mutation at position 236 of the other Fc polypeptide.

[0165] In certain embodiments, the heterodimer Fc variant includes an asymmetric mutation at position 236, resulting in increased selectivity for FcγRIIb compared to the parental Fc. The asymmetric mutation at position 236 includes a mutation selected from G236N and G236D. In some embodiments, the heterodimer Fc variant includes an asymmetric mutation at position 236, where one Fc polypeptide includes the mutation G236N or G236D, and the other Fc polypeptide does not include the mutation at position 236. In some embodiments, the heterodimer Fc variant includes an asymmetric mutation at position 236, where one Fc polypeptide includes the mutation G236N, and the other Fc polypeptide includes the mutation G236D.

[0166] In a particular embodiment, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where one Fc polypeptide comprises the mutation G236N and the other Fc polypeptide comprises the mutation G236D, G236K, or G236S, or does not contain the mutation at position 236.

[0167] In a particular embodiment, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where one Fc polypeptide comprises the mutation G236D and the other Fc polypeptide comprises the mutations G236N, G236Q, G236K, G236E, or G236H, or does not contain the mutation at position 236.

[0168] In certain embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236 as described in any one of the embodiments above and one or more additional mutations in the CH2 domain. The one or more additional mutations in the CH2 domain may be symmetric or asymmetric and may increase the selectivity of the heterodimer Fc variant to FcγRIIb, or increase the affinity of the heterodimer Fc variant to FcγRIIb, or increase both the selectivity and affinity of the heterodimer Fc variant to FcγRIIb. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236 as described in any one of the embodiments above and one or more additional asymmetric mutations in the CH2 domain.

[0169] In a particular embodiment, the heterodimer Fc variant includes 1 to 20 mutations in the CH2 domain, including an asymmetric mutation at position 236. In some embodiments, the heterodimer Fc variant includes an asymmetric mutation at position 236 and 1 to 18 additional mutations in the CH2 domain, e.g., 1 to 17 additional mutations, 1 to 16 additional mutations, or 1 to 15 additional mutations in the CH2 domain.

[0170] Joint enhancer In certain embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236 as described in any one of the embodiments above, and further comprises one or more “binding enhancers” as described above. In some embodiments, one or more binding enhancers are selected from S239D, S239E, V266I, V266L, S267A, S267I, S267V, S267Q, and H268D. In some embodiments, one or more binding enhancers are selected from S239D, S239E, V266L, S267A, S267I, S267V, and H268D.

[0171] In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236 selected from G236N and G236D, and further comprises one or more binding enhancers described above. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, wherein the first Fc polypeptide comprises the mutation G236N or G236D, and the second Fc polypeptide does not contain a mutation at position 236, wherein the second Fc polypeptide further comprises one or more binding enhancers described above. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, wherein the first Fc polypeptide comprises the mutation G236N or G236D, and the second Fc polypeptide comprises a different mutation at position 236, wherein the second Fc polypeptide further comprises one or more binding enhancers described above. In some embodiments, one or more coupling enhancers are selected from S239D, S239E, V266L, S267A, S267I, S267V, and H268D.

[0172] In certain embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, the second Fc polypeptide comprises the mutation G236D, G236K, or G236S, and the second Fc polypeptide further comprises one or more binding enhancers as described above. In some embodiments, one or more binding enhancers are selected from S239D, S239E, V266L, S267A, S267I, S267V, and H268D.

[0173] In certain embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, wherein the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, G236K, or G236S, wherein the second Fc polypeptide further comprises the binding enhancer (i) S239D or S239E, and / or (ii) H268D. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, wherein the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, G236K, or G236S, wherein the second Fc polypeptide further comprises the mutations S239D and H268D.

[0174] In certain embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutations G236D, G236K, or G236S, where the second Fc polypeptide further comprises the binding enhancers (i) S239D or S239E, and / or (ii) H268D, and / or (iii) S267A, S267I, or S267V. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutations G236D, G236K, or G236S, where the second Fc polypeptide further comprises the mutations S239D, H268D, and S267V. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutations G236D, G236K, or G236S, where the second Fc polypeptide further comprises the mutations S239D, H268D, and S267A.

[0175] In certain embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, the second Fc polypeptide comprises the mutation G236D, and the second Fc polypeptide further comprises one or more binding enhancers as described above. In some embodiments, one or more binding enhancers are selected from S239D, S239E, V266L, S267A, S267I, S267V, and H268D.

[0176] In certain embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, where the second Fc polypeptide further comprises the binding enhancer (i) S239D or S239E, and / or (ii) H268D. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, where the second Fc polypeptide further comprises the mutations S239D and H268D.

[0177] In certain embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, where the second Fc polypeptide further comprises the binding enhancer (i) S239D or S239E, and / or (ii) H268D, and / or (iii) S267A, S267I, or S267V. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, where the second Fc polypeptide further comprises the mutations S239D, H268D, and S267V. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, where the second Fc polypeptide further comprises the mutations S239D, H268D, and S267A. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, where the second Fc polypeptide further comprises the mutations S239D, H268D, and S267I.

[0178] In certain embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236D, and the second Fc polypeptide comprises the mutations G236N, G236Q, G236K, G236E, or G236H, where the second Fc polypeptide further comprises one or more binding enhancers as described above. In some embodiments, one or more binding enhancers are selected from S239D, S239E, V266L, S267A, S267I, S267V, and H268D.

[0179] In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236D, and the second Fc polypeptide comprises the mutations G236N, G236Q, G236K, G236E, or G236H, where the second Fc polypeptide further comprises the binding enhancers (i) S239D or S239E, and / or (ii) H268D.

[0180] In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236D, and the second Fc polypeptide comprises the mutations G236N, G236Q, G236K, G236E, or G236H, where the second Fc polypeptide further comprises the binding enhancer (i) S239D or S239E, and / or (ii) H268D, and / or (iii) S267A, S267I, or S267V. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236D, and the second Fc polypeptide comprises the mutations G236N, G236Q, G236K, G236E, or G236H, where the second Fc polypeptide further comprises the mutations S239D, H268D, and S267V.

[0181] In a particular embodiment, the heterodimer Fc variant includes the amino acid mutations described in Core Set 1 below: Core Set 1 First Fc polypeptide: G236N The second Fc polypeptide is: G236D_S239D_H268D.

[0182] In a particular embodiment, the heterodimer Fc variant includes the amino acid mutations described in the following core set 1A: Core Set 1A First Fc polypeptide: G236N The second Fc polypeptide is: G236D_S239D_S267A / I / V_H268D.

[0183] In a particular embodiment, the heterodimer Fc variant contains one of the variants listed under “Asymmetric 236 Mutations” as listed in Table 5A.

[0184] Additional CH2 domain mutations As described in the examples provided herein, various in silico approaches were employed to identify Fc variants that increase selectivity for FcγRIIb. Experimental validation and refinement of the initially identified variants led to the identification of two lead variants, Lead 1 and Lead 2 (see Example 3 and Table 4), which increased selectivity for FcγRIIb, each containing an asymmetric mutation at position 236 and one or more binding enhancers, along with additional CH2 domain mutations. Further refinement of these lead variants (see Example 4) yielded launch modules 1 and 2 (see Table 4), each also containing an asymmetric mutation at position 236, one or more binding enhancers, and additional CH2 domain mutations. To further improve FcγRIIb selectivity and / or affinity, a series of additional investigations based on launch modules 1 and 2 identified alternative amino acid substitutions that can be made at the mutated CH2 domain position in these launch modules and additional CH2 domain mutations that can be included in heterodimeric Fc variants (see Example 6). Accordingly, certain embodiments of this disclosure relate to heterodimeric Fc variants comprising an asymmetric mutation at position 236, one or more binding enhancers, and one or more additional CH2 domain mutations.

[0185] [Table 4]

[0186] Strategy 1 / 3 variant Further optimization of Launch Module 1 was performed, yielding additional heterodimeric Fc variants with improved selectivity for FcγRIIb. These are collectively referred to as “Strategy 1 / 3 Variants” in the following sections. As used herein, the term “Strategy 1 / 3 Variant” refers to a heterodimeric Fc variant comprising (a) an asymmetric mutation at position 236 as described above, (b) an asymmetric loop substitution in the CH2 domain, (c) one or more optionally bound enhancers as described above, and (d) one or more optionally additional mutations in the CH2 domain. Therefore, this term is not limited to the heterodimeric Fc variants explicitly referred to as “Strategy 1 Variant” and “Strategy 3 Variant” in the Examples. In a particular embodiment, the strategy 1 / 3 variant is a heterodimer Fc variant comprising (a) an asymmetric mutation at position 236 as described above, (b) an asymmetric loop substitution in the CH2 domain, (c) one or more binding enhancers as described above, and (d) one or more optional additional mutations in the CH2 domain.

[0187] In certain embodiments, the heterodimer Fc variant is a strategy 1 / 3 variant. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236 as described in any one of the embodiments above, and further comprises an asymmetric loop substitution in the CH2 domain. In some embodiments, the asymmetric loop substitution included in the heterodimer Fc variant comprises a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above, for the native loop at positions 325-331 in one of the Fc polypeptides, as an "asymmetric loop substitution".

[0188] In certain embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236 selected from G236N and G236D, further comprising a substitution of a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length for the native loop at positions 325-331 as described in any one of the embodiments provided above as an "asymmetric loop substitution". In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N or G236D, and the second Fc polypeptide comprises a different mutation at position 236, further comprising a substitution in a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above, as an "asymmetric loop substitution" for the native loop at positions 325-331.

[0189] In some particular embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, G236K, or G236S, where the second Fc polypeptide further comprises a substitution of a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above as an "asymmetric loop substitution" for the native loop at positions 325-331. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236D, and the second Fc polypeptide comprises the mutation G236N, G236Q, G236K, G236E, or G236H, where the second Fc polypeptide further comprises a substitution in a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above, as an "asymmetric loop substitution" for the native loop at positions 325-331.

[0190] In a particular embodiment, the heterodimer Fc variant is a strategy 1 / 3 variant and includes the following amino acid mutations (referred to as core set 2): Core Set 2 First Fc polypeptide: G236N Second Fc polypeptide: G236D_loop substitution (325~331).

[0191] In certain embodiments, the substitution loop included in the Strategy 1 / 3 variant is a polypeptide loop containing an amino acid sequence described in any one of the following formulas, as described above as an "asymmetric loop substitution": formula (I), formula (Ia), formula (Ib), formula (II), formula (III), formula (IV), formula (V), or formula (VI). In some embodiments, the polypeptide loop contains an amino acid sequence described in any one of the sequences shown in Tables 3A and 3B (SEQ ID NOs. 4-172). In some embodiments, the polypeptide loop contains an amino acid sequence described in any one of the SEQ ID NOs. 4-90 (see Table 3A above). In some embodiments, the polypeptide loop is represented by sequence numbers 6, 8, 9, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , containing any one of the amino acid sequences listed in 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 (see Table 3A above).

[0192] In a particular embodiment, the heterodimer Fc variant is a strategic 1 / 3 variant comprising the amino acid mutations described in core set 2, wherein the second Fc polypeptide further comprises (a) an amino acid mutation at position 239 selected from S239D and S239E, (b) an amino acid mutation at position 267 selected from S267I, S267Q and S267V, and (c) an amino acid mutation at position 268 selected from H268A, H268D, H268E, H268F, H268I, H268K, H268L, H268N, H268P, H268Q, H268T, H268V, H268W and H268Y.

[0193] In a particular embodiment, the heterodimer Fc variant is a strategy 1 / 3 variant comprising (a) an asymmetric mutation at position 236 as described in any one of the embodiments above, (b) a substitution of a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above as an "asymmetric loop substitution" for the native loop at positions 325-331 in one of the Fc polypeptides, and (c) one or more binding enhancers as described in any one of the embodiments above.

[0194] In a particular embodiment, the heterodimer Fc variant is a strategy 1 / 3 variant comprising (a) an asymmetric mutation at position 236, wherein the first Fc polypeptide comprises the mutation G236N and the second Fc polypeptide comprises the mutation G236D, G236K, or G236S; (b) a substitution in the second Fc polypeptide for the native loop at positions 325–331, in a polypeptide loop of 7–15 amino acid length or 8–15 amino acid length as described in any one of the embodiments provided above, and (c) one or more binding enhancers in the second Fc polypeptide as described in any one of the embodiments above.

[0195] In a particular embodiment, the heterodimer Fc variant is a strategy 1 / 3 variant comprising (a) an asymmetric mutation at position 236, wherein the first Fc polypeptide comprises mutation G236N and the second Fc polypeptide comprises mutation G236D; (b) a substitution in the second Fc polypeptide for the native loop at positions 325–331, in a polypeptide loop of 7–15 amino acid length or 8–15 amino acid length as described in any one of the embodiments provided above, and (c) one or more binding enhancers in the second Fc polypeptide as described in any one of the embodiments above.

[0196] In a particular embodiment, the heterodimer Fc variant is a strategy 1 / 3 variant comprising (a) an asymmetric mutation at position 236, wherein the first Fc polypeptide comprises mutation G236D and the second Fc polypeptide comprises mutation G236N, G236Q, G236K, G236E, or G236H; (b) a substitution in the second Fc polypeptide for the native loop at positions 325–331, in a polypeptide loop of 7–15 amino acid length or 8–15 amino acid length as described in any one of the embodiments provided above, and (c) one or more binding enhancers in the second Fc polypeptide as described in any one of the embodiments above.

[0197] In a particular embodiment, the heterodimer Fc variant is a strategy 1 / 3 variant comprising amino acid mutations described in core set 2, and the second Fc polypeptide further comprises one or more binding enhancers.

[0198] In certain embodiments, one or more binding enhancers included in the strategy 1 / 3 heterodimer Fc variant are selected from S239D, S239E, V266I, S267I, S267Q, S267V, and H268D. In some embodiments, one or more binding enhancers are (i) S239D or S239E, and / or (ii) H268D, and / or (iii) S267I or S267V. In some embodiments, one or more binding enhancers are S239D and H268D. In some embodiments, one or more binding enhancers are S239D, H268D, and S267V.

[0199] In some embodiments, the heterodimer Fc variant is a strategy 1 / 3 variant and includes the following amino acid mutation (referred to as core set 2A): Core Set 2A First Fc polypeptide: G236N Second Fc polypeptide: G236D_S239D_H268D_loop substitution (325~331).

[0200] In some embodiments, the heterodimer Fc variant is a strategy 1 / 3 variant and includes the following amino acid mutation (referred to as core set 2B): Core Set 2B First Fc polypeptide: G236N Second Fc polypeptide: G236D_S239D_S267I / V_H268D_loop substitution (325~331).

[0201] In a particular embodiment, the heterodimer Fc variant is a strategy 1 / 3 variant comprising the amino acid mutation described in core set 2A, wherein the asymmetric mutation at position 236 is modified as shown in core sets 2C and 2D below. Core Set 2C First Fc polypeptide: G236N Second Fc polypeptide: G236D, E, K, or T +S239D_H268D_Loop substitution (325~331). Core Set 2D First Fc polypeptide: G236N, A, E, F, H, I, L, P, Q, S, T, V, W or Y, or no G236 mutation. Second Fc polypeptide: G236D_S239D_H268D_loop substitution (325~331).

[0202] In some embodiments, the heterodimer Fc variant comprises amino acid mutations described in core set 2C, wherein the second Fc polypeptide contains mutation G236D or G236K.

[0203] In a particular embodiment, the heterodimer Fc variant is a strategy 1 / 3 variant comprising the amino acid mutation described in core set 2B, wherein the asymmetric mutation at position 236 is modified as shown in core sets 2E and 2F below. Core Set 2E First Fc polypeptide: G236N Second Fc polypeptide: G236D, E, K, or T +S239D_S267I / V_H268D_Loop substitution (325~331). Core Set 2F First Fc polypeptide: G236N, A, E, F, H, I, L, P, Q, S, T, V, W or Y, or no G236 mutation. Second Fc polypeptide: G236D_S239D_S267I / V_H268D_loop substitution (325~331).

[0204] In some embodiments, the heterodimer Fc variant comprises amino acid mutations described in core set 2E, wherein the second Fc polypeptide contains mutation G236D or G236K.

[0205] Introducing an aspartic acid (D) or asparagine (N) residue at position 236 in the heterodimeric Fc variant can introduce a deamidation site to Fc, as the G236D / N mutation precedes the native glycine (G) residue at position 237. Therefore, in certain embodiments in which the heterodimeric Fc variant contains the mutant G236D and / or mutant G236N, the heterodimeric Fc variant may optionally further include an amino acid mutation at position G237.

[0206] In some embodiments, where the heterodimer Fc variant is a strategic 1 / 3 variant and one of the Fc polypeptides contains the mutation G236D, the Fc polypeptide may further contain an amino acid mutation at position G237 selected from G237F, G237I, G237K, G237L, G237Q, G237T, G237V, and G237Y. In some embodiments, where the heterodimer Fc variant is one of the Fc polypeptides containing the mutation G236D, the Fc polypeptide may further contain the amino acid mutation G237F.

[0207] In some embodiments, where the heterodimer Fc variant is a strategic 1 / 3 variant and one of the Fc polypeptides contains the mutation G236N, the Fc polypeptide may further contain amino acid mutations at position G237 selected from G237A, G237D, G237F, G237H, G237L, G237N, G237P, G237S, G237V, G237W, and G237Y. In some embodiments, where the heterodimer Fc variant is one of the Fc polypeptides containing the mutation G236N, the Fc polypeptide may further contain the amino acid mutation G237A.

[0208] In a particular embodiment, where the heterodimer Fc variant is a strategy 1 / 3 variant containing the mutation G236N in the first Fc polypeptide, the first Fc polypeptide may further contain additional CH2 mutations at one or more of positions 234, 235, 237, and 239.

[0209] In some embodiments, the heterodimer Fc variant is a strategy 1 / 3 variant comprising an amino acid mutation described in any one of core sets 2, 2A, 2B, 2C, 2D, 2E, or 2F, wherein the first Fc polypeptide may further comprise an additional CH2 mutation at one or more of positions 234, 235, 237, and 239.

[0210] In some embodiments, the first Fc polypeptide further comprises an additional CH2 mutation at one or more of positions 234, 235, 237, and 239. (i) The mutation at position 234 is selected from L234A, L234D, L234E, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235I, L235N, L235P, L235Q, L235S, L235T, L235V, L235W, and L235Y. (iii) The mutation at position 237 is selected from G237A, G237D, G237F, G237H, G237L, G237N, G237P, G237S, G237V, G237W and G237Y, (iv) The mutation at position 239 is selected from S239A, S239D, S239E, S239F, S239G, S239H, S239I, S239L, S239N, S239Q, S239R, S239T, S239V, S239W, and S239Y.

[0211] In some embodiments, the first Fc polypeptide further comprises an additional CH2 mutation at one or more of positions 234, 235, 237, and 239. (i) The mutation at position 234 is selected from L234D, L234F, L234Q, L234T, and L234W. (ii) The mutation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235R, L235W, and L235Y. (iii) The mutation at position 237 is selected from G237A, G237D, G237L, and G237N. (iv) The mutation at position 239 is selected from S239A, S239G, S239H, S239T, and S239Y.

[0212] In some embodiments, the heterodimer Fc polypeptide is a strategy 1 / 3 variant containing the mutation G236N in the first Fc polypeptide, and the first Fc polypeptide further contains the mutation L234D.

[0213] In some embodiments, the heterodimer Fc variant is a strategy 1 / 3 variant comprising an amino acid mutation described in any one of core sets 2, 2A, 2B, 2C, 2D, 2E, or 2F, wherein the first Fc polypeptide further comprises the mutation L234D.

[0214] In some embodiments, the heterodimeric Fc polypeptide is a strategy 1 / 3 variant containing the mutation G236N in the first Fc polypeptide, and the first Fc polypeptide further contains the mutation L235F.

[0215] In some embodiments, the heterodimer Fc variant is a strategy 1 / 3 variant comprising an amino acid mutation described in any one of core sets 2, 2A, 2B, 2C, 2D, 2E, or 2F, wherein the first Fc polypeptide further comprises the mutation L235F.

[0216] In a particular embodiment, the heterodimer Fc variant is a strategy 1 / 3 variant comprising the mutation G236D and a loop substitution at positions 325–331 in the second Fc polypeptide, the second Fc polypeptide may further comprise an additional CH2 mutation at one or more of positions 234, 235, 237, 240, 263, 264, 266, 269, 271, 273, 323, and 332.

[0217] In some embodiments, the heterodimer Fc variant is a strategy 1 / 3 variant comprising an amino acid mutation described in any one of core sets 2, 2A, 2B, 2C, 2D, 2E, or 2F, and the second Fc polypeptide may further comprise an additional CH2 mutation at one or more of positions 234, 235, 237, 240, 263, 264, 266, 269, 271, 273, 323, and 332.

[0218] In some embodiments, the second Fc polypeptide further comprises an additional CH2 mutation at one or more of the positions 234, 235, 237, 240, 263, 264, 266, 269, 271, 273, 323, and 332, (i) The mutation at position 234 is selected from L234A, L234E, L234F, L234G, L234H, L234I, L234K, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 235 is selected from L235A, L235D, L235F, L235G, L235N, L235S, L235W, and L235Y. (iii) The mutation at position 237 is selected from G237F, G237I, G237K, G237L, G237Q, G237T, G237V, and G237Y. (iv) The mutation at position 240 was selected from V240I and V240L, (v) The mutation at position 263 is V263T, (vi) The mutation at position 264 is V264T, (vii) The mutation at position 266 is V266I, (viii) The mutation at position 269 is E269Q, (ix) The mutation at position 271 is P271D, The mutation at position (x)273 was selected from V273A and V273I. (xi) The mutation at position 323 was selected from V323A and V323I. (xii) The mutation at position 332 is selected from I332F and I332L.

[0219] In some embodiments, the second Fc polypeptide further comprises an additional CH2 mutation at one or more of the positions 271, 323, and 332, (i) The mutation at position 271 is P271D, (ii) The mutation at position 323 is V323A, (iii) The mutation at position 332 is selected from I332F and I332L.

[0220] In certain embodiments, the heterodimer Fc variant is a strategy 1 / 3 variant and includes the amino acid mutations listed in Tables 5A, 5B, and 5C for any one of the variants listed under “Strategy 1 / 3” and “Strategy 1 / 3 + Strategy 2 combination.” In certain embodiments, the heterodimer Fc variant is a strategy 1 / 3 variant and includes the amino acid mutations listed for any one of the variants shown in Tables 6.22, 6.24, 6.25, and 6.27. In some embodiments, the heterodimer Fc variant is a strategy 1 / 3 variant and includes the amino acid mutations listed for any one of the variants shown in Tables 6.22 and 6.24.

[0221] In certain embodiments, the heterodimer Fc variant is a strategy 1 / 3 variant and includes one amino acid mutation from any of the variants shown in Tables 6.17, 6.19, and 6.20, having a "IIb selectivity ratio relative to control" value > 0.5 and a "IIb ratio relative to control" value > 0.5 ("Criterion B"). In some embodiments, the heterodimer Fc variant includes one amino acid mutation from any of the variants shown in Tables 6.17, 6.19, and 6.20, having a "IIb selectivity ratio relative to control" value > 1.0 and a "IIb ratio relative to control" value > 0.3 ("Criterion C"). In certain embodiments, the heterodimer Fc variant includes one amino acid mutation from any of the variants shown in Tables 6.17, 6.19, and 6.20, having a "IIb selectivity ratio relative to control" value > 1.0 and a "IIb ratio relative to control" value > 0.5 ("Criterion D"). In a particular embodiment, the heterodimer Fc variant includes one amino acid mutation from any of the variants shown in Tables 6.17, 6.19, and 6.20, having a "ratio of IIb selectivity to control" value > 1.5 and a "ratio of IIb to control" value > 0.3 ("reference A").

[0222] Strategy 2 Variant Further optimization of Launch Module 2 was performed to obtain additional heterodimeric Fc variants with improved selectivity for FcγRIIb. These are referred to herein as “Strategy 2 variants.” As used herein, the term “Strategy 2 variant” refers to a heterodimeric Fc variant comprising (a) an asymmetric mutation at position 236 as described above, (b) one or more binding enhancers as described above, (c) one or more IgG4-based mutations, and (d) one or more optional additional mutations in the CH2 domain. Therefore, this term is not limited to the description of heterodimeric Fc variants explicitly referred to as “Strategy 2 variants” in the examples.

[0223] In certain embodiments, the heterodimer Fc variant is a strategy 2 variant. In certain embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236 as described in any one of the embodiments above, one or more binding enhancers as described in any one of the embodiments above, and a mutation at one or more positions selected from 234, 268, 327, 330, and 331. In some embodiments, the heterodimer Fc variant comprises an asymmetric mutation at position 236 as described in any one of the embodiments above, one or more binding enhancers as described in any one of the embodiments above in one Fc polypeptide, and a mutation at one or more positions selected from 234, 268, 327, 330, and 331 in the other Fc polypeptide.

[0224] In a particular embodiment, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236 as described in any one of the embodiments above, one or more binding enhancers selected from S239D, S239E, V266L, S267A, S267I, S267Q, S267V and H268D in one Fc polypeptide, and one or more mutations at positions 234, 268, 327, 330 and 331 in the other Fc polypeptide.

[0225] In a particular embodiment, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236 selected from G236N and G236D, one or more binding enhancers selected from S239D, S239E, V266L, S267A, S267I, S267Q, S267V and H268D in one Fc polypeptide, and one or more mutations at positions 234, 268, 327, 330 and 331 in the other Fc polypeptide.

[0226] In a particular embodiment, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, G236K, or G236S, where the second Fc polypeptide further comprises one or more binding enhancers selected from S239D, S239E, V266L, S267A, S267I, S267Q, S267V, and H268D, and the first Fc polypeptide further comprises mutations at one or more positions selected from 234, 268, 327, 330, and 331.

[0227] In a particular embodiment, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, where the second Fc polypeptide further comprises one or more binding enhancers selected from S239D, S239E, V266L, S267A, S267I, S267Q, S267V and H268D, and the first Fc polypeptide further comprises mutations at one or more positions selected from 234, 268, 327, 330 and 331.

[0228] In a particular embodiment, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, where the second Fc polypeptide further comprises one or more binding enhancers selected from S239D, S239E, V266L, S267A, S267I, S267Q and S267V, and a mutation at position 268 selected from H268A, H268D, H268E, H268F, H268N, H268Q, H268S, H268V, H268W and H268Y, and the first Fc polypeptide further comprises a mutation at one or more positions selected from 234, 268, 327, 330 and 331.

[0229] In a particular embodiment, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236D, and the second Fc polypeptide comprises the mutation G236N, G236Q, G236K, G236E, or G236H, where the second Fc polypeptide further comprises one or more binding enhancers selected from S239D, S239E, V266L, S267A, S267I, S267Q, S267V, and H268D, and the first Fc polypeptide further comprises a mutation at one or more positions selected from 234, 268, 327, 330, and 331.

[0230] In some embodiments, the heterodimeric Fc variant is a strategy 2 variant, and the amino acid mutations of core set 1 described above: Core Set 1 First Fc polypeptide: G236N The second Fc polypeptide contains: G236D_S239D_H268D, Here, the first Fc polypeptide further comprises a mutation at one or more positions selected from 234, 268, 327, 330, and 331.

[0231] In some embodiments, the heterodimer Fc variant is a strategy 2 variant comprising an amino acid mutation of core set 1, where the first Fc polypeptide further comprises a mutation at one or more positions selected from 234, 268, 327, 330, and 331, and the second Fc polypeptide further comprises the amino acid mutation S267A or S267Q.

[0232] In certain embodiments, one or more binding enhancers included in the strategy 2 heterodimer Fc variant are selected from S239D, V266L, S267A, S267Q, and H268D. In some embodiments, one or more binding enhancers include mutant S239D and / or H268D. In some embodiments, one or more binding enhancers include mutant S239D and H268D. In some embodiments, one or more binding enhancers include mutant S239D, H268D, and (i) mutant V266L, or (ii) mutant S267A / Q, or (iii) mutant V266L and S267A / Q. In some embodiments, one or more binding enhancers include mutant S239D, H268D, V266L, and S267A. In some embodiments, one or more binding enhancers include mutations S239D, H268D, V266L, and S267Q.

[0233] In a particular embodiment, the mutations at one or more positions selected from 234, 268, 327, 330, and 331 in the first Fc polypeptide of the Strategy 2 variant are one or more of the following: (i) A mutation at position 234 selected from L234A, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, (ii) A variation at position 268 selected from H268A, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268N, H268P, H268Q, H268R, H268S, H268T, H268V, H268W and H268Y, (iii) Variation at position 327 selected from A327E and A327G, (iv) Variations at position 330 selected from A330K, A330H, A330Q, A330R, A330S and A330T, and (v) A mutation at position 331 selected from P331A, P331D, P331E, P331H, P331Q, and P331S.

[0234] In a particular embodiment, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236 as described in any one of the embodiments above, wherein one Fc polypeptide comprises one or more binding enhancers selected from S239D, S239E, V266L, S267A, S267I, S267Q, S267V and H268D, and the other Fc polypeptide comprises a mutation at position 234 selected from L234A, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, and an optional mutation at one or more positions 268, 327, 330 and 331. In some embodiments, one or more binding enhancers are S239D, H268D, optionally (i) V266L, or (ii) S267A / Q, or (iii) V266L and S267A / Q. In some embodiments, the mutation at position 234 is L234F.

[0235] In a particular embodiment, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236 as described in any one of the embodiments above, where one Fc polypeptide comprises one or more binding enhancers selected from S239D, S239E, V266L, S267A, S267I, S267Q, S267V and H268D, and the other Fc polypeptide The lipeptide comprises a mutation at position 268 selected from H268A, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268N, H268P, H268Q, H268R, H268S, H268T, H268V, H268W, and H268Y, and an optional mutation at one or more of positions 234, 327, 330, and 331. In some embodiments, one or more binding enhancers are S239D, H268D, optionally (i)V266L, or (ii)S267A / Q, or (iii)V266L and S267A / Q. In some embodiments, the mutation at position 268 is H268Q.

[0236] In some embodiments, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236 as described in any one of the embodiments above, where one Fc polypeptide comprises one or more binding enhancers selected from S239D, S239E, V266L, S267A, S267I, S267Q, S267V, and H268D, and the other Fc polypeptide comprises a mutation at position 327 selected from A327E and A327G, and an optional mutation at one or more positions 234, 268, 330, and 331. In some embodiments, the one or more binding enhancers are S239D, H268D, and optionally, (i) V266L, or (ii) S267A / Q, or (iii) V266L and S267A / Q. In some embodiments, the mutation at position 327 is A327G.

[0237] In some embodiments, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236 as described in any one of the embodiments above, where one Fc polypeptide comprises one or more binding enhancers selected from S239D, S239E, V266L, S267A, S267I, S267Q, S267V and H268D, and the other Fc polypeptide comprises a mutation at position 330 selected from A330K, A330H, A330Q, A330R, A330S and A330T, and an optional mutation at one or more positions 234, 268, 327 and 331. In some embodiments, one or more binding enhancers are S239D, H268D, and optionally (i) V266L, or (ii) S267A / Q, or (iii) V266L and S267A / Q. In some embodiments, the mutation at position 330 is A330K or A330T. In some embodiments, the mutation at position 330 is A330K.

[0238] In some embodiments, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236 as described in any one of the embodiments above, wherein one Fc polypeptide comprises one or more binding enhancers selected from S239D, S239E, V266L, S267A, S267I, S267Q, S267V and H268D, and the other Fc polypeptide comprises a mutation at position 331 selected from P331A, P331D, P331E, P331H, P331Q and P331S, and an optional mutation at one or more positions 234, 268, 327 and 330. In some embodiments, the one or more binding enhancers are S239D, H268D, and optionally, (i) V266L, or (ii) S267A / Q, or (iii) V266L and S267A / Q. In some embodiments, the mutation at position 331 is P331S.

[0239] In a particular embodiment, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, where the second Fc polypeptide further comprises the binding enhancers S239D, H268D, and optionally (i) V266L, or (ii) S267A / Q, or (iii) V266L and S267A / Q, and the first Fc polypeptide further comprises one or more mutations selected from the following: (i) A mutation at position 234 selected from L234A, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, (ii) A variation at position 268 selected from H268A, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268N, H268P, H268Q, H268R, H268S, H268T, H268V, H268W and H268Y, (iii) Variation at position 327 selected from A327E and A327G, (iv) Variations at position 330 selected from A330K, A330H, A330Q, A330R, A330S and A330T, and (v) A mutation at position 331 selected from P331A, P331D, P331E, P331H, P331Q, and P331S.

[0240] In a particular embodiment, the heterodimer Fc variant is a strategy 2 variant comprising an asymmetric mutation at position 236, where the first Fc polypeptide comprises the mutation G236N, and the second Fc polypeptide comprises the mutation G236D, where the second Fc polypeptide further comprises the binding enhancers S239D, H268D, and optionally (i) V266L, or (ii) S267A / Q, or (iii) V266L and S267A / Q, and the first Fc polypeptide further comprises the following mutations: (i) A mutation at position 234 selected from L234A, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, (ii) A variation at position 268 selected from H268A, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268N, H268P, H268Q, H268R, H268S, H268T, H268V, H268W and H268Y, (iii) Variation at position 327 selected from A327G and A327E, (iv) Variations at position 330 selected from A330K, A330H, A330Q, A330R, A330S and A330T, and (v) A mutation at position 331 selected from P331A, P331D, P331E, P331H, P331Q, and P331S.

[0241] In some embodiments, the mutation at position 234 is L234F. In some embodiments, the mutation at position 268 is H268Q. In some embodiments, the mutation at position 327 is A327G. In some embodiments, the mutation at position 330 is A330K or A330T. In some embodiments, the mutation at position 331 is P331S.

[0242] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at one or more of the positions 235, 237, 239, 264, 266, 267, 269, 270, 271, 272, 273, 323, 326 and / or 332.In some embodiments, the mutation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235I, L235P, L235Q, L235S, L235T, L235V, L235W, and L235Y; the mutation at position 237 is selected from G237A, G237F, G237L, G237N, G237T, G237W, and G237Y; and the mutation at position 239 is selected from S239A, S239D, S239E, S239G, S239I, S239L, S239N, S239Q, and S239R The mutations at position 264 are selected from V264A, V264F, V264I, V264L and V264T, the mutation at position 266 is V266I, the mutation at position 267 is selected from S267A, S267G, S267H, S267I, S267N, S267P, S267T and S267V, and the mutation at position 269 is E269A, E269D, E269F, E269G, E269H, E269I, E269K, E269L, E269N, E269P, E269Q, E269R, E26 The mutations at position 270 are selected from 9S, E269T, E269V, E269W and E269Y, the mutations at position 270 are selected from D270A, D270E, D270F, D270H, D270I, D270N, D270Q, D270S, D270T, D270W and D270Y, the mutations at position 271 are selected from P271D, P271E, P271G, P271H, P271I, P271K, P271L, P271N, P271Q, P271R, P271V and P271W, and the mutations at position 272 are E272A, E272D and E272 The mutations at position 273 are selected from F, E272G, E272H, E272I, E272L, E272N, E272S, E272T, E272V, E272W, and E272Y, the mutation at position 273 is V273A, the mutation at position 323 is selected from V323A, V323I, and V323L, the mutation at position 326 is selected from K326A, K326D, K326H, K326N, K326Q, K326R, K326S, and K326T, and the mutation at position 332 is selected from I332A, I332L, I332T, and I332V.

[0243] In certain embodiments, the heterodimer Fc variant is the Strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 235 selected from L235A, L235D, L235E, L235F, L235H, L235I, L235P, L235Q, L235S, L235T, L235V, L235W, and L235Y. In some embodiments, the mutation at position 235 is L235D.

[0244] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 237 selected from G237A, G237F, G237L, G237N, G237T, G237W, and G237Y.

[0245] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 239 selected from S239A, S239D, S239E, S239G, S239I, S239L, S239N, S239Q, S239R and S239V.

[0246] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 264 selected from V264A, V264F, V264I, V264L, and V264T.

[0247] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises mutation V266I.

[0248] In certain embodiments, the heterodimer Fc variant is the Strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 267 selected from S267A, S267G, S267H, S267I, S267N, S267P, S267T, and S267V. In some embodiments, the mutation at position 267 is S267A.

[0249] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 269 selected from E269A, E269D, E269F, E269G, E269H, E269I, E269K, E269L, E269N, E269P, E269Q, E269R, E269S, E269T, E269V, E269W and E269Y.

[0250] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 270 selected from D270A, D270E, D270F, D270H, D270I, D270N, D270Q, D270S, D270T, D270W, and D270Y.

[0251] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 271 selected from P271D, P271E, P271G, P271H, P271I, P271K, P271L, P271N, P271Q, P271R, P271V and P271W.

[0252] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 272 selected from E272A, E272D, E272F, E272G, E272H, E272I, E272L, E272N, E272S, E272T, E272V, E272W and E272Y.

[0253] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises mutation V273A.

[0254] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 323 selected from V323A, V323I, and V323L.

[0255] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 326 selected from K326A, K326D, K326H, K326N, K326Q, K326R, K326S and K326T.

[0256] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the first Fc polypeptide further comprises a mutation at position 332 selected from I332A, I332L, I332T, and I332V.

[0257] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the second Fc polypeptide further comprises a mutation at one or more of the positions 234, 235, 237, 240, 264, 269, 271, 272 and / or 273. In some embodiments, the mutation at position 234 is selected from L234A, L234D, L234E, L234F, L234G, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, the mutation at position 235 is selected from L235A, L235D, L235F, L235G, L235H, L235N, L235W and L235Y, and the mutation at position 237 is selected from G237A, G237D, G237E, G237F, G237H, G237I, G237K, G237L, G237N, G237Q, G237R, G237S, G237T, The mutation at position 240 is selected from G237V, G237W, and G237Y; the mutation at position 240 is selected from V240I, V240L, and V240T; the mutation at position 264 is selected from V264L and V264T; the mutation at position 269 is selected from E269D, E269T, and E269V; the mutation at position 271 is P271G; the mutation at position 272 is selected from E272A, E272D, E272I, E272K, E272L, E272P, E272Q, E272R, E272T, and E272V; and the mutation at position 273 is selected from V273A, V273I, V273L, and V273T.

[0258] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the second Fc polypeptide further comprises a mutation at position 234 selected from L234A, L234D, L234E, L234F, L234G, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y.

[0259] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the second Fc polypeptide further comprises a mutation at position 235 selected from L235A, L235D, L235F, L235G, L235H, L235N, L235W, and L235Y.

[0260] In certain embodiments, the heterodimer Fc variant is the Strategy 2 variant described in any one of the embodiments above, where the second Fc polypeptide further comprises a mutation at position 237 selected from G237A, G237D, G237E, G237F, G237H, G237I, G237K, G237L, G237N, G237Q, G237R, G237S, G237T, G237V, G237W, and G237Y. In some embodiments, the mutation at position 237 is G237D or G237L.

[0261] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the second Fc polypeptide further comprises a mutation at position 240 selected from V240I, V240L, and V240T.

[0262] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the second Fc polypeptide further comprises a mutation at position 264 selected from V264L and V264T.

[0263] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the second Fc polypeptide further comprises a mutation at position 269 selected from E269D, E269T, and E269VL.

[0264] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the second Fc polypeptide further comprises mutant P271G.

[0265] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the second Fc polypeptide further comprises a mutation at position 272 selected from E272A, E272D, E272I, E272K, E272L, E272P, E272Q, E272R, E272T and E272V.

[0266] In a particular embodiment, the heterodimer Fc variant is the strategy 2 variant described in any one of the embodiments above, where the second Fc polypeptide further comprises a mutation at position 273 selected from V273A, V273I, V273L, and V273T.

[0267] In a particular embodiment, the heterodimer Fc variant is a strategy 2 variant as described in any one of the embodiments described above, further comprising a substitution of a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above, for the native loop at positions 325-331 in the second Fc polypeptide.

[0268] In certain embodiments, the polypeptide loop contained in the second Fc polypeptide of the Strategy 2 variant includes an amino acid sequence described in any one of formulas (I), (Ia), (Ib), (II), (III), (IV), (V), or (VI), as described above as an "asymmetric loop substitution." In some embodiments, the polypeptide loop contained in the second Fc polypeptide includes an amino acid sequence described in any one of the sequences shown in Tables 3A and 3B (SEQ ID NOs. 4-172). In some embodiments, the polypeptide loop contained in the second Fc polypeptide includes an amino acid sequence described in any one of SEQ ID NOs. 4-90 (see Table 3A above). In some embodiments, the polypeptide loop contained in the second Fc polypeptide is sequence numbers 6, 8, 9, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, Contains an amino acid sequence listed in any one of the following: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 (see Table 3A above).

[0269] In certain embodiments, the heterodimer Fc variant is a strategy 2 variant and includes the amino acid mutations listed in Tables 5A, 5B, and 5C for any one of the variants listed under “Strategy 2” and “Strategy 1 / 3 + Strategy 2 combinations”. In certain embodiments, the heterodimer Fc variant is a strategy 2 variant and includes the amino acid mutations listed for any one of the variants shown in Table 6.23 or Table 6.26. In some embodiments, the heterodimer Fc variant is a strategy 2 variant and includes the amino acid mutations listed for any one of the variants shown in Table 6.23.

[0270] In certain embodiments, the heterodimer Fc variant is a strategy 2 variant and includes one amino acid mutation from any of the variants shown in Table 6.18, having a "IIb selectivity ratio relative to control" value > 0.5 and a "IIb ratio relative to control" value > 0.5 ("Criterion B"). In some embodiments, the heterodimer Fc variant includes one amino acid mutation from any of the variants shown in Table 6.18, having a "IIb selectivity ratio relative to control" value > 1.0 and a "IIb ratio relative to control" value > 0.3 ("Criterion C"). In certain embodiments, the heterodimer Fc variant includes one amino acid mutation from any of the variants shown in Table 6.18, having a "IIb selectivity ratio relative to control" value > 1.0 and a "IIb ratio relative to control" value > 0.5 ("Criterion D"). In a particular embodiment, the heterodimer Fc variant includes one amino acid mutation from any of the variants shown in Table 6.18, having a “ratio of IIb selectivity to control” value > 1.5 and a “ratio of IIb to control” value > 0.3 (“Criterion A”).

[0271] Combination variant As described in the examples provided herein, mutations contained in strategy 1 / 3 variants can be combined with mutations contained in strategy 2 variants to provide heterodimer Fc variants having increased selectivity for FcγRIIb, and optionally, increased affinity. In certain embodiments, the heterodimer Fc variant is a combination variant, in which one Fc polypeptide contains a mutation from strategy 1 / 3 variant and the other Fc polypeptide contains a mutation from strategy 2 variant.

[0272] In a particular embodiment, the heterodimer Fc variant is a combinational variant, (a) A first Fc polypeptide comprising a mutation of a strategy 2 variant, wherein the mutation comprises mutation G236N and mutations at one or more positions selected from 234, 268, 327, 330 and 331, where, (i) The mutation at position 234 is selected from L234A, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 268 is selected from H268A, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268N, H268P, H268Q, H268R, H268S, H268T, H268V, H268W and H268Y, (iii) The mutation at position 327 is selected from A327G and A327E, (iv) The mutation at position 330 was selected from A330K, A330H, A330Q, A330R, A330S, and A330T. (v) The mutation at position 331 is a first Fc polypeptide containing a strategy 2 variant mutation selected from P331A, P331D, P331E, P331H, P331Q and P331S, (b) A second Fc polypeptide comprising a mutation of the strategy 1 / 3 variant, wherein the mutation comprises mutation G236D and a substitution in a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above as an "asymmetric loop substitution" for the native loop at positions 325-331.

[0273] In some embodiments, the heterodimer Fc variant is a combination variant. (a) A first Fc polypeptide comprising a mutation of a strategy 2 variant, wherein the mutation comprises a mutation G236N and a mutation at one or more positions selected from 234, 268, 327, 330 and 331 as described above, (b) A second Fc polypeptide comprising a mutation of the strategy 1 / 3 variant, wherein the mutation comprises mutation G236D and a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above as an "asymmetric loop substitution" for the native loop at positions 325-331, and one or more binding enhancers as described above.

[0274] In some embodiments, the heterodimer Fc variant is a combination variant. (a) A first Fc polypeptide comprising a mutation of a strategy 2 variant, wherein the mutation comprises a mutation G236N and a mutation at one or more positions selected from 234, 268, 327, 330 and 331 as described above, (b) A second Fc polypeptide comprising a mutation of a strategy 1 / 3 variant, wherein the mutation comprises a mutation G236D and a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above as an "asymmetric loop substitution" for a native loop at positions 325-331, and one or more binding enhancers selected from S239D, S239E, V266I, S267I, S267Q, S267V and H268D.

[0275] In some embodiments, the heterodimer Fc variant is a combination variant. (a) A first Fc polypeptide comprising a mutation of a strategy 2 variant, wherein the mutation comprises a mutation G236N and a mutation at one or more positions selected from 234, 268, 327, 330 and 331 as described above, (b) A second Fc polypeptide comprising a mutation of a strategy 1 / 3 variant, wherein the mutation comprises mutation G236D and a substitution in a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above as an "asymmetric loop substitution" for the native loop at positions 325-331, and (i) mutation S239D or S239E, and / or (ii) mutation H268D, and / or (iii) mutation S267I or S267V, comprising a second Fc polypeptide comprising a mutation of a strategy 1 / 3 variant.

[0276] In some embodiments, the heterodimer Fc variant is a combination variant. (a) A first Fc polypeptide comprising a mutation of a strategy 2 variant, wherein the mutation comprises a mutation G236N and a mutation at one or more positions selected from 234, 268, 327, 330 and 331 as described above, (b) A second Fc polypeptide comprising a mutation of the strategy 1 / 3 variant, wherein the mutation comprises mutation G236D and a substitution in a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above as an "asymmetric loop substitution" for the native loop at positions 325-331, and mutations S239D and H268D.

[0277] In some embodiments, the heterodimer Fc variant is a combination variant. (a) A first Fc polypeptide comprising a mutation of a strategy 2 variant, wherein the mutation comprises a mutation G236N and a mutation at one or more positions selected from 234, 268, 327, 330 and 331 as described above, (b) A second Fc polypeptide comprising a mutation of the strategy 1 / 3 variant, wherein the mutation comprises mutation G236D and a substitution in a polypeptide loop of 7-15 amino acid length or 8-15 amino acid length as described in any one of the embodiments provided above as an "asymmetric loop substitution" for the native loop at positions 325-331, and mutations S239D, H268D and S267V, comprising a second Fc polypeptide comprising a mutation of the strategy 1 / 3 variant.

[0278] In certain embodiments, the mutation at position 234 in the first Fc polypeptide of the combination variant is L234F. In some embodiments, the mutation at position 268 in the first Fc polypeptide of the combination variant is H268Q. In some embodiments, the mutation at position 327 in the first Fc polypeptide of the combination variant is A327G. In some embodiments, the mutation at position 330 in the first Fc polypeptide of the combination variant is A330K or A330T. In some embodiments, the mutation at position 331 in the first Fc polypeptide of the combination variant is P331S.

[0279] In certain embodiments, the polypeptide loop contained in the second Fc polypeptide of the combination variant includes an amino acid sequence that is a variant of the sequence described in any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, where the variant includes 1, 2, 3, 4, or 5 amino acid mutations. In some embodiments, the polypeptide loop contained in the second Fc polypeptide of the combination variant includes an amino acid sequence described in any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the polypeptide loop contained in the second Fc polypeptide includes an amino acid sequence described in any one of the following formulas, as described above as an "asymmetric loop substitution": formula (I), formula (Ia), formula (Ib), formula (II), formula (III), formula (IV), formula (V), or formula (VI). In some embodiments, the polypeptide loop contained in the second Fc polypeptide includes an amino acid sequence described in any one of the sequences shown in Tables 3A and 3B (SEQ ID NOs: 4 to 172). In some embodiments, the polypeptide loop contained in the second Fc polypeptide contains the amino acid sequence described in any one of SEQ ID NOs: 4-90 (see Table 3A above). In some embodiments, the polypeptide loop contained in the second Fc polypeptide contains the amino acid sequence described in SEQ ID NOs: 6, 8, 9, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, Contains an amino acid sequence listed in any one of the following: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 (see Table 3A above).

[0280] In a particular embodiment, the heterodimer Fc variant is a combination variant containing one of the amino acid mutations listed in Table 5A or Table 5C for any one of the variants listed under "Combination of Strategy 1 / 3 + Strategy 2".

[0281] [Table 5A] TIFF2026143609000030.tif204165TIFF2026143609000031.tif188165TIFF20261436090 00032.tif188165TIFF2026143609000033.tif198165TIFF2026143609000034.tif188165 TIFF2026143609000035.tif153165TIFF2026143609000036.tif157165TIFF20261436090 00037.tif193165TIFF2026143609000038.tif192165TIFF2026143609000039.tif150165

[0282] [Table 5B] TIFF2026143609000041.tif198165TIFF2026143609000042.tif188165TIFF2026143609000043.tif162165TIFF20261436090 00044.tif174165TIFF2026143609000045.tif188165TIFF2026143609000046.tif162165TIFF2026143609000047.tif122165

[0283] [Table 5C] TIFF2026143609000049.tif215165

[0284] Stability-enhancing mutations In certain embodiments, the heterodimer Fc variant may further contain one or more mutations that enhance the thermal stability of the variant ("stability-enhancing mutations"). Including one or more stability-enhancing mutations may be particularly useful if the heterodimer Fc variant exhibits a lower CH2 domain Tm compared to the melting temperature (Tm) of the wild-type IgG1 CH2 domain, which is typically about 69°C to about 73°C as measured by differential scanning calorimetry (DSC).

[0285] As described herein, the following mutations have been shown to enhance the thermal stability of heterodimeric Fc variants while maintaining FcγRIIb selectivity: A287F, T250V, L309Q, M428F, A287F / M428F, A287F / T250V, M428F / T250V, and T250V / L309Q. Thus, in certain embodiments, the heterodimeric Fc variant may further comprise one or more stability-enhancing mutations selected from A287F, T250V, L309Q, and M428F. In some embodiments, the heterodimeric Fc variant may comprise two stability-enhancing mutations selected from A287F, T250V, L309Q, and M428F. In some embodiments, the heterodimer Fc variant includes one stability-enhancing mutation selected from A287F, T250V, L309Q, and M428F. In some embodiments, the heterodimer Fc variant includes two stability-enhancing mutations selected from A287F / M428F, A287F / T250V, M428F / T250V, and T250V / L309Q.

[0286] If the heterodimeric Fc variant contains one or more stability-enhancing mutations described above, the mutation(s) are introduced symmetrically into Fc, i.e., the mutation(s) are present in both the first and second Fc polypeptides of the heterodimeric Fc variant.

[0287] Other mutations that are known to enhance the thermal stability of Fc and may be included in heterodimer Fc variants of several embodiments include those described in U.S. Patent Application Publication No. 2015 / 0210763.

[0288] CH3 domain mutation In certain embodiments, the heterodimer Fc variant described herein comprises a modified CH3 domain containing one or more asymmetric amino acid mutations that promote the formation of heterodimer Fc rather than homodimer Fc.

[0289] Various amino acid mutations that can be made to the CH3 domain of Fc to promote the formation of heterodimer Fc are known in the art, for example, as described in International Patent Application Publication No. WO 96 / 027011 ("Knob-into-Hole"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("Electrostatic Steering"), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (Strand Exchange Operation Domain (SEED) Technology), and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab Arm Exchange). Another example is the approach of combining positive and negative design strategies to obtain a stable asymmetrically modified Fc region, as described in international patent application publication numbers WO 2012 / 058768 and WO2013 / 063702.

[0290] In certain embodiments, the heterodimer Fc variant comprises a modified CH3 domain containing mutations based on a "knob-into-hole" approach. In some embodiments, the heterodimer Fc variant comprises a modified CH3 domain, where one Fc polypeptide contains amino acid mutations Y349C, T366S, L368A, and Y407V, and the other Fc polypeptide contains amino acid mutations S354C and T366W.

[0291] In certain embodiments, the heterodimer Fc variant includes a modified CH3 domain containing mutations based on an "electrostatic steering" approach. In some embodiments, the heterodimer Fc variant includes a modified CH3 domain, where one Fc polypeptide contains amino acid mutations K392D and K409D, and the other Fc polypeptide contains amino acid mutations E356K and D399K.

[0292] In certain embodiments, the heterodimer Fc variant comprises a modified CH3 domain as described in international patent application publication numbers WO2012 / 058768 or WO2013 / 063702.

[0293] In certain embodiments, the heterodimer Fc variant comprises a modified CH3 domain, where one Fc polypeptide contains amino acid mutations at positions F405 and Y407, and the other Fc polypeptide contains amino acid mutations at positions T366 and T394. In some embodiments, the amino acid mutation at position F405 is F405A, F405S, F405T, or F405V. In some embodiments, the amino acid mutation at position Y407 is Y407I or Y407V. In some embodiments, the amino acid mutation at position T366 is T366I, T366L, or T366M. In some embodiments, the amino acid mutation at position T366 is T366I or T366L. In some embodiments, the amino acid mutation at position T394 is T394W.

[0294] In some embodiments, one Fc polypeptide comprises amino acid mutations at positions F405 and Y407 as described above, and further comprises an amino acid mutation at position L351. In some embodiments, the amino acid mutation at position L351 is L351Y.

[0295] In some embodiments, one Fc polypeptide comprises amino acid mutations at positions T366 and T394 as described above, and further comprises an amino acid mutation at position K392. In some embodiments, the amino acid mutation at position K392 is K392F, K392L, or K392M. In some embodiments, the amino acid mutation at position K392 is K392L or K392M.

[0296] In some embodiments, the heterodimer Fc variant comprises a modified CH3 domain, where, as described above, one Fc polypeptide comprises amino acid mutations at positions F405 and Y407, optionally further comprising an amino acid mutation at position L351, and the other Fc polypeptide comprises amino acid mutations at positions T366 and T394, optionally further comprising an amino acid mutation at position K392, and one or both of the Fc polypeptides further comprising the amino acid mutation T350V.

[0297] In certain embodiments, the heterodimer Fc variant comprises a modified CH3 domain, where one Fc polypeptide contains amino acid mutations F405A, F405S, F405T, or F405V together with amino acid mutations Y407I or Y407V, and optionally further comprising amino acid mutation L351Y, and the other Fc polypeptide contains amino acid mutations T366I or T366L together with amino acid mutation T394W, and optionally further comprising amino acid mutations K392L or K392M. In some embodiments, one or both Fc polypeptides further comprise amino acid mutation T350V. In some embodiments, both Fc polypeptides further comprise amino acid mutation T350V.

[0298] In a particular embodiment, the heterodimer Fc variant comprises a modified CH3 domain, where, as described above, the first Fc polypeptide comprises amino acid modifications at positions F405 and Y407, optionally further comprising an amino acid modification at position L351, the second Fc polypeptide comprises amino acid modifications at positions T366 and T394, optionally further comprising an amino acid modification at position K392, and the first Fc polypeptide comprises an amino acid modification at one or both positions S400 or Q347. The second Fc polypeptide further comprises amino acid modifications and / or further comprises amino acid modifications at one or both positions K360 or N390, where the amino acid modification at position S400 is S400E, S400D, S400R or S400K, the amino acid modification at position Q347 is Q347R, Q347E or Q347K, the amino acid modification at position K360 is K360D or K360E, and the amino acid modification at position N390 is N390R, N390K or N390D.

[0299] In a particular embodiment, the heterodimer Fc variant includes a modified CH3 domain comprising an amino acid modification described in any one of Variant 1, Variant 2, Variant 3, Variant 4, or Variant 5 in Table 6.

[0300] [Table 6]

[0301] Assay for testing activity The heterodimer Fc variants of this disclosure exhibit increased selectivity for FcγRIIb compared to the parent Fc region. “Increased selectivity for FcγRIIb” means that the heterodimer Fc variant exhibits a greater improvement in affinity for FcγRIIb than any improvement in affinity for FcγRIIaR compared to the parent Fc region. In certain embodiments, the heterodimer Fc variant exhibits greater affinity for FcγRIIb than for FcγRIIaR compared to the parent Fc region.

[0302] Candidate heterodimeric Fc variants can be tested for FcγRIIb selectivity using standard methods known in the art. For example, the binding affinity of heterodimeric Fc variants to each of the Fcγ receptors can be measured by methods based on surface plasmon resonance (SPR), SPR imaging (SPRi), biolayer interferometry (BLI), ELISA, binding equilibrium exclusion (KinExA®), or Meso Scale Discovery® (MSD®) (see, for example, Current Protocols in Immunology: Ligand-Receptor Interactions in the Immune System, Eds. J. Coligan et al., 2018 & updates, Wiley Inc., Hoboken, NJ; Yang et al., 2016, Analytical Biochem, 508:78-96) and compared to the binding affinity of the parental Fc variant to the Fcγ receptor. Typically, binding affinity is the dissociation constant (K) of the binding of heterodimeric Fc variants to the Fcγ receptor. D It is expressed as ).

[0303] Selectivity can be expressed as an increase in the fold difference of FcγRIIb selectivity relative to the parent Fc region. In this disclosure, the fold difference of FcγRIIb selectivity is calculated as follows: First, the K of the binding of the heterodimeric Fc variant and the parent Fc region to FcγRIIb is calculated. DDetermine the factor difference of the FcγRIIb affinity of the variant according to equation [4]: K D FcγRIIb(parent) / K D FcγRIIb(variant) = FcγRIIb affinity factor difference[4]

[0304] K regarding the binding of heterodimeric Fc variants and parent Fc regions to FcγRIIaR D Determine the factor difference of the FcγRIIaR affinity of the variant according to equation [5]: K D FcγRIIaR(parent) / K D FcγRIIaR(variant) = FcγRIIaR affinity factor difference[5]

[0305] Next, according to equation [6], the multiplier difference in the FcγRIIb selectivity of the heterodimer Fc variant relative to the parent Fc region can be calculated: FcγRIIb affinity ratio difference / FcγRIIaR affinity ratio difference = FcγRIIb selectivity ratio difference [6] Here, results >1 indicate increased FcγRIIb selectivity for the parent Fc region, and results <1 indicate decreased FcγRIIb selectivity for the parent Fc region.

[0306] In certain embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 1.5 times compared to the parent Fc region. In some embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 2 times compared to the parent Fc region. In some embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 3 times, for example, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times or at least 9 times compared to the parent Fc region.

[0307] In some embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 10 times compared to the parent Fc region. In some embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, or at least 50 times compared to the parent Fc region.

[0308] In certain embodiments, the heterodimeric Fc variant also exhibits increased affinity for FcγRIIb compared to the parent Fc region. “Increased affinity for FcγRIIb” means that the heterodimeric Fc variant shows increased affinity for FcγRIIb compared to the affinity of the parent Fc for FcγRIIb. Affinity is, for example, related to the dissociation constant (K). D ) can be measured by determining it using the standard techniques described above.

[0309] The increased affinity of the heterodimeric Fc variant to FcγRIIb can be expressed as an increased ratio relative to the affinity of the parent Fc region. In this disclosure, the increased ratio can be calculated as outlined above. Specifically, the K ratios relating to the binding of the heterodimeric Fc variant and the parent Fc region to FcγRIIb are calculated. D Determine the factor difference of FcγRIIb affinity for the variant according to equation [4]: K D FcγRIIb(parent) / K D FcγRIIb(variant) = FcγRIIb affinity factor difference[4] Here, results >1 indicate an increase in FcγRIIb affinity to the parent Fc region, and results <1 indicate a decrease in FcγRIIb affinity to the parent Fc region.

[0310] In certain embodiments, the heterodimer Fc variant has an affinity for FcγRIIb that is at least 5 times greater than that of the parent Fc region. In some embodiments, the heterodimer Fc variant has an affinity for FcγRIIb that is at least 10 times greater than that of the parent Fc region, for example, at least 15 times, at least 20 times, or at least 25 times greater than that of the parent Fc region. In some embodiments, the heterodimer Fc variant has an affinity for FcγRIIb that is at least 30 times greater than that of the parent Fc region, at least 40 times greater than that of the parent Fc region, or at least 50 times greater than that of the parent Fc region. In some embodiments, the heterodimer Fc variant has an affinity for FcγRIIb that is at least 100 times greater than that of the parent Fc region.

[0311] In certain embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 5 times compared to the parent Fc region and affinity for FcγRIIb increased by at least 5 times compared to the parent Fc region. In some embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 5 times compared to the parent Fc region and affinity for FcγRIIb increased by at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, or at least 50 times compared to the parent Fc region.

[0312] In certain embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased at least 10-fold compared to the parent Fc region and affinity for FcγRIIb increased at least 5-fold compared to the parent Fc region. In some embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased at least 10-fold compared to the parent Fc region and affinity for FcγRIIb increased at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, or at least 50-fold compared to the parent Fc region.

[0313] In certain embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 20 times compared to the parent Fc region and affinity for FcγRIIb increased by at least 5 times compared to the parent Fc region. In some embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 20 times compared to the parent Fc region and affinity for FcγRIIb increased by at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, or at least 50 times compared to the parent Fc region.

[0314] In certain embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 30 times compared to the parent Fc region and affinity for FcγRIIb increased by at least 5 times compared to the parent Fc region. In some embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 30 times compared to the parent Fc region and affinity for FcγRIIb increased by at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, or at least 50 times compared to the parent Fc region.

[0315] In certain embodiments, the heterodimeric Fc variant has selectivity for FcγRIIb increased by at least 40 times compared to the parent Fc region and affinity for FcγRIIb increased by at least 5 times compared to the parent Fc region. In some embodiments, the heterodimeric Fc variant has selectivity for FcγRIIb increased by at least 40 times compared to the parent Fc region and affinity for FcγRIIb increased by at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, or at least 50 times compared to the parent Fc region.

[0316] In certain embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 50 times compared to the parent Fc region and affinity for FcγRIIb increased by at least 5 times compared to the parent Fc region. In some embodiments, the heterodimer Fc variant has selectivity for FcγRIIb increased by at least 50 times compared to the parent Fc region and affinity for FcγRIIb increased by at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, or at least 50 times compared to the parent Fc region.

[0317] In a particular embodiment, K is used to determine the FcγRIIb affinity and selectivity of a heterodimeric Fc variant. D The value is determined by SPR. Various formats can be employed in SPR assays to evaluate antibody Fc-FcγR binding. For example, the assay may utilize immobilizing the receptor on a biosensor chip and flowing the antibody in solution onto the chip; the assay may utilize immobilizing the antibody on a biosensor chip and flowing the receptor in solution onto the chip; or the assay may utilize immobilizing the target antigen on a biosensor chip and first flowing the antibody in solution onto the chip, followed by flowing the receptor in solution. In a particular embodiment, K is used to determine the FcγRIIb affinity and selectivity of a heterodimeric Fc variant. D The value is determined by SPR, which uses a format in which the target antigen is immobilized on a biosensor chip, and the antibody in solution is first flowed onto the chip, followed by the receptor in solution.

[0318] To further characterize heterodimeric Fc variants, other assays using standard techniques may be performed at the discretion of the user. For example, heterodimeric Fc variants may be evaluated for purity, FcRn binding, aggregation, thermal stability, and / or C1q binding. Purity and aggregation may be evaluated, for example, by liquid chromatography-mass spectrometry (LC-MS) or size exclusion chromatography (SEC). FcRn binding may be evaluated using standard techniques, such as those outlined above for FcγR binding. Thermal stability may be evaluated, for example, by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorescence (DSF). C1q binding may be evaluated, for example, by ELISA or surface plasmon resonance (SPR). Exemplary methods for evaluating various properties of heterodimeric Fc variants are described in the examples provided herein.

[0319] polypeptide Certain embodiments of this disclosure relate to polypeptides comprising heterodimeric Fc variants described herein. Typically, a polypeptide comprises one or more additional protein moieties fused to or covalently bound to the heterodimeric Fc variant, for example, via a linker. For example, a polypeptide may be an Fc fusion protein or an antibody or antibody fragment. Examples of protein moieties that can be fused to or bound to a heterodimeric Fc variant include, but are not limited to, antigen-binding domains, ligands, receptors, receptor fragments, cytokines, and antigens.

[0320] If a polypeptide contains more than one additional protein moiety, these moieties may be the same or different. One or more additional protein moieties may be fused or covalently attached to one or both of the N-terminuses, C-terminuses, or both the N-terminuses and C-terminuses of the Fc polypeptide. In some embodiments, the polypeptide contains one or more additional protein moieties fused or covalently attached to one or both of the N-terminuses of the Fc polypeptide. In some embodiments, the polypeptide contains one additional protein moiety fused or covalently attached to one of the N-terminuses of the Fc polypeptide. In some embodiments, the polypeptide contains two additional protein moieties, one of which is fused or covalently attached to the N-terminus of a first Fc polypeptide and the other which is fused or covalently attached to the N-terminus of a second Fc polypeptide. In some embodiments, the two additional protein moieties contained in the polypeptide may be linked in tandem.

[0321] In some embodiments, the polypeptide comprises a heterodimeric Fc variant fused or covalently bound to one or more protein portions that are antigen-binding domains. In some embodiments, the polypeptide comprises a heterodimeric Fc variant and one or more antigen-binding domains. In some embodiments, the polypeptide comprises a heterodimeric Fc variant and two or more antigen-binding domains, for example, 2, 3, 4, 5, 6, 7, or 8 antigen-binding domains. When the polypeptide comprises a heterodimeric Fc variant and two or more antigen-binding domains, the antigen-binding domains may bind to the same antigen or to different antigens.

[0322] In some embodiments, the polypeptide comprises one or more protein moieties that are antigen-binding domains and one or more heterodimeric Fc variants fused or covalently bound to one or more other protein moieties. Examples of other protein moieties in this context include, but are not limited to, receptors, receptor fragments (such as extracellular components), ligands, and cytokines.

[0323] In some embodiments, the polypeptide may be an antibody or antibody fragment, where at least one of one or more protein moieties is an antigen-binding domain. For example, the antigen-binding domain may be a Fab fragment, an Fv fragment, a single-chain Fv fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the polypeptide may be a monospecific antibody. In some embodiments, the polypeptide may be a monospecific antibody containing one antigen-binding domain. In some embodiments, the polypeptide may be a monospecific antibody containing two antigen-binding domains. In some embodiments, the polypeptide may be a monospecific antibody containing two or more antigen-binding domains. In some embodiments, the polypeptide may be a bispecific or multispecific antibody containing a heterodimeric Fc variant and two or more antigen-binding domains, where the two or more antigen-binding domains bind to different antigens.

[0324] In some embodiments, polypeptides can be agonist antibodies. The agonist activity of antibodies against TNF receptor family members (such as CD40, DR4, DR5, CD30, and CD137) has been reported to require interaction with FcγRIIb (see, e.g., White, et al., 2011, J Immunol., 187:1754-1763). Therefore, in some embodiments, heterodimeric Fc variants can be used as the Fc region of an agonist antibody against a TNF receptor family member to enhance the agonist activity of the antibody. Certain embodiments of this disclosure relate to an agonist antibody comprising a heterodimeric Fc variant described herein, wherein the agonist antibody comprises one or more antigen-binding domains that bind to a TNF receptor family member.

[0325] In some embodiments, the polypeptide comprises a heterodimeric Fc variant and one or more antigen-binding domains, where at least one of the antigen-binding domains binds to a tumor-associated antigen or a tumor-specific antigen.

[0326] In some embodiments, the polypeptide may be an Fc fusion protein in which one or more protein moieties may be, for example, a ligand for a cell surface receptor, a soluble fragment of a cell surface receptor, a biologically active peptide, a cytokine, a growth factor, a hormone, or an enzyme. Examples of protein moieties that may be included in the Fc fusion proteins described herein include, but are not limited to, ligands such as tumor necrosis factor (TNF), PD-L1, ICOS-L, VEGF, and LFA-3; extracellular ligand-binding moieties for cell surface receptors such as TNFR, PD-1, CTLA-4, ICOS, VEGFR, and IL-1R; biologically active peptides such as thrombopoietin-binding peptides; hormones such as erythropoietin (Epo); cytokines such as interferon-α or interferon-β; or enzymes such as factor IX.

[0327] Preparation of heterodimer Fc variants The heterodimeric Fc variants and polypeptides comprising the heterodimeric Fc variants described herein can be prepared using standard recombinant methods. Recombinant production of heterodimeric Fc variants and polypeptides generally involves synthesizing one or more polynucleotides encoding the heterodimeric Fc variant or polypeptide, cloning one or more polynucleotides into one or more suitable vectors, and introducing the vector(s) into host cells suitable for the expression of the heterodimeric Fc variant or polypeptide. Recombinant protein production is known in the art and can be achieved using standard techniques described, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, (1987 & updates), John Wiley & Sons, New York, NY; and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990).

[0328] Accordingly, certain embodiments of this disclosure relate to isolated polynucleotides or a series of polynucleotides encoding a heterodimer Fc variant described herein or a polypeptide comprising a heterodimer Fc variant described herein. In this context, a polynucleotide may encode all or part of a heterodimer Fc variant or polypeptide.

[0329] The terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used herein without distinction and refer to macromolecular forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-exclusive examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, isolated DNA, isolated RNA, nucleic acid probes, and primers.

[0330] A given polynucleotide that "encodes" a polypeptide is a polynucleotide that, under the control of appropriate regulatory sequences, is transcribed in vivo (in the case of DNA) and translated into a polypeptide (in the case of mRNA). The boundaries of the coding sequence are determined by the 5' (amino) terminal start codon and the 3' (carboxy) terminal translation termination codon. The transcription termination sequence may be located 3' to the coding sequence.

[0331] One or more polynucleotides encoding a heterodimeric Fc variant or polypeptide can be inserted into a suitable expression vector either directly or after one or more subcloning steps using standard ligation techniques. Examples of suitable vectors include, but are not limited to, plasmids, phagemids, cosmids, bacteriophages, baculoviruses, retroviruses, or DNA viruses. The vector is typically selected to function in the specific host cell in which it is employed; that is, the vector is compatible with the host cell's mechanisms and capable of amplifying and / or expressing the polynucleotide(s). In this regard, the selection of a suitable vector-host cell combination is within the ordinary art of those skilled in the art.

[0332] Accordingly, certain embodiments of this disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding a heterodimeric Fc variant or a polypeptide comprising a heterodimeric Fc variant. The polynucleotide(s) may be contained in a single vector or in multiple vectors. In some embodiments, the polynucleotide(s) are contained in a multicistronic vector.

[0333] Typically, an expression vector contains one or more regulatory elements for plasmid maintenance and the cloning and expression of an exogenous polynucleotide sequence. Examples of such regulatory elements include promoters, enhancer sequences, origins of replication, termination sequences, donor and acceptor splice sites, leader sequences for polypeptide secretion, ribosome binding sites, polyadenylation sequences, polylinker regions for inserting polynucleotides encoding the polypeptide to be expressed, and selection markers.

[0334] Regulatory elements can be homogeneous (i.e., from the same species and / or lineage as the host cell), heterogeneous (i.e., from a species other than the host cell's species or lineage), hybrid (i.e., a combination of sequences from multiple sources), or synthetic. Thus, the source of the regulatory element may be any prokaryote or eukaryote, as long as its sequence functions within and can be activated by the host cell mechanism in which it is employed.

[0335] Optionally, the vector may contain a “tag” coding sequence, i.e., a nucleic acid sequence located at the 5' or 3' end of the coding sequence that codes for a heterologous peptide sequence such as polyHis (e.g., 6xHis), FLAG®, HA (hemagglutinin influenza virus), myc, metalloaffinity, avidin / streptavidin, glutathione-S-transferase (GST), or biotin tag. This tag typically remains fused to the expressed protein and can function as a means of protein affinity purification or detection. Optionally, this tag can then be removed from the purified protein by various means, such as using a specific cleavage peptidase.

[0336] Various expression vectors are readily available from private suppliers. Alternatively, if a commercially available vector containing all the desired regulatory elements is not available, an expression vector may be constructed using a commercially available vector as a starting vector. If one or more of the desired regulatory elements are not already present in the vector, they can be obtained individually and ligated into the vector. Various methods for obtaining regulatory elements are well known to those skilled in the art.

[0337] Once an expression vector containing a polynucleotide (or more) encoding a heterodimeric Fc variant or polypeptide is constructed, the vector can be inserted into a host cell suitable for amplification and / or protein expression. Transformation of selected host cells with the expression vector can be achieved by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran transfection, and other known techniques. The chosen method is determined in part by the type of host cell used. These methods and other suitable methods are well known to those skilled in the art (see, for example, Sambrook, et al., ibid.).

[0338] When host cells are cultured under appropriate conditions, they express the protein encoded by the vector, and the protein can then be recovered from the culture medium (if the host cell secretes the protein) or directly from the host cell producing the protein (if the protein is not secreted). The host cell can be a prokaryote (e.g., a bacterial cell) or a eukaryote (e.g., a yeast, fungus, plant, or mammalian cell). The selection of an appropriate host cell can be easily carried out by those skilled in the art, taking into account various factors such as the desired expression level, polypeptide modifications desirable or required for activity (e.g., glycosylation or phosphorylation), and ease of folding into a bioactive molecule.

[0339] Accordingly, certain embodiments of this disclosure relate to a host cell or one or more vectors comprising polynucleotides(or polynucleotides). In certain embodiments, the host cell is a eukaryotic cell.

[0340] For example, eukaryotic microorganisms such as filamentous fungi or yeasts may be used as host cells, including fungal and yeast strains in which the glycosylation pathway has been "humanized" (see, e.g., Gerngross, (2004), Nat. Biotech., 22:1409-1414, and Li et al., (2006), Nat. Biotech., 24:210-215). Plant cells can also be used as host cells (see, e.g., U.S. Patents No. 5,959,177; No. 6,040,498; No. 6,420,548; No. 7,125,978, and No. 6,417,429 (described in relation to PLANTIBODIES® technology)).

[0341] In some embodiments, the host cell is a mammalian cell. Various mammalian cell lines can also be used as host cells. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney cell line CV1 (COS-7), human fetal kidney cell line 293 (e.g., HEK293 cells described in Graham, et al., (1977), J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, (1980), Biol. Reprod., 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT 060562), and TRI cells (e.g., Mather, et al., 1982, Annals). (as described in NYAcad.Sci., 383:44-68), MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (DHFR as described in Urlaub, et al., 1980, Proc. Natl. Acad.Sci. USA, 77:4216) - Examples include, but are not limited to, CHO cells and myeloma cells (Y0, NS0, and Sp2 / 0, etc.). See also Yazaki and Wu, 2003, Methods in Molecular Biology, Vol.248, pp.255-268 (BKCLo, ed., Humana Press, Totowa, NJ).

[0342] Certain embodiments of this disclosure relate to a method for preparing a heterodimeric Fc variant described herein or a polypeptide comprising a heterodimeric Fc variant described herein, comprising transfecting a host cell with one or more polynucleotides encoding the heterodimeric Fc variant or polypeptide, for example, as one or more vectors comprising polynucleotides; and culturing the host cell under conditions suitable for the expression of the encoded heterodimeric Fc variant or polypeptide.

[0343] Typically, heterodimeric Fc variants or polypeptides can be isolated from host cells after expression and optionally purified. Methods for isolating and purifying expressed proteins are known in the art. Standard purification methods include, for example, chromatographic techniques such as ion exchange, hydrophobic interaction, affinity, size, gel filtration, or reversed phase, which can be performed at atmospheric pressure or medium-high pressure using systems such as FPLC, MPLC, and HPLC. Other purification methods include electrophoresis, immunological methods, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration may also be useful.

[0344] Various native proteins that bind to the Fc region or other regions of antibodies are known in the art, and therefore these proteins can be used for the purification of Fc-containing proteins. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification can often be made possible by specific fusion partners or affinity tags as described above. For example, antibodies can bind to glutathione resin when GST fusion is used, or to Ni when His tags are used. +2Purification can be performed using affinity chromatography, or, if FLAG tags are used, immobilized anti-flag antibodies. Examples of useful purification techniques are found in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), and Protein Purification: Principles and Practice, 3. rd It is described in Ed., Scopes, Springer-Verlag, NY (1994).

[0345] How to use Certain embodiments of this disclosure relate to the therapeutic use of heterodimeric Fc variants and polypeptides comprising heterodimeric Fc variants as described herein.

[0346] For example, the heterodimeric Fc variants and polypeptides described herein that selectively activate FcγRIIb in some embodiments may be used to suppress the activation of B cells, mast cells, dendritic cells, and / or basophils. B cell activation includes proliferation, IgE production, IgM production, and IgA production. Certain embodiments of this disclosure relate to a polypeptide comprising a heterodimeric Fc variant and one or more antigen-binding domains that bind to molecules such as CD19 or CD79b expressed on the surface of B cells. Such polypeptides may be particularly useful for suppressing B cell activation by crosslinking FcγRIIb with B cells.

[0347] Certain embodiments relate to the use of the heterodimeric Fc variants and polypeptides described herein in the treatment of inflammatory diseases and disorders. In some embodiments, the heterodimeric Fc variants and polypeptides described herein may be used in the treatment of autoimmune diseases or disorders. Those skilled in the art will understand that some diseases and disorders may be characterized as both inflammatory and autoimmune, and therefore these two categories are not mutually exclusive. Examples of diseases and disorders that may be characterized as inflammatory and / or autoimmune include, but are not limited to, Addison's disease, ankylosing spondylitis, autoimmune vasculitis, celiac disease, type 1 diabetes mellitus, type 2 diabetes mellitus, gout, gouty arthritis, Graves' disease, Hashimoto's thyroiditis, inflammatory bowel disease (IBD), multiple sclerosis, myasthenia gravis, myositis, pernicious anemia, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, and systemic lupus erythematosus (SLE).

[0348] Certain embodiments relate to the use of heterodimeric Fc variants and polypeptides disclosed herein in the treatment of cancer. In this context, treatment with heterodimeric Fc variants or polypeptides may result in one or more of the following: reduction of tumor size, slowing or preventing tumor size growth, increased disease-free survival from tumor disappearance or removal to recurrence, prevention of subsequent tumor development (e.g., metastasis), increased time to progression, reduction of one or more tumor-related adverse symptoms, or increased overall survival in subjects with cancer.

[0349] Examples of cancers that can be treated or stabilized according to certain embodiments include hematological malignancies (including leukemia, myeloma, and lymphoma), carcinomas (including adenocarcinoma and squamous cell carcinoma), melanoma, and sarcoma. Carcinomas and sarcomas are also often referred to as “solid tumors.” Common examples of solid tumors include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, and uterine cancer, non-small cell lung cancer, and colorectal cancer. Various forms of lymphoma can also lead to the formation of solid tumors, and therefore are often considered solid tumors as well.

[0350] As described above, it is known that increased FcγRIIb binding of an agonist antibody enhances the antibody's agonist activity and thus its antitumor effect. Accordingly, some embodiments of this disclosure relate to methods for treating cancer using polypeptides, which are agonist antibodies against receptors of the TNF receptor family and include heterodimeric Fc variants described herein.

[0351] Pharmaceutical composition For therapeutic use, heterodimeric Fc variants and polypeptides may be provided in the form of compositions comprising a heterodimeric Fc variant or polypeptide and a pharmaceutically acceptable carrier or diluent. Compositions may be prepared by known procedures using well-known and readily available components and may be formulated for administration to a subject by, for example, oral (including, e.g., buccal or sublingual), topical, parenteral, rectal or vaginal routes, or by inhalation or spray. As used herein, the term “parenteral” includes injection or infusion by subcutaneous, intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal or intrathecal routes.

[0352] The composition is typically formulated in a form suitable for administration to a target via a selected route, such as a syrup, elixir, tablet, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injection, or liquid. The composition may also be provided as a unit dosage form.

[0353] Pharmacokinetically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used. Examples of such carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkylparabens (such as methyl or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than approximately 10 residues) polypeptides; and serum albumin. Examples of such substances include, but are not limited to, proteins such as gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and nonionic surfactants such as polyethylene glycol (PEG).

[0354] In certain embodiments, the composition may be in the form of an aqueous or oily solution or suspension for sterile injection. Such suspensions may be formulated using suitable dispersants or wetting agents and / or suspending agents known in the art. The solution or suspension for sterile injection may contain a heterodimeric Fc variant or polypeptide in an orally acceptable, non-toxic diluent or solvent. Acceptable diluents and solvents that may be employed include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile non-volatile oils may be employed as solvents or suspension media. For this purpose, various brands of non-volatile oils, including synthetic monoglycerides or diglycerides, may be employed. In addition, fatty acids such as oleic acid are also used in the preparation of injections. Auxiliaries such as local anesthetics, preservatives, and / or buffers known in the art may also be included in the solution or suspension for injection.

[0355] Other pharmaceutical compositions and methods for preparing them are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0356] Embodiment Exemplary, non-limiting embodiments of this disclosure include:

[0357] 1. A heterodimeric Fc variant comprising a first Fc polypeptide and a second Fc polypeptide, wherein the selectivity for binding to FcγRIIb is increased compared to the parent Fc region, One of the Fc polypeptides includes a substitution of all or part of the native loop in the CH2 domain of the Fc polypeptide with an alternative amino acid sequence such that the length of the native loop is extended, and at least one of the amino acid residues of the alternative amino acid sequence is within a heavy atom distance of 3 Å from the target amino acid residue in FcγRIIb when the heterodimeric Fc variant is bound to FcγRIIb. Heterodimer Fc variants are variants of immunoglobulin G (IgG) Fc. Heterodimer Fc variant.

[0358] 2. The heterodimer Fc polypeptide according to Embodiment 1, wherein the natural loop comprises amino acids 325-331 of the Fc polypeptide, and the amino acid numbering follows the EU index.

[0359] 3. The heterodimer Fc variant according to Embodiment 2, wherein the alternative amino acid sequence is a polypeptide with a length of 7 to 15 amino acids.

[0360] 4. The heterodimer Fc variant according to Embodiment 2, wherein the alternative amino acid sequence is a polypeptide with a length of 8 to 15 amino acids.

[0361] 5. A heterodimeric Fc variant according to any one of Embodiments 1 to 4, wherein the target amino acid residue in FcγRIIb is Ser135.

[0362] 6. A heterodimer Fc variant comprising a first Fc polypeptide and a second Fc polypeptide, One of the Fc polypeptides contains the substitution of amino acids 325-331 by a polypeptide with a length of 8-15 amino acids. The heterodimeric Fc variant exhibits increased selectivity for binding to FcγRIIb compared to the parent Fc region. Heterodimer Fc variants are variants of immunoglobulin G (IgG)Fc, The amino acid numbering follows the EU index. Heterodimer Fc variant.

[0363] 7. The heterodimer Fc variant according to Embodiment 6, wherein the polypeptide is derived from the sequence of the loop-forming segment of the second protein.

[0364] 8. The heterodimer Fc variant according to Embodiment 7, wherein the loop-forming segment is immobilized on the second protein by a beta-strand.

[0365] 9. In the native conformation within the second protein, the loop-forming segment has the following properties, as described in Embodiment 7 or 8, for the heterodimer Fc variant: i) The loop-forming segment contains one or more beta-strand amino acids at both its N-terminus and C-terminus; ii) One or more beta-strand amino acids at the C-terminus of the loop-forming segment do not form hydrogen bonds with any amino acids in the parent protein, except for the beta-strand amino acid at the N-terminus of the loop-forming segment; iii) The mean squared deviation (RMSD) of the skeletal heavy atoms of one or more beta-strand amino acids at the N-terminus of the loop-forming segment for one or more amino acids ending at position 324 is <0:85 Å; and iv) For one or more amino acids starting at position 332, the RMSD of the backbone heavy atoms of one or more beta-strand amino acids at the C-terminus of the loop-forming segment is <0:85 Å.

[0366] 10. The heterodimer Fc variant according to Embodiment 9, wherein the loop-forming segment further comprises the following properties: The loop-forming segment contains at least one hydrogen bond between the beta-strand amino acids at both ends of the loop-forming segment.

[0367] 11. Loop-forming segments are (a) The amino acid sequence described in any one of sequence numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or (b) an amino acid sequence which is a variant of the sequence described in any one of sequence numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, wherein the variant contains 1, 2, 3, 4, or 5 amino acid mutations. A heterodimer Fc variant according to any one of embodiments 7 to 10, including the above.

[0368] 12. Heterodimer Fc variant according to Embodiment 6, wherein the polypeptide comprises the amino acid sequence of formula (I), formula (Ia), formula (Ib), formula (II), formula (III), formula (IV), formula (V), or formula (VI): Equation (I): X 1 X 2 WX 3 X 4 X 5 GX 6 X 7 T(I) (In the formula, X 1 is A, D, N, or S, X 2 is A, D, E, F, H, I, L, N, Q, S, T, V, W, or Y, X 3 is A, D, E, F, H, I, N, Q, S, T, V, W, or Y, X 4 is D, E, G, I, L, P, or Q, X 5 is A, D, E, G, H, K, N, R, S, T, or Y, X 6 is A, D, E, F, H, P, W, or Y, X 7 (The letters are A, D, E, F, G, H, K, L, N, Q, or R.) Equation (Ia): X 1 X 2 WX 3 X 4 X 5 GYX 6 T(Ia) (In the formula, X 1is A, D, N, or S, X 2 is A, D, E, F, H, I, L, N, Q, S, T, V, W, or Y, X 3 is A, D, E, F, H, I, N, Q, S, T, V, W, or Y, X 4 is D, E, G, I, L, P, or Q, X 5 is A, D, E, G, H, K, N, R, S, T, or Y, X 6 (The letters are A, D, E, F, G, H, K, L, N, Q, or R.) Formula (Ib): X 1 X 2 WX 3 X 4 GGYX 5 T(Ib) (In the formula, X 1 is A or S, X 2 is A, D, E, F, H, I, L, N, Q, T, V, or W. X 3 is D, E, F, H, N, Q, S, T, or Y, X 4 is D, G, I, or L, X 5 (This is A, F, H, K, L, or N) Formula (II): X 1 LDX 2 X 3 GKGX 4 V(II) (In the formula, X 1 is F or G, X 2 is E, H, Q, or T, X 3 is E, N, R, S, or T, X 4 (is A, Y, or V) Formula (III): X 1 TDEX2 GKGX 3 T(III) (In the formula, X 1 is F or G, X 2 is either E or N, X 3 (is A or V) Formula (IV): X 1 FX 2 X 3 X 4 X 5 GEVV(IV) (In the formula, X 1 is either A or D, X 2 is either D or N, X 3 is D, E, H, N, P, Q, S, or T, X 4 is D, E, N, S, or T, X 5 (is D or Q) Formula (V): X 1 TDX 2 X 3 X 4 GEVT(V) (In the formula, X 1 is either A or D, X 2 is D, P, or Q, X 3 is D, E, or N, X 4 (is D or Q) Equation (VI): LTDX 1 X 2 GX 3 PX 4 R(VI) (In the formula, X 1 is E or H, X 2 is D, E, or N, X 3 is R or S, X 4 (is I, Q, or Y).

[0369] 13. The heterodimer Fc variant according to Embodiment 12, wherein the polypeptide comprises the amino acid sequence of formula (I).

[0370] 14.X 1 A heterodimer Fc variant according to Embodiment 13, wherein is A or S.

[0371] 15.X 2 but, (i) A, D, E, F, H, I, L, N, Q, T, V or W, or (ii) H or T, The heterodimer Fc variant according to Embodiment 13 or 14.

[0372] 16.X 3 but, (i) A, F, H, I, S, T, V, W or Y, or (ii) D, E, F, H, N, Q, S, T or Y, (iii) F, H, S, T or Y, (iv) E, F, H, Q, S or T, (v) F, H, S or T, (vi) E, F or S, (vii) It is either F or S. A heterodimer Fc variant according to any one of embodiments 13 to 15.

[0373] 17.X 4 but, (i) D, G, I or L, or (ii) D or G, A heterodimer Fc variant according to any one of embodiments 13 to 16.

[0374] 18.X 5 but, (i) A, D, E, G, H, K or R, (ii) G is A heterodimer Fc variant according to any one of embodiments 13 to 17.

[0375] 19.X 6 but, (i) F, W or Y, (ii) Y is A heterodimer Fc variant according to any one of embodiments 13 to 18.

[0376] 20.X 7 but, (i) A, D, E, G, H, K, L, N, Q or R, (ii) A, F, H, K, L or N, (iii) A, H, K, L or N, (iv) A or N, A heterodimer Fc variant according to any one of embodiments 13 to 19.

[0377] 21. The heterodimer Fc variant according to Embodiment 12, wherein the polypeptide comprises the amino acid sequence of formula (Ia).

[0378] 22.X 1 A heterodimer Fc variant according to Embodiment 21, wherein is A or S.

[0379] 23.X 2 but, (i) A, D, E, F, H, I, L, N, Q, T, V or W, or (ii) H or T, The heterodimer Fc variant according to Embodiment 21 or 22.

[0380] 24.X 3 but, (i) A, F, H, I, S, T, V, W or Y, or (ii) D, E, F, H, N, Q, S, T or Y, (iii) F, H, S, T or Y, (iv) E, F, H, Q, S or T, (v) F, H, S or T, (vi) E, F or S, (vii) It is either F or S. A heterodimer Fc variant according to any one of embodiments 21 to 23.

[0381] 25.X 4 but, (i) D, G, I or L, or (ii) D or G, A heterodimer Fc variant according to any one of embodiments 21 to 24.

[0382] 26.X 5 but, (i) A, D, E, G, H, K or R, (ii) G is A heterodimer Fc variant according to any one of embodiments 21 to 25.

[0383] 27.X 6 but, (i) A, D, E, G, H, K, L, N, Q or R, (ii) A, F, H, K, L or N, (iii) A, H, K, L or N, (iv) A or N, A heterodimer Fc variant according to any one of embodiments 21 to 26.

[0384] 28. The heterodimer Fc variant according to Embodiment 12, wherein the polypeptide comprises the amino acid sequence of formula (Ib).

[0385] 29.X 2 A heterodimer Fc variant according to Embodiment 28, wherein is H or T.

[0386] 30.X 3 but, (i) F, H, S or Y, or (ii) E, F, H, Q, S or T, (iii) F, H or S, (iv) E, F or S, (v) F or S, The heterodimer Fc variant according to Embodiment 28 or 29.

[0387] 31.X 4 A heterodimer Fc variant according to any one of embodiments 28 to 30, wherein is D or G.

[0388] 32.X 5 but, (i) A, F, H, K or L, (ii) A or N, or (iii) A is, A heterodimer Fc variant as described in any one of embodiments 28 to 31.

[0389] 33. The heterodimer Fc variant according to Embodiment 12, wherein the polypeptide comprises the amino acid sequence of formula (II).

[0390] 34.X 2 The heterodimer Fc variant according to Embodiment 33, wherein E is present.

[0391] 35.X 3 A heterodimer Fc variant according to embodiment 33 or 34, wherein is E, N, R, or S.

[0392] 36.X 3 A heterodimer Fc variant according to embodiment 33 or 34, wherein is E or N.

[0393] 37. The heterodimer Fc variant according to Embodiment 12, wherein the polypeptide comprises the amino acid sequence of formula (III).

[0394] 38. The heterodimer Fc variant according to Embodiment 12, wherein the polypeptide comprises the amino acid sequence of formula (IV).

[0395] 39.X 1 The heterodimer Fc variant according to Embodiment 38, wherein D is present.

[0396] 40.X 2 A heterodimer Fc variant according to embodiment 38 or 39, wherein is D.

[0397] 41.X 3 A heterodimer Fc variant according to any one of embodiments 38 to 40, wherein is E, H, N, S, or T.

[0398] 42.X 4 A heterodimer Fc variant according to any one of embodiments 38 to 41, wherein is D, N, S, or T.

[0399] 43. The heterodimer Fc variant according to Embodiment 12, wherein the polypeptide comprises the amino acid sequence of formula (V).

[0400] 44. The heterodimer Fc variant according to Embodiment 12, wherein the polypeptide comprises the amino acid sequence of formula (VI).

[0401] 45.X 1 The heterodimer Fc variant according to Embodiment 44, wherein E is present.

[0402] 46.X 4 A heterodimer Fc variant according to embodiment 44 or 45, wherein is I or Y.

[0403] 47. The heterodimer Fc variant according to Embodiment 12, wherein the polypeptide comprises the amino acid sequence described in any one of SEQ ID NOs: 4 to 172.

[0404] 48. The heterodimer Fc variant according to Embodiment 12, wherein the polypeptide comprises an amino acid sequence described in any one of SEQ ID NOs: 4 to 90.

[0405] 49. Polypeptides (a) Sequence IDs 6, 8, 9, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, The amino acid sequence described in any one of the following: 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90, or (b) The amino acid sequence described in one of the following sequence numbers: 6, 8, 47, 68, or 73 A heterodimer Fc variant according to Embodiment 12, including the above.

[0406] 50. A heterodimer Fc variant according to any one of embodiments 6 to 49, further comprising one or more additional amino acid mutations in the CH2 domain of the heterodimer Fc variant.

[0407] 51. The heterodimer Fc variant according to Embodiment 50, wherein one or more additional amino acid mutations include a mutation at position 236.

[0408] 52. The heterodimer Fc variant according to Embodiment 51, wherein both the first Fc polypeptide and the second Fc polypeptide contain a mutation at position 236.

[0409] 53. The heterodimer Fc variant according to Embodiment 52, wherein the mutations at position 236 of the first and second Fc polypeptides are asymmetric.

[0410] 54. The heterodimer Fc variant according to Embodiment 53, wherein the mutation at position 236 is selected from G236D, G236N, and G236K.

[0411] 55. The heterodimer Fc variant according to Embodiment 53, wherein the mutation at position 236 is G236D or G236N.

[0412] 56. A heterodimer Fc variant according to Embodiment 51 or 52, wherein the mutations at position 236 of the first and second Fc polypeptides are asymmetric.

[0413] 57. A heterodimer Fc variant according to Embodiment 56, wherein the substitution of amino acids 325-331 is present in the second Fc polypeptide, the first Fc polypeptide contains a mutation at position 236 selected from G236A, G236D, G236E, G236F, G236H, G236I, G236L, G236N, G236P, G236Q, G236S, G236T, G236V, G236W and G236Y, and the second Fc polypeptide contains a mutation at position 236 selected from G236D, G236E, G236K, G236N and G236T.

[0414] 58. A heterodimer Fc variant according to Embodiment 56, wherein the substitution of amino acids 325-331 is present in the second Fc polypeptide, the first Fc polypeptide contains a mutation at position 236 selected from G236A, G236D, G236E, G236F, G236H, G236I, G236L, G236N, G236P, G236Q, G236S, G236T, G236V, G236W and G236Y, and the second Fc polypeptide contains the mutation G236D or does not contain the mutation at position 236.

[0415] 59. The heterodimer Fc variant according to Embodiment 56, wherein the amino acid substitution between 325 and 331 is present in the second Fc polypeptide, the first Fc polypeptide contains the G236N mutation or does not contain the mutation at position 236, and the second Fc polypeptide contains the mutation at position 236 selected from G236D, G236E, G236K, G236N, and G236T.

[0416] 60. A heterodimer Fc variant according to Embodiment 56, wherein the substitution of amino acids 325-331 is present in the second Fc polypeptide, the first Fc polypeptide contains a mutation at position 236 selected from G236D, G236K, and G236N, and the second Fc polypeptide contains a mutation at position 236 selected from G236D and G236N, or does not contain a mutation at position 236.

[0417] 61. The heterodimer Fc variant according to Embodiment 56, wherein the amino acid substitutions at 325-331 are present in the second Fc polypeptide, the first Fc polypeptide contains mutant G236N, and the second Fc polypeptide contains mutant G236D.

[0418] 62. A heterodimer Fc variant according to any one of embodiments 6 to 61, wherein the substitution of amino acids 325 to 331 is present in the second Fc polypeptide, and the second Fc polypeptide further comprises one or more mutations selected from S239D, S239E, V266I, V266L, S267A, S267I, S267V, S267Q and H268D.

[0419] 63. A heterodimer Fc variant according to any one of embodiments 6 to 61, wherein the amino acid substitution at 325 to 331 is present in the second Fc polypeptide, and the second Fc polypeptide further comprises one or more mutations selected from S239D, S239E, V266L, S267A, S267I, S267V, and H268D.

[0420] 64. The heterodimer Fc variant according to Embodiment 63, wherein the second Fc polypeptide comprises (i) mutation S239D or S239E, and / or (ii) mutation H268D, and / or (iii) mutation S267A, S267I, or S267V.

[0421] 65. The heterodimer Fc variant according to Embodiment 63, wherein the second Fc polypeptide comprises mutants S239D, H268D, and S267V.

[0422] 66. A heterodimer Fc variant according to any one of Embodiments 6 to 65, wherein the substitution of amino acids 325 to 331 is present in the second Fc polypeptide, and the first Fc polypeptide further comprises a mutation at one or more of the positions 234, 235, 237, and 239.

[0423] 67.(i) The mutation at position 234 is selected from L234A, L234D, L234E, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235I, L235N, L235P, L235Q, L235S, L235T, L235V, L235W, and L235Y, (iii) The mutation at position 237 is selected from G237A, G237D, G237F, G237H, G237L, G237N, G237P, G237S, G237V, G237W, and G237Y, (iv) The mutation at position 239 is selected from S239A, S239D, S239E, S239F, S239G, S239H, S239I, S239L, S239N, S239Q, S239R, S239T, S239V, S239W, and S239Y. The heterodimer Fc variant described in Embodiment 66.

[0424] 68.(i) The mutation at position 234 is selected from L234D, L234F, L234Q, L234T, and L234W. (ii) The mutation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235R, L235W, and L235Y, (iii) The mutation at position 237 is selected from G237A, G237D, G237L, and G237N. (iv) The mutation at position 239 is selected from S239A, S239G, S239H, S239T, and S239Y. The heterodimer Fc variant described in Embodiment 66.

[0425] 69. The heterodimer Fc variant according to Embodiment 66, wherein the first Fc polypeptide comprises mutant L234D and / or L235F.

[0426] 70. A heterodimer Fc variant according to any one of Embodiments 6 to 69, wherein the amino acid substitution is located at positions 325 to 331 in the second Fc polypeptide, and the second Fc polypeptide further comprises a mutation at one or more of the positions 234, 235, 237, 240, 263, 264, 266, 269, 271, 273, 323, and 332.

[0427] 71.(i) The mutation at position 234 is selected from L234A, L234E, L234F, L234G, L234H, L234I, L234K, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 235 is selected from L235A, L235D, L235F, L235G, L235N, L235S, L235W, and L235Y. (iii) The mutation at position 237 is selected from G237F, G237I, G237K, G237L, G237Q, G237T, G237V, and G237Y, (iv) The mutation at position 240 is selected from V240I and V240L, (v) The mutation at position 263 is V263T, (vi) The mutation at position 264 is V264T, (vii) The mutation at position 266 is V266I, (viii) The mutation at position 269 is E269Q, (ix) The mutation at position 271 is P271D, (x) The mutation at position 273 was selected from V273A and V273I. (xi) The mutation at position 323 is selected from V323A and V323I, (xii) The mutation at position 332 is selected from I332F and I332L. The heterodimer Fc variant described in Embodiment 70.

[0428] 72. A heterodimer Fc variant according to Embodiment 70 or 71, wherein the second Fc polypeptide comprises a mutation at one or more of the positions 271, 323, and 332.

[0429] 73.(i) The mutation at position 271 is P271D, (ii) The mutation at position 323 is V323A, (iii) The mutation at position 332 is selected from I332F and I332L. The heterodimer Fc variant described in Embodiment 72.

[0430] 74. A heterodimer Fc variant according to any one of Embodiments 6 to 73, wherein the first Fc polypeptide and the second Fc polypeptide further comprise one or more mutations selected from A287F, T250V, L309Q, and M428F.

[0431] 75. The heterodimer Fc variant according to Embodiment 74, wherein the first Fc polypeptide and the second Fc polypeptide further comprise mutant A287F / M428F, A287F / T250V, M428F / T250V, or T250V / L309Q.

[0432] 76. The heterodimer Fc variant according to Embodiment 6, wherein the heterodimer Fc variant comprises an amino acid mutation described in any one of the variants shown in Table 6.22, Table 6.24, Table 6.25, or Table 6.27.

[0433] 77.(i) The first Fc polypeptide contains the mutation G236N_G237D, and the second Fc polypeptide contains the mutation template 1(D329 * I) Includes +G236D_G237F_S239D_S267V_H268D (variant 31186), (ii) The first Fc polypeptide contains the mutation L235F_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D (variant 31187), (iii) The first Fc polypeptide contains the mutation L235F_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (G330 * K)+G236D_G237F_S239D_S267V_H268D (Variant 31188) or (iv) The first Fc polypeptide contains the mutation G236N_G237D, and the second Fc polypeptide contains the mutation template 7(E328 * H_E329 * R_A331 * BY)+G236D_G237F_S239D_S267V_H268D (Variant 31191) or (v) The first Fc polypeptide contains the mutation L235F_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 31213), (vi) The first Fc polypeptide contains the mutation L235F_G236N_G237A_T250V_A287F, and the second Fc polypeptide contains the mutation template 1 (D329 *I) Includes +G236D_G237F_S239D_T250V_S267V_H268D_A287F (Variant 31274), (vii) The first Fc polypeptide contains the mutation L235F_G236N_G237A_T250V_M428F, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_T250V_S267V_H268D_M428F (variant 31275), (viii) The first Fc polypeptide contains the mutation L235F_G236N_G237A_A287F_M428F, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_A287F_M428F (variant 31276), (ix) The first Fc polypeptide contains the mutation G236N_G237D, and the second Fc polypeptide contains the mutation template 1(D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32210), (x) The first Fc polypeptide contains the mutation G236N_G237E, and the second Fc polypeptide contains the mutation template 1(D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32211), (xi) The first Fc polypeptide contains mutant G236N, and the second Fc polypeptide contains mutant template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32212), (xii) The first Fc polypeptide contains the mutation L235D_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32226), (xiii) The first Fc polypeptide contains the mutation L235E_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32227), (xiv) The first Fc polypeptide contains the mutation L235V_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32230), (xv) The first Fc polypeptide contains the mutation L235Y_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32231), (xvi) The first Fc polypeptide contains the mutation G236N_G237A_S239P, and the second Fc polypeptide contains the mutation template 1(D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32242), (xvii) The first Fc polypeptide contains the mutation L234D_G236N_G237A, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32282), (xviii) The first Fc polypeptide contains the mutation L235D_G236N_G237A, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32284), (xix) The first Fc polypeptide contains the mutation G236N_G237A_S239G, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32287), (xx) The first Fc polypeptide contains the mutation G236N_G237A_S239H, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32288), (xxi) The first Fc polypeptide contains the mutation G236N_G237E, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32296), (xxii) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D (Variant 31192), (xxiii) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32292), (xxiv) The first Fc polypeptide contains the mutation L234F_G236N_S267A_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32293), (xxv) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_A330T_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32294), or (xxvi) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_P329I_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32295), The heterodimer Fc variant described in Embodiment 6.

[0434] 78. A heterodimer Fc variant according to any one of Embodiments 1 to 77, wherein the heterodimer Fc variant is a variant of IgG1 Fc.

[0435] 79. The heterodimer Fc variant according to Embodiment 78, wherein the heterodimer Fc variant is a variant of human IgG1 Fc.

[0436] 80. The selectivity of the heterodimeric Fc variant to binding to FcγRIIb is increased by at least 1.5 times or at least 2 times compared to the parent Fc region, where, FcγRIIb selectivity ratio difference = FcγRIIb affinity ratio difference / FcγRIIaR affinity ratio difference And in the formula, FcγRIIb affinity factor difference = K D FcγRIIb(parent) / K D FcγRIIb (variant) and FcγRIIaR affinity factor difference = K D FcγRIIaR(parent) / K D FcγRIIaR (variant) The heterodimer Fc variant described in any one of Embodiments 1 to 79.

[0437] 81. A heterodimer Fc variant according to any one of Embodiments 1 to 80, wherein the heterodimer Fc variant has increased binding affinity to FcγRIIb compared to the parent Fc region.

[0438] 82. The binding affinity of the heterodimer Fc variant to FcγRIIb is increased by at least 10 times compared to the parent Fc region, where, FcγRIIb affinity factor difference = K D FcγRIIb(parent) / K D FcγRIIb (variant) The heterodimer Fc variant described in Embodiment 81.

[0439] 83. A polypeptide comprising a heterodimeric Fc variant described in any one of Embodiments 1 to 82 and one or more protein moieties fused to or covalently bonded to the heterodimeric Fc variant.

[0440] 84. The polypeptide according to Embodiment 83, wherein the polypeptide is an antibody, and one or more protein portions are one or more antigen-binding domains.

[0441] 85. The polypeptide according to Embodiment 84, wherein at least one of the antigen-binding domains binds to a tumor-associated antigen or a tumor-specific antigen.

[0442] 86. A pharmaceutical composition comprising a heterodimer Fc variant according to any one of Embodiments 1 to 82 or a polypeptide according to any one of Embodiments 83 to 85, and a pharmaceutically acceptable carrier or diluent.

[0443] 87. A polypeptide according to any one of embodiments 83 to 85 for use in therapeutic purposes.

[0444] 88. Polypeptide according to Embodiment 85 for use in the treatment of cancer.

[0445] 89. A nucleic acid encoding a heterodimer Fc variant according to any one of Embodiments 1 to 82, or a polypeptide according to any one of Embodiments 83 to 85.

[0446] 90. A host cell comprising the nucleic acid described in Embodiment 89.

[0447] 91. A method for preparing a heterodimer Fc variant according to any one of Embodiments 1 to 82, or a polypeptide according to any one of Embodiments 83 to 85, comprising expressing a nucleic acid encoding the heterodimer Fc variant or polypeptide in a host cell.

[0448] 92. A method for preparing a heterodimer Fc variant comprising a first Fc polypeptide and a second Fc polypeptide, which exhibits increased selectivity for a target receptor compared to the parent Fc region, (a) Using an in silico model of the parental Fc region complexed with the target receptor, (i) To provide variant candidates, insert a sequence of one or more amino acid residues into one of the natural loops of the Fc polypeptide such that the length of the natural loop is extended. (ii) Determine the distance of at least one amino acid residue of the insertion sequence from the target amino acid residue in the receptor, (iii) If at least one amino acid residue of the insertion sequence is within a heavy atom distance of 3 Å from the target amino acid residue in the receptor, the candidate variant is selected as a heterodimeric Fc variant, (b) preparing nucleic acids encoding heterodimeric Fc variants, (c) Expressing nucleic acids in host cells to provide heterodimeric Fc variants Includes, Here, the target receptor is FcγRIIb. method.

[0449] 93. A heterodimeric Fc variant comprising a first Fc polypeptide and a second Fc polypeptide, having increased selectivity for binding to FcγRIIb compared to the parent Fc region, and containing an asymmetric mutation at position 236, One of the Fc polypeptides contains the mutation G236N or G236D. Heterodimer Fc variants are variants of immunoglobulin G (IgG)Fc, The amino acid numbering follows the EU index. Heterodimer Fc variant.

[0450] 94. The heterodimer Fc variant according to Embodiment 93, wherein the first Fc polypeptide comprises the mutation G236N or G236D, and the second Fc polypeptide does not contain the mutation at position 236.

[0451] 95. The heterodimer Fc variant according to Embodiment 93, wherein the first Fc polypeptide comprises the mutation G236N or G236D, and the second Fc polypeptide comprises a different mutation at position 236.

[0452] 96. The heterodimer Fc variant according to Embodiment 95, wherein the first Fc polypeptide comprises mutant G236N and the second Fc polypeptide comprises mutant G236D, G236K, or G236S.

[0453] 97. The heterodimer Fc variant according to Embodiment 95, wherein the first Fc polypeptide contains mutant G236N and the second Fc polypeptide contains mutant G236D.

[0454] 98. The heterodimer Fc variant according to Embodiment 95, wherein the first Fc polypeptide comprises mutant G236D, and the second Fc polypeptide comprises mutant G236N, G236Q, G236K, G236E, or G236H.

[0455] 99. A heterodimer Fc variant according to any one of embodiments 93 to 98, wherein the first Fc polypeptide and / or the second Fc polypeptide further comprises one or more additional amino acid mutations in the CH2 domain of the heterodimer Fc variant.

[0456] 100. The heterodimer Fc variant according to Embodiment 99, wherein the second Fc polypeptide further comprises one or more mutations selected from S239D, S239E, V266I, V266L, S267A, S267I, S267V, S267Q, and H268D.

[0457] 101. The heterodimer Fc variant according to Embodiment 99, wherein the second Fc polypeptide further comprises one or more mutations selected from S239D, S239E, V266L, S267A, S267I, S267Q, S267V, and H268D.

[0458] 102. The second Fc polypeptide is a) Mutation S239D or S239E; or b) Mutation H268D, or c) Mutations S239D or S239E, and mutation H268D The heterodimer Fc variant according to embodiment 99, further comprising the above.

[0459] 103. The heterodimer Fc variant according to Embodiment 99, wherein the second Fc polypeptide further comprises mutants S239D and H268D.

[0460] 104. A heterodimer Fc variant according to any one of embodiments 93 to 103, wherein the heterodimer Fc variant is a strategic 1 / 3 variant.

[0461] 105. A heterodimer Fc variant according to any one of embodiments 93 to 104, wherein the second Fc polypeptide further comprises the mutation S267A, S267I, or S267V.

[0462] 106. A heterodimer Fc variant according to any one of embodiments 93 to 105, wherein amino acids 325 to 331 in the second Fc polypeptide are replaced with a polypeptide having a length of 8 to 15 amino acids.

[0463] 107. A heterodimer Fc variant according to Embodiment 106, wherein the polypeptide is derived from the loop-forming segment of the second protein.

[0464] 108. The heterodimer Fc variant according to Embodiment 107, wherein the loop-forming segment is immobilized on a second protein by a beta-strand.

[0465] 109. In the native conformation within the second protein, the loop-forming segment of the heterodimer Fc variant according to Embodiment 107 or 108 has the following properties: i) The loop-forming segment contains one or more beta-strand amino acids at both the N-terminus and C-terminus of the loop; ii) One or more beta-strand amino acids at the C-terminus of the loop-forming segment do not form hydrogen bonds with any amino acids in the parent protein, except for the beta-strand amino acid at the N-terminus of the loop-forming segment; iii) The mean squared deviation (RMSD) of the skeletal heavy atoms of one or more beta-strand amino acids at the N-terminus of the loop-forming segment for one or more amino acids ending at position 324 is <0:85 Å; and iv) For one or more amino acids starting at position 332, the RMSD of the backbone heavy atoms of one or more beta-strand amino acids at the C-terminus of the loop-forming segment is <0:85 Å.

[0466] 110. The heterodimer Fc variant according to Embodiment 109, wherein the loop-forming segment further comprises the following properties: The loop-forming segment contains at least one hydrogen bond between the beta-strand amino acids at both ends of the loop-forming segment.

[0467] 111. Polypeptides, (a) The amino acid sequence described in any one of sequence numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or (b) an amino acid sequence which is a variant of the sequence described in any one of sequence numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, wherein the variant contains 1, 2, 3, 4, or 5 amino acid mutations. A heterodimer Fc variant according to any one of embodiments 106 to 110, including the above.

[0468] 112. Heterodimer Fc variant according to Embodiment 106, wherein the polypeptide comprises the amino acid sequence of formula (I), formula (Ia), formula (Ib), formula (II), formula (III), formula (IV), formula (V), or formula (VI): Equation (I): X 1 X 2 WX 3 X 4 X 5 GX 6 X 7 T(I) (In the formula, X 1 is A, D, N, or S, X 2 is A, D, E, F, H, I, L, N, Q, S, T, V, W, or Y, X 3 is A, D, E, F, H, I, N, Q, S, T, V, W, or Y, X 4 is D, E, G, I, L, P, or Q, X 5 is A, D, E, G, H, K, N, R, S, T, or Y, X 6 is A, D, E, F, H, P, W, or Y, X 7 (The letters are A, D, E, F, G, H, K, L, N, Q, or R.) Equation (Ia): X 1 X 2 WX 3 X 4 X 5 GYX 6 T(Ia) (In the formula, X 1 is A, D, N, or S, X 2 is A, D, E, F, H, I, L, N, Q, S, T, V, W, or Y, X 3 is A, D, E, F, H, I, N, Q, S, T, V, W, or Y, X 4 is D, E, G, I, L, P, or Q, X 5 is A, D, E, G, H, K, N, R, S, T, or Y, X 6 (The letters are A, D, E, F, G, H, K, L, N, Q, or R.) Formula (Ib): X 1 X 2 WX 3 X 4 GGYX 5 T(Ib) (In the formula, X 1 is A or S, X 2 is A, D, E, F, H, I, L, N, Q, T, V, or W. X 3 is D, E, F, H, N, Q, S, T, or Y, X 4 is D, G, I, or L, X 5 (This is A, F, H, K, L, or N) Formula (II): X 1 LDX 2 X 3 GKGX 4 V(II) (In the formula, X 1 is F or G, X 2 is E, H, Q, or T, X 3 is E, N, R, S, or T, X 4 (is A, Y, or V) Formula (III): X 1 TDEX 2 GKGX 3 T(III) (In the formula, X 1 is F or G, X 2 is either E or N, X 3 (is A or V) Formula (IV): X1 FX 2 X 3 X 4 X 5 GEVV(IV) (In the formula, X 1 is either A or D, X 2 is either D or N, X 3 is D, E, H, N, P, Q, S, or T, X 4 is D, E, N, S, or T, X 5 (is D or Q) Formula (V): X 1 TDX 2 X 3 X 4 GEVT(V) (In the formula, X 1 is either A or D, X 2 is D, P, or Q, X 3 is D, E, or N, X 4 (is D or Q) Equation (VI): LTDX 1 X 2 GX 3 PX 4 R(VI) (In the formula, X 1 is E or H, X 2 is D, E, or N, X 3 is R or S, X 4 (is I, Q, or Y).

[0469] 113. The heterodimer Fc variant according to Embodiment 112, wherein the polypeptide comprises the amino acid sequence of formula (I).

[0470] 114.X 1A heterodimer Fc variant according to Embodiment 113, wherein is A or S.

[0471] 115.X 2 but, (i) A, D, E, F, H, I, L, N, Q, T, V or W, or (ii) H or T The heterodimer Fc variant described in embodiment 113 or 114.

[0472] 116.X 3 but, (i) A, F, H, I, S, T, V, W or Y, or (ii) D, E, F, H, N, Q, S, T or Y, (iii) F, H, S, T or Y, (iv) E, F, H, Q, S or T, (v) F, H, S or T, (vi) E, F or S, (vii)F or S The heterodimer Fc variant described in any one of embodiments 113 to 115.

[0473] 117.X 4 but, (i) D, G, I or L, or (ii) D or G The heterodimer Fc variant described in any one of embodiments 113 to 116.

[0474] 118.X 5 but, (i) A, D, E, G, H, K or R, (ii)G The heterodimer Fc variant described in any one of embodiments 113 to 117.

[0475] 119.X 6 but, (i) F, W or Y, (ii)Y The heterodimer Fc variant described in any one of embodiments 113 to 118.

[0476] 120.X 7 but, (i) A, D, E, G, H, K, L, N, Q or R, (ii) A, F, H, K, L or N, (iii) A, H, K, L or N, (iv) A or N The heterodimer Fc variant described in any one of embodiments 113 to 119.

[0477] 121. The heterodimer Fc variant according to Embodiment 112, wherein the polypeptide comprises the amino acid sequence of formula (Ia).

[0478] 122.X 1 A heterodimer Fc variant according to Embodiment 121, wherein is A or S.

[0479] 123.X 2 but, (i) A, D, E, F, H, I, L, N, Q, T, V or W, or (ii) H or T The heterodimer Fc variant according to embodiment 121 or 122.

[0480] 124.X 3 but, (i) A, F, H, I, S, T, V, W or Y, or (ii) D, E, F, H, N, Q, S, T or Y, (iii) F, H, S, T or Y, (iv) E, F, H, Q, S or T, (v) F, H, S or T, (vi) E, F or S, (vii)F or S The heterodimer Fc variant described in any one of embodiments 121 to 123.

[0481] 125.X 4 but, (i) D, G, I or L, or (ii) D or G The heterodimer Fc variant described in any one of embodiments 121 to 124.

[0482] 126.X 5 but, (i) A, D, E, G, H, K or R, (ii)G The heterodimer Fc variant described in any one of embodiments 121 to 125.

[0483] 127.X 6 but, (i) A, D, E, G, H, K, L, N, Q or R, (ii) A, F, H, K, L or N, (iii) A, H, K, L or N, (iv) A or N The heterodimer Fc variant described in any one of embodiments 121 to 126.

[0484] 128. The heterodimer Fc variant according to Embodiment 112, wherein the polypeptide comprises the amino acid sequence of formula (Ib).

[0485] 129.X 2 A heterodimer Fc variant according to Embodiment 126, wherein is H or T.

[0486] 130.X 3 but, (i) F, H, S or Y, or (ii) E, F, H, Q, S or T, (iii) F, H or S, (iv) E, F or S, (v) F or S The heterodimer Fc variant according to embodiment 128 or 129.

[0487] 131.X 4 A heterodimer Fc variant as described in any one of embodiments 128 to 130, wherein is D or G.

[0488] 132.X 5 but, (i) A, F, H, K or L, (ii) A or N, or (iii) A A heterodimer Fc variant as described in any one of embodiments 128 to 131.

[0489] 133. The heterodimer Fc variant according to Embodiment 112, wherein the polypeptide comprises the amino acid sequence of formula (II).

[0490] 134.X 2 A heterodimer Fc variant according to Embodiment 133, wherein E is present.

[0491] 135.X 3 A heterodimer Fc variant according to embodiment 133 or 134, wherein is E, N, R, or S.

[0492] 136.X 3 A heterodimer Fc variant according to embodiment 133 or 134, wherein is E or N.

[0493] 137. The heterodimer Fc variant according to Embodiment 112, wherein the polypeptide comprises the amino acid sequence of formula (III).

[0494] 138. The heterodimer Fc variant according to Embodiment 112, wherein the polypeptide comprises the amino acid sequence of formula (IV).

[0495] 139.X 1The heterodimer Fc variant according to Embodiment 138, wherein D is present.

[0496] 140.X 2 A heterodimer Fc variant according to embodiment 138 or 139, wherein is D.

[0497] 141.X 3 A heterodimer Fc variant according to any one of embodiments 138 to 140, wherein is E, H, N, S, or T.

[0498] 142.X 4 A heterodimer Fc variant according to any one of embodiments 138 to 141, wherein is D, N, S, or T.

[0499] 143. The heterodimer Fc variant according to Embodiment 112, wherein the polypeptide comprises the amino acid sequence of formula (V).

[0500] 144. The heterodimer Fc variant according to Embodiment 112, wherein the polypeptide comprises the amino acid sequence of formula (VI).

[0501] 145.X 1 A heterodimer Fc variant according to Embodiment 144, wherein E is present.

[0502] 146.X 4 A heterodimer Fc variant according to embodiment 144 or 145, wherein is I or Y.

[0503] 147. The heterodimer Fc variant according to Embodiment 106, wherein the polypeptide comprises the amino acid sequence described in any one of SEQ ID NOs: 4 to 172.

[0504] 148. The heterodimer Fc variant according to Embodiment 106, wherein the polypeptide comprises the amino acid sequence described in any one of SEQ ID NOs: 4 to 90.

[0505] 149. polypeptide is sequence numbers 6, 8, 9, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 5 A heterodimer Fc variant according to Embodiment 106, comprising an amino acid sequence described in any one of 6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90.

[0506] 150. A heterodimer Fc variant according to any one of embodiments 93 to 149, wherein the second Fc polypeptide further comprises the mutant S267V.

[0507] 151. A heterodimer Fc variant according to any one of embodiments 93 to 150, wherein the first Fc polypeptide and / or the second Fc polypeptide further comprises a mutation at position 237.

[0508] 152. The heterodimer Fc variant according to Embodiment 151, wherein the first or second Fc polypeptide comprises the mutation G236N, and the same Fc polypeptide further comprises a mutation at position 237 selected from G237A, G237D, G237F, G237H, G237L, G237N, G237P, G237S, G237V, G237W, and G237Y.

[0509] 153. The heterodimer Fc variant according to Embodiment 151, wherein the first or second Fc polypeptide comprises mutant G236N, and the same Fc polypeptide further comprises mutant G237A.

[0510] 154. The heterodimer Fc variant according to Embodiment 151, wherein the first or second Fc polypeptide comprises the mutation G236D, and the same Fc polypeptide further comprises a mutation at position 237 selected from G237F, G237I, G237K, G237L, G237Q, G237T, G237V, and G237Y.

[0511] 154. The heterodimer Fc variant according to Embodiment 151, wherein the first or second Fc polypeptide comprises mutant G236D, and the same Fc polypeptide further comprises mutant G237F.

[0512] 155. A heterodimer Fc variant according to any one of embodiments 93 to 154, wherein the first Fc polypeptide comprises the mutation G236N, and the first Fc polypeptide further comprises a mutation at one or more of the positions 234, 235, 237, and 239.

[0513] 156.(i) The mutation at position 234 is selected from L234A, L234D, L234E, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235I, L235N, L235P, L235Q, L235S, L235T, L235V, L235W, and L235Y, (iii) The mutation at position 237 is selected from G237A, G237D, G237F, G237H, G237L, G237N, G237P, G237S, G237V, G237W, and G237Y, (iv) The mutation at position 239 is selected from S239A, S239D, S239E, S239F, S239G, S239H, S239I, S239L, S239N, S239Q, S239R, S239T, S239V, S239W, and S239Y. The heterodimer Fc variant described in Embodiment 155.

[0514] 157.(i) A mutation at position 234 is selected from L234D, L234F, L234Q, L234T, and L234W. (ii) The mutation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235R, L235W, and L235Y, (iii) The mutation at position 237 is selected from G237A, G237D, G237L, and G237N. (iv) The mutation at position 239 is selected from S239A, S239G, S239H, S239T, and S239Y. The heterodimer Fc variant described in Embodiment 155.

[0515] 158. The heterodimer Fc variant according to Embodiment 155, wherein the first Fc polypeptide further comprises the mutation L234D.

[0516] 159. The heterodimer Fc variant according to Embodiment 155 or 158, wherein the first Fc polypeptide further comprises mutant L235F.

[0517] 160. A heterodimer Fc variant according to any one of embodiments 93 to 159, wherein the second Fc polypeptide comprises the mutation G236D, and the second Fc polypeptide further comprises a mutation at one or more of the positions 234, 235, 237, 240, 263, 264, 266, 269, 271, 273, 323, and 332.

[0518] 161.(i) The mutation at position 234 is selected from L234A, L234E, L234F, L234G, L234H, L234I, L234K, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 235 is selected from L235A, L235D, L235F, L235G, L235N, L235S, L235W, and L235Y. (iii) The mutation at position 237 is selected from G237F, G237I, G237K, G237L, G237Q, G237T, G237V, and G237Y, (iv) The mutation at position 240 is selected from V240I and V240L, (v) The mutation at position 263 is V263T, (vi) The mutation at position 264 is V264T, (vii) The mutation at position 266 is V266I, (viii) The mutation at position 269 is E269Q, (ix) The mutation at position 271 is P271D, (x) The mutation at position 273 was selected from V273A and V273I. (xi) The mutation at position 323 is selected from V323A and V323I, (xii) The mutation at position 332 is selected from I332F and I332L. The heterodimer Fc variant described in Embodiment 160. 162.(i) The mutation at position 271 is P271D, (ii) The mutation at position 323 is V323A, (iii) The mutation at position 332 is selected from I332F and I332L. The heterodimer Fc variant described in Embodiment 160.

[0519] 163. The heterodimer Fc variant according to Embodiment 93, wherein the heterodimer Fc variant comprises an amino acid mutation described in any one of the variants shown in Table 6.22, Table 6.24, Table 6.25, or Table 6.27.

[0520] 164.(i) The first Fc polypeptide contains the mutation G236N_G237D, and the second Fc polypeptide contains the mutation template 1(D329 * I) Includes +G236D_G237F_S239D_S267V_H268D (variant 31186), (ii) The first Fc polypeptide contains the mutation L235F_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D (variant 31187), (iii) The first Fc polypeptide contains the mutation L235F_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (G330 * K)+G236D_G237F_S239D_S267V_H268D (Variant 31188) or (iv) The first Fc polypeptide contains the mutation G236N_G237D, and the second Fc polypeptide contains the mutation template 7(E328 * H_E329 * R_A331 * BY)+G236D_G237F_S239D_S267V_H268D (Variant 31191) or (v) The first Fc polypeptide contains the mutation L235F_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 31213), (vi) The first Fc polypeptide contains the mutation L235F_G236N_G237A_T250V_A287F, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_T250V_S267V_H268D_A287F (Variant 31274), (vii) The first Fc polypeptide contains the mutation L235F_G236N_G237A_T250V_M428F, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_T250V_S267V_H268D_M428F (variant 31275), (viii) The first Fc polypeptide contains the mutation L235F_G236N_G237A_A287F_M428F, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_A287F_M428F (variant 31276), (ix) The first Fc polypeptide contains the mutation G236N_G237D, and the second Fc polypeptide contains the mutation template 1(D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32210), (x) The first Fc polypeptide contains the mutation G236N_G237E, and the second Fc polypeptide contains the mutation template 1(D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32211), (xi) The first Fc polypeptide contains mutant G236N, and the second Fc polypeptide contains mutant template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32212), (xii) The first Fc polypeptide contains the mutation L235D_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32226), (xiii) The first Fc polypeptide contains the mutation L235E_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32227), (xiv) The first Fc polypeptide contains the mutation L235V_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 *I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32230), (xv) The first Fc polypeptide contains the mutation L235Y_G236N_G237A, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32231), (xvi) The first Fc polypeptide contains the mutation G236N_G237A_S239P, and the second Fc polypeptide contains the mutation template 1(D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32242), (xvii) The first Fc polypeptide contains the mutation L234D_G236N_G237A, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32282), (xviii) The first Fc polypeptide contains the mutation L235D_G236N_G237A, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32284), (xix) The first Fc polypeptide contains the mutation G236N_G237A_S239G, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32287), (xx) The first Fc polypeptide contains the mutation G236N_G237A_S239H, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32288), (xxi) The first Fc polypeptide contains the mutation G236N_G237E, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32296), (xxii) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D (Variant 31192), (xxiii) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32292), (xxiv) The first Fc polypeptide contains the mutation L234F_G236N_S267A_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32293), (xxv) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_A330T_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32294), or (xxvi) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_P329I_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32295), The heterodimer Fc variant described in Embodiment 93.

[0521] 165. A heterodimer Fc variant according to any one of embodiments 93 to 103, wherein the heterodimer Fc variant is a strategy 2 variant.

[0522] 166. A heterodimer Fc variant according to any one of embodiments 93 to 103 and 165, wherein the first Fc polypeptide further comprises a mutation at one or more positions selected from 234, 268, 327, 330, and 331.

[0523] 167.(i) The mutation at position 234 is selected from L234A, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 268 is selected from H268A, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268N, H268P, H268Q, H268R, H268S, H268T, H268V, H268W and H268Y, (iii) The mutation at position 327 is selected from A327E and A327G, (iv) The mutation at position 330 is selected from A330K, A330H, A330Q, A330R, A330S, and A330T. (v) The mutation at position 331 is selected from P331A, P331D, P331E, P331H, P331Q, and P331S. The heterodimer Fc variant described in Embodiment 166.

[0524] 168. A heterodimer Fc variant according to Embodiment 166 or 167, wherein the first Fc polypeptide further comprises a mutation at position 234 selected from L234A, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y.

[0525] 169. The heterodimer Fc variant according to Embodiment 168, wherein the mutation at position 234 is L234F.

[0526] 170. A heterodimer Fc variant according to any one of embodiments 166 to 169, wherein the first Fc polypeptide further comprises a mutation at position 268 selected from H268A, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268N, H268P, H268Q, H268R, H268S, H268T, H268V, H268W and H268Y.

[0527] 171. The heterodimer Fc variant according to Embodiment 170, wherein the mutation at position 268 is H268Q.

[0528] 172. A heterodimer Fc variant according to any one of embodiments 166 to 171, wherein the first Fc polypeptide further comprises a mutation at position 327 selected from A327E and A327G.

[0529] 173. The heterodimer Fc variant according to Embodiment 172, wherein the mutation at position 327 is A327G.

[0530] 174. A heterodimer Fc variant according to any one of embodiments 166 to 173, wherein the first Fc polypeptide further comprises a mutation at position 330 selected from A330K, A330H, A330Q, A330R, A330S, and A330T.

[0531] 175. The heterodimer Fc variant according to Embodiment 174, wherein the mutation at position 330 is A330K or A330T.

[0532] 176. The heterodimer Fc variant according to Embodiment 174, wherein the mutation at position 330 is A330K.

[0533] 177. A heterodimer Fc variant according to any one of embodiments 166 to 176, wherein the first Fc polypeptide further comprises a mutation at position 331 selected from P331A, P331D, P331E, P331H, P331Q, and P331S.

[0534] 178. The heterodimer Fc variant according to Embodiment 177, wherein the mutation at position 331 is P331S.

[0535] 179. A heterodimer Fc variant according to any one of embodiments 93-103 and 165-178, wherein the second Fc polypeptide further comprises the mutation S267A or S267Q.

[0536] 180. A heterodimer Fc variant according to any one of embodiments 93-103 and 165-179, wherein the second Fc polypeptide further comprises mutant V266L.

[0537] 181. A heterodimer Fc variant according to any one of embodiments 93-103 and 165-180, wherein the first Fc polypeptide further comprises a mutation at one or more of the positions 235, 237, 239, 264, 266, 267, 269, 270, 271, 272, 273, 323, 326 and / or 332.

[0538] 182.(i) The mutation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235I, L235P, L235Q, L235S, L235T, L235V, L235W, and L235Y. (ii) The mutation at position 237 is selected from G237A, G237F, G237L, G237N, G237T, G237W, and G237Y. (iii) The mutation at position 239 is selected from S239A, S239D, S239E, S239G, S239I, S239L, S239N, S239Q, S239R and S239V, (iv) A mutation at position 264 is selected from V264A, V264F, V264I, V264L, and V264T. (v) The mutation at position 266 is V266I, (vi) The mutation at position 267 is selected from S267A, S267G, S267H, S267I, S267N, S267P, S267T, and S267V. (vii) The mutation at position 269 is selected from E269A, E269D, E269F, E269G, E269H, E269I, E269K, E269L, E269N, E269P, E269Q, E269R, E269S, E269T, E269V, E269W, and E269Y. (viii) The mutation at position 270 is selected from D270A, D270E, D270F, D270H, D270I, D270N, D270Q, D270S, D270T, D270W and D270Y, (ix) The mutation at position 271 is selected from P271D, P271E, P271G, P271H, P271I, P271K, P271L, P271N, P271Q, P271R, P271V, and P271W. (x) The mutation at position 272 is selected from E272A, E272D, E272F, E272G, E272H, E272I, E272L, E272N, E272S, E272T, E272V, E272W, and E272Y. (xi) The mutation at position 273 is V273A, (xii) The mutation at position 323 was selected from V323A, V323I, and V323L. (xiii) The mutation at position 326 is selected from K326A, K326D, K326H, K326N, K326Q, K326R, K326S, and K326T. (xiv) The mutation at position 332 is selected from I332A, I332L, I332T, and I332V. The heterodimer Fc variant described in Embodiment 181.

[0539] 183. A heterodimer Fc variant according to Embodiment 181 or 182, wherein the first Fc polypeptide further comprises a mutation at position 235.

[0540] 184. The heterodimer Fc variant according to Embodiment 183, wherein the mutation at position 235 is L235D.

[0541] 185. A heterodimer Fc variant according to any one of embodiments 181 to 184, wherein the first Fc polypeptide further comprises a mutation at position 267.

[0542] 186. The heterodimer Fc variant according to Embodiment 185, wherein the mutation at position 267 is S267A.

[0543] 187. A heterodimer Fc variant according to any one of embodiments 93-103 and 165-186, wherein the second Fc polypeptide further comprises a mutation at one or more positions selected from 234, 235, 237, 240, 264, 269, 271, 272, and 273.

[0544] 188.(i) The mutation at position 234 is selected from L234A, L234D, L234E, L234F, L234G, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 235 is selected from L235A, L235D, L235F, L235G, L235H, L235N, L235W, and L235Y. (iii) The mutation at position 237 is selected from G237A, G237D, G237E, G237F, G237H, G237I, G237K, G237L, G237N, G237Q, G237R, G237S, G237T, G237V, G237W, and G237Y, (iv) A mutation at position 240 is selected from V240I, V240L, and V240T. (v) The mutation at position 264 is selected from V264L and V264T, (vi) The mutation at position 269 is selected from E269D, E269T, and E269V. (vii) The mutation at position 271 is P271G, (viii) The mutation at position 272 is selected from E272A, E272D, E272I, E272K, E272L, E272P, E272Q, E272R, E272T, and E272V, (ix) The mutation at position 273 is selected from V273A, V273I, V273L, and V273T. The heterodimer Fc variant described in Embodiment 187.

[0545] 189. The heterodimer Fc variant according to Embodiment 187 or 188, wherein the second Fc polypeptide further comprises a mutation at position 237.

[0546] 190. The heterodimer Fc variant according to Embodiment 189, wherein the mutation at position 237 is G237D or G237L.

[0547] 191. A heterodimer Fc variant according to any one of embodiments 93-103 and 165-190, wherein amino acids 325-331 in the second Fc polypeptide are replaced with a polypeptide having a length of 8-15 amino acids.

[0548] 192. The polypeptide originates from the loop-forming segment of the second protein, and the loop-forming segment is (a) The amino acid sequence described in any one of sequence numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or (b) an amino acid sequence which is a variant of the sequence described in any one of sequence numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, wherein the variant contains 1, 2, 3, 4, or 5 amino acid mutations. A heterodimer Fc variant according to Embodiment 191, including the above.

[0549] 193. The heterodimer Fc variant according to Embodiment 93, wherein the heterodimer Fc variant comprises an amino acid mutation described in either Table 6.23 or Table 6.26.

[0550] 194.(i) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation G236D_G237L_S239D_V266L_S267A_H268D (variant 31190), (ii) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_P329I_A330K_P331S, and the second Fc polypeptide contains the mutation G236D_G237D_S239D_V266L_S267A_H268D (variant 31256), (iii) The first Fc polypeptide contains the mutant L234F_G236N_H268Q_A327G_P329A_A330K_P331S, and the second Fc polypeptide contains the mutant G236D_G237L_S239D_V266L_S267A_H268D (variant 32274), (iv) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D (Variant 31192), (v) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32292), (vi) The first Fc polypeptide contains the mutation L234F_G236N_S267A_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32293), (vii) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_A330T_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32294), or (viii) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_P329I_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32295), The heterodimer Fc variant described in Embodiment 93.

[0551] 195.(a) The first Fc polypeptide comprises the mutation G236N and a mutation at one or more positions selected from 234, 268, 327, 330, and 331, wherein, (i) The mutation at position 234 is selected from L234A, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W, and L234Y. (ii) The mutation at position 268 is selected from H268A, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268N, H268P, H268Q, H268R, H268S, H268T, H268V, H268W and H268Y, (iii) The mutation at position 327 is selected from A327G and A327E, (iv) The mutation at position 330 was selected from A330K, A330H, A330Q, A330R, A330S, and A330T. (v) The mutation at position 331 was selected from P331A, P331D, P331E, P331H, P331Q, and P331S. (b) The second Fc polypeptide, (i) Mutation G236D and (ii) A substitution of the native loop at positions 325-331 with a polypeptide of 8-15 amino acid length, wherein the polypeptide is derived from the loop-forming segment of the second protein, and the loop-forming segment comprises the amino acid sequence described in any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or a variant thereof containing 1, 2, 3, 4, or 5 amino acid mutations, (iii) One or more mutations selected from S239D, S239E, V266I, S267I, S267Q, S267V and H268D including, The heterodimer Fc variant described in Embodiment 93.

[0552] 196. The second Fc polypeptide, (i) Mutation G236D and (ii) A substitution of the native loop at positions 325-331 with a polypeptide of 8-15 amino acid length, wherein the polypeptide is derived from the loop-forming segment of the second protein, and the loop-forming segment comprises the amino acid sequence described in any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or a variant thereof containing 1, 2, 3, 4, or 5 amino acid mutations, (iii) mutation S239D or S239E, and / or mutation H268D, and / or mutation S267I or S267V A heterodimer Fc variant according to Embodiment 195, including the above.

[0553] 197. The second Fc polypeptide, (i) Mutation G236D and (ii) A substitution of the native loop at positions 325-331 with a polypeptide of 8-15 amino acid length, wherein the polypeptide is derived from the loop-forming segment of the second protein, and the loop-forming segment comprises the amino acid sequence described in any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or a variant thereof containing 1, 2, 3, 4, or 5 amino acid mutations, (iii) Mutations S239D, H268D and S267V A heterodimer Fc variant according to Embodiment 195, including the above.

[0554] 198. A heterodimer Fc variant according to any one of embodiments 195 to 197, wherein the mutation at position 234 in the first Fc polypeptide is L234F.

[0555] 199. A heterodimer Fc variant according to any one of embodiments 195 to 198, wherein the mutation at position 268 in the first Fc polypeptide is H268Q.

[0556] 200. A heterodimer Fc variant according to any one of embodiments 195 to 199, wherein the mutation at position 327 in the first Fc polypeptide is A327G.

[0557] 201. A heterodimer Fc variant according to any one of Embodiments 195 to 200, wherein the mutation at position 330 in the first Fc polypeptide is A330K or A330T.

[0558] 202. A heterodimer Fc variant according to any one of embodiments 195 to 201, wherein the mutation at position 331 in the first Fc polypeptide is P331S.

[0559] 203.(i) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D (Variant 31192), (ii) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 *I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32292), (iii) The first Fc polypeptide contains the mutation L234F_G236N_S267A_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32293), (iv) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_A330T_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32294), or (v) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_P329I_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 * I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32295), The heterodimer Fc variant described in Embodiment 195.

[0560] 204. A heterodimer Fc variant according to any one of embodiments 93 to 203, wherein the first Fc polypeptide and the second Fc polypeptide further comprise one or more mutations selected from A287F, T250V, L309Q, and M428F.

[0561] 205. The heterodimer Fc variant according to Embodiment 204, wherein the first Fc polypeptide and the second Fc polypeptide further comprise the mutant A287F / M428F, A287F / T250V, M428F / T250V, or T250V / L309Q.

[0562] 206. A heterodimer Fc variant according to any one of embodiments 93 to 205, wherein the heterodimer Fc variant is a variant of IgG1 Fc.

[0563] 207. The heterodimer Fc variant according to Embodiment 206, wherein the heterodimer Fc variant is a variant of human IgG1 Fc.

[0564] 208. The selectivity of the heterodimer Fc variant to binding to FcγRIIb is increased by at least 1.5 times or at least 2 times compared to the parent Fc region, where, Increase in FcγRIIb selectivity = Difference in FcγRIIb affinity / Difference in FcγRIIaR affinity And in the formula, FcγRIIb affinity factor difference = K D FcγRIIb(parent) / K D FcγRIIb (variant) and FcγRIIaR affinity factor difference = K D FcγRIIaR(parent) / K D FcγRIIaR (variant) The heterodimer Fc variant described in any one of embodiments 93 to 207.

[0565] 209. A heterodimer Fc variant according to any one of embodiments 93 to 208, wherein the heterodimer Fc variant has increased binding affinity to FcγRIIb compared to the parent Fc region.

[0566] 210. The binding affinity of the heterodimer Fc variant to FcγRIIb is increased by at least 10 times compared to the parent Fc region, where, FcγRIIb affinity factor difference = K D FcγRIIb(parent) / K D FcγRIIb (variant) The heterodimer Fc variant described in Embodiment 209.

[0567] 211. A polypeptide comprising a heterodimeric Fc variant described in any one of embodiments 93 to 210 and one or more protein moieties fused to or covalently bonded to the heterodimeric Fc variant.

[0568] 212. The polypeptide according to Embodiment 211, wherein the polypeptide is an antibody, and one or more protein portions are one or more antigen-binding domains.

[0569] 213. The polypeptide according to Embodiment 212, wherein at least one of the antigen-binding domains binds to a tumor-associated antigen or a tumor-specific antigen.

[0570] 214. A pharmaceutical composition comprising a heterodimer Fc variant described in any one of Embodiments 93 to 210 or a polypeptide described in any one of Embodiments 211 to 213, and a pharmaceutically acceptable carrier or diluent.

[0571] 215. A polypeptide according to any one of embodiments 211 to 213, for use in therapeutic purposes.

[0572] 216. The polypeptide according to Embodiment 213 for use in the treatment of cancer.

[0573] 217. A nucleic acid encoding a heterodimer Fc variant according to any one of embodiments 93 to 210, or a polypeptide according to any one of embodiments 211 to 213.

[0574] 218. A host cell comprising the nucleic acid described in Embodiment 217.

[0575] 219. A method for preparing a heterodimer Fc variant according to any one of embodiments 93 to 210, or a polypeptide according to any one of claims 211 to 213, comprising expressing a nucleic acid encoding the heterodimer Fc variant or polypeptide in a host cell.

[0576] The following examples are provided for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. [Examples]

[0577] Overview Figure 1 provides an overview of the strategies employed to generate FcγRIIb-specific variants. Various steps are described in detail in the following examples. Briefly, two approaches were employed to identify the first variant exhibiting higher selectivity for FcγRIIb than wild-type IgG1 Fc. Then, the variants obtained from each of these approaches were combined and further refined to generate optimized FcγRIIb-selective variants. Since both approaches leverage the asymmetry of the interaction between the Fc region and FcγRIIb, a heterodimer Fc capable of distinguishing between the two strands of Fc was required as a starting scaffold.

[0578] The two approaches we used to identify the initial variant were as follows:

[0579] (1) Asymmetric 1x approach (Figure 2A): This approach utilizes the asymmetry of the interaction between the Fc region and FcγRIIb to screen for mutations in the CH2 and hinge regions.

[0580] (2) Loop substitution approach (Figure 2B): In this approach, loop 3 (L3) on one of the Fc regions was replaced and lengthened. The L3 loop is normally too far from FcγRIIb and does not participate in binding (see Figure 2B). The loop substitution approach extended this region to be closer to position 135 of FcγRIIb. The amino acid at position 135 of FcγRIIb is serine (S), while in FcγRIIa, the amino acid at the corresponding position is leucine (L). By creating an additional interaction at this position, the selectivity of Fc to FcγRIIb was improved.

[0581] The overall strategy described herein yielded a library of variants with increased FcγRIIb selectivity. These variants exhibit both broad FcγRIIb selectivity and FcγRIIb affinity, resulting in a variety of effector profiles. Therefore, the library allows for the selection of variants with the optimal activity profile for the desired application.

[0582] General Methods Variant preparation Variants and controls were prepared by site-directed mutagenesis and / or restriction / ligation using standard methods. The final DNA was subcloned into vector pTT5 (see U.S. Patent No. 9,353,382). The following scaffolds were used for variant preparation:

[0583] Scaffold 1: Full-size antibody (FSA) based on trastuzumab containing homodimer IgG1 Fc

[0584] Scaffold 2: A one-arm antibody (OAA) scaffold containing one trastuzumab Fab and a heterodimer IgG1 Fc containing the following mutations: Chain A:T350V_L351Y_F405A_Y407V Chain B: T350V_T366L_K392L_T394W

[0585] The corresponding array is provided below.

[0586] Heavy chain A, upper hinge, CH2 and CH3 domains: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG[Sequence ID 2]

[0587] Heavy chain B, upper hinge, CH2 and CH3 domains: EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG[Sequence ID 3]

[0588] Scaffold 3: A full-size antibody (FSA) based on trastuzumab containing the same heterodimer Fc as scaffold 2.

[0589] Scaffold 4: A full-size antibody (FSA) based on the 4G7 anti-CD19 antibody (Meeker, et al., 1984, Hybridoma, 3:305-320) containing the same heterodimer Fc as scaffold 2. The sequence used is described in U.S. Patent No. 8,524,867.

[0590] Scaffold 5: A full-size antibody (FSA) based on the CP-870,893 anti-CD40 antibody (Gladue, et al., 2011, Cancer Immunol Immunother, 60:1009-1017) containing the same heterodimer Fc as scaffold 2. The variable domain sequence was obtained from international patent application publication number WO2013 / 132044.

[0591] Expression - Protocol 1 Expression was performed in 2 mL, 50 mL, or 500 mL of CHO3E7 cells. Transfection of CHO cells in the exponential growth phase (15-2 million cells / mL) was performed using a 1 mg / mL solution of 25 kDa polyethyleneimine (PEI) solution. proTransfection was performed using Polyplus Transfection SA (Illkirch, France) with a PEI:DNA ratio of 2.5:1 (Delafosse, et al., 2016, J. Biotechnol., 227:103-111). DNA was transfected with a predetermined optimal DNA ratio of heavy chain A (HC-A), light chain (LC), and heavy chain B (HC-B) to enable heterodimer formation (e.g., HC-A / HC-B / LC ratio = 25:25:50%). Transfected cells were harvested after 5-6 days. After centrifugation at 4000 rpm, the culture medium was collected and clarified using a 0.45 μm filter.

[0592] The clarified culture medium was loaded onto a MabSelect® SuRe® (GE Healthcare, Baie-d'Urfe, QC, Canada) Protein A column and washed with 10 column volumes of PBS buffer (pH 7.2). The antibody was eluted with 10 column volumes of citrate buffer (pH 3.6), and the antibody-containing pool fraction was neutralized with TRIS (pH 11). The Protein A purified antibody was further purified by size exclusion chromatography (SEC). For gel filtration, 3.5 mg of the antibody mixture was concentrated to 1.5 mL and loaded onto a Sephadex 200 HiLoad® 16 / 600 200 pg column (GE Healthcare) equilibrated with PBS (pH 7.4) at a flow rate of 1 mL / min using an AKTA Express FPLC. The fraction corresponding to the purified antibody was recovered, concentrated to approximately 1 mg / mL, and stored at -80°C.

[0593] Expression - Protocol 2 Expression was performed using HEK293-6E cells (NRC, Canada) in either small (1 mL) or large (30 mL or more) tanks.

[0594] For 1 mL scale expression, 1 μg DNA / mL cells were transfected into HEK293-6E cells in the exponential growth phase (1.5–2 million cells / mL) using DNA pre-complexed with the cationic lipid 293Fectin® (Life Technologies, Paisley, UK). The heavy and light chain DNA were mixed in a ratio of 47.5:52.5%, and the DNA and 293Fectin® were complexed with a final concentration of 11.7 μg / mL DNA and 1.65% (v / v) 293Fectin®. This mixture was then incubated at ambient temperature for 30 minutes before being added to the cells. To achieve optimal heterodimerization, the DNA ratio of heavy chain A to heavy chain B in the transfection mix was set to either 50:50% or a slightly variable range. Cells were cultured for 5-6 days in a humidified, shaking incubator at 37°C with 5% carbon dioxide in 96-well deep-well plates sealed with gas-permeable seals. The culture medium was then collected after centrifugation at 1600 × g.

[0595] For large-scale expression, 1 μg DNA / mL cells were transfected into HEK293-6E cells in the exponential growth phase (1.5–2 million cells / mL) using DNA pre-complexed with Gemini cationic lipids (Camilleri et al., 2000, Chem.Commun., 1253-1254). Heavy and light chain DNA were mixed in a 50:50 ratio, and the DNA and Gemini were complexed with a final concentration of 10 μg / mL DNA and 40 μg / mL Gemini. The mixture was then incubated at ambient temperature for 15–30 minutes before being added to the cells. The DNA ratio of heavy chain A to heavy chain B in the transfection mix was as described above. Cells were cultured in appropriately sized Erlenmeyer flasks or BioReactor tubes in a humidified shaking incubator at 37°C and 5% carbon dioxide for up to 10 days. Next, the culture medium was collected after centrifugation at 2750 × g and clarified using a 0.22 μm filter.

[0596] The clarified culture medium was loaded onto a MabSelect® SuRe® (GE Healthcare, Little Chalfont, UK) Protein A column, washed with 3–10 column volumes of Tris acetate buffer (pH 7.5), then eluted with 2–5 column volumes of acetic acid (pH 2.6), and the eluted fraction was neutralized with TRIS. Selected samples were further purified by size exclusion chromatography (Superdex® 200 column (GE Healthcare, Little Chalfont, UK), using PBS running buffer) and / or cation exchange (ReSource® S column (GE Healthcare, Little Chalfont, UK)). The purified Protein A antibodies were buffer-exchanged with PBS.

[0597] Preparation of Fcγ receptors Protocol 1 As previously described, FcγRIIaH, IIaR, IIb, IIIaF, and IIIaV were produced in HEK293-6E cells, and FcγRIa was produced in CHO-3E7 cells (Dorian-Thibaudeau, et al., 2014, J.Immunol.Methods, 408:24-34). Human FcRn was also expressed in HEK293-6E cells by cotransfection with a 1:1 ratio of the extracellular domain of the alpha subunit (p51) containing a TEV-cleaved C-terminal His tag to β2-microglobulin. After purification as described by Dorian-Thibaudeau et al. (ibid.), the C-terminal His tag was removed by TEV cleavage.

[0598] Protocol 2 Soluble FcγRI extracellular domains containing a 6xHis tag at the C-terminus were purchased from R&D Systems (catalog number 1257-Fc). Soluble FcγRIIaH, IIaR, IIb, IIIaF, and IIIaV extracellular domains containing a 10xHis tag at the C-terminus were produced in HEK293-6E cells. Using DNA pre-complexed with Gemini cationic lipids, cells in the exponential growth phase (15-2 million cells / mL) were transfected with 1 μg DNA / mL cells (Camilleri et al., 2000, Chem.Commun., 1253-1254). Cells were cultured in appropriately sized Erlenmeyer flasks in a humidified shaking incubator at 37°C and 5% carbon dioxide for up to 7 days. The point of harvesting was determined when cell viability fell below 50%. Next, the culture medium was collected after centrifugation at 2750 × g and clarified using a 0.22 μm filter.

[0599] The clarified culture medium was buffer-exchanged to a pH 7.7 load buffer containing 25 mM imidazole by dialysis or tangential flow filtration, and then filtered on a Ni-Sepharose 6 column (GE Healthcare, Little Chalfont, UK). Elution was then performed by increasing the imidazole concentration in the buffer to 300 mM. The eluted protein was concentrated, buffer-exchanged to PBS by diafiltration, and then further purified by size exclusion chromatography (Superdex® 75 column (GE Healthcare, Little Chalfont, UK)).

[0600] Soluble human FcRn extracellular domains were expressed in HEK293-6E cells by cotransfection with an alpha subunit containing a 6xHis tag at the C-terminus and β2-microglobulin in a 1:1 ratio, and FcγR was expressed as described separately. The pH of the clarified medium was adjusted to pH 5.3 with citrate, and then loaded onto an IgG Sepharose column (GE Healthcare, Little Chalfont, UK). The bound proteins were eluted with pH 7.7 HEPES buffer. The eluted proteins were concentrated, buffered with PBS by diafiltration, and then further purified by size exclusion chromatography (Superdex® 75 column (GE Healthcare, Little Chalfont, UK)).

[0601] To eliminate the interaction between FcγR and the Fc domain, soluble FcγRIIb and FcγRIIaR extracellular domains, genetically fused via the C-terminus to human IgG1 Fc containing the CH2 mutation L234A_L235A_D265S, were expressed as described above for the His-tagged extracellular domain. The clarified medium was loaded onto a MabSelect® SuRe® protein A column (GE Healthcare, Little Chalfont, UK), washed with 3–10 column volumes of Tris acetate buffer (pH 7.5), then eluted with 2–5 column volumes of acetate (pH 2.6), and the eluted fraction was neutralized with TRIS. The sample was then buffer-exchanged with PBS and further purified by size exclusion chromatography using PBS running buffer (Superdex® 200 column (GE Healthcare, Little Chalfont, UK)).

[0602] Fcγ receptor binding: Surface plasmon resonance (SPR) Protocol 1 The affinity of FcγR for antibody Fc was measured by SPR using ProteOn®XPR36 at 25°C, with PBS containing 150 mM NaCl, 3.4 mM EDTA, and 0.05% Tween20 (pH 7.4) as the running buffer. For trastuzumab variants, recombinant HER2 was immobilized on a GLM sensor chip using standard amine coupling with a BioRad amine coupling kit. Briefly, the GLM sensor chip was activated with NHS / EDC, followed by injection of HER2 at 4.0 μg / mL in 10 mM NaOAc (pH 4.5) until approximately 3000 resonance units (RUs) were immobilized. The remaining active groups were quenched with ethanolamine. Next, a 40 μg / mL purified antibody solution was injected in the ligand direction at 25 μL / min for 240 seconds to indirectly capture the wild-type trastuzumab variant on the SPR surface, resulting in a surface area of ​​approximately 500 RU. After establishing a stable baseline by injecting buffer in the analyte direction, the analyte was injected at 50 μL / min for 120 seconds, followed by a 180-second dissociation phase, to obtain a series of binding sensograms. Five concentrations of FcγR in a 3-fold dilution series, with a nominal maximum concentration of 10 μM for all receptors except FcγR1a (30 nM), were used, and buffer was included for dual reference. The resulting Kd (affinity) values ​​were determined from the alignment and referenced sensograms using the Equilibrium Fit model in ProteOn™ Manager v3.1.0. The values ​​reported were the average of two or three independent runs.

[0603] Protocol 2 The affinity of FcγR for antibody Fc was measured by SPR using Biacore® 4000 (GE Healthcare, Little Chalfont, UK) at 25°C, with PBSTE (PBS containing 0.05% Tween-20 and 3.4 mM EDTA) as the running buffer. For anti-HER2 antibodies, recombinant HER2 extracellular domains (Merck, Darmstadt, Germany or ThermoFisher Scientific, Loughborough, UK) were immobilized on CM5 chips (GE Healthcare, Little Chalfont, UK) using amine coupling (EDC / NHS chemistry). Briefly, the CM5 sensor chip was activated with NHS / EDC, followed by injection of HER2 at 10.0 μg / mL in 10 mM NaOAc (pH 4.5). The immobilization level ranged from 1000 to 4000 RU. The remaining active groups were then quenched with ethanolamine. First, the antibody was captured on the immobilized surface of the tip by injecting it into the spot and flow cell at a flow rate of 10 μl / min for 35 seconds at approximately 15 μg / ml, with spot 3 serving as a blank for reference subtraction. The receptor was diluted in PBSTE buffer to a concentration range defined according to the expected affinity. Six concentrations, including zero, were used for each analyte. The contact time of the analytes was optimized according to the receptor used and its expected kinetics. For example, for FcγRIIb and FcγRIIaR, the contact time was 18 seconds at 30 μl / min. After injection of each analyte concentration, the tip surface was regenerated with 87 mM phosphate. Before testing, the tips were prepared by injecting 87 mM phosphate for 3 × 18 seconds. Double reference subtraction was performed (reference spot 3 and receptor concentration 0) to normalize the binding response at the antibody capture level. Samples were analyzed using either kinetics or a steady-state (equilibrium) fit model.

[0604] FcγRIIb binding and selectivity: Competitive electrochemiluminescence assay The relative affinity and selectivity of Fc variants to FcγRIIb compared to FcγRIIaR were measured by a competitive electrochemiluminescence assay using an MSD SECTOR 6000 Imager (Meso Scale Diagnostics, Rockville, USA). MSD standard-bound 384-well plates were coated overnight at 4°C with 10 nM soluble HER2 extracellular domain in PBS (Speed ​​Biosystems, Gaithersburg, USA), and then blocked for 1 hour with 3% bovine serum albumin in PBS containing 0.05% Tween-20 (Sigma Aldrich, Gillingham, UK). Test antibody variants in PBS (assay buffer) containing 0.5% BSA and 0.05% Tween-20 were applied to the plates at 100 nM and allowed to bind for 1 hour. After washing, biotinylated FcγRIIb extracellular domain-Fc fusions in assay buffer were added to each sample well and incubated for 1 hour in or without FcγRIIaR extracellular domain-Fc fusions. After washing, streptavidin-sulfotag (Meso Scale Diagnostics) in assay buffer diluted 1:2000 was added to each sample and the plate was incubated for 60 minutes. The plate was washed again, 1x Read Buffer T (Meso Scale Diagnostics) was added to each well, and the plate was read immediately. Data were analyzed for both the signal of samples incubated with biotinylated FcγRIIb-Fc receptor alone and compared to the control (considered an indicator of relative affinity of FcγRIIb) and the percentage of the same signal measured in the presence of non-biotinylated FcγRIIaR-Fc (considered an indicator of the selectivity of the Fc variant for FcγRIIb over FcγRIIaR). The experiments were conducted using both dose-response curves with a constant FcγRIIb-Fc concentration and varying FcγRIIaR-Fc concentrations, and "single-shot" assays with single concentrations of both FcγRIIb-Fc and FcγRIIaR-Fc.To screen for multiple variants, the receptor concentrations used in the single-shot assay were 10 nM biotinylated FcγRIIb-Fc and 100 nM FcγRIIaR-Fc.

[0605] FcRn binding The affinity of FcRn for antibody Fc was measured by SPR using a Biacore® T200 (GE Healthcare, Little Chalfont, UK) at 25°C, with HBS-EP+pH7.4 or MES pH6.0 as the running buffer. Samples were captured on immobilized protein L CM5 chips (GE Healthcare), but the 4G7 anti-CD19 antibody failed to capture. First, the antibody was captured on the immobilized surface of the chip by injecting it into spot and flow cells at a flow rate of 5 μl / min at approximately 15 μg / ml for 60 seconds. The receptor was diluted to the defined concentration range in HBS-EP+pH7.4 or MES pH6.0 buffer. Three concentrations (4096, 512, and 0 nM) were used for each analyte at pH7.4, and four concentrations (512, 64, 8, and 0 nM) were used for each analyte at pH6.0. After injecting each analyte concentration, the chip surface was regenerated with 10 mM glycine (pH 1.5). Results were analyzed using Biacore® T200 Evaluation V2 software and a 1:1 coupled kinetics model.

[0606] Differential Scanning Calorimetry (DSC) Protocol 1 Each antibody construct was diluted to 0.2 mg / mL in PBS, and a total of 400 μL was used for DSC analysis using VP-Capillary DSC (GE Healthcare). At the start of each DSC run, five buffer blank injections were performed to stabilize the baseline, and buffer injections were set up before each antibody injection for reference. Each sample was scanned from 20°C to 100°C at a rate of 60°C / hour using low feedback, an 8-second filter, a 5-minute preTstat, and a nitrogen pressure of 70 psi. The resulting thermograms were referenced and analyzed using Origin7 software (OriginLab Corporation, Northampton, MA).

[0607] Protocol 2 Antibody constructs were evaluated using the same method as described above for Protocol 1, except that antibody concentrations of 0.1 to 1.0 mg / ml were used, with concentrations of 0.4 mg / ml or higher being preferred.

[0608] Differential scanning fluorescence (DSF) 20 μL of purified sample (0.2–1.0 mg / mL) was added to 10 μL of SYPRO® Orange (Invitrogen, Paisley, UK), diluted from 5000x stock to 20x reverse osmosis (RO) water, and placed in a 96-well PCR plate with clear walls. The sample was incubated at 40°C for 5 minutes, and then the fluorescence emission of SYPRO® Orange was measured using a BioRad CFX Connect® RT-PCR instrument (BioRad, Watford, UK) at a rate of 15°C / hour between 40 and 95°C. Peaks were analyzed using Bio-Rad CFX Manager® version 3.1 to derive the temperature of the protein unfolding event, and then correlated this temperature with the unfolding of known domains within the protein.

[0609] Size exclusion chromatography (SEC) Using an Agilent 1100 HPLC system (Agilent, Stockport, UK), 10 μL of purified sample (concentration range of 0.2–2 mg / mL) was injected into a Supelco TSKgel® G3000 SWXL size exclusion column (Tosoh, Reading, UK). A mobile phase of 400 mM sodium phosphate and 200 mM NaCl (pH 6.8) was continuously flowed at 0.5 mL / min, with each sample run for 30 minutes. A diode array detector was connected to the flow line after the column, and UV / vis absorption at 210 nm and 280 nm was recorded. The obtained traces were integrated using Chemstation software (Agilent, Stockport, UK), and then analyzed using ChromView® software. Sample purity was recorded by classifying the area % of the main peak compared to the total % peak area of ​​molecular weights higher than the main peak and the total % peak area of ​​molecular weights lower than the main peak.

[0610] C1q binding The binding of the antibody construct to human C1q was evaluated by ELISA. The test antibody construct was coated into the wells of a 96-well flat-bottom Nunc Maxisorp® plate (Invitrogen, Paisley, UK) by adding 100 μl of 10 μg / ml test antibody in PBS to each well. The plate was sealed and incubated at 4°C for 16 hours. The plate was washed three times with 300 μl of 0.05% (v / v) Tween-20-containing PBS. The plate surface was then blocked by adding 200 μl of 1% (w / v) bovine serum albumin per well. The plate was incubated at ambient temperature for 1 hour and then washed in the same manner. Recombinant human C1q (C1740, Sigma Aldrich, Gillingham, UK) was diluted in 50 mM carbonic acid / bicarbonate buffer (C3041, Sigma Aldrich) to the final assay concentration and added at a rate of 100 μl per well. The samples were incubated at ambient temperature for 2 hours, and the plates were washed as before. Next, 100 μl of sheep anti-human C1q-HRP (Ab46191, AbCam, Cambridge, UK), diluted to 2 μg / ml in PBS, was added per well, and the samples were incubated at ambient temperature for 1 hour, after which the plates were washed as before. For detection, 100 μl of Sureblue® TMB (52-00-01, Seracare Life Sciences Inc., Milford, MA) was added per well, and the samples were incubated at ambient temperature for 20 minutes with stirring. The reaction was stopped by adding 100 μl of 1 M HCl to each well. The absorbance of each sample well was then measured at 450 nm using an M5e SpectraMax® plate reader (Molecular Devices, Wokingham, UK). For each antibody variant, seven C1q concentrations ranging from 2 μg / ml to 6 ng / ml with a half-log step, along with a control without C1q, were tested in duplicate. Data were analyzed using Prism (GraphPad, San Diego, CA). A four-parameter nonlinear regression model using absorbance and logarithmically transformed C1q concentrations was used to fit the coupled curves.The C1q concentration at which binding exceeds the threshold absorbance (0.5 OD, 17% of the maximum signal) was interpolated from the fitted curve. For screening, inter-sample comparisons were made using the signal at 2 μg / ml C1q as the baseline. Data were normalized as a percentage of WT.

[0611] Stress test Samples with normalized concentrations were subjected to stress for two weeks in both acidic and neutral buffers at 40°C (stressed condition) or 4°C (unstressed condition). Afterward, the 40°C samples were returned to 4°C. Changes in aggregation and fragmentation were evaluated by analytical SEC, and changes in binding to FcγRIIb were evaluated by SPR for both stressed and unstressed samples.

[0612] Aggregation and fragmentation were evaluated using a method similar to the SEC method described above. Briefly, using an Agilent 1100 HPLC system (Agilent, Stockport, UK), 10 μL of purified sample (1 mg / mL concentration) was injected into an ACQUITY® ULC® Protein BEH 200 4.6 × 150 mm size exclusion column (Waters Corporation, Elstree, UK), and 100 mM sodium phosphate and 350 mM NaCl (pH 6.8) were flowed as the mobile phase. A diode array detector was connected to the flow line after the column, and UV / vis absorption at 214 nm and 280 nm was recorded.

[0613] The binding of the sample to the FcγRIIb antigen was evaluated by SPR using Biacore® 8K+ (GE Healthcare, Little Chalfont, UK) at 25°C. This method evaluates the R of antigen binding to the capture antibody. maxThe effective concentration of the active sample is evaluated by utilizing the binding signal and comparing this signal to the signal of a standard curve of representative samples captured at different concentrations. In the data reported herein, the reference antibody was a heterodimer anti-CD19 antibody containing the symmetric E233D_G237D_P238D_H268D_P271G_A330R CH2 mutation, and was evaluated over a concentration range of 2.5 to 20 μg / ml. Test samples were each evaluated at a concentration of 10 μg / ml. The antibody was captured by injecting it into the surface of a Sensor Chip Protein A (GE Healthcare, Little Chalfont, UK) chip at 10 μl / min for 60 seconds. Subsequently, 20 μg / ml of FcγRIIb was injected onto the chip at 30 μl / min for 60 seconds. max The following was reported. Using the reference antibody value, standard curves were generated for both the antibody capture step and the antigen binding step. Then, the R of the test sample was derived from the standard curve. max The values ​​were interpolated and multiplied by the dilution factor required to dilute the sample from its original concentration to 10 ug / ml to obtain estimated antibody concentrations (from the antibody capture step) and relative antigen binding concentrations (from the antigen binding step). The loss of binding activity was calculated based on the difference in relative antigen binding concentrations of the sample under stress and non-stress conditions.

[0614] Example 1: Asymmetric point mutation 1.1 1x symmetric mutation Based on in silico analysis of the structure of the IgG1 Fc region bound to various Fcγ receptors, the lower hinge residue was identified as a candidate site for introducing mutations that alter the affinity and selectivity of FcγR. Variants containing the selected mutation in this region were constructed using a symmetric homodimer scaffold (scaffold 1), and the affinity and selectivity of these variants for FcγRIIb, FcγRIIaR, FcγRIIaH, and FcγRIIIa were experimentally determined by SPR (see General Methods, Protocol 1).

[0615] Table 1.1 shows the top mutations identified in this screening. G236 was identified as the most promising location in the lower hinge for introducing a mutation that drives FcγRIIb selectivity.

[0616] [Table 1.1]

[0617] 1.2 Asymmetric single CH2 mutation 1) System analysis of the Fc / FcγRIIb interface Using the crystal structure of a complex containing IgG1 Fc bound to FcγRIIb, we created a model suitable for in silico systematic screening. A cartoon representation of this model is shown in Figure 3.

[0618] A systematic systemic analysis of the Fc region and FcγRIIb interface was performed using a variety of in silicometrics, including sequence scoring, residue contact, and affinity degradation. Sequence scoring is based on the sequence identity of a given residue in the CH2 domain across various species and isotypes, with high sequence scores assigned to residues exhibiting high sequence conservation across species and isotypes. Residues with high sequence scores are often important for functionality, protein folding / stability, or both. Residue contact evaluates the interbinding affinity between residues. Residues located at the interface and exhibiting high binding affinity are considered hot spots ("H"), while residues located at the interface but exhibiting low binding affinity are considered cold spots ("C"). Affinity degradation expresses the contribution of each residue to the Fc / FcγRIIb complex using an energy term (kcal / mol). -1) This is how it is quantified. Residues with negative energy strengthen the complex, while high positive energy reflects the repulsive force between the residue and FcγRIIb.

[0619] The results of the system analysis are shown in Table 1.2.

[0620] [Table 1.2] TIFF2026143609000053.tif41165

[0621] 2) In silico 1x scan To identify residues that can increase the selectivity of the Fc region for FcγRIIb, systematic 1X scans were performed in silico. Numerous metrics were simultaneously evaluated, including AMBER energy (a combination of van der Waals (VdW) and Coulomb interactions) and knowledge-based latent metrics reflecting the likelihood of residues being in the same environment, based on insights from large databases such as the Protein Data Bank (PDB).

[0622] Table 1.3 summarizes the sites identified as potentially useful with this approach, along with the mutations at these sites that yielded favorable in silico metrics for selectivity to FcγRIIb compared to FcγRIIaR.

[0623] [Table 1.3]

[0624] 3) Mutations based on IgG4 The reported binding affinities of IgG1 and IgG4 to the Fcγ receptor indicate that IgG4 exhibits significant selectivity for FcγRIIb (see Table 1.4 below).

[0625] [Table 1.4]

[0626] Aligning the sequences of IgG1 and IgG4 reveals many differences in the lower hinge and CH2 region (see Figure 4).

[0627] Based on the above, the selectivity of IgG4 for FcγRIIb was investigated by selecting the following mutations and combinations of mutations: 1. Loop 3 mutations: A327G, A330S, P331S 2. Hinge mutation: L234F 3. Loop 1 mutations: H268Q, Q274K 4. Loop 3 mutation + Loop 1 mutation 5. Loop 3 mutation + Loop 1 mutation + Hinge mutation 6. Loop 3 mutation + Loop 1 mutation + Hinge mutation + Loop 2 mutation (F296Y)

[0628] 1.3 Deconvolution of Asymmetric Bonds The symmetry of homodimeric Fc antibodies and the structure of the Fc / FcγR complex revealed that there are at least two modes of binding of Fc to its receptor (see Figure 5). In asymmetric designs, the effect of asymmetric mutations must be evaluated for the binding modes of both the designed variant and its mirror variant. In silico data have shown that negative designs of asymmetric variants that prevent binding to a specific receptor often do not produce the same effect with mirror variants. Therefore, if the second binding mode is still acceptable, the specificity obtained by the asymmetric mutation may be lost.

[0629] The E269K mutation in the CH2 domain of the Fc region is known to inactivate binding to the Fcγ receptor when introduced symmetrically into both strands of the CH2 domain. When this mutation is introduced asymmetrically into only one of the two strands of the CH2 domain, it acts as a "polar driver," blocking FcγR binding on the side where the mutation is present, while the other side of Fc interacts with FcγR in the normal manner.

[0630] For each selected variant, the binding of the variant to FcγRIIb was deconvoluted and tested with the E269K polarity driver (PD) to determine whether the mutation was effective on strand A or strand B of Fc. A total of three constructs were required for each mutation, as shown in Table 1.5. X is the mutation being evaluated, and PD is the polarity driver.

[0631] [Table 1.5]

[0632] The wild-type P329 residue was identified as a hotspot mutation in Example 1.2 Part 1). Therefore, mutations at position P329 were tested not only with PD but also in the presence of binding enhancers. Mutations H268D and S267E have been shown to be binding enhancers for FcγRIIb, and the combination of these two mutations improves binding by 100-fold. Therefore, when testing P329 mutations, these two mutations were used as binding enhancers. PD is expected to reduce this 100-fold improvement in binding to 50-fold. Therefore, for P329 mutations, we evaluated their ability to reduce binding to FcγRIIaR / FcγRIIaH to below wild-type levels, while simultaneously reducing binding to FcγRIIb to near wild-type levels in the presence of binding enhancers and PD. The constructs tested for P329 mutations are shown in Table 1.6.

[0633] [Table 1.6]

[0634] The contribution of a given mutation to FcγR binding in each chain was determined, as described below with reference to Figure 6. The contribution of a given mutation was deconvoluted using three constructs. In Figure 6, mutation G236A was used as an exemplary mutation. G236A showed increased binding to the FcγRIIb receptor, but how this mutation drives selectivity was unclear. In all constructs shown in Figure 6, E269K was used as the polarity driver. This blocks binding to FcγR only in the binding mode closest to the receptor position L135 (and R134). This binding mode is indicated by an "x" in Figure 6. The names of chains A and B used below and in Figure 6 are based on the structure of the human IgG1 Fc / FcγRIII complex available in Protein Data Bank (PDB) ID 1E4K (see Figure 10; chain A features the hotspot P329 and chain B features the hotspot D270).

[0635] In Construct 1 of Figure 6, the G236A mutation is located in a different heavy chain than PD(E269K), so only a binding mode in which G236A is closer to the L135 residue of the receptor is permitted, as shown in the upper structure. In Construct 2, the G236A mutation is located in the same heavy chain as PD, so only a binding mode in which G236A is closer to the F163 residue of the receptor is permitted, as shown in the lower structure. In Construct 3, PD is tested alone, and binding is permitted only when PD is closer to the F163 residue of the receptor.

[0636] By comparing the binding of the three constructs, it is possible to deconvolve the contribution of the G236A mutation. If the mutation drives "strand A," construct 2 will show higher binding than construct 3, and the same should be true for construct 1. If the mutation drives "strand B," construct 1 will show higher binding than constructs 3 and 2. If the mutation is important for both strands, both constructs 1 and 2 will show better binding than construct 3. This analysis assumes independent, additive contributions. In the case of synergistic contributions, constructs 1 and 2 will both show the same binding as construct 3, but the symmetric construct will outperform all other constructs. The various possible results described above are summarized in Table 1.7.

[0637] [Table 1.7]

[0638] Variants containing asymmetric mutations were constructed using a one-arm antibody scaffold (Scaffold 2), and their FcγR binding was tested by SPR as described in the general method (Protocol 1). The thermal stability of the variants was also tested by DSF as described in the general method.

[0639] Table 1.8 shows the most selective variants identified using the above approach. Table 1.9 shows the results regarding the deconvolution of the mutations contained in these variants.

[0640] [Table 1.8]

[0641] [Table 1.9] TIFF2026143609000061.tif115165

[0642] Example 2: Loop Replacement The L3(FG) loop in the CH2 domain of the IgG Fc chain B (referred to herein as the "B / 325 loop") is not directly involved in FcγR binding (see Figure 2B) and contributes only minimally to CH2 domain stability. Therefore, this loop is an attractive target for manipulating FcγRIIb selectivity. Analysis of available crystal structures and anisosolar MD simulations performed on wild-type Fc / FcγR complexes have shown that residues in the B / 325 loop are typically far from position 135 of FcγR ("C / 135 site"). The typical minimum C between the target C / 135 site and the nearest residue on the B / 325 loop is... β -C β The distance was determined to be approximately 10 Å. Based on this structural analysis, the B / 325 loop was engineered to extend the loop to directly interact with the receptor near site C / 135, thereby driving selective binding to FcγRIIb. The engineering of the B / 325 loop was carried out according to the steps described below.

[0643] 2.1 Template Search The B / 325 loop in Fc can be extended by either inserting residues into the wild-type (WT) loop sequence or by replacing the WT loop with an entirely new loop or a combination of the loop and secondary structure. The approach taken here was to replace the entire L3 loop (positions 325-331) of WT Fc with a novel protein insert or “template.” A “template” is a polypeptide segment originating from an existing protein structure available from the Protein Data Bank (PDB). Figure 7 shows the names used to refer to the various parts, including templates.

[0644] The initial template identification process was intended to identify protein elements from their wild-type sequences that possess a conformation in which, when Fc binds, a portion of the...

Claims

1. A heterodimeric Fc variant comprising a first Fc polypeptide and a second Fc polypeptide, wherein the selectivity for binding to FcγRIIb is increased compared to the parent Fc region, One of the Fc polypeptides comprises a substitution of all or part of the native loop in the CH2 domain of the Fc polypeptide with an alternative amino acid sequence such that the length of the native loop is extended, and at least one of the amino acid residues of the alternative amino acid sequence is within a heavy atom distance of 3 Å from the target amino acid residue in FcγRIIb when the heterodimer Fc variant is bound to FcγRIIb. The aforementioned heterodimer Fc variant is a variant of immunoglobulin G (IgG) Fc. The aforementioned heterodimer Fc variant.

2. The heterodimer Fc variant according to claim 1, wherein the alternative amino acid sequence is a polypeptide having a length of 7 to 15 amino acids or 8 to 15 amino acids.

3. A heterodimer Fc variant comprising a first Fc polypeptide and a second Fc polypeptide, One of the Fc polypeptides includes substitution of amino acids 325 to 331 by a polypeptide having a length of 8 to 15 amino acids. The aforementioned heterodimer Fc variant exhibits increased selectivity for binding to FcγRIIb compared to the parent Fc region. The heterodimer Fc variant is a variant of immunoglobulin G (IgG)Fc, The amino acid numbering follows the EU index. The aforementioned heterodimer Fc variant.

4. The polypeptide described above, (a) an amino acid sequence described in any one of sequence numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (b) an amino acid sequence which is a variant of the sequence described in any one of sequence numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, wherein the variant comprises 1, 2, 3, 4 or 5 amino acid mutations. A heterodimer Fc variant according to claim 3, comprising:

5. The heterodimer Fc variant according to claim 3, wherein the polypeptide comprises the amino acid sequence of formula (I), formula (Ia), formula (Ib), formula (II), formula (III), formula (IV), formula (V), or formula (VI): Equation (I): X １ X ２ WX ３ X ４ X ５ GX ６ X ７ T(I) (In the formula, X １ is A, D, N, or S, X ２ is A, D, E, F, H, I, L, N, Q, S, T, V, W, or Y, X ３ is A, D, E, F, H, I, N, Q, S, T, V, W, or Y, X ４ is D, E, G, I, L, P, or Q, X ５ is A, D, E, G, H, K, N, R, S, T, or Y, X ６ is A, D, E, F, H, P, W, or Y, X ７ (The letters are A, D, E, F, G, H, K, L, N, Q, or R.) Equation (Ia): X １ X ２ WX ３ X ４ X ５ GYX ６ T(Ia) (In the formula, X １ is A, D, N, or S, X ２ is A, D, E, F, H, I, L, N, Q, S, T, V, W, or Y, X ３ is A, D, E, F, H, I, N, Q, S, T, V, W, or Y, X ４ is D, E, G, I, L, P, or Q, X ５ is A, D, E, G, H, K, N, R, S, T, or Y, X ６ (The letters are A, D, E, F, G, H, K, L, N, Q, or R.) Formula (Ib): X １ X ２ WX ３ X ４ GGYX ５ T(Ib) (In the formula, X １ is A or S, X ２ is A, D, E, F, H, I, L, N, Q, T, V, or W. X ３ is D, E, F, H, N, Q, S, T, or Y, X ４ is D, G, I, or L, X ５ (The letters are A, F, H, K, L, or N.) Formula (II): X １ LDX ２ X ３ GKGX ４ V(II) (In the formula, X １ is F or G, X ２ is E, H, Q, or T, X ３ is E, N, R, S, or T, X ４ (This is A, Y, or V) Formula (III): X １ TDEX ２ GKGX ３ T(III) (In the formula, X １ is F or G, X ２ is E or N, X ３ (is A or V) Formula (IV): X １ FX ２ X ３ X ４ X ５ GEVV(IV) (In the formula, X １ is A or D, X ２ is either D or N, X ３ is D, E, H, N, P, Q, S, or T, X ４ is D, E, N, S, or T, X ５ (This is either D or Q) Formula (V): X １ TDX ２ X ３ X ４ GEVT(V) (In the formula, X １ is A or D, X ２ is D, P, or Q, X ３ is D, E, or N, X ４ (This is either D or Q) Equation (VI): LTD6 １ 6 ２ GX ３ PX ４ R (VI) (In the formula, X １ is E or H, X ２ is D, E, or N, X ３ is R or S, X ４ (This is I, Q, or Y).

6. The polypeptide described above, (a) an amino acid sequence described in any one of SEQ ID NOs: 4 to 172, or (b) an amino acid sequence described in any one of Sequence IDs 4 to 90, or (c) Sequence numbers 6, 8, 9, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, An amino acid sequence described in any one of the following: 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90, or (d) The amino acid sequence described in any one of the following sequence numbers: 6, 8, 47, 68, or 73 The heterodimer Fc variant according to claim 5, comprising:

7. The heterodimer Fc variant according to any one of claims 3 to 6, further comprising one or more additional amino acid mutations in the CH2 domain of the heterodimer Fc variant.

8. The heterodimer Fc variant according to claim 7, wherein the one or more additional amino acid mutations include a mutation at position 236.

9. The heterodimer Fc variant according to claim 8, wherein the mutations at position 236 of the first and second Fc polypeptides are symmetrical.

10. The heterodimer Fc variant according to claim 9, wherein the mutation at position 236 is selected from G236D, G236N, and G236K.

11. The heterodimer Fc variant according to claim 8, wherein the mutations at position 236 of the first and second Fc polypeptides are asymmetric.

12. The above substitutions of amino acids 325-331 are present in the second Fc polypeptide. (a) The first Fc polypeptide contains a mutation at position 236 selected from G236A, G236D, G236E, G236F, G236H, G236I, G236L, G236N, G236P, G236Q, G236S, G236T, G236V, G236W and G236Y, and the second Fc polypeptide contains a mutation at position 236 selected from G236D, G236E, G236K, G236N and G236T, or (b) The first Fc polypeptide contains a mutation at position 236 selected from G236A, G236D, G236E, G236F, G236H, G236I, G236L, G236N, G236P, G236Q, G236S, G236T, G236V, G236W and G236Y, and the second Fc polypeptide contains the mutation G236D or does not contain the mutation at position 236. The heterodimer Fc variant according to claim 11.

13. The heterodimer Fc variant according to any one of claims 3 to 12, wherein the substitution of amino acids 325 to 331 is in the second Fc polypeptide, and the second Fc polypeptide further comprises one or more mutations selected from S239D, S239E, V266I, V266L, S267A, S267I, S267V, S267Q and H268D.

14. The heterodimer Fc variant according to any one of claims 3 to 13, wherein the substitution of amino acids 325 to 331 is in the second Fc polypeptide, and the first Fc polypeptide further comprises a mutation at one or more positions 234, 235, 237, and 239.

15. (i) The variation at position 234 is selected from L234A, L234D, L234E, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, (ii) The variation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235I, L235N, L235P, L235Q, L235S, L235T, L235V, L235W and L235Y, (iii) The variation at position 237 is selected from G237A, G237D, G237F, G237H, G237L, G237N, G237P, G237S, G237V, G237W and G237Y, (iv) The variation at position 239 is selected from S239A, S239D, S239E, S239F, S239G, S239H, S239I, S239L, S239N, S239Q, S239R, S239T, S239V, S239W and S239Y. The heterodimer Fc variant according to claim 14.

16. The heterodimer Fc variant according to any one of claims 3 to 15, wherein the substitution of amino acids 325 to 331 is present in the second Fc polypeptide, and the second Fc polypeptide further comprises a mutation at one or more of the positions 234, 235, 237, 240, 263, 264, 266, 269, 271, 273, 323, and 332.

17. (i) The variation at position 234 is selected from L234A, L234E, L234F, L234G, L234H, L234I, L234K, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, (ii) The variation at position 235 is selected from L235A, L235D, L235F, L235G, L235N, L235S, L235W and L235Y, (iii) The variation at position 237 is selected from G237F, G237I, G237K, G237L, G237Q, G237T, G237V and G237Y, (iv) The mutation at position 240 is selected from V240I and V240L, (v) The variation at position 263 is V263T, (vi) The variation at position 264 is V264T, (vii) The mutation at position 266 is V266I, (viiii) The mutation at position 269 is E269Q, The mutation at position (ix) 271 is P271D, (x) The variation at position 273 is selected from V273A and V273I, (xi) The variation at position 323 is selected from V323A and V323I, (xii) The variation at position 332 is selected from I332F and I332L. The heterodimer Fc variant according to claim 16.

18. A method for preparing a heterodimer Fc variant comprising a first Fc polypeptide and a second Fc polypeptide, which exhibit increased selectivity for a target receptor compared to the parent Fc region, (a) Using an in silico model of the parent Fc region complexed with the target receptor, (i) To provide a variant candidate, insert a sequence of one or more amino acid residues into one of the natural loops of the Fc polypeptide such that the length of the natural loop is extended. (ii) Determine the distance from the target amino acid residue in the receptor to at least one of the amino acid residues of the insertion sequence, (iii) If at least one amino acid residue of the insertion sequence is within a heavy atom distance of 3 Å from the target amino acid residue in the receptor, the variant candidate is selected as the heterodimer Fc variant, (b) preparing nucleic acids encoding the heterodimer Fc variant, (c) Expressing the nucleic acid in a host cell in order to provide the heterodimer Fc variant Includes, Here, the target receptor is FcγRIIb. The aforementioned method.

19. A heterodimeric Fc variant comprising a first Fc polypeptide and a second Fc polypeptide, having increased selectivity for binding to FcγRIIb compared to the parent Fc region, and including an asymmetric mutation at position 236, One of the Fc polypeptides comprises mutant G236N or G236D, The heterodimer Fc variant is a variant of immunoglobulin G (IgG)Fc, The amino acid numbering follows the EU index. The aforementioned heterodimer Fc variant.

20. (a) The first Fc polypeptide contains mutation G236N or G236D, and the second Fc polypeptide does not contain the mutation at position 236, or (b) The first Fc polypeptide contains mutation G236N or G236D, and the second Fc polypeptide contains a different mutation at position 236, or (c) The first Fc polypeptide contains mutant G236N, and the second Fc polypeptide contains mutant G236D, G236K, or G236S, or (d) The first Fc polypeptide contains mutant G236N and the second Fc polypeptide contains mutant G236D, or (e) The first Fc polypeptide comprises mutant G236D, and the second Fc polypeptide comprises mutant G236N, G236Q, G236K, G236E, or G236H The heterodimer Fc variant according to claim 19.

21. The heterodimer Fc variant according to claim 19 or 20, wherein the first Fc polypeptide and / or the second Fc polypeptide further comprises one or more additional amino acid mutations in the CH2 domain of the heterodimer Fc variant.

22. The heterodimer Fc variant according to claim 21, wherein the second Fc polypeptide further comprises one or more mutations selected from S239D, S239E, V266I, V266L, S267A, S267I, S267V, S267Q, and H268D.

23. The second Fc polypeptide is a) Mutation S239D or S239E; or b) Mutation H268D, or c) Mutations S239D or S239E, and mutation H268D The heterodimer Fc variant according to claim 22, further comprising:

24. A heterodimer Fc variant according to any one of claims 19 to 23, which is a strategy 1 / 3 variant.

25. The heterodimer Fc variant according to any one of claims 19 to 24, wherein the second Fc polypeptide further comprises mutant S267A, S267I, or S267V.

26. The heterodimer Fc variant according to any one of claims 19 to 25, wherein amino acids 325 to 331 in the second Fc polypeptide are replaced with polypeptides having a length of 8 to 15 amino acids.

27. The polypeptide described above, (a) an amino acid sequence described in any one of sequence numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (b) an amino acid sequence which is a variant of the sequence described in any one of sequence numbers 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, wherein the variant comprises 1, 2, 3, 4 or 5 amino acid mutations. The heterodimer Fc variant according to claim 26, comprising:

28. The heterodimer Fc variant according to claim 26, wherein the polypeptide comprises the amino acid sequence of formula (I), formula (Ia), formula (Ib), formula (II), formula (III), formula (IV), formula (V), or formula (VI): Equation (I): X １ X ２ WX ３ X ４ X ５ GX ６ X ７ T(I) (In the formula, X １ is A, D, N, or S, X ２ is A, D, E, F, H, I, L, N, Q, S, T, V, W, or Y, X ３ is A, D, E, F, H, I, N, Q, S, T, V, W, or Y, X ４ is D, E, G, I, L, P, or Q, X ５ is A, D, E, G, H, K, N, R, S, T, or Y, X ６ is A, D, E, F, H, P, W, or Y, X ７ is A, D, E, F, G, H, K, L, N, Q or R) Equation (Ia): X １ X ２ WX ３ X ４ X ５ GYX ６ T(Ia) (In the formula, X １ is A, D, N or S, X ２ is A, D, E, F, H, I, L, N, Q, S, T, V, W, or Y, X ３ is A, D, E, F, H, I, N, Q, S, T, V, W, or Y, X ４ is D, E, G, I, L, P, or Q, X ５ is A, D, E, G, H, K, N, R, S, T, or Y, X ６ (The letters are A, D, E, F, G, H, K, L, N, Q, or R.) Formula (Ib): X １ X ２ WX ３ X ４ GGYX ５ T(Ib) (In the formula, X １ is A or S, X ２ is A, D, E, F, H, I, L, N, Q, T, V, or W. X ３ is D, E, F, H, N, Q, S, T, or Y, X ４ is D, G, I, or L, X ５ (The letters are A, F, H, K, L, or N.) Formula (II): X １ LDX ２ X ３ GKGX ４ V(II) (In the formula, X １ is F or G, X ２ is E, H, Q, or T, X ３ is E, N, R, S, or T, X ４ (This is A, Y, or V) Formula (III): X １ TDEX ２ GKGX ３ T(III) (In the formula, X １ is F or G, X ２ is E or N, X ３ (is A or V) Formula (IV): X １ FX ２ X ３ X ４ X ５ GEVV(IV) (In the formula, X １ is A or D, X ２ is either D or N, X ３ is D, E, H, N, P, Q, S, or T, X ４ is D, E, N, S, or T, X ５ (This is either D or Q) Formula (V): X １ TDX ２ X ３ X ４ GEVT(V) (In the formula, X １ is A or D, X ２ is D, P, or Q, X ３ is D, E, or N, X ４ (This is either D or Q) Equation (VI): LTD6 １ 6 ２ GX ３ PX ４ R (VI) (In the formula, X １ is E or H, X ２ is D, E, or N, X ３ is R or S, X ４ (This is I, Q, or Y).

29. The polypeptide described above, (a) an amino acid sequence described in any one of SEQ ID NOs: 4 to 172, or (b) an amino acid sequence described in any one of Sequence IDs 4 to 90, or (c) Sequence numbers 6, 8, 9, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, An amino acid sequence described in any one of the following: 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90, or (d) The amino acid sequence described in any one of the following sequence numbers: 6, 8, 47, 68, or 73 The heterodimer Fc variant according to claim 26, comprising:

30. The heterodimer Fc variant according to any one of claims 19 to 29, wherein the second Fc polypeptide further comprises the mutant S267V.

31. The heterodimer Fc variant according to any one of claims 19 to 30, wherein the first Fc polypeptide and / or the second Fc polypeptide further comprises a mutation at position 237.

32. (a) The first Fc polypeptide or the second Fc polypeptide contains the G236N mutation, and the same Fc polypeptide further contains a mutation selected from G237A, G237D, G237F, G237H, G237L, G237N, G237P, G237S, G237V, G237W and G237Y, or (b) The first Fc polypeptide or the second Fc polypeptide comprises the mutation G236D, and the same Fc polypeptide further comprises a mutation selected from G237F, G237I, G237K, G237L, G237Q, G237T, G237V and G237Y. The heterodimer Fc variant according to claim 31.

33. The heterodimer Fc variant according to any one of claims 19 to 31, wherein the first Fc polypeptide comprises mutation G236N, and the first Fc polypeptide further comprises a mutation at one or more of positions 234, 235, 237, and 239.

34. (i) The variation at position 234 is selected from L234A, L234D, L234E, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, (ii) The variation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235I, L235N, L235P, L235Q, L235S, L235T, L235V, L235W and L235Y, (iii) The variation at position 237 is selected from G237A, G237D, G237F, G237H, G237L, G237N, G237P, G237S, G237V, G237W and G237Y, (iv) The variation at position 239 is selected from S239A, S239D, S239E, S239F, S239G, S239H, S239I, S239L, S239N, S239Q, S239R, S239T, S239V, S239W and S239Y. The heterodimer Fc variant according to claim 33.

35. The heterodimer Fc variant according to any one of claims 19 to 31, 33, and 34, wherein the second Fc polypeptide comprises mutation G236D, and the second Fc polypeptide further comprises a mutation at one or more of the positions 234, 235, 237, 240, 263, 264, 266, 269, 271, 273, 323, and 332.

36. (i) The variation at position 234 is selected from L234A, L234E, L234F, L234G, L234H, L234I, L234K, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, (ii) The variation at position 235 is selected from L235A, L235D, L235F, L235G, L235N, L235S, L235W and L235Y, (iii) The variation at position 237 is selected from G237F, G237I, G237K, G237L, G237Q, G237T, G237V and G237Y, (iv) The mutation at position 240 is selected from V240I and V240L, (v) The variation at position 263 is V263T, (vi) The variation at position 264 is V264T, (vii) The mutation at position 266 is V266I, (viiii) The mutation at position 269 is E269Q, The mutation at position (ix) 271 is P271D, (x) The variation at position 273 is selected from V273A and V273I, (xi) The variation at position 323 is selected from V323A and V323I, (xi) The variation at position 332 is selected from I332F and I332L. The heterodimer Fc variant according to claim 35.

37. A heterodimer Fc variant according to claim 3 or 19, comprising an amino acid mutation described in any one of the variants shown in Table 6.22, Table 6.24, Table 6.25, or Table 6.

27.

38. (i) The first Fc polypeptide contains mutation G236N_G237D, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D (Variant 31186), (ii) The first Fc polypeptide contains mutation L235F_G236N_G237A, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D (Variant 31187), (iii) The first Fc polypeptide contains mutation L235F_G236N_G237A, and the second Fc polypeptide contains mutation template 1 (G330 ＊ K) + G236D_G237F_S239D_S267V_H268D (Variant 31188) or (iv) The first Fc polypeptide contains mutation G236N_G237D, and the second Fc polypeptide contains mutation template 7 (E328 ＊ H_E329 ＊ R_A331 ＊ BY) + G236D_G237F_S239D_S267V_H268D (Variant 31191) or (v) The first Fc polypeptide contains mutation L235F_G236N_G237A, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 31213), (vi) The first Fc polypeptide contains mutation L235F_G236N_G237A_T250V_A287F, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_T250V_S267V_H268D_A287F (Variant 31274), (vii) The first Fc polypeptide contains mutation L235F_G236N_G237A_T250V_M428F, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_T250V_S267V_H268D_M428F (Variant 31275), (viiii) The first Fc polypeptide contains mutation L235F_G236N_G237A_A287F_M428F, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_A287F_M428F (variant 31276), (ix) The first Fc polypeptide contains mutation G236N_G237D, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32210), (x) The first Fc polypeptide contains mutation G236N_G237E, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32211), (xi) The first Fc polypeptide contains mutation G236N, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32212), (xi) The first Fc polypeptide contains mutation L235D_G236N_G237A, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32226), (xiii) The first Fc polypeptide contains mutation L235E_G236N_G237A, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32227), (xiv) The first Fc polypeptide contains mutation L235V_G236N_G237A, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32230), (xv) The first Fc polypeptide contains mutation L235Y_G236N_G237A, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32231), (xvi) The first Fc polypeptide contains the mutation G236N_G237A_S239P, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (variant 32242), (xvii) The first Fc polypeptide contains mutation L234D_G236N_G237A, and the second Fc polypeptide contains mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32282), (xviiii) The first Fc polypeptide contains mutation L235D_G236N_G237A, and the second Fc polypeptide contains mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32284), (xix) The first Fc polypeptide contains the mutation G236N_G237A_S239G, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32287), (xx) The first Fc polypeptide contains the mutation G236N_G237A_S239H, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32288), (xxi) The first Fc polypeptide contains the mutation G236N_G237E, and the second Fc polypeptide contains the mutation template 7+G236D_G237F_S239D_S267V_H268D (variant 32296), (xxii) The first Fc polypeptide contains mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D (Variant 31192), (xxiii) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32292), (xxiv) The first Fc polypeptide contains the mutation L234F_G236N_S267A_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32293), (xxv) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_A330T_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32294), or (xxvi) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_P329I_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Including +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32295), The heterodimer Fc variant according to claim 3 or 19.

39. A heterodimer Fc variant according to any one of claims 19 to 23, which is a strategy 2 variant.

40. The heterodimer Fc variant according to any one of claims 19 to 23 and 39, wherein the first Fc polypeptide further comprises a mutation at one or more positions selected from 234, 268, 327, 330, and 331.

41. (i) The variation at position 234 is selected from L234A, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, (ii) The mutation at position 268 is selected from H268A, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268N, H268P, H268Q, H268R, H268S, H268T, H268V, H268W and H268Y, (iii) The variation at position 327 is selected from A327E and A327G, (iv) The variation at position 330 is selected from A330K, A330H, A330Q, A330R, A330S and A330T, (v) The variation at position 331 is selected from P331A, P331D, P331E, P331H, P331Q and P331S. The heterodimer Fc variant according to claim 40.

42. The heterodimer Fc variant according to any one of claims 19-23 and 39-41, wherein the second Fc polypeptide further comprises mutant S267A or S267Q.

43. The heterodimer Fc variant according to any one of claims 19-23 and 39-42, wherein the second Fc polypeptide further comprises mutant V266L.

44. The heterodimer Fc variant according to any one of claims 19 to 23 and 39 to 43, wherein the first Fc polypeptide further comprises a mutation at one or more of the positions 235, 237, 239, 264, 266, 267, 269, 270, 271, 272, 273, 323, 326 and / or 332.

45. (i) The variation at position 235 is selected from L235A, L235D, L235E, L235F, L235H, L235I, L235P, L235Q, L235S, L235T, L235V, L235W and L235Y, (ii) The variation at position 237 is selected from G237A, G237F, G237L, G237N, G237T, G237W, and G237Y, (iii) The variation at position 239 is selected from S239A, S239D, S239E, S239G, S239I, S239L, S239N, S239Q, S239R and S239V, (iv) The mutation at position 264 is selected from V264A, V264F, V264I, V264L, and V264T. (v) The variation at position 266 is V266I, (vi) The variation at position 267 is selected from S267A, S267G, S267H, S267I, S267N, S267P, S267T and S267V, (vii) The variation at position 269 is selected from E269A, E269D, E269F, E269G, E269H, E269I, E269K, E269L, E269N, E269P, E269Q, E269R, E269S, E269T, E269V, E269W and E269Y, (viiii) The variation at position 270 is selected from D270A, D270E, D270F, D270H, D270I, D270N, D270Q, D270S, D270T, D270W and D270Y, (ix) The variation at position 271 is selected from P271D, P271E, P271G, P271H, P271I, P271K, P271L, P271N, P271Q, P271R, P271V and P271W, (x) The variation at position 272 is selected from E272A, E272D, E272F, E272G, E272H, E272I, E272L, E272N, E272S, E272T, E272V, E272W and E272Y, (xi) The variation at position 273 is V273A, (xi) The variation at position 323 is selected from V323A, V323I and V323L, (xiii) The variation at position 326 is selected from K326A, K326D, K326H, K326N, K326Q, K326R, K326S and K326T, (xiv) The mutation at position 332 is selected from I332A, I332L, I332T, and I332V. The heterodimer Fc variant according to claim 44.

46. The heterodimer Fc variant according to any one of claims 19-23 and 39-45, wherein the second Fc polypeptide further comprises a mutation at one or more positions selected from 234, 235, 237, 240, 264, 269, 271, 272, and 273.

47. (i) The variation at position 234 is selected from L234A, L234D, L234E, L234F, L234G, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, (ii) The variation at position 235 is selected from L235A, L235D, L235F, L235G, L235H, L235N, L235W and L235Y, (iii) The variation at position 237 is selected from G237A, G237D, G237E, G237F, G237H, G237I, G237K, G237L, G237N, G237Q, G237R, G237S, G237T, G237V, G237W and G237Y, (iv) The mutation at position 240 is selected from V240I, V240L and V240T, (v) The variation at position 264 is selected from V264L and V264T, (vi) The variation at position 269 is selected from E269D, E269T and E269V, (vii) The mutation at position 271 is P271G, (viiii) The variation at position 272 is selected from E272A, E272D, E272I, E272K, E272L, E272P, E272Q, E272R, E272T and E272V, (ix) The variation at position 273 is selected from V273A, V273I, V273L, and V273T. The heterodimer Fc variant according to claim 46.

48. The heterodimer Fc variant according to any one of claims 19-23 and 39-47, wherein amino acids 325-331 in the second Fc polypeptide are replaced with polypeptides having a length of 8-15 amino acids.

49. The heterodimer Fc variant according to claim 19, comprising an amino acid mutation described in either Table 6.23 or Table 6.

26.

50. (i) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation G236D_G237L_S239D_V266L_S267A_H268D (variant 31190), (ii) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_P329I_A330K_P331S, and the second Fc polypeptide contains the mutation G236D_G237D_S239D_V266L_S267A_H268D (variant 31256), (iii) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_P329A_A330K_P331S, and the second Fc polypeptide contains the mutation G236D_G237L_S239D_V266L_S267A_H268D (variant 32274), (iv) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D (Variant 31192), (v) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32292), (vi) The first Fc polypeptide contains the mutation L234F_G236N_S267A_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32293), (vii) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_A330T_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32294), or (viiii) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_P329I_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Including +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32295), The heterodimer Fc variant according to claim 19.

51. (a) The first Fc polypeptide comprises the mutation G236N and a mutation at one or more positions selected from 234, 268, 327, 330, and 331, wherein, (i) The variation at position 234 is selected from L234A, L234F, L234G, L234H, L234I, L234N, L234P, L234Q, L234S, L234T, L234V, L234W and L234Y, (ii) The mutation at position 268 is selected from H268A, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268N, H268P, H268Q, H268R, H268S, H268T, H268V, H268W and H268Y, (iii) The variation at position 327 is selected from A327G and A327E, (iv) The variation at position 330 is selected from A330K, A330H, A330Q, A330R, A330S and A330T, (v) The variation at position 331 is selected from P331A, P331D, P331E, P331H, P331Q and P331S, (b) The second Fc polypeptide is (i) Mutation G236D and (ii) A substitution of the native loop at positions 325-331 with a polypeptide of 8-15 amino acid length, wherein the polypeptide is derived from a loop-forming segment of a second protein, and the loop-forming segment comprises an amino acid sequence described in any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid mutations, (iii) One or more mutations selected from S239D, S239E, V266I, S267I, S267Q, S267V and H268D including, The heterodimer Fc variant according to claim 19.

52. (i) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D (Variant 31192), (ii) The first Fc polypeptide contains the mutation L234F_L235D_G236N_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32292), (iii) The first Fc polypeptide contains the mutation L234F_G236N_S267A_H268Q_A327G_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32293), (iv) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_A330T_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Includes +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32294), or (v) The first Fc polypeptide contains the mutation L234F_G236N_H268Q_A327G_P329I_A330K_P331S, and the second Fc polypeptide contains the mutation template 1 (D329 ＊ I) Including +G236D_G237F_S239D_S267V_H268D_I332L (Variant 32295), The heterodimer Fc variant according to claim 51.

53. The heterodimer Fc variant according to claim 19, comprising an amino acid mutation described in any one of the variants shown in Table 13.

1.

54. The heterodimer Fc variant according to any one of claims 1 to 17 and 19 to 53, wherein the first Fc polypeptide and the second Fc polypeptide further comprise one or more mutations selected from A287F, T250V, L309Q, and M428F.

55. The heterodimer Fc variant according to claim 54, wherein the first Fc polypeptide and the second Fc polypeptide further comprise the mutant A287F / M428F, A287F / T250V, M428F / T250V, or T250V / L309Q.

56. A heterodimer Fc variant according to any one of claims 1 to 17 and 19 to 55, which is a variant of IgG1 Fc.

57. A heterodimer Fc variant according to claim 56, which is a variant of human IgG1 Fc.

58. The selectivity of the heterodimer Fc variant to binding to FcγRIIb is increased by at least 1.5 times compared to the parent Fc region, where, FcγRIIb selectivity ratio (Fold Difference) = FcγRIIb affinity ratio / FcγRIIaR affinity ratio ratio And, During the ceremony, FcγRIIb affinity factor difference = K Ｄ FcγRIIb (parent) / K Ｄ FcγRIIb (variant) and FcγRIIaR affinity factor difference = K Ｄ FcγRIIaR (parent) / K Ｄ FcγRIIaR (variant) The heterodimer Fc variant according to any one of claims 1 to 17 and 19 to 57.

59. A heterodimer Fc variant according to any one of claims 1 to 17 and 19 to 58, wherein the binding affinity to FcγRIIb is increased compared to the parent Fc region.

60. The binding affinity of the heterodimer Fc variant to FcγRIIb is increased by at least 10 times compared to the parent Fc region, where, FcγRIIb affinity factor difference = K Ｄ FcγRIIb (parent) / K Ｄ FcγRIIb (variant) The heterodimer Fc variant according to claim 59.

61. A polypeptide comprising a heterodimer Fc variant according to any one of claims 1 to 17 and 19 to 60, and one or more protein moieties fused to or covalently bonded to the heterodimer Fc variant.

62. The polypeptide according to claim 61, wherein the polypeptide is an antibody, and the one or more protein portions are one or more antigen-binding domains.

63. The polypeptide according to claim 62, wherein at least one of the antigen-binding domains binds to a tumor-associated antigen or a tumor-specific antigen.

64. A pharmaceutical composition comprising a heterodimer Fc variant according to any one of claims 1 to 17 and 19 to 60 or a polypeptide according to any one of claims 61 to 63, and a pharmaceutically acceptable carrier or diluent.

65. A polypeptide according to any one of claims 61 to 63, for use in treatment.

66. The polypeptide according to claim 63 for use in the treatment of cancer.

67. A method for treating cancer in a subject in need thereof, comprising administering an effective amount of the polypeptide described in claim 63 to the subject.

68. A nucleic acid encoding a heterodimer Fc variant according to any one of claims 1 to 17 and 19 to 60, or a polypeptide according to any one of claims 61 to 63.

69. A host cell comprising the nucleic acid described in claim 68.

70. A method for preparing a heterodimer Fc variant according to any one of claims 1 to 17 and 19 to 60, or a polypeptide according to any one of claims 61 to 63, the method comprising expressing a nucleic acid encoding the heterodimer Fc variant or the polypeptide in a host cell.

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