Immunoglobulin Fc region variants containing stability-enhancing mutations

Abstract

Fc variants are described that comprise one or more amino acid mutations that increase the stability of the Fc variant compared to a parent Fc that does not contain the one or more amino acid mutations, as well as polypeptides comprising the Fc variants and polynucleotides encoding the Fc variants.

Description

[Technical Field]

[0001] Field The present disclosure relates to the field of immunoglobulins, and in particular to immunoglobulin Fc variants that contain stability-enhancing mutations. [Background technology]

[0002] background Immunoglobulin-based drugs are becoming an increasingly important therapeutic approach, and monoclonal antibodies have been recognized as the primary treatment modality for a variety of diseases over the past 25 years.

[0003] A significant amount of research has been directed at engineering immunoglobulins to improve various functions. For example, modifications have been made to the antibody Fc region to improve pharmacokinetics, enhance or reduce antibody-dependent cellular cytotoxicity (ADCC) activity, enhance or reduce selectivity for specific Fcγ or FcRn receptors, or improve the formation of heterodimeric Fc regions in bispecific antibodies. However, such modifications may adversely affect other properties of the antibody, including thermal stability.

[0004] Efforts to improve the stability of engineered antibodies include the introduction of mutations that provide new disulfide bonds (Gong et al., 2009, J. Biol. Chem., 284(21):14203-14210 (Non-Patent Document 1); Jacobsen et al., 2017, J. Biol. Chem., 292(5):1865-1875 (Non-Patent Document 2)) and the introduction of combinations of point mutations (International Patent Application Publication No. WO2012 / 032080 (Patent Document 1)). A method for improving the stability of antibody Fc regions by introducing various amino acid substitutions into the loop regions of the antibody Fc region has also been described (U.S. Patent Application Publication No. 2015 / 0210763 (Patent Document 2)).

[0005] This background information is provided for the purpose of providing known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily made that any of the preceding information is intended, nor should it be construed, to constitute prior art against the claimed invention. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Patent Application Publication No. WO2012 / 032080 [Patent Document 2] U.S. Patent Application Publication No. 2015 / 0210763 [Non-patent literature]

[0007] [Non-Patent Document 1] Gong et al.,2009,J Biol Chem,284(21):14203-14210 [Non-patent document 2] Jacobsen et al.,2017,J Biol Chem,292(5):1865-1875 Summary of the Invention

[0008] overview Immunoglobulin Fc region variants comprising stability-enhancing mutations are described herein. One embodiment of the present disclosure includes a mutation at position 250, wherein the mutation is a substitution of the amino acid at position 250 with Ala, Ile, or Val; a mutation at position 250, wherein the mutation is a substitution of the amino acid at position 287 with Phe, His, Met, Trp, or Tyr; a mutation at position 287, wherein the mutation is a substitution of the amino acid at position 308 with Ile; a mutation at position 308, wherein the mutation is a substitution of the amino acid at position 309 with Gln or Thr. The present invention relates to an Fc variant comprising one or more stability-enhancing amino acid mutations selected from a mutation at position 309, a mutation at position 428 where the mutation is a substitution of the amino acid at position 428 with Phe, and a pair of mutations at positions 242 and 336 where both mutations are substitutions with Cys, wherein the Fc variant has an increased CH2 domain melting temperature (Tm) compared to a parent Fc that does not contain the one or more stability-enhancing amino acid mutations.

[0009] Another embodiment of the present disclosure is an Fc variant comprising one to three stability-enhancing amino acid mutations, the mutations being one or more selected from (a) a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with He, and a mutation at position 309 that is a substitution with Gln or Thr, or (b) a mutation at position 250 that is a substitution with Ala, He, or Val, a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with He, a mutation at position 309 that is a substitution with Gln or Thr, and a mutation at position 428 that is a substitution with Phe, and both are substitutions with Cys. or (c) a pair of mutations at positions 242 and 336, both of which are substitutions with Cys, and three or more mutations including a mutation at position 250 which is substitution with Ala, Ile, or Val, a mutation at position 287 which is substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 which is substitution with Ile, a mutation at position 309 which is substitution with Gln or Thr, and a mutation at position 428 which is substitution with Phe, wherein the Fc variant has an increased CH2 domain melting temperature (Tm) compared to a parent Fc that does not contain one or more stability-enhancing amino acid mutations.

[0010] Another aspect of the present disclosure relates to a polypeptide comprising an Fc variant described herein and one or more proteinaceous moieties fused or covalently linked to the Fc variant.

[0011] Another aspect of the present disclosure pertains to a polynucleotide or set of polynucleotides that encode the Fc variants described herein.

[0012] Another aspect of the present disclosure relates to a polynucleotide or set of polynucleotides that encodes a polypeptide comprising an Fc variant described herein and one or more proteinaceous moieties fused or covalently linked to the Fc variant.

[0013] Another aspect of the present disclosure pertains to a vector or set of vectors comprising one or more polynucleotides encoding the Fc variants described herein.

[0014] Another aspect of the present disclosure relates to a vector or set of vectors comprising one or more polynucleotides encoding a polypeptide comprising an Fc variant described herein and one or more proteinaceous moieties fused or covalently linked to the Fc variant.

[0015] Another aspect of the present disclosure pertains to host cells comprising one or more polynucleotides encoding the Fc variants described herein.

[0016] Another aspect of the present disclosure relates to host cells comprising one or more polynucleotides encoding a polypeptide comprising an Fc variant described herein and one or more proteinaceous moieties fused or covalently linked to the Fc variant.

[0017] Another aspect of the present disclosure relates to a method of preparing an Fc variant described herein, comprising transfecting a host cell with one or more polynucleotides encoding the Fc variant and culturing the host cell under conditions suitable for expression of the Fc variant.

[0018] Another aspect of the present disclosure relates to a method for preparing a polypeptide comprising an Fc variant described herein and one or more proteinaceous moieties fused or covalently linked to the Fc variant, the method comprising transfecting a host cell with one or more polynucleotides encoding the polypeptide and culturing the host cell under conditions suitable for expression of the polypeptide.

[0019] Another aspect of the present disclosure relates to pharmaceutical compositions comprising the Fc variants described herein.

[0020] Another aspect of the present disclosure relates to pharmaceutical compositions comprising an Fc variant described herein and a polypeptide comprising one or more proteinaceous moieties fused or covalently linked to the Fc variant.

[0021] Another embodiment of the present disclosure is a method for increasing the CH2 domain melting temperature (Tm) of an Fc, comprising introducing one or more stability-enhancing amino acid mutations into a parent Fc to provide an Fc variant with an increased CH2 domain Tm compared to the parent Fc, the mutations including a mutation at position 250 where the amino acid at position 250 is substituted with Ala, Ile, or Val; a mutation at position 287 where the amino acid at position 287 is substituted with Phe, His, Met, Trp, or Tyr; a mutation at position 308, wherein the mutation at position 308 is a substitution of the amino acid at position 308 with Ile; a mutation at position 309, wherein the mutation at position 309 is a substitution of the amino acid at position 309 with Gln or Thr; a mutation at position 428, wherein the mutation at position 428 is a substitution of the amino acid at position 428 with Phe; and a pair of mutations at positions 242 and 336, wherein both mutations are substitutions with Cys.

[0022] Another aspect of the present disclosure is a method for increasing the CH2 domain melting temperature (Tm) of an Fc, comprising introducing one to three stability-enhancing amino acid mutations into a parent Fc to provide an Fc variant with an increased CH2 domain Tm compared to the parent Fc, wherein the mutations are one or more mutations selected from (a) a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with He, and a mutation at position 309 that is a substitution with Gln or Thr, or (b) a mutation at position 250 that is a substitution with Ala, He, or Val, a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with He, and a mutation at position 309 that is a substitution with He. or (c) two or more mutations selected from a mutation at position 242 that is a substitution with Ala, He, or Val, a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with He, a mutation at position 309 that is a substitution with Gln or Thr, a mutation at position 428 that is a substitution with Phe, and a pair of mutations at positions 242 and 336 that are both substitutions with Cys; or (d) three or more mutations including a pair of mutations at positions 242 and 336 that are both substitutions with Cys and a mutation at position 250 that is a substitution with Ala, He, or Val, a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with He, a mutation at position 309 that is a substitution with Gln or Thr, and a mutation at position 428 that is a substitution with Phe. [The present invention 1001] a mutation at position 250, wherein the mutation is a substitution of the amino acid at position 250 with Ala, Ile, or Val; a mutation at position 287, wherein the mutation is a substitution of the amino acid at position 287 with Phe, His, Met, Trp, or Tyr; a mutation at position 308, wherein the mutation is a substitution of the amino acid at position 308 with He; a mutation at position 309, wherein the mutation is a substitution of the amino acid at position 309 with Gln or Thr; a mutation at position 428, wherein the mutation is a substitution of the amino acid at position 428 with Phe; and A pair of mutations at positions 242 and 336, both of which are substitutions with Cys. an Fc variant comprising one or more stability-enhancing amino acid mutations selected from: the Fc variant has an increased CH2 domain melting temperature (Tm) compared to a parent Fc that does not comprise said one or more stability-enhancing amino acid mutations; Amino acid numbering is according to the EU index, The Fc variant. [The present invention 1002] 1001. An Fc variant of the present invention comprising a single stability-enhancing amino acid mutation selected from a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with Ile, and a mutation at position 309 that is a substitution with Gln or Thr. [The present invention 1003] 1001. An Fc variant of the present invention comprising two or more stability-enhancing amino acid mutations selected from a mutation at position 250 that is a substitution with Ala, Ile, or Val, a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with Ile, a mutation at position 309 that is a substitution with Gln or Thr, a mutation at position 428 that is a substitution with Phe, and a pair of mutations at positions 242 and 336 that are both substitutions with Cys. [The present invention 1004] 1001. An Fc variant of the present invention, wherein the Fc variant comprises three or more stability-enhancing amino acid mutations, the mutations comprising a pair of mutations at positions 242 and 336, both of which are substitutions with Cys, and a mutation selected from a mutation at position 250 which is substitution with Ala, Ile, or Val, a mutation at position 287 which is substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 which is substitution with Ile, a mutation at position 309 which is substitution with Gln or Thr, and a mutation at position 428 which is substitution with Phe. [The present invention 1005] An Fc variant comprising one to three stability-enhancing amino acid mutations, wherein the mutations are: (a) one or more mutations selected from a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with He, and a mutation at position 309 that is a substitution with Gln or Thr, or (b) two or more mutations selected from a mutation at position 250 that is a substitution with Ala, Ile, or Val, a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with Ile, a mutation at position 309 that is a substitution with Gln or Thr, a mutation at position 428 that is a substitution with Phe, and a pair of mutations at positions 242 and 336 that are both substitutions with Cys; or (c) three or more mutations, including a pair of mutations at positions 242 and 336, both of which are substitutions with Cys, and a mutation at position 250, which is substitution with Ala, Ile, or Val; a mutation at position 287, which is substitution with Phe, His, Met, Trp, or Tyr; a mutation at position 308, which is substitution with Ile; a mutation at position 309, which is substitution with Gln or Thr; and a mutation at position 428, which is substitution with Phe. Including, the Fc variant has an increased CH2 domain melting temperature (Tm) compared to a parent Fc that does not comprise said one or more stability-enhancing amino acid mutations; Amino acid numbering is according to the EU index, The Fc variant. [The present invention 1006] 6. The Fc variant of any of claims 1001 to 1005, comprising a mutation at position 287 which is a substitution with Phe, His, Met, Trp, or Tyr. [The present invention 1007] The Fc variant of the present invention 1006, wherein the mutation at position 287 is a substitution with Phe. [The present invention 1008] 1006. The Fc variant of any of claims 1001 to 1005, comprising a mutation at position 308 which is a substitution with Ile. [The present invention 1009] 1006. The Fc variant of any of claims 1001 to 1005, comprising a mutation at position 309 which is a substitution with Gln or Thr. [The present invention 1010] The Fc variant of the present invention 1009, wherein the mutation at position 309 is a substitution with Gln. [The present invention 1011] An Fc variant of any of claims 1001 and 1003 to 1005, comprising a mutation at position 250 which is a substitution with Ala, Ile, or Val, and a mutation at position 287 which is a substitution with Phe, His, Met, Trp, or Tyr. [The present invention 1012] The Fc variant of the present invention 1011, wherein the mutation at position 250 is a substitution with Val. [The present invention 1013] The Fc variant of the present invention 1011 or 1012, wherein the mutation at position 287 is a substitution with Phe. [The present invention 1014] An Fc variant of the present invention 1001 and any one of the present inventions 1003 to 1005, comprising a mutation at position 250 which is a substitution with Ala, Ile, or Val, and a mutation at position 309 which is a substitution with Gln or Thr. [The present invention 1015] The Fc variant of the present invention 1014, wherein the mutation at position 250 is a substitution with Val. [The present invention 1016] The Fc variant of the present invention 1014 or 1015, wherein the mutation at position 309 is a substitution with Gln. [The present invention 1017] An Fc variant of the present invention 1001 and any one of the present inventions 1003 to 1005, comprising a mutation at position 250 which is a substitution with Ala, Ile, or Val, and a mutation at position 428 which is a substitution with Phe. [The present invention 1018] The Fc variant of the present invention 1017, wherein the mutation at position 250 is a substitution with Val. [The present invention 1019] An Fc variant of any one of 1001 and 1003 to 1005 of the present invention, comprising a mutation at position 287 which is a substitution with Phe, His, Met, Trp, or Tyr, and a mutation at position 428 which is a substitution with Phe. [The present invention 1020] An Fc variant of 1019 of the present invention, wherein the mutation at position 287 is a substitution with Phe. [The present invention 1021] An Fc variant of the present invention 1001 and any one of 1003 to 1005, comprising a pair of mutations at positions 242 and 336, both of which are substitutions with Cys. [The present invention 1022] An Fc variant of any of 1001, 1004 or 1005 of the invention comprising a pair of mutations at positions 242 and 336, both of which are substitutions with Cys, and a mutation at position 308, which is substitution with Ile. [The present invention 1023] An Fc variant of the present invention 1001 or 1005, wherein the stability-enhancing mutation contained in the Fc variant is selected from 250V, 287F, 308I, 309Q, 428F, 242C_336C, 287F / 428F, 250V / 287F, 250V / 309Q, 250V / 428F, and 242C_336C / 308I. [The present invention 1024] An Fc variant of the present invention 1001 or 1005, wherein the stability-enhancing mutations contained in the Fc variant are selected from 287F, 308I, 309Q, 242C_336C, 287F / 428F, 250V / 287F, 250V / 309Q, 250V / 428F, and 242C_336C / 308I. [The present invention 1025] An Fc variant of any of claims 1001 to 1024 of the present invention based on IgG, IgA, IgD, IgE, or IgM Fc. [The present invention 1026] An Fc variant of the present invention 1025 based on human IgG, IgA, IgD, IgE, or IgM Fc. [The present invention 1027] An Fc variant of any one of 1001 to 1024 of the present invention based on IgG Fc. [The present invention 1028] The Fc variant of the present invention 1027, wherein the IgG Fc is IgG1 Fc. [The present invention 1029] The Fc variant of the present invention 1027 or 1028, wherein the IgG Fc is human IgG Fc. [The present invention 1030] The Fc variant of any of claims 1001 to 1029, wherein the parent Fc comprises one or more amino acid mutations that improve the function of the Fc region. [The present invention 1031] the parent Fc is one or more amino acid mutations that improve the function of the Fc region and decrease the CH2 domain Tm of the corresponding wild-type Fc An Fc variant of any of 1001 to 1029 of the present invention, comprising: [The present invention 1032] The Fc variant of any of claims 1001 to 1031, wherein the Tm of the CH2 domain of the Fc variant is increased by at least 0.5°C compared to the parent Fc. [The present invention 1033] The Fc variant of the present invention 1032, wherein the CH2 domain Tm of the Fc variant is increased by at least 1.0°C, at least 2.0°C, or at least 3.0°C compared to the parent Fc. [The present invention 1034] The Fc variant of any of claims 1001 to 1031, wherein the Tm of the CH2 domain of the Fc variant is increased by 0.5°C to 9.0°C compared to the parent Fc. [This invention 1035] The Fc variant of any of claims 1001 to 1031, wherein the Tm of the CH2 domain of the Fc variant is increased by 2.0°C to 10.5°C compared to the parent Fc. [The present invention 1036] A polypeptide comprising an Fc variant of any of claims 1001 to 1035 of the present invention and one or more proteinaceous moieties fused or covalently linked to the Fc variant. [This invention 1037] 1036. The polypeptide of claim 1036, wherein said one or more proteinaceous moieties comprise an antigen-binding domain, a ligand, a receptor, a receptor fragment, a cytokine, or an antigen. [The present invention 1038] 1037. The polypeptide of the invention, wherein at least one of said one or more proteinaceous moieties is an antigen-binding domain. [This invention 1039] The polypeptide of the present invention 1037, wherein the antigen-binding domain is Fab or scFv. [The present invention 1040] The polypeptide of any one of 1036 to 1039 of the present invention, which is an antibody or an antigen-binding antibody fragment. [This invention 1041] A polypeptide of the invention 1040 which is a therapeutic antibody or antibody fragment. [The present invention 1042] A polynucleotide or a set of polynucleotides encoding any one of the Fc variants 1001 to 1035 of the present invention. [This invention 1043] A polynucleotide or a set of polynucleotides encoding any one of the polypeptides of the present invention 1036 to 1041. [This invention 1044] A vector or a set of vectors comprising one or more polynucleotides encoding any one of the Fc variants of the present inventions 1001 to 1035 or any one of the polypeptides of the present inventions 1036 to 1041. [This invention 1045] A host cell comprising one or more polynucleotides encoding any one of the Fc variants of the present inventions 1001 to 1035 or any one of the polypeptides of the present inventions 1036 to 1041. [The present invention 1046] A method for preparing any of the Fc variants of the present invention 1001 to 1035, comprising transfecting a host cell with one or more polynucleotides encoding the Fc variant, and culturing the host cell under conditions suitable for expression of the Fc variant. [This invention 1047] A method for preparing any of the polypeptides of the present invention 1036 to 1041, comprising transfecting a host cell with one or more polynucleotides encoding said polypeptide, and culturing said host cell under conditions suitable for expression of said polypeptide. [This invention 1048] A pharmaceutical composition comprising any one of the Fc variants of the present invention 1001 to 1035 or any one of the polypeptides of the present invention 1036 to 1041. [This invention 1049] 1. A method for increasing the CH2 domain melting temperature (Tm) of an Fc, comprising introducing one or more stability-enhancing amino acid mutations into a parent Fc to provide an Fc variant having an increased CH2 domain Tm compared to the parent Fc, wherein the mutations are: a mutation at position 250, wherein the mutation is a substitution of the amino acid at position 250 with Ala, Ile, or Val; a mutation at position 287, wherein the mutation is a substitution of the amino acid at position 287 with Phe, His, Met, Trp, or Tyr; a mutation at position 308, wherein the mutation is a substitution of the amino acid at position 308 with He; a mutation at position 309, wherein the mutation is a substitution of the amino acid at position 309 with Gln or Thr; a mutation at position 428, wherein the mutation is a substitution of the amino acid at position 428 with Phe; and A pair of mutations at positions 242 and 336, both of which are substitutions with Cys. is selected from Amino acid numbering is according to the EU index, The method. [The present invention 1050] 1049. The method of claim 1049, comprising introducing a single stability-enhancing amino acid mutation into the parent Fc, wherein the mutation is selected from a mutation at position 287 that is a substitution with Phe, His, Met, Trp or Tyr, a mutation at position 308 that is a substitution with Ile, and a mutation at position 309 that is a substitution with Gln or Thr. [This invention 1051] 1049. The method of claim 1049, comprising introducing two or more stability-enhancing amino acid mutations into the parent Fc, wherein the mutations are selected from a mutation at position 250 that is a substitution with Ala, Ile or Val, a mutation at position 287 that is a substitution with Phe, His, Met, Trp or Tyr, a mutation at position 308 that is a substitution with Ile, a mutation at position 309 that is a substitution with Gln or Thr, a mutation at position 428 that is a substitution with Phe, and a pair of mutations at positions 242 and 336, both of which are substitutions with Cys. [This invention 1052] 1049. The method of claim 1049, comprising introducing three or more stability-enhancing amino acid mutations into the parent Fc, wherein the mutations include a pair of mutations at positions 242 and 336, both of which are substitutions with Cys, and a mutation at position 250, which is substitution with Ala, Ile or Val, a mutation at position 287, which is substitution with Phe, His, Met, Trp or Tyr, a mutation at position 308, which is substitution with Ile, a mutation at position 309, which is substitution with Gln or Thr, and a mutation at position 428, which is substitution with Phe. [This invention 1053] 1. A method for increasing the CH2 domain melting temperature (Tm) of an Fc, comprising introducing one to three stability-enhancing amino acid mutations into a parent Fc to provide an Fc variant having an increased CH2 domain Tm compared to the parent Fc, wherein the mutations are: (a) one or more mutations selected from a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with He, and a mutation at position 309 that is a substitution with Gln or Thr, or (b) two or more mutations selected from a mutation at position 250 that is a substitution with Ala, Ile, or Val, a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with Ile, a mutation at position 309 that is a substitution with Gln or Thr, a mutation at position 428 that is a substitution with Phe, and a pair of mutations at positions 242 and 336 that are both substitutions with Cys, or (c) three or more mutations, including a pair of mutations at positions 242 and 336, both of which are substitutions with Cys, and a mutation at position 250, which is substitution with Ala, Ile, or Val; a mutation at position 287, which is substitution with Phe, His, Met, Trp, or Tyr; a mutation at position 308, which is substitution with Ile; a mutation at position 309, which is substitution with Gln or Thr; and a mutation at position 428, which is substitution with Phe. Including, Amino acid numbering is according to the EU index, The method. [This invention 1054] The method of any of claims 1049 to 1053, wherein the Tm of the CH2 domain of the Fc variant is increased by at least 0.5°C compared to the parent Fc. [This invention 1055] 1054. The method of claim 1054, wherein the CH2 domain Tm of said Fc variant is increased by at least 1.0°C, at least 2.0°C, or at least 3.0°C compared to the parent Fc. [The present invention 1056] Any of the methods of claims 1049 to 1053, wherein the Tm of the CH2 domain of the Fc variant is increased by 0.5°C to 9.0°C compared to the parent Fc. [This invention 1057] Any of the methods of claims 1049 to 1053, wherein the Tm of the CH2 domain of the Fc variant is increased by 2.0°C to 10.5°C compared to the parent Fc. [This invention 1058] Any of the methods of claims 1049 to 1057, wherein an Fc variant exhibiting reduced aggregation under weakly acidic conditions compared to the parent Fc region is provided by introducing the stability-enhancing amino acid mutation into the parent Fc. [This invention 1059] 1058. The method of claim 1058, wherein the stability-enhancing amino acid mutations include 250V and 287F. [Brief explanation of the drawings]

[0023] [Figure 1] (A) Provides the sequence of the IgG1 Fc region sequence [SEQ ID NO: 1]. (B) Provides a structural diagram of the IgG1 Fc region (PDB ID: 4BSV) showing the locations of exemplary stability-enhancing designs T250V, A287F, and M428F. [Figure 2A] Figure 1 shows the improvement in CH2 domain melting temperature (Tm) resulting from the introduction of exemplary stability-enhancing mutations into various Fc scaffolds. (A) Mutations into scaffold 3 contain asymmetric mutations to promote heterodimeric Fc formation. Scaffold 1 is a homodimeric IgG1 Fc. [Figure 2B](B) Improvements in CH2 domain melting temperature (Tm) resulting from the introduction of exemplary stability-enhancing mutations into various Fc scaffolds. (C) Mutations into scaffold 6, including the N297A mutation. Scaffold 1 is a homodimeric IgG1 Fc. [Figure 2C] Figure 1 shows the improvement in CH2 domain melting temperature (Tm) resulting from the introduction of exemplary stability-enhancing mutations into various Fc scaffolds. (C) Mutations into scaffold 7, including S239D / I332E mutations. Scaffold 1 is a homodimeric IgG1 Fc. [Figure 3A] (A) Sequence alignment of the CH2 domains of IgA, IgD, and IgG with the CH3 domains of IgE and IgM. Positions corresponding to IgG1 T250, A287, and M428 are boxed. [Figure 3B] (B) Sequence alignment of the CH3 domains of IgA, IgD, and IgG with the CH4 domains of IgE and IgM. Positions corresponding to IgG1 T250, A287, and M428 are boxed. [Figure 4A] Correlation between aggregation induced by incubation at 40°C for 2 weeks in acidic or neutral conditions and thermal stability of the CH2 domain for antibody variants with and without the stability-enhancing mutation T250V / A287F. (A) Standard scale x-axis, showing correlation under incubation in weakly acidic conditions. Variants showing small or no negative change in aggregation throughout the study are omitted. The parent sequence (non-stabilizing) is shown as a circle, stabilized variants as a square, and each non-stabilized variant and the corresponding stabilized variant are connected by an arrow. [Figure 4B](B) Correlation between aggregation induced by incubation at 40°C for 2 weeks in acidic or neutral conditions and thermal stability of the CH2 domain for antibody variants with or without the stability-enhancing mutation T250V / A287F. (C) Standard scale x-axis, showing correlation under incubation under neutral conditions. Variants showing small or no negative change in aggregation throughout the study are omitted. The parent sequence (non-stabilized) is shown as a circle, stabilized variants as a square, and each non-stabilized variant and the corresponding stabilized variant are connected by an arrow. [Figure 4C] Correlation between aggregation induced by incubation at 40°C for 2 weeks in acidic or neutral conditions and thermal stability of the CH2 domain for antibody variants with and without the stability-enhancing mutation T250V / A287F. (C) Logarithmic scale x-axis, showing correlation under incubation in weakly acidic conditions. Variants showing small or no negative change in aggregation throughout the study are omitted. The parent sequence (non-stabilized) is shown as a circle, stabilized variants as a square, and each non-stabilized variant and the corresponding stabilized variant are connected by an arrow. [Figure 4D] (D) Correlation between aggregation induced by incubation at 40°C for 2 weeks in acidic or neutral conditions and thermal stability of the CH2 domain for antibody variants with and without the stability-enhancing mutation T250V / A287F. (E) Logarithmic scale x-axis, showing correlation under incubation under neutral conditions. Variants showing small or no negative change in aggregation throughout the study are omitted. The parent sequence (non-stabilized) is shown as a circle, stabilized variants as a square, and each non-stabilized variant and the corresponding stabilized variant are connected by an arrow. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description Described herein are Fc variants that contain one or more amino acid mutations that increase the stability of the Fc variant compared to a parent Fc that does not contain the one or more amino acid mutations. These mutations are referred to herein as "stability-enhancing amino acid mutations" or "stability-enhancing mutations."

[0025] In certain embodiments, the one or more stability-enhancing amino acid mutations comprised by the Fc variant are: a mutation at position 250, wherein the mutation is a substitution of the amino acid at position 250 with Ala, Ile or Val; a mutation at position 287, wherein the mutation is a substitution of the amino acid at position 287 with Phe, His, Met, Trp or Tyr; a mutation at position 308, which is a substitution of the amino acid at position 308 with He; a mutation at position 309, wherein the mutation is a substitution of the amino acid at position 309 with Gln or Thr; a mutation at position 428, which is a substitution of the amino acid at position 428 with Phe, and A mutation at position 242 and a mutation at position 336 are selected, both mutations being substitutions with Cys.

[0026] Certain embodiments of the present disclosure relate to polypeptides comprising the Fc variants described herein. Examples of such polypeptides include, but are not limited to, antibodies, antibody fragments, and Fc fusion proteins. Polypeptides comprising the Fc variants described herein can be used as therapeutic, diagnostic, or research tools.

[0027] Certain embodiments of the present disclosure relate to polynucleotides encoding the Fc variants described herein and to polynucleotides encoding polypeptides comprising the Fc variants, as well as host cells comprising the polynucleotides and methods of using the polynucleotides and host cells to prepare Fc variants or polypeptides comprising the Fc variants.

[0028] Certain embodiments of the present disclosure relate to methods of stabilizing an Fc (parent Fc) by introducing one or more stability-enhancing mutations described herein into the Fc. Some embodiments of the present disclosure relate to methods of increasing the CH2 domain melting temperature (Tm) of an Fc (parent Fc) by introducing one or more stability-enhancing mutations described herein into the Fc. The parent Fc can be a wild-type Fc or a variant Fc that already contains one or more amino acid mutations, for example, to improve the function of the Fc region.

[0029] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0030] As used herein, the term "about" refers to about a ±10% variation from a given value, and it is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0031] The use of the term "a" or "an," when used herein in conjunction with the term "comprising," can mean "one," but it is also synonymous with "one or more," "at least one," or "one or more."

[0032] As used herein, the terms "comprising," "having," "including," and "containing," and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps. The term "consisting essentially of," when used herein in connection with a composition, use, or method, means that additional elements and / or method steps may be present, but these additions do not materially affect the manner in which the recited composition, method, or use functions. When used herein in connection with a composition, use, or method, the term "consisting of" excludes the presence of additional elements and / or method steps. A composition, use, or method described herein as including certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments, and may consist of those elements and / or steps in other embodiments, whether or not these embodiments are specifically referred to.

[0033] The term "isolated," as used herein with respect to a material, means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials 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 still be said to be isolated in that such a vector or composition is not part of its natural environment.

[0034] The terms "Fc region" and "Fc," when used interchangeably herein, refer to the C-terminal region of an immunoglobulin heavy chain. The human IgG heavy chain Fc region sequence, for example, is typically defined as extending from position 239 to the C-terminus of the heavy chain. The "Fc polypeptide" of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., the polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. The Fc region typically comprises a CH2 domain and a CH3 domain. The Fc region may also be considered to encompass the hinge region in certain embodiments.

[0035] The "CH2 domain" of the human IgG Fc region is usually defined to extend from positions 239 to 340. The "CH3 domain" is usually defined to include the C-terminal amino acid residues of the CH2 domain of the Fc region, i.e., amino acid residues 341 to 447. The "hinge region" of human IgG1 is generally defined to extend from positions 216 to 238 (Burton, 1985, Molec. Immunol., 22:161-206). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by aligning the first and last cysteine ​​residues that form intra-heavy chain disulfide bonds.

[0036] Unless otherwise specified herein, numbering of amino acid residues in the Fc region is according to the EU numbering system, also referred to 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).

[0037] Naturally occurring amino acids are identified throughout by their conventional three-letter or one-letter abbreviations, which are generally accepted in the art and recommended by the IUPAC-IUB Biochemical Nomenclature Commission, and are set out in Table A below.

[0038] [Table A]

[0039] It should be understood that the positive listing of a feature in one embodiment serves as a basis for excluding features in alternative embodiments. For example, where a list of options is presented for a given embodiment or claim, it should be understood that one or more options may be deleted from the list, and that such shortened list may form an alternative embodiment, whether or not specifically mentioned.

[0040] It is contemplated that any embodiment disclosed herein can be implemented with respect to a method, use, or composition disclosed herein, and vice versa.

[0041] Fc variants The Fc variants of the present disclosure comprise one or more amino acid mutations ("stability-enhancing mutations") that increase the stability of the Fc variant compared to the parent Fc. The increased stability may result in increased thermal stability of the CH2 domain, reduced aggregation potential, increased serum half-life, improved manufacturability, or a combination thereof. In certain embodiments, the one or more stability-enhancing mutations contained in the Fc variant increase the thermal stability (Tm) of the CH2 domain compared to the parent Fc.

[0042] In some embodiments, the one or more stability-enhancing mutations contained in the Fc variant increase the thermal stability (Tm) of the CH2 domain relative to the parent Fc and reduce aggregation of the Fc region. In some embodiments, the one or more stability-enhancing mutations contained in the Fc variant increase the thermal stability (Tm) of the CH2 domain relative to the parent Fc and reduce aggregation of the Fc region at low pH. "Low pH" in this context refers to a pH of about 4.0 to 7.5.

[0043] In some embodiments, the one or more stability-enhancing mutations contained in the Fc variant increase the thermal stability (Tm) of the CH2 domain compared to the parent Fc and reduce aggregation of the Fc region under mildly acidic conditions, which include a pH below neutral. In some embodiments, the mildly acidic conditions include a pH of about 4.0 to 7.0. In some embodiments, the mildly acidic conditions include a pH of about 4.0 to 6.5.

[0044] The Fc variant may comprise one stability-enhancing mutation, or may comprise one or more stability-enhancing mutations. In certain embodiments, the Fc variant comprises one to five stability-enhancing mutations. In some embodiments, the Fc variant comprises one to four stability-enhancing mutations. In some embodiments, the Fc variant comprises one to three stability-enhancing mutations. In some embodiments, the Fc variant comprises one, two, or three stability-enhancing mutations.

[0045] In certain embodiments, the CH2 domain Tm of the Fc variant is increased by at least 0.5° C. compared to the parent Fc. In some embodiments, the CH2 domain Tm of the Fc variant is increased by at least 1.0° C., at least 1.5° C., at least 2.0° C., at least 2.5° C., or at least 3.0° C. compared to the parent Fc. In some embodiments, the CH2 domain Tm of the Fc variant is increased by at least 5.0° C., at least 5.5° C., at least 6.0° C., at least 6.5° C., or at least 7.0° C. compared to the parent Fc.

[0046] In certain embodiments, the CH2 domain Tm of the Fc variant is increased by about 0.5°C to about 6.5°C compared to the parent Fc. In some embodiments, the CH2 domain Tm of the Fc variant is increased by about 0.5°C to about 9.0°C compared to the parent Fc. In some embodiments, the CH2 domain Tm of the Fc variant is increased by about 1.0°C to about 9.0°C, about 2.0°C to about 9.0°C, or about 3.0°C to about 9.0°C compared to the parent Fc. In some embodiments, the CH2 domain Tm of the Fc variant is increased by about 2.0°C to about 10.5°C, or about 3.0°C to about 10.5°C compared to the parent Fc.

[0047] In certain embodiments, the CH2 domain Tm is measured by DSC or DSF.

[0048] The parent Fc may be a wild-type Fc or a variant Fc that already contains one or more amino acid mutations, for example, to improve the function of the Fc region. In some embodiments, the parent Fc may be an Fc containing one or more amino acid mutations that functionally enhance the Fc region. In some embodiments, the parent Fc may be an Fc containing one or more amino acid mutations that functionally enhance the Fc region but result in reduced stability compared to the wild-type Fc. In some embodiments, the parent Fc may contain one or more amino acid mutations that functionally enhance the Fc region but reduce the thermal stability of the CH2 domain compared to the wild-type Fc.

[0049] Examples of amino acid mutations that functionally enhance the Fc region but decrease the thermal stability of the CH2 domain compared to wild-type Fc include mutations that promote heterodimeric Fc formation (e.g., knobs-into-holes or electrostatic steering mutations described by Atwell et al., 1997, J Biol Chem, 270:26-35 and Gunasekaran et al., 2010, J Biol Chem, 285(25):19637-19646), mutations that generate aglycosylated Fc (e.g., the N297A mutation described by Lund et al., 1995, FASEB, 9(1):115-119; Leabman et al., 2013, mAbs, 5(6):896-903; and Jacobsen et al., 2017, JBC, 292(5):1865-1875), and mutations that alter FcγR selectivity (e.g., the N297A mutation described by Lazar et al., 2017, JBC, 292(5):1865-1875). Examples of amino acid mutations that functionally enhance the Fc region but decrease the thermal stability of the CH2 domain compared to wild-type or parent Fc include, but are not limited to, the S239D / I332E or S239D / A330L / I332E mutations, which increase affinity for FcγRIIIa, as described by Oganesyan et al., 2008, Molec Immunol, 45(7):1872-1882, or the E233D / G237D / P238D / H268D / P271G / A330R mutations, which increase selectivity for FcγRIIb, as described by Mimoto et al., 2013, Protein Eng. Des. Sel., 26:589-598. Additional examples of amino acid mutations that functionally enhance the Fc region but decrease the thermal stability of the CH2 domain compared to wild-type or parent Fc are described in the Examples section of this specification.

[0050] The parent Fc into which stability-enhancing mutations are introduced can be IgG Fc, IgA Fc, IgD Fc, IgE Fc, or IgM Fc. While the amino acid numbering used herein refers to IgG Fc, one of skill in the art can readily determine the corresponding positions of mutations in other Ig Fc sequences by sequence alignment using one of several sequence alignment tools known in the art. Thus, when a specific position in the Fc region for a stability-enhancing mutation is mentioned herein, it is intended to encompass the specific position in IgG Fc, as well as the corresponding position in the IgA, IgD, IgE, or IgM Fc region. Sequence alignments of the CH2 domains of IgA, IgD, and IgG with the CH3 domains of IgE and IgM, and the CH3 domains of IgA, IgD, and IgG with the CH4 domains of IgE and IgM are shown in Figures 3A and 3B.

[0051] In certain embodiments, the Fc variants are based on IgG, IgA, IgD, IgE, or IgM Fc. In some embodiments, the Fc variants are based on human IgG, IgA, IgD, IgE, or IgM Fc. In some embodiments, the Fc variants are based on IgG or IgA Fc. In some embodiments, the Fc variants are based on human IgG or IgA Fc. In some embodiments, the Fc variants are based on IgG Fc. In some embodiments, the Fc variants are based on human IgG Fc.

[0052] In certain embodiments, the Fc variants are based on IgG Fc, which may be IgG1, IgG2, IgG3, or IgG4 Fc. In some embodiments, the Fc variants are based on human IgG1, IgG2, IgG3, or IgG4 Fc. Sequence alignments of the CH2 and CH3 domains of human IgG1, IgG2, IgG3, and IgG4 are shown in Figures 3A and 3B. In some embodiments, the Fc variants are based on IgG1 Fc. In some embodiments, the Fc variants are based on human IgG1 Fc.

[0053] Stability-enhancing mutations As described herein, in silico and bioinformatics approaches were used to identify positions within the Fc region that can be mutated to improve Fc stability. These approaches identified the mutations shown in Table 1 as stability-enhancing mutations.

[0054] [Table 1]

[0055] In particular, the in silico approach identified the following amino acid mutations as mutations that increased Fc stability when introduced as single mutations: A mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y, and Mutation M428F.

[0056] Furthermore, a bioinformatics approach identified the following amino acid mutations as mutations that increase Fc stability when introduced as single mutations: a mutation at position 250 selected from T250A, T250I and T250V, Mutation V308I, and A mutation at position 309 selected from L309Q and L309T.

[0057] Additionally, the mutation pairs L242C_I336C, V240C_I332C, and V263C_V302C, which each introduce an additional disulfide bond in the Fc region, were shown to increase Fc stability in the absence of any additional stability-enhancing mutations.

[0058] Combinations of stability-enhancing mutations shown in Table 1 were also shown to further increase the stability of the Fc variants.

[0059] Thus, in certain embodiments, the Fc variant comprises one or more stability-enhancing mutations, where at least one mutation is selected from the mutations shown in Table 1. In the same embodiment, the Fc variant comprises one or more stability-enhancing mutations selected from the mutations shown in Table 1.

[0060] In some embodiments, the Fc variant comprises one or more stability-enhancing mutations, at least one of which is selected from the following: a mutation at position 287, wherein the mutation is a substitution of the amino acid at position 287 with Phe, His, Met, Trp or Tyr; a mutation at position 308, which is a substitution of the amino acid at position 308 with He, and A mutation at position 309, wherein the mutation is a substitution of the amino acid at position 309 with Gln or Thr.

[0061] In certain embodiments, the Fc variant comprises a single stability-enhancing mutation. In some embodiments, the Fc variant comprises a single stability-enhancing mutation selected from the following: a mutation at position 287, wherein the mutation is a substitution of the amino acid at position 287 with Phe, His, Met, Trp or Tyr; a mutation at position 308, wherein the mutation is a substitution of the amino acid at position 308 with He, and A mutation at position 309, wherein the mutation is a substitution of the amino acid at position 309 with Gln or Thr.

[0062] In some embodiments, the mutation at position 287 comprised in the Fc variant is a substitution of the amino acid at position 287 with Phe. In some embodiments, the mutation at position 309 comprised in the Fc variant is a substitution of the amino acid at position 309 with Gln.

[0063] In certain embodiments, the Fc variant comprises a pair of stability-enhancing mutations, each of which introduces a cysteine ​​residue that allows for the formation of a new disulfide bond in the Fc region. In some embodiments, the pair of stability-enhancing mutations is selected from 242C_336C, 240C_332C, and 263C_302C. In some embodiments, the pair of stability-enhancing mutations is 242C_336C or 240C_332C. In some embodiments, the pair of stability-enhancing mutations is 242C_336C.

[0064] In certain embodiments, the Fc variant comprises two or more stability-enhancing mutations. In some embodiments, the Fc variant comprises two or more stability-enhancing mutations selected from the following: a mutation at position 250, wherein the mutation is a substitution of the amino acid at position 250 with Ala, Ile or Val; a mutation at position 287, wherein the mutation is a substitution of the amino acid at position 287 with Phe, His, Met, Trp, or Tyr; a mutation at position 308, wherein the mutation is a substitution of the amino acid at position 308 with He; a mutation at position 309, wherein the mutation is a substitution of the amino acid at position 309 with Gln or Thr; a mutation at position 428, wherein the mutation is a substitution of the amino acid at position 428 with Phe, and A pair of mutations at positions 224 and 336, each of which is a substitution to Cys, and a pair of mutations at positions 242 and 336.

[0065] In certain embodiments, the Fc variant comprises two stability-enhancing mutations. In some embodiments, the Fc variant comprises: two stability-enhancing mutations selected from a mutation at position 250, a mutation at position 287, a mutation at position 308, a mutation at position 309, and a mutation at position 428, wherein the mutation at position 250 is a substitution with Ala, He, or Val, the mutation at position 287 is a substitution with Phe, His, Met, Trp, or Tyr, the mutation at position 308 is a substitution with He, the mutation at position 309 is a substitution with Gln or Thr, and the mutation at position 428 is a substitution with Phe; or A pair of mutations at positions 242 and 336, each of which is a substitution with Cys.

[0066] In some embodiments, the Fc variant comprises: a mutation at position 250 and a mutation at position 287, wherein the mutation at position 250 is a substitution with Ala, Ile or Val, and the mutation at position 287 is a substitution with Phe, His, Met, Trp or Tyr, and a mutation at position 308; a mutation at position 250 and a mutation at position 308, wherein the mutation at position 250 is a substitution with Ala, Ile or Val and the mutation at position 308 is a substitution with Ile; a mutation at position 250 and a mutation at position 309, wherein the mutation at position 250 is a substitution with Ala, Ile or Val, and the mutation at position 309 is a substitution with Gln or Thr; a mutation at position 250 and a mutation at position 428, wherein the mutation at position 250 is a substitution with Ala, Ile or Val and the mutation at position 428 is a substitution with Phe; a mutation at position 287 and a mutation at position 308, wherein the mutation at position 287 is a substitution with Phe, His, Met, Trp or Tyr, and the mutation at position 308 is a substitution with Ile; a mutation at position 287 and a mutation at position 309, wherein the mutation at position 287 is a substitution with Phe, His, Met, Trp or Tyr, and the mutation at position 309 is a substitution with Gln or Thr; a mutation at position 287 and a mutation at position 428, wherein the mutation at position 287 is a substitution with Phe, His, Met, Trp or Tyr, and the mutation at position 428 is a substitution with Phe; a mutation at position 308 and a mutation at position 309, wherein the mutation at position 308 is a substitution with Ile and the mutation at position 309 is a substitution with Gln or Thr; a mutation at position 308 and a mutation at position 428, wherein the mutation at position 308 is a substitution with Ile and the mutation at position 428 is a substitution with Phe; a mutation at position 309 and a mutation at position 428, wherein the mutation at position 309 is a substitution with Gln or Thr and the mutation at position 428 is a substitution with Phe; or A pair of mutations at positions 242 and 336, each of which is a substitution with Cys.

[0067] In some embodiments, the Fc variant comprises: a mutation at position 250 and a mutation at position 287, wherein the mutation at position 250 is a substitution with Ala, Ile or Val, and the mutation at position 287 is a substitution with Phe, His, Met, Trp or Tyr; a mutation at position 250 and a mutation at position 309, wherein the mutation at position 250 is a substitution with Ala, Ile or Val, and the mutation at position 309 is a substitution with Gln or Thr; a mutation at position 250 and a mutation at position 428, wherein the mutation at position 250 is a substitution with Ala, Ile or Val and the mutation at position 428 is a substitution with Phe; a mutation at position 287 and a mutation at position 428, wherein the mutation at position 287 is a substitution with Phe, His, Met, Trp or Tyr and the mutation at position 428 is a substitution with Phe; or A pair of mutations at positions 242 and 336, each of which is a substitution with Cys.

[0068] In some embodiments, the Fc variant comprises a mutation at position 250 that is a substitution with Val. In some embodiments, the Fc variant comprises a mutation at position 287 that is a substitution with Phe. In some embodiments, the Fc variant comprises a mutation at position 309 that is a substitution with Gln.

[0069] In some embodiments, the Fc variant comprises the stability-enhancing mutations 250V / 287F, 250V / 308I, 250V / 309Q, 250V / 428F, 287F / 308I, 287F / 309Q, 287F / 428F, 308I / 309Q, 308I / 428F, 309Q / 428F, or 242C / 336C.

[0070] In some embodiments, the Fc variant comprises the stability-enhancing mutations 250V / 287F, 250V / 309Q, 250V / 428F, 287F / 428F, or 242C_336C.

[0071] In some embodiments, the Fc variant comprises the stability-enhancing mutations 250V / 287F, 250V / 309Q, 250V / 428F, or 287F / 428F.

[0072] In some embodiments, the stability-enhancing mutations comprised in the Fc variant are selected from 250V, 287F, 308I, 309Q, 428F, 242C_336C, 287F / 428F, 250V / 287F, 250V / 309Q, 250V / 428F and 242C_336C / 308I.

[0073] In some embodiments, the stability-enhancing mutations comprised in the Fc variant are selected from 287F, 308I, 309Q, 242C_336C, 287F / 428F, 250V / 287F, 250V / 309Q, 250V / 428F and 242C_336C / 308I.

[0074] In certain embodiments, the Fc variant comprises three or more stability-enhancing mutations. In some embodiments, the Fc variant comprises three or more stability-enhancing mutations selected from the following: A mutation at position 250 which is a substitution with Ala, Ile or Val, a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr, a mutation at position 308 which is a substitution with Ile, a mutation at position 309 which is a substitution with Gln or Thr, a mutation at position 428 which is a substitution with Phe, and a pair of mutations at positions 242 and 336 which are both substitutions with Cys.

[0075] In some embodiments, the Fc variant comprises three stability-enhancing mutations: In some embodiments, the Fc variant comprises three stability-enhancing mutations selected from the following: A mutation at position 250 which is a substitution with Ala, Ile or Val, a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr, a mutation at position 308 which is a substitution with Ile, a mutation at position 309 which is a substitution with Gln or Thr, a mutation at position 428 which is a substitution with Phe, and a pair of mutations at positions 242 and 336 which are both substitutions with Cys.

[0076] In some embodiments, the Fc variant comprises: A pair of mutations at positions 242 and 336, both substitutions with Cys, and a mutation at position 250 which is a substitution with Ala, Ile or Val, a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr, a mutation at position 308 which is a substitution with Ile, a mutation at position 309 which is a substitution with Gln or Thr, and a mutation at position 428 which is a substitution with Phe. Includes:

[0077] In certain embodiments, the Fc variant comprises one to three stability-enhancing mutations. one or more mutations selected from a mutation at position 287 that is a substitution with Phe, His, Met, Trp or Tyr, a mutation at position 308 that is a substitution with He, and a mutation at position 309 that is a substitution with Gln or Thr, or two or more mutations selected from a mutation at position 250 which is a substitution with Ala, Ile, or Val, a mutation at position 287 which is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 which is a substitution with Ile, a mutation at position 309 which is a substitution with Gln or Thr, a mutation at position 428 which is a substitution with Phe, and a pair of mutations at positions 242 and 336 which are both substitutions with Cys, or three or more mutations including a mutation at position 242 and a mutation at position 336, both of which are substitutions with Cys, and a mutation selected from a mutation at position 250 which is substitution with Ala, Ile, or Val, a mutation at position 287 which is substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 which is substitution with Ile, a mutation at position 309 which is substitution with Gln or Thr, and a mutation at position 428 which is substitution with Phe Includes:

[0078] In certain embodiments, the Fc variant is an IgG Fc variant. In certain embodiments, the IgG Fc variant comprises one or more stability-enhancing mutations. In some embodiments, the IgG Fc variant comprises one or more stability-enhancing mutations, at least one of which is a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y; Mutation V308I, and A mutation at position 309 selected from L309Q and L309T is selected from.

[0079] In certain embodiments, the IgG Fc variant comprises a single stability-enhancing mutation. In some embodiments, the IgG Fc variant comprises: a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y; Mutation V308I, and A mutation at position 309 selected from L309Q and L309T The stability-enhancing mutation is selected from the group consisting of:

[0080] In some embodiments, the mutation at position 287 comprised in the IgG Fc variant is A287F. In some embodiments, the mutation at position 309 comprised in the IgG Fc variant is L309Q.

[0081] In certain embodiments, the Fc variant comprises a pair of stability-enhancing mutations, each of which introduces a cysteine ​​residue that allows for the formation of a new disulfide bond into the Fc region. In some embodiments, the pair of stability-enhancing mutations is selected from L242C_I336C, V240C_I332C, and V263C_V302C. In some embodiments, the pair of stability-enhancing mutations is L242C_I336C or V240C_I332C. In some embodiments, the pair of stability-enhancing mutations is L242C_I336C.

[0082] In certain embodiments, the IgG Fc variant comprises two or more stability-enhancing mutations. In some embodiments, the IgG Fc variant comprises two or more stability-enhancing mutations selected from the following: a mutation at position 250 selected from T250A, T250I and T250V, a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y; Mutation V308I, a mutation at position 309 selected from L309Q and L309T, Mutation M428F, and L242C and I336C mutations.

[0083] In certain embodiments, the IgG Fc variant comprises two stability-enhancing mutations: two stability-enhancing mutations selected from a mutation at position 250, a mutation at position 287, a mutation at position 308, a mutation at position 309, and a mutation at position 428, wherein the mutation at position 250 is selected from T250A, T250I, and T250V; the mutation at position 287 is selected from A287F, A287H, A287M, A287W, and A287Y; the mutation at position 308 is V308I; the mutation at position 309 is selected from L309Q and L309T; and the mutation at position 428 is M428F; or Mutations L242C and I336C.

[0084] In some embodiments, the IgG Fc variant comprises: a mutation at position 250 selected from T250A, T250I and T250V, and a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y; a mutation at position 250 selected from T250A, T250I and T250V, and the mutation V308I, a mutation at position 250 selected from T250A, T250I and T250V, and a mutation at position 309 selected from L309Q and L309T; a mutation at position 250 selected from T250A, T250I and T250V, and the mutation M428F, a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y, and the mutation V308I, a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y, and a mutation at position 309 selected from L309Q and L309T; a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y, as well as the mutation M428F, the mutation V308I and a mutation at position 309 selected from L309Q and L309T, Mutation V308I, and mutation M428F, a mutation at position 309 selected from L309Q and L309T, and the mutation M428F, or Mutations L242C and I336C.

[0085] In some embodiments, the IgG Fc variant comprises: a mutation at position 250 selected from T250A, T250I and T250V, and a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y; a mutation at position 250 selected from T250A, T250I and T250V, and a mutation at position 309 selected from L309Q and L309T; a mutation at position 250 selected from T250A, T250I and T250V, and the mutation M428F, a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y, and the mutation M428F, or Mutations L242C and I336C.

[0086] In some embodiments, the IgG Fc variant comprises a mutation at position 250 is T250V. In some embodiments, the IgG Fc variant comprises a mutation at position 287 is A287F. In some embodiments, the IgG Fc variant comprises a mutation at position 309 is L309Q.

[0087] In some embodiments, the IgG Fc variant comprises the stability-enhancing mutations T250V / A287F, T250V / V308I, T250V / L309Q, T250V / M428F, A287F / V308I, A287F / L309Q, A287F / M428F, V308I / L309Q, V308I / M428F, L309Q / M428F or L242C_I336C.

[0088] In some embodiments, the IgG Fc variant comprises the stability-enhancing mutations T250V / A287F, T250V / L309Q, T250V / M428F, A287F / M428F or L242C_I336C.

[0089] In some embodiments, the IgG Fc variant comprises the stability-enhancing mutations T250V / A287F, T250V / L309Q, T250V / M428F or A287F / M428F.

[0090] In some embodiments, the stability-enhancing mutations comprised in the Fc variant are selected from T250V, A287F, V308I, L309Q, M428F, L242C_I336C, A287F / M428F, T250V / A287F, T250V / L309Q, T250V / M428F and L242C_I336C / V308I.

[0091] In some embodiments, the stability-enhancing mutations comprised in the Fc variant are selected from A287F, V308I, L309Q, L242C_I336C, A287F / M428F, T250V / A287F, T250V / L309Q, T250V / M428F and L242C_I336C / V308I.

[0092] In certain embodiments, the IgG Fc variant comprises three or more stability-enhancing mutations. In some embodiments, the IgG Fc variant comprises three or more stability-enhancing mutations selected from the following: a mutation at position 250 selected from T250A, T250I and T250V, a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y, a mutation at position 309 selected from the mutation V308I, the mutation L309Q and L309T, the mutation M428F, and the mutations L242C and I336C.

[0093] In some embodiments, the IgG Fc variant comprises three stability-enhancing mutations: In some embodiments, the IgG Fc variant comprises three stability-enhancing mutations selected from the following: a mutation at position 250 selected from T250A, T250I and T250V, a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y, a mutation at position 309 selected from the mutation V308I, the mutation L309Q and L309T, the mutation M428F, and the mutations L242C and I336C.

[0094] In some embodiments, the IgG Fc variant comprises the mutations L242C and I336C and a mutation at position 250 selected from T250A, T250I, and T250V; a mutation at position 287 selected from A287F, A287H, A287M, A287W, and A287Y; the mutation V308I; a mutation at position 309 selected from L309Q and L309T, and the mutation M428F.

[0095] In certain embodiments, the IgG Fc variant comprises one to three stability-enhancing mutations. one or more mutations selected from a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y, a mutation V308I, and a mutation at position 309 selected from L309Q and L309T, two or more mutations selected from a mutation at position 250 selected from T250A, T250I and T250V, a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y, a mutation at position 309 selected from the mutation V308I, L309Q and L309T, the mutation M428F, and the mutations L242C and I336C, or Three or more mutations, including the mutations L242C and I336C, a mutation at position 250 selected from T250A, T250I and T250V, a mutation at position 287 selected from A287F, A287H, A287M, A287W and A287Y, a mutation at position 309 selected from the mutations V308I, L309Q and L309T, and a mutation selected from the mutation M428F.

[0096] Certain stability-enhancing mutations are known in the art. For example, the introduction of additional disulfide bonds by including mutations L242C_K334C, L240C_K334C, A287C_L306C, V259C_L306C, R292C_V302C or V323C_I332C in the Fc region has been shown to increase stability (Gong et al., 2009, J Biol Chem, 284(21):14203-14210; Jacobsen et al., 2017, J Boil Chem, 292(5):1865-1875). Other stability-enhancing mutations are described in US Patent Application Publication No. 2015 / 0210763. Certain embodiments of the present disclosure contemplate Fc variants that include a combination of one or more of the stability-enhancing mutations disclosed herein with one or more mutations previously shown to increase the stability of the Fc region.

[0097] method Certain embodiments of the present disclosure relate to methods of stabilizing an Fc region (parent Fc) by introducing one or more stability-enhancing mutations described herein into the parent Fc to provide an Fc variant.

[0098] Some embodiments of the present disclosure relate to methods of increasing the CH2 domain melting temperature (Tm) of an Fc region (parent Fc) by introducing one or more stability-enhancing mutations described herein into the parent Fc to provide an Fc variant having a CH2 domain Tm that is increased by at least 0.5°C compared to the parent Fc.

[0099] Some embodiments of the present disclosure relate to methods for increasing the CH2 domain Tm of a parent Fc, the methods comprising introducing one or more stability-enhancing mutations described herein into the Fc to provide an Fc variant, wherein the Fc variant has a CH2 domain Tm that is at least 0.5°C higher than the CH2 domain Tm of the parent Fc.

[0100] The parent Fc can be a wild-type Fc or a variant Fc that already contains one or more amino acid mutations, for example, to improve the function of the Fc region, hi some embodiments, the parent Fc can contain one or more amino acid mutations that improve the function of the Fc region but simultaneously decrease the CH2 domain Tm.

[0101] Some embodiments of the present disclosure relate to a method for increasing the CH2 domain Tm of a parent Fc having a lower CH2 domain Tm than a corresponding wild-type Fc, the method comprising introducing one or more stability-enhancing mutations described herein into the Fc to provide an Fc variant, wherein the Fc variant has a CH2 domain Tm that is at least 0.5°C higher than the CH2 domain Tm of the parent Fc.

[0102] In some embodiments, the method provides Fc variants with a CH2 domain Tm that is at least 1.0°C, at least 2.0°C, or at least 3.0°C higher than the CH2 domain Tm of the parent Fc.

[0103] In some embodiments, the method provides an Fc variant having a CH2 domain Tm that is about 0.5°C to about 6.5°C higher than the CH2 domain Tm of the parent Fc. In some embodiments, the CH2 domain Tm of the Fc variant is about 0.5°C to about 9.0°C, about 1.0°C to about 9.0°C, about 2.0°C to about 9.0°C, or about 3.0°C to about 9.0°C higher than the CH2 domain Tm of the parent Fc. In some embodiments, the CH2 domain Tm of the Fc variant is about 2.0°C to about 10.5°C, or about 3.0°C to about 10.5°C higher than the CH2 domain Tm of the parent Fc.

[0104] In certain embodiments, the method further comprises measuring the CH2 domain Tm of the Fc variant, hi some embodiments, the method further comprises measuring the CH2 domain Tm of the Fc variant by DSC or DSF.

[0105] In certain embodiments, the method comprises introducing one to five stability-enhancing mutations described herein into the parent Fc. In some embodiments, the method comprises introducing one to four stability-enhancing mutations described herein into the parent Fc. In some embodiments, the method comprises introducing one to three stability-enhancing mutations described herein into the parent Fc. In some embodiments, the method comprises introducing one, two, or three stability-enhancing mutations into the parent Fc.

[0106] In some embodiments, the method comprises introducing into the parent Fc one or more stability-enhancing mutations selected from the following: a mutation at position 250, wherein the mutation is a substitution of the amino acid at position 250 with Ala, Ile or Val; a mutation at position 287, wherein the mutation is a substitution of the amino acid at position 287 with Phe, His, Met, Trp or Tyr; a mutation at position 308, wherein the mutation is a substitution of the amino acid at position 308 with He; a mutation at position 309, wherein the mutation is a substitution of the amino acid at position 309 with Gln or Thr; a mutation at position 428, wherein the mutation is a substitution of the amino acid at position 428 with Phe, and A mutation at position 242 and a mutation at position 336, both mutations being substitutions with Cys.

[0107] In some embodiments, the method comprises introducing into the parent Fc a single stability-enhancing amino acid mutation selected from: a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr; a mutation at position 308 that is a substitution with He; or a mutation at position 309 that is a substitution with Gln or Thr.

[0108] In some embodiments, the method comprises introducing into the parent Fc two or more stability-enhancing amino acid mutations selected from: a mutation at position 250 that is a substitution with Ala, Ile, or Val; a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr; a mutation at position 308 that is a substitution with Ile; a mutation at position 309 that is a substitution with Gln or Thr; a mutation at position 428 that is a substitution with Phe; and mutations at positions 242 and 336, both of which are substitutions with Cys.

[0109] In some embodiments, the method comprises introducing into the parent Fc three or more stability-enhancing amino acid mutations, including a mutation at position 242 and a mutation at position 336, both of which are substitutions with Cys, and a mutation at position 250, which is substitution with Ala, He, or Val, a mutation at position 287, which is substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308, which is substitution with He, a mutation at position 309, which is substitution with Gln or Thr, and a mutation at position 428, which is substitution with Phe.

[0110] Certain embodiments relate to a method for increasing the CH2 domain Tm of an Fc region (parent Fc), comprising introducing one to three stability-enhancing amino acid mutations into the parent Fc to provide an Fc variant having an increased CH2 domain Tm compared to the parent Fc region, wherein the one to three stability-enhancing mutations include: (a) one or more mutations selected from a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with He, and a mutation at position 309 that is a substitution with Gln or Thr; or (b) two or more mutations selected from a mutation at position 250 that is a substitution with Ala, Ile, or Val, a mutation at position 287 that is a substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 that is a substitution with Ile, a mutation at position 309 that is a substitution with Gln or Thr, a mutation at position 428 that is a substitution with Phe, and a mutation at position 242 and a mutation at position 336 that are both substitutions with Cys; or (c) Three or more mutations including a mutation at position 242 and a mutation at position 336, both of which are substitutions with Cys, and a mutation selected from a mutation at position 250 which is substitution with Ala, Ile, or Val, a mutation at position 287 which is substitution with Phe, His, Met, Trp, or Tyr, a mutation at position 308 which is substitution with Ile, a mutation at position 309 which is substitution with Gln or Thr, and a mutation at position 428 which is substitution with Phe.

[0111] In some embodiments, the method comprises introducing stability-enhancing mutations 250V / 287F, 250V / 308I, 250V / 309Q, 250V / 428F, 287F / 308I, 287F / 309Q, 287F / 428F, 308I / 309Q, 308I / 428F, 309Q / 428F, or 242C_336C into a parent Fc. In some embodiments, the method comprises introducing stability-enhancing mutations 250V / 287F, 250V / 309Q, 250V / 428F, 287F / 428F, or 242C_336C into a parent Fc. In some embodiments, the method comprises introducing stability-enhancing mutations 250V / 287F, 250V / 309Q, 250V / 428F, or 287F / 428F into a parent Fc.

[0112] In certain embodiments, the method comprises introducing stability-enhancing mutations into a parent Fc selected from: 250V, 287F, 308I, 309Q, 428F, 242C_336C, 287F / 428F, 250V / 287F, 250V / 309Q, 250V / 428F, and 242C_336C / 308I. In some embodiments, the method comprises introducing stability-enhancing mutations into a parent Fc selected from: 287F, 308I, 309Q, 242C_336C, 287F / 428F, 250V / 287F, 250V / 309Q, 250V / 428F, and 242C_336C / 308I.

[0113] In certain embodiments, the parent Fc is an IgG, IgA, IgD, IgE, or IgM Fc, such as a human IgG, IgA, IgD, IgE, or IgM Fc. In some embodiments, the parent Fc is an IgG or IgA Fc, such as a human IgG or IgA Fc. In some embodiments, the parent Fc is an IgG Fc, such as a human IgG Fc.

[0114] In certain embodiments, the parent Fc is an IgG1, IgG2, IgG3, or IgG4 Fc, e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc. In some embodiments, the parent Fc is an IgG1 Fc, e.g., a human IgG1 Fc.

[0115] In certain embodiments, the method provides Fc variants having a CH2 domain Tm increased by at least 0.5°C compared to the parent Fc, and exhibit reduced aggregation of the Fc region compared to the parent Fc. In some embodiments, the Fc variants produced by the method have a CH2 domain Tm increased by at least 0.5°C compared to the parent Fc, and exhibit reduced aggregation of the Fc region at low pH compared to the parent Fc. In some embodiments, the Fc variants produced by the method have a CH2 domain Tm increased by at least 0.5°C compared to the parent Fc, and exhibit reduced aggregation under mildly acidic conditions compared to the parent Fc.

[0116] Assay The Fc variants of the present disclosure have increased stability compared to the parent Fc, which may result in increased thermal stability of the CH2 domain, reduced aggregation, increased serum half-life, increased manufacturability, or a combination thereof.

[0117] In certain embodiments, the Fc variant has increased thermal stability over the parent Fc, as determined by CH2 domain melting temperature (Tm). The CH2 domain Tm of the Fc variant and parent Fc can be measured, for example, by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorometry (DSF) using standard techniques. In certain embodiments, the Fc variant has increased stability over the parent Fc, as determined by the CH2 domain Tm, and the CH2 domain Tm is measured by DSC or DSF.

[0118] In certain embodiments, stability-enhancing mutations, when introduced into an Fc as single mutations, result in an increase in the CH2 domain Tm of the Fc variant of at least 0.5°C over the parent Fc. In some embodiments, stability-enhancing mutations, when introduced into an Fc as single mutations, result in an increase in the CH2 domain Tm of the Fc variant of at least 1.0°C, at least 1.5°C, at least 2.0°C, at least 2.5°C, or at least 3.0°C over the parent Fc. In some embodiments, stability-enhancing mutations, when introduced into an Fc as single mutations, result in an increase in the CH2 domain Tm of the Fc variant of about 0.5°C to about 6.5°C over the parent Fc.

[0119] In certain embodiments, stability-enhancing mutations, when introduced into an Fc as a combination of two or more mutations, result in an increase in the CH2 domain Tm of the Fc variant of at least 0.5°C over the parent Fc. In some embodiments, stability-enhancing mutations, when introduced into an Fc as a combination of two or more mutations, result in an increase in the CH2 domain Tm of the Fc variant of at least 1.0°C, at least 1.5°C, at least 2.0°C, at least 2.5°C, or at least 3.0°C over the parent Fc. In some embodiments, stability-enhancing mutations, when introduced into an Fc as a combination of two or more mutations, result in an increase in the CH2 domain Tm of the Fc variant of at least 5.0°C, at least 5.5°C, at least 6.0°C, at least 6.5°C, or at least 7.0°C over the parent Fc. In some embodiments, stability-enhancing mutations, when introduced into an Fc as a combination of two or more mutations, result in an increase in the CH2 domain Tm of the Fc variant of about 0.5°C to about 9.0°C over the parent Fc. In some embodiments, stability-enhancing mutations, when introduced into an Fc as a combination of two or more mutations, result in an increase in the CH2 domain Tm of the Fc variant by about 1.0° C. to about 9.0° C., about 2.0° C. to about 9.0° C., or about 3.0° C. to about 9.0° C. compared to the parent Fc. In some embodiments, stability-enhancing mutations, when introduced into an Fc as a combination of two or more mutations, result in an increase in the CH2 domain Tm of the Fc variant by about 2.0° C. to about 10.5° C., or about 3.0° C. to about 10.5° C. compared to the parent Fc.

[0120] In certain embodiments, increased stability of an Fc variant results in reduced aggregation and / or increased serum half-life of the Fc variant compared to the parent Fc. Aggregation and serum half-life can be measured by a variety of standard techniques known in the art. For example, aggregation of an Fc variant and parent Fc can be assessed by size exclusion chromatography (SEC) or dynamic light scattering (DLS). Serum half-life of an Fc variant and parent Fc can be assessed, for example, by pharmacokinetic studies in animal models.

[0121] In certain embodiments, the Fc variants have increased thermal stability (Tm) relative to the parent Fc, as determined by CH2 domain melting temperature (Tm), and also exhibit reduced aggregation. In some embodiments, the Fc variants have increased thermal stability (Tm) relative to the parent Fc, and also exhibit reduced aggregation at low pH. In some embodiments, the Fc variants have increased thermal stability (Tm) relative to the parent Fc, and also exhibit reduced aggregation under mildly acidic conditions.

[0122] To further characterize the Fc variants, other assays can optionally be performed using standard techniques. For example, the Fc variants can be assessed for purity, FcR binding, FcRn binding, aggregation, and / or C1q binding. Purity and aggregation can be assessed, for example, by liquid chromatography-mass spectrometry (LC-MS) and size exclusion chromatography (SEC), respectively. FcR and FcRn binding can be measured, for example, by surface plasmon resonance (SPR), SPR imaging (SPRi), biolayer interferometry (BLI), ELISA, kinetic exclusion assay (KinExA®), or Meso Scale Discovery™ (MSD™)-based methods (see, e.g., Current Protocols in Immunology: Ligand-Receptor Interactions in the Immune System, Eds. J. Coligan et al., 2018 & updates, Wiley Inc., Hoboken, NJ, and Yang et al., 2016, Analytical Biochem, 508:78-96). C1q binding can be assessed, for example, by ELISA or SPR.

[0123] In certain embodiments, the Fc variants are IgG Fc variants and may be assessed for FcγR binding and / or FcRn binding. Typically, binding affinity is measured using the dissociation constant (K) for binding of the Fc variant to FcγR or FcRn. DIn some embodiments where the Fc variant is an IgG Fc variant, the Fc variant retains substantially the same binding to each of the Fcγ receptors as the parent Fc. In some embodiments where the Fc variant is an IgG Fc variant, the Fc variant retains substantially the same binding to FcRn as the parent Fc. "Substantially the same binding" in this context refers to a decrease in K compared to the parent Fc. D This means a change of less than three-fold in

[0124] Polypeptides Certain embodiments of the present disclosure relate to polypeptides comprising the Fc variants described herein. Typically, the polypeptide comprises one or more additional proteinaceous moieties fused to or covalently attached to the Fc variant, e.g., by a linker. For example, the polypeptide can be an Fc fusion protein or an antibody or antibody fragment. Examples of proteinaceous moieties that can be fused to or attached to the Fc variant include, but are not limited to, antigen-binding domains, ligands, receptors, receptor fragments, cytokines, and antigens.

[0125] When a polypeptide comprises two or more additional proteinaceous moieties, these moieties can be the same or different. The one or more additional proteinaceous moieties can be fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus of one or both Fc polypeptides. In some embodiments, a polypeptide comprises one or more additional proteinaceous moieties fused to the N-terminus of one or both Fc polypeptides. In some embodiments, a polypeptide comprises one additional proteinaceous moiety fused to the N-terminus of one of the Fc polypeptides. In some embodiments, a polypeptide comprises two additional proteinaceous moieties, one fused to the N-terminus of a first Fc polypeptide and the other fused to the N-terminus of a second Fc polypeptide. In some embodiments, the two additional proteinaceous moieties comprised by a polypeptide can be linked in tandem.

[0126] In some embodiments, the polypeptide comprises an Fc variant fused to one or more proteinaceous moieties that are antigen-binding domains. In some embodiments, the polypeptide comprises an Fc variant and one or more antigen-binding domains. In some embodiments, the polypeptide comprises an Fc variant and two or more antigen-binding domains, for example, 2, 3, 4, 5, 6, 7, or 8 antigen-binding domains. When a polypeptide comprises an Fc variant and two or more antigen-binding domains, the antigen-binding domains may bind to the same antigen or they may bind to different antigens.

[0127] In some embodiments, the polypeptide comprises one or more proteinaceous moieties that are antigen-binding domains and an Fc variant fused to one or more other proteinaceous moieties. In some embodiments, the polypeptide comprises an antigen-binding domain and an Fc variant fused to one or more other proteinaceous moieties. Examples of other proteinaceous moieties in this context include, but are not limited to, receptors, receptor fragments (such as extracellular portions), ligands, and cytokines.

[0128] In some embodiments, the polypeptide may be an antibody or antibody fragment, wherein at least one of the one or more proteinaceous 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 comprising one antigen-binding domain. In some embodiments, the polypeptide may be a monospecific antibody comprising two antigen-binding domains. In some embodiments, the polypeptide may be a monospecific antibody comprising more than two antigen-binding domains. In some embodiments, the polypeptide may be a bispecific or multispecific antibody comprising an Fc variant and two or more antigen-binding domains, wherein the two or more antigen-binding domains bind to different antigens.

[0129] In some embodiments, the polypeptide may be a therapeutic or diagnostic antibody or antibody fragment, in which at least one of the one or more proteinaceous moieties is an antigen-binding domain.

[0130] In some embodiments, the polypeptide comprises an Fc variant and one or more antigen-binding domains that bind to a tumor-associated or tumor-specific antigen.

[0131] Preparation of Fc variants The Fc variants described herein and polypeptides comprising the Fc variants described herein can be prepared using standard recombinant methods. Recombinant production of Fc variants and polypeptides generally involves synthesizing one or more polynucleotides encoding the Fc variant or polypeptide, cloning the one or more polynucleotides into one or more appropriate vectors, and introducing the vector(s) into a suitable host cell for expression of the Fc variant or polypeptide. Recombinant production of proteins is well known in the art and can be accomplished using standard techniques described, for example, in 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).

[0132] Accordingly, certain embodiments of the present disclosure relate to isolated polynucleotides or sets of polynucleotides that encode an Fc variant described herein or that encode a polypeptide comprising an Fc variant described herein. A polynucleotide in this context may encode all or part of an Fc variant or polypeptide.

[0133] The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, isolated DNA, isolated RNA, nucleic acid probes, and primers.

[0134] A polynucleotide "encoding" a given polypeptide is one that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0135] One or more polynucleotides encoding Fc variants or polypeptides can be inserted into one or more suitable expression vectors using standard ligation techniques, either directly or after one or more subcloning steps. Examples of suitable vectors include, but are not limited to, plasmids, phagemids, cosmids, bacteriophages, baculoviruses, retroviruses, or DNA viruses. A vector is typically selected to be functional in the particular host cell to be used, i.e., the vector is compatible with the host cell machinery and allows amplification and / or expression of the polynucleotide(s). In this regard, selection of an appropriate vector and host cell combination is well within the ordinary skill of one of ordinary skill in the art.

[0136] Thus, certain embodiments of the present disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding an Fc variant or a polypeptide comprising an Fc variant. The polynucleotide(s) may comprise a single vector or more than one vector. In some embodiments, the polynucleotides are comprised in a multicistronic vector.

[0137] Typically, expression vectors contain one or more regulatory elements for plasmid maintenance and for the cloning and expression of exogenous polynucleotide sequences. Examples of such regulatory elements include a promoter, an enhancer sequence, an origin of replication, a transcription termination sequence, donor and acceptor splice sites, a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a polynucleotide encoding a polypeptide to be expressed, and a selectable marker.

[0138] Regulatory elements can be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of regulatory elements from two or more sources), or synthetic. Thus, the source of the regulatory elements can be any prokaryotic or eukaryotic organism, provided that they are functional in and can be activated by the machinery of the host cell used.

[0139] Optionally, the vector may also contain a "tag" coding sequence. A tag coding sequence is a nucleic acid sequence located at the 5' or 3' end of the coding sequence that encodes a heterologous peptide sequence such as polyHis (e.g., 6xHis), FLAG®, HA (influenza virus hemagglutinin), myc, metal affinity, avidin / streptavidin, glutathione-S-transferase (GST), or biotin tag. This tag typically remains fused to the expressed polypeptide and can serve as a means for affinity purification or detection of the polypeptide. Optionally, the tag can then be removed from the purified polypeptide by various means, such as using specific peptidases for cleavage.

[0140] A variety of expression vectors are readily available from commercially available sources. Alternatively, if a commercially available vector containing all the desired regulatory elements is not available, an expression vector can 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 individually obtained and ligated into the vector. Methods and sources for obtaining various regulatory elements are well known to those skilled in the art.

[0141] After construction of the expression vector(s) containing the polynucleotide(s) encoding the Fc variant or polypeptide, the vector(s) can be inserted into a suitable host cell for amplification and / or protein expression. Transfection of the expression vector into the selected host cell can be achieved by well-known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, and other known techniques. The method selected will depend, in part, on the type of host cell used. These and other suitable methods are well known to those of skill in the art (see, e.g., Sambrook, et al., ibid.).

[0142] When cultured under appropriate conditions, host cells express the polypeptide encoded by the vector, and the polypeptide can then be collected from the culture medium (if the host cells secrete the polypeptide) or directly from the host cells that produce it (if the polypeptide is not secreted). Host cells can be prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast, fungal, plant, or mammalian) cells. Selection of an appropriate host cell can be readily made by one of ordinary skill in the art, taking into consideration various factors, such as the desired expression level, polypeptide modifications desired or necessary for activity (such as glycosylation or phosphorylation), and ease of folding into a biologically active molecule.

[0143] Accordingly, certain embodiments of the present disclosure relate to host cells comprising polynucleotide(s) encoding an Fc variant or a polypeptide comprising an Fc variant, or one or more vectors comprising the polynucleotide(s). In some embodiments, the host cell is a eukaryotic cell.

[0144] For example, eukaryotic microorganisms such as filamentous fungi or yeast can be used as host cells, including fungal and yeast strains in which glycosylation pathways have 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 utilized as host cells (see, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, which describe PLANTIBODIES™ technology).

[0145] In some embodiments, the eukaryotic host cell is a mammalian cell. A variety of mammalian cell lines can be used as host cells. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney line CV1 (COS-7), human embryonic kidney line 293 (e.g., HEK293 as described in Graham et al., (1977), J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, (1980), Biol. Reprod., 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (e.g., Mather et al., 1982, Annals of 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 of suitable cell lines include, but are not limited to, human myeloma cell lines (including CHO cells), and myeloma cell lines (such as Y0, NS0, and Sp2 / 0). See also Yazaki and Wu, 2003, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKC Lo, ed., Humana Press, Totowa, NJ).

[0146] Certain embodiments of the present disclosure relate to methods of preparing an Fc variant described herein or a polypeptide comprising an Fc variant described herein, comprising transfecting a host cell with one or more polynucleotides encoding the Fc variant or polypeptide, e.g., in the form of one or more vectors comprising the polynucleotide(s), and culturing the host cell under conditions suitable for expression of the encoded Fc variant or polypeptide.

[0147] Typically, Fc variants or polypeptides are isolated from host cells after expression and can optionally be purified. Methods for isolating and purifying expressed proteins are well known in the art. Standard purification methods include chromatographic techniques such as ion exchange, hydrophobic interaction, affinity, sizing, gel filtration, or reversed phase, which can be performed at atmospheric pressure or at medium or high pressure using systems such as FPLC, MPLC, and HPLC. Other purification methods include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques in combination with protein concentration can also be useful.

[0148] Various natural proteins are known in the art to bind to the Fc region of antibodies, and therefore these proteins can be used to purify Fc-containing proteins. For example, bacterial proteins A and G bind to the Fc region. Purification can often be enabled by specific fusion partners or affinity tags, as described above. For example, antibodies can be purified by binding to glutathione resins when GST fusions are used, or Ni-tags when His-tags are used.+2 Purification can be performed using affinity chromatography, or, if a FLAG tag is used, using 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 Ed., Scopes, Springer-Verlag, NY (1994).

[0149] Pharmaceutical Compositions Certain embodiments of the present disclosure relate to therapeutic uses of Fc variants or polypeptides comprising Fc variants. For therapeutic use, the Fc variants and polypeptides may be provided in the form of compositions comprising the Fc variant or polypeptide and a pharmaceutically acceptable carrier or diluent. The compositions may be prepared by known procedures using well-known and readily available ingredients. For example, they may be formulated for administration to patients via oral (e.g., buccal or sublingual), topical, parenteral, rectal, or vaginal routes, or by inhalation or aerosol. The term "parenteral," as used herein, includes injection or infusion via subcutaneous, intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal, or intrathecal routes.

[0150] The compositions are typically formulated in a form suitable for administration to a subject by a selected route, for example, as a syrup, elixir, tablet, troche, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable solution, or solution. The compositions may be provided as unit dose formulations.

[0151] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include buffers, such as phosphate, citric acid, and other organic acids, antioxidants, such as ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (e.g., methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 amino acids) polypeptides, proteins, such as serum albumin. or 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 dextrins, 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 non-ionic surfactants such as polyethylene glycol (PEG).

[0152] In certain embodiments, the composition may be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents known in the art. The sterile injectable solution or suspension may contain the Fc variant or polypeptide in a non-toxic, parenterally acceptable diluent or solvent. Acceptable diluents and solvents that may be used include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic saline. In addition, sterile, fixed oils may be used as solvents or suspending agents. For this purpose, various mild, fixed oils, including synthetic mono- or diglycerides, may be used. In addition, fatty acids such as oleic acid have been found to be useful in injectable preparations. Adjuvants such as local anesthetics, preservatives, and / or buffers known in the art may also be included in the injectable solution or suspension.

[0153] Other pharmaceutical compositions and methods for preparing pharmaceutical compositions are known in the art; see, e.g., "Remington: The Science and Practice of Pharmacy" (formerly "Remington's Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0154] How to use Certain embodiments of the present disclosure relate to the use of Fc variants or polypeptides comprising Fc variants as therapeutic, diagnostic or research tools. Some embodiments relate to the therapeutic use of Fc variants and polypeptides comprising Fc variants.

[0155] Polypeptides comprising the Fc variants and one or more antigen binding domains, e.g., antibodies or antibody fragments, described herein are particularly useful as diagnostics and therapeutics. Accordingly, some embodiments relate to methods of using polypeptides comprising an Fc variant and one or more antigen binding domains in the diagnosis of a disease or disorder in a patient. Some embodiments relate to methods of using polypeptides comprising an Fc variant and one or more antigen binding domains in the treatment of a disease or disorder in a patient in need thereof.

[0156] The disease or disorder to be diagnosed or treated depends on the antigen or antigens targeted by the antigen-binding domain. Examples of diseases and disorders that can be diagnosed or treated include, but are not limited to, inflammatory diseases and disorders, autoimmune diseases and disorders, and proliferative diseases and disorders such as various cancers.

[0157] The following examples are offered for illustrative purposes and are not intended to limit the scope of the claimed invention in any way. [Example]

[0158] General method Preparation of variants Variants and controls were prepared by site-directed mutagenesis and / or restriction / ligation using standard methods. The final DNA was subcloned into the vector pTT5 (see U.S. Patent No. 9,353,382). All scaffolds used for the preparation of variants were based on IgG1 Fc. The sequence of the IgG1 Fc region is provided in Figure 1A. In certain clones, the C-terminal lysine residue was omitted from the Fc sequence.

[0159] The following scaffolds were used:

[0160] Scaffold 1: Trastuzumab-based full-size antibody (FSA) with homodimeric IgG1 Fc (SEQ ID NO: 1).

[0161] Scaffold 2: An array-armed antibody (OAA) scaffold comprising trastuzumab Fab and a heterodimeric IgG1 Fc containing the following mutations: Chain A:T350V_L351Y_F405A_Y407V Chain B: T350V_T366L_K392L_T394W

[0162] Scaffold 3: a full-size antibody (FSA) based on trastuzumab containing the same heterodimeric Fc as scaffold 2.

[0163] Scaffold 4: Full-size antibody (FSA) based on the 4G7 anti-CD19 antibody (Meeker, et al., 1984, Hybridoma, 3:305-320; U.S. Patent No. 8,524,867) containing the same heterodimeric Fc as scaffold 2.

[0164] 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 heterodimeric Fc as scaffold 2. Variable domain sequences were obtained from International Patent Application Publication No. WO2013 / 132044.

[0165] Scaffold 6: A full-size antibody (FSA) based on trastuzumab containing the N297A mutation (Leabman, et al, 2013, mAb, 5(6):896-903), which results in an aglycosylated Fc and abrogates binding to all FcγRs.

[0166] Scaffold 7: A full-size antibody (FSA) based on trastuzumab containing the S239D and I332E mutations (Lazar, et al, 2006, PNAS, 103:4005-4010), which results in increased FcγRIIIa binding.

[0167] Scaffold 8: An array-armed antibody (OAA) scaffold comprising trastuzumab Fab and a heterodimeric IgG1 Fc containing the following mutations that result in increased FcγRIIb selectivity: TIFF0007766049000003.tif41165

[0168] "Template 1" shows the substitution of amino acid residues at positions 325 to 331 with the following sequence: STWFDGGYAT [SEQ ID NO: 2].

[0169] Scaffold 9: An array-armed antibody (OAA) scaffold comprising trastuzumab Fab and a heterodimeric IgG1 Fc containing the following mutations that result in increased FcγRIIb selectivity: TIFF0007766049000004.tif35165

[0170] Expression - Protocol 1 Expression was carried out in 200 mL of CHO3E7 cells. CHO cells were incubated with 1 mg / mL of aqueous 25 kDa polyethyleneimine (PEI pro Cells were transfected in exponential growth phase (1.5-2 million cells / mL) at a PEI:DNA ratio of 2.5:1 using Polyplus Transfection SA (Illkirch, France) (Delafosse, et al., 2016, J. Biotechnol., 227:103-111). To determine the optimal concentration range for heterodimer formation, DNA was transfected at the optimal DNA ratio of heavy chain A (HC-A), light chain (LC), and heavy chain B (HC-B) that allowed heterodimer formation (e.g., HC-A / HC-B / LC ratio = 30:30:40%). For homodimer expression, a 50:50% ratio of HC:LC was used. Transfected cells were harvested after 5-6 days, with the medium collected after centrifugation at 4000 rpm and clarified using a 0.45 μm filter.

[0171] The clarified 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 at pH 7.2. The antibody was eluted with TRIS at pH 11, with pooled fractions containing neutralized antibody being washed with 10 column volumes of citrate buffer at pH 3.6. The sample was then buffer-exchanged into PBS pH 7.4 and stored at -80°C.

[0172] Expression - Protocol 2 Expression was carried out at either small scale (1 mL) or large scale (30 mL or more) using HEK293-6E cells (NRC, Canada).

[0173] For 1 mL-scale expression, HEK293-6E cells were transfected in exponential growth phase (1.5–2 million cells / mL) using DNA precomplexed with the cationic lipid 293Fectin™ (Life Technologies, Paisley, UK) at 1 μg DNA / mL. Heavy and light chain DNA were mixed at a ratio of 47.5:52.5%, and DNA was complexed with 293Fectin™ to a final concentration of 11.7 μg / mL DNA and 1.65% (v / v) 293Fectin™, then incubated for 30 min at ambient temperature before addition to cells. The DNA ratio of HC-A and HC-B in the transfection mixture to achieve optimal heterodimer formation was either 50:50%, or slightly varied. Cells were cultured in 96-well deep-well plates sealed with gas-permeable seals in a humidified shaking incubator at 37°C and 5% carbon dioxide for 5-6 days, and the medium was then collected after centrifugation at 1600 × g.

[0174] For large-scale expression, HEK293-6E cells were transfected at exponential growth phase (1.5–2 million cells / mL) using DNA precomplexed with Gemini cationic lipids at 1 μg DNA / mL (Camilleri et al., 2000, Chem. Commun., 1253–1254). Heavy and light chain DNA were mixed at a 50:50 ratio, and the DNA was complexed with Gemini to 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 addition to the cells. The ratio of HC-A and HC-B DNA in the transfection mixture was as described above. Cells were placed in appropriately sized Erlenmeyer flasks or BioReactor tubes and cultured at 37°C and 5% carbon dioxide in a humidified shaking incubator for up to 10 days. The culture medium was then harvested after centrifugation at 2750×g and clarified using a 0.22 μm filter.

[0175] Clarified culture fluid was loaded onto a MabSelect™ SuRe™ (GE Healthcare, Little Chalfont, UK) Protein A column, washed with 3–10 column volumes of Tris-Acetate buffer at pH 7.5, and then eluted with 2–5 column volumes of acetic acid at pH 2.6, with the elution fraction neutralized with TRIS. Further purification by size exclusion chromatography (Superdex™ 200 column (GE Healthcare, Little Chalfont, UK) with PBS running buffer) and / or cation exchange (ReSource™ S column (GE Healthcare, Little Chalfont, UK)) was utilized for selected samples. Protein A-purified antibodies were buffer-exchanged into PBS.

[0176] Preparation of Fcγ and FcRn receptors Protocol 1 FcγRIIaH, IIaR, IIb, IIIaF, and IIIaV were produced in HEK293-6E cells, while FcγRIa was produced in CHO-3E7 cells as previously described (see Dorian-Thibaudeau, et al., 2014, J. Immunol. Methods, 408:24-34). Human FcRn was also expressed in HEK293-6E cells by cotransfecting the alpha subunit (p51) extracellular domain containing a TEV-cleavable C-terminal His tag with β2-microglobulin at a 1:1 ratio. After purification as described in Dorian-Thibaudeau et al. (ibid.), the C-terminal His tag was removed by TEV cleavage.

[0177] Protocol 2 Soluble FcγRI extracellular domains with a C-terminal 6xHis tag were purchased from R&D Systems (catalog no. 1257-Fc). Soluble FcγRIIaH, IIaR, IIb, IIIaF, and IIIaV extracellular domains were produced in HEK293-6E cells with a C-terminal 10xHis tag. Cells were transfected in exponential growth phase (1.5–2 million cells / mL) at 1 μg DNA / mL using DNA precomplexed with Gemini cationic lipids (Camilleri et al., 2000, Chem. Commun., 1253–1254). Cells were plated in appropriately sized Erlenmeyer flasks and cultured at 37°C and 5% carbon dioxide in a humidified shaking incubator for up to 7 days. The harvest time was determined when cell viability fell below 50%. The culture medium was then harvested after centrifugation at 2750×g and clarified using a 0.22 μm filter.

[0178] The clarified medium was exchanged into a loading buffer containing 25 mM imidazole, pH 7.7, by dialysis or tangential flow filtration, applied to a Ni Sepharose 6 column (GE Healthcare, Little Chalfont, UK), and then eluted by increasing the buffer imidazole concentration to 300 mM. The eluted protein was concentrated and buffer-exchanged into PBS by diafiltration, and then further purified by size-exclusion chromatography on a Superdex® 75 column (GE Healthcare, Little Chalfont, UK).

[0179] Soluble human FcRn extracellular domain was expressed in HEK293-6E cells by cotransfection of the alpha subunit containing a C-terminal 6xHis tag with β2-microglobulin at a 1:1 ratio; FcγR was expressed as described elsewhere. The pH of the clarified culture medium was adjusted to pH 5.3 with citrate and then loaded onto an IgG Sepharose column (GE Healthcare, Little Chalfont, UK). Bound protein was eluted with HEPES buffer at pH 7.7. The eluted protein was concentrated and buffer-exchanged into PBS by diafiltration, then further purified by size-exclusion chromatography (Superdex® 75 column (GE Healthcare, Little Chalfont, UK)).

[0180] Fcγ receptor binding (surface plasmon resonance (SPR)) Protocol 1 The affinity of FcγRs for antibody Fc was measured by SPR using a ProteOn™ XPR36 at 25°C with PBS containing 150 mM NaCl, 3.4 mM EDTA, and 0.05% Tween 20 at 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 4.0 μg / mL of HER2 prepared in 10 mM NaOAc (pH 4.5) until approximately 3,000 resonance units (RU) were immobilized. The remaining activated groups were then quenched with ethanolamine. Wild-type trastuzumab variants were indirectly captured on these SPR surfaces by injecting 40 μg / mL of solution-purified antibody in the ligand direction at 25 μL / min for 240 seconds, resulting in approximately 500 RU on the surface. Following a buffer injection to establish a stable baseline in the analyte direction, the analyte was injected at 50 μL / min for 120 seconds with a 180-second dissociation phase to obtain a set of binding sensorgrams. A three-fold dilution series of FcγRs was used at five concentrations, with a top nominal concentration of 10 μM for all receptors, except for FcγR1a, which was 30 nM. Buffer was also included for double referencing. The resulting Kd (affinity) values ​​were determined from the alignment and reference sensorgrams using an equilibrium-fit model in ProteOn™ Manager v3.1.0 and reported as the average of two or three independent runs.

[0181] Protocol 2 The affinity of FcγRs to antibody Fc was measured by SPR using a Biacore™ 4000 (GE Healthcare, Little Chalfont, UK) at 25°C with PBSTE (PBS with 0.05% Tween-20 and 3.4 mM EDTA) as the running buffer. For anti-HER2 antibodies, CM5 chips (GE Healthcare, Little Chalfont, UK) were immobilized with recombinant HER2 extracellular domain (Merck, Darmstadt, Germany, or ThermoFisher Scientific, Loughborough, UK) using amine coupling (EDC / NHS chemistry). Briefly, the CM5 sensor chip was activated with NHS / EDC, followed by injection of 10.0 μg / mL HER2 prepared in 10 mM NaOAc (pH 4.5). Immobilization levels ranged from 1,000 to 4,000 RU. Any remaining active groups were then quenched with ethanolamine. Antibodies were first captured on the immobilized surface of the chip by injecting approximately 15 μg / ml across the spot and flow cell for 35 seconds at a flow rate of 10 μl / min. Spot 3 was left blank for baseline subtraction. Receptors were diluted in PBSTE buffer to a defined concentration range depending on the expected affinity. Six concentrations per analyte, including zero, were used. Analyte contact times were optimized depending on the receptor used and its expected kinetics. For example, the contact time for FcγRIIB and FcγRIIaR was 18 seconds at 30 μl / min. The chip surface was regenerated with 87 mM phosphoric acid after each analyte concentration injection. Prior to testing, the chip was prepared by injecting 87 mM phosphoric acid for 3 × 18 seconds. Double reference subtraction was performed (reference spot 3 and zero receptor concentration), and binding responses were normalized by antibody capture level. Samples were analyzed using either kinetic and / or steady-state (equilibrium) fitting models.

[0182] FcRn binding (surface plasmon resonance (SPR)) Protocol 1 The affinity of FcRn for antibody variant Fc was measured by SPR using a ProteOn™ XPR36 at 25°C in HBS-EP+ (10 mM HEPES, 150 mM NaCl, 0.003% EDTA, and 0.05% v / v surfactant P20 (Teknova, Hollister, USA)) at pH 7.4 or pH 6.0 as the running buffer. Protein L (ThermoScientific, Loughborough, UK) was immobilized onto a GLM sensor chip using standard amine coupling with a GE Healthcare coupling kit. Briefly, the GLM sensor chip was activated with NHS / EDC, followed by injection of 50 μg / mL of Protein L prepared in 10 mM NaOAc (pH 4.5) until approximately 3000 resonance units (RU) were immobilized. The remaining activated groups were then quenched with ethanolamine. Antibody variants were indirectly captured on the SPR surface by injecting a 50 μg / mL purified antibody solution in the ligand direction at 30 μL / min for 120 seconds. Following a buffer injection to establish a stable baseline in the analyte direction, the analyte was injected at 40 μL / min for 300 seconds, with a 600-second dissociation phase to obtain a set of binding sensorgrams. Five concentrations of a four-fold dilution series of FcRn with a top nominal concentration of 2048 nM were used, and buffer was included for double referencing. The resulting Kd (affinity) values ​​were determined from the alignment and reference sensorgrams using an equilibrium-fit model in ProteOn™ Manager v3.1.0.

[0183] Protocol 2 The affinity of FcRn for antibody variant Fc was measured by SPR using a Biacore™ T200 (GE Healthcare, Little Chalfont, UK) at 25°C with HBS-EP + pH 7.4 or MES pH 6.0 as the running buffer. Samples were captured on an immobilized Protein L CM5 chip (GE Healthcare), but the 4G7 anti-CD19 antibody failed to capture. The antibody was first captured on the immobilized surface of the chip by injecting approximately 15 μg / ml into the spot and flow cell for 60 seconds at a flow rate of 5 μl / min. The receptor was diluted to a range of concentrations in HBS-EP + pH 7.4 or MES pH 6.0 buffer. Three concentrations (4096, 512, and 0 nM) were used per sample at pH 7.4, and four concentrations (512, 64, 8, and 0 nM) were used per sample at pH 6.0. The chip surface was regenerated with 10 mM glycine pH 1.5 after each analyte concentration injection. Results were analyzed using Biacore™ T200 Evaluation V2 software and a 1:1 binding kinetics model.

[0184] Protocol 3 FcRn affinity was measured by SPR using an IBIS MX96 (IBIS Technologies, Enschede, The Netherlands) at 25°C with HBS-EP + pH 7.4 or MES pH 6.0 as the running buffer. Samples were diluted with pH 4.5 acetate buffer and then captured onto a SensEye® G Easy2Spot® sensor chip (SensEye, Enschede, The Netherlands) using a continuous-flow microspotter (Carterra, Salt Lake City, USA). The receptor was diluted to a predetermined concentration range in HBS-EP + pH 7.4 or MES pH 6.0 buffer. Three concentrations (4096, 512, and 0 nM) were used per analyte at pH 7.4, and four concentrations (512, 64, 8, and 0 nM) were used per analyte at pH 6.0. The chip surface was regenerated with 10 mM glycine pH 2.0 after injection of each analyte concentration. Results were analyzed using Scrubber V2 (BioLogic Software, Campbell, Australia) and a kinetic fitting model.

[0185] Protocol 4 Antibodies were screened for FcRn binding using a Biacore™ T200 (GE Healthcare) surface plasmon resonance instrument. Experiments were performed at 25°C using a running buffer containing PBS with 0.05% Tween® 20 and 3.4 mM EDTA at pH 6. Biotinylated FcRn (produced by Protocol 1 above) was captured on a CM-5 sensor chip with neutravidin (Thermo Fisher, Waltham, MA) previously immobilized on blank and capture surfaces using standard amine coupling. Antibody dilutions were then run over the FcRn and control surfaces. Using the immobilization wizard within the Biacore control software, neutravidin prepared in 10 mM sodium acetate pH 4.5 buffer was added at 25 μg / mL to each NHS / EDC-activated surface until 2000 RU was reached. To generate the FcRn surface, biotinylated FcRn was diluted to 2 μg / mL in PBS containing 0.05% Tween® 20 and 3.4 mM EDTA at pH 7.4 and injected over the capture surface at a flow rate of 25 μg / mL for 110 seconds until 32 RU were captured. This FcRn surface was used for all antibodies. Antibodies were screened in duplicate using single-cycle kinetics. Five concentrations between 900 and 11.1 nM were injected at a flow rate of 25 μL / min with 90 seconds of association and 180 seconds of dissociation, using a 3-fold dilution in pH 6 running buffer. The FcRn surface was regenerated between different antibody variants by injecting pH 7.4 buffer at 30 μL / min for 30 seconds. Sensorgrams were double-referenced against a blank control surface and fitted using an affinity binding model to generate KD values ​​for each antibody-FcRn interaction.

[0186] Differential scanning calorimetry 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 a VP-Capillary DSC (GE Healthcare). At the beginning of each DSC run, five buffer blank injections were performed to stabilize the baseline, and a buffer injection was set before each sample injection for reference. Each sample was scanned from 20 to 100 °C at a rate of 60 °C / h using low feedback, an 8-second filter, a 5-minute pre-scan thermostat, and 70 psi nitrogen pressure. The resulting thermograms were referenced and analyzed using Origin7 software (OriginLab Corporation, Northampton, MA).

[0187] Protocol 2 The antibody constructs were evaluated by the same method as described for Protocol 1 above, except that antibody concentrations of 0.1-1.0 mg / ml were used, preferably 0.4 mg / ml or higher.

[0188] Differential scanning fluorimetry Protocol 1 20 μL of purified sample (0.2–1.0 mg / mL) was added to 10 μL of SYPRO® Orange (Invitrogen, Paisley, UK), diluted 20-fold from a 5000x stock with reverse osmosis (RO) water, and placed in a clear-walled 96-well PCR plate. Samples were incubated at 40°C for 5 min, and then SYPRO® Orange fluorescence was measured from 40°C to 95°C at a rate of 15°C / h using a BioRad CFX Connect™ RT-PCR machine (BioRad, Watford, UK). Bio-Rad CFX Manager™ version 3.1 was used to analyze peaks of protein unfolding events, which correlated with the unfolding of known domains within the protein, and to derive temperatures.

[0189] Protocol 2 Ten microliters of purified sample (0.2–1.0 mg / mL) was loaded into a Prometheus NT.Plex nanoDSF standard-grade glass capillary (PR-AC002, NanoTemper Technologies, London, UK) and analyzed in a Prometheus NT.Plex nanoDSF (NanoTemper Technologies, London, UK) at 20–95 °C using a rate of 60 °C / h. PR.stability Analysis software version 1.02 was used to analyze peaks of protein unfolding events, which correlated with the unfolding of known domains within the protein, and to derive temperatures.

[0190] Size Exclusion Chromatography: Ten microliters of purified sample (within a concentration range of 0.2–2 mg / mL) was injected onto a Supelco TSKgel® G3000 SWXL size-exclusion column (Tosoh, Reading, UK) using an Agilent 1100 HPLC system (Agilent, Stockport, UK) running a 400 mM sodium phosphate, 200 mM NaCl, pH 6.8 mobile phase at a constant rate of 1.0 mL / min for a run time of 15 min per sample. A diode array detector was connected along the column, and UV / vis absorption at 210 nm and 280 nm was recorded. The resulting traces were integrated using Chemstation software (Agilent, Stockport, UK) and subsequently analyzed using ChromView™ software. Sample purity was recorded by classifying the area percent of the main peak relative to the total area percent of peaks with higher molecular weights and peaks with lower molecular weights.

[0191] Liquid chromatography mass spectrometry Mass spectrometry was used to confirm the identity of the samples. To remove N-linked glycosylation, 10 μl of 80 μg / mL PNGase F in 5% glycerol was added to 50 μL of antibody sample (within a concentration range of 0.2–2 mg / mL), and the mixture was incubated overnight at 30°C. Just prior to MS analysis, 5 μL of 0.5 M DTT was added to each sample. Using an Agilent 1200 HPLC system (Agilent Technologies, Stockport, UK), 3–5 μg of sample was injected onto a reversed-phase guard column. After washing with 0.1% formate, 5% acetonitrile, the sample was eluted with 0.1% formate, 90% acetonitrile at a flow rate of 0.5 mL / min. The eluate was directed to a 6224 Accurate-Mass TOF LC / MS mass spectrometer (Agilent Technologies) controlled from a PC running MassHunter software (Agilent Technologies). The masses of the samples were determined by charge envelope deconvolution using MassHunter.

[0192] C1q binding The binding of the antibody constructs to human C1q was assessed by ELISA. The test antibody constructs were coated onto the wells of a 96-well flat-bottom Nunc Maxisorp® plate (Invitrogen, Paisley, UK) by adding 100 μl per well of 10 μg / ml of test antibody dissolved in PBS. The plate was sealed and incubated at 4° C. for 16 hours. The plate was washed three times with 300 μl of PBS containing 0.05% (v / v) Tween® 20. 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 washed as described above. Recombinant human C1q (C1740, Sigma Aldrich, Gillingham, UK) was diluted to the final assay concentration in 50 mM carbonate / bicarbonate buffer (C3041, Sigma Aldrich) and added at 100 μl per well. Samples were incubated at ambient temperature for 2 hours, and the plates were washed as described above. Then, 100 μl of sheep anti-human C1q-HRP (Ab46191, AbCam, Cambridge, UK) diluted to 2 μg / ml in PBS was added per well, samples were incubated at ambient temperature for 1 hour, and the plates were then washed as before. For detection, 100 μl of Sureblue™ TMB (52-00-01, Seracare, Milford, USA) was added per well, and samples were incubated at ambient temperature for 20 minutes with agitation. 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). Each antibody variant 7 C1q concentration was tested in duplicate at half-log steps from 2 μg / mL to 6 ng / mL, plus a no C1q control. Data were analyzed using Prism (GraphPad, San Diego, USA). Binding curves were fitted using a four-parameter nonlinear regression model of absorbance and log-transformed C1q concentration.The concentration of C1q at which binding exceeded the threshold absorbance was interpolated from the fitted curve.

[0193] Example 1: Stability mutations identified with in silico prediction tools Using the three-dimensional structure of FcγRIIb bound to IgG1 Fc, single mutations were generated in silico at each position in the CH2 domain and at specific positions in the CH3 domain, which is located within the two shells of the CH2 domain. The first shell residues are residues that directly interact with the CH2 domain, and the second shell residues are residues that interact with at least one first shell residue. All possible amino acid substitutions except proline or cysteine ​​were made at each position. Identified mutations that enhance the thermal stability of the Fc are referred to as stability-enhancing mutations.

[0194] We first investigated the relevance of stability-enhancing mutations to FcγRIIb-selective Fc variants. Because binding of FcγRIIb to IgG Fc results in an asymmetric complex, only mutations that improved in silico stability for both chains of the Fc were selected for testing to ensure that the stability mutations were compatible with FcγRIIb-selective variants, as well as other antibody therapeutics.

[0195] All in silico-generated models were analyzed using multiple molecular modeling tools, including in silico mutagenesis and packing modeling with in-house software tools. All models were scored and ranked based on multiple factors, including knowledge-based and physics-based probabilities for folding and complex formation, as well as energy contributions that combine electrostatics, solvent screening, Lennard-Jones, and hydrophobic interactions.

[0196] The results were further filtered to select the best stability-enhancing mutations, preferably with the following characteristics: No solvent exposure (less than 30% solvent accessible surface area (SASA) preferred, less than 50% acceptable) ●Away from the FcγR interface No known adverse effects on FcRn binding No known adverse effects on C1q binding • It does not affect N-glycosylation of Fc at position N297.

[0197] The above process identified the following mutations as potential stability-enhancing mutations: A287F, T289W, A339W, A339Q, A378W, and M428F. Six variants of trastuzumab (Scaffold 1), each containing one of the above-identified mutations, were constructed as described in General Methods. Each variant was evaluated for expression, aggregation, thermal stability, and binding affinity for FcγRIIa, FcγRIIb, and FcRn as described in General Methods. Specifically, aggregation was evaluated by analytical SEC, thermal stability by DSC (Protocol 2) and DSF (Protocol 1), FcγRIIa and FcγRIIb binding by Biacore binding (Protocol 2), and FcRn binding by Protocol 1. The results are shown in Tables 1.1 and 1.2.

[0198] [Table 1.1]

[0199] [Table 1.2]

[0200] Mutations A287F and M428F were selected and further evaluated based on the following criteria: T by DSC exceeding 2°C for point mutations m Increase in Retains wild-type-like properties for FcγRIIb and FcγRIIa binding (WT value ±30%) Retention of binding to FcRn (K relative to WT) D (less than a two-fold difference in •More than 95% monomer content by analytical SEC.

[0201] The stabilization by the A287F mutation is energetically favorable and likely results from the creation of stacking π-π interactions with W277 and the burying of the hydrogen bond between W277 and S304. Therefore, alternating mutations at these positions with amino acids with similar aromatic and hydrophobic properties are predicted to increase and enhance stability. These include the mutations A287Y, A287W, and A287H, as well as A287M. The latter mutation is predicted to bury the hydrogen bond but is likely to result in lower stabilization due to the loss of π-π stacking interactions.

[0202] FIG. 1B shows positions A287 and M428 in the IgG Fc region.

[0203] Example 2: Stability mutations identified by bioinformatics analysis Sequences from multiple organisms and IgG subtypes (59 nonredundant sequences extracted from Uniprot) were aligned with ClustalX (Larkin, et al., 2007, Bioinformatics, 23:2947-2948) to differentiate functional residues from those that are conserved or potentially substituted for folding. Non-surface-exposed positions with some sequence identity across all IgGs were identified, along with alternative common residue(s) found in other species or subtypes. Additional filtering based on relative SASA at selected positions was included to reduce the risk of immunogenicity.

[0204] The results were filtered to select the best stability-enhancing mutations, preferably with the following characteristics: No solvent exposure (less than 30% solvent accessible surface area (SASA) preferred, less than 50% acceptable) Partially conserved (less than 85% sequence identity across all CH2IgGs is preferred, and less than 95% is acceptable) ●Away from the FcγR interface No known adverse effects on FcRn binding No known adverse effects on C1q binding • It does not affect N-glycosylation of Fc at position N297.

[0205] The amino acid substitutions found in mouse IgG2a are in the CH2 domain T m Mouse IgG2a (T m = approximately 80°C) for human IgG1 (T m = 72 °C), it was preferred over the other species. The stability of the CH2 domain for the other species was not considered.

[0206] The above approach identified the mutations L242I, T250V, F275I, V279I, V308I, Y319F, P247I, M252S, L309Q, L314M, and K334R as potential stability-enhancing mutations. Eleven variants of trastuzumab (Scaffold 1) were constructed as described in General Methods, each containing one of the identified mutations. Each variant was evaluated for expression in mammalian cells, aggregation after purification, thermal stability, and binding affinity for FcγRIIb, FcγRIIa, and FcRn, as described in Example 1. The results are shown in Tables 2.1 and 2.2.

[0207] [Table 2.1]

[0208] [Table 2.2]

[0209] The mutations T250V, L309Q and V308I were selected for further evaluation based on the following criteria: T by DSC exceeding 2°C for point mutations m Increase in Retains wild-type-like properties for FcγRIIb and FcγRIIa binding (WT value ±30%) Retention of binding to FcRn (K less than WT) D (2-fold difference in • Monomer content by analytical SEC greater than 95%.

[0210] Alternating mutations at these positions, such as T250I, T250A, or L309T, are predicted to increase stability due to similar amino acid properties in terms of size and hydrophobicity. Small differences in amino acid size (V vs. I or A) and side chain branching (Cβ-branched vs. unbranched residues) can result in small variations in stabilizing effect.

[0211] FIG. 1B shows the location of position T250 in the IgG Fc region.

[0212] Example 3: Stabilizing mutations containing non-native disulfide bonds Using the three-dimensional structure of FcγRIIb bound to IgG1 Fc, distances between all Cα and Cβ atom pairs in the CH2 domain were calculated and averaged across both chains of the Fc to determine where disulfide bonds could be introduced. Filtering was based on a maximum distance of 7.5 Å for Cα-Cα pairs and 5.0 Å for Cβ-Cβ pairs.

[0213] Because binding of FcγRIIb to IgG Fc results in an asymmetric complex, only mutations that improved stability in silico for both chains of the Fc were selected for testing, which ensured that stabilizing mutations matched the selective variants for FcγRIIb and the non-binding Fc.

[0214] In silico models were generated for all possible artificial disulfide bonds and energy minimized to determine the one with the lowest energy. Models were visually inspected and scored based on the relative solvent-accessible surface area (SASA) of the cysteine ​​residues in both chains, the root mean square deviation (RMSD) of the main chain, backbone, and side chains relative to the wild-type structure, improvements in knowledge-based and physics-based possibilities for affinity and stability, steric clashes, and disulfide strain energy (DSE) scores (Katz & Kossiakoff, 1986, J Biol Chem, 261(33):15480-15485).

[0215] The results were filtered to select the best stability-enhancing mutations, preferably with the following characteristics: No exposure to solvents (relative SASA greater than 30% preferred, less than 50% acceptable) ●Away from the FcγR interface No known adverse effects on FcRn binding No known adverse effects on C1q binding Does not affect N-glycosylation of Fc at position N297 Low backbone and side chain RMSD (RMSD<0.5Å for backbone, RMSD<1.0Å for side chain) Equivalent or improved knowledge-based and physics-based possibilities for stability • Low DSE score (less than 30 (DSE) preferred, less than 50 acceptable).

[0216] Through the above process, the following mutation pairs were identified as potential stability-enhancing mutations: D249C-P257C, F275C-S304C, V263C-V302C, L242C-I336C, T289C-S304C, F243C-T260C, V266C-Y300C, V240C-I332C, and W277C-V284C. Thirteen variants of trastuzumab (Scaffold 1) were constructed as described in General Methods, each containing one or more pairs of identified mutations previously reported in the literature to improve stability through the introduction of disulfide bonds (Jacobsen, et al., 2017, J Biol Chem, 292(5):1865-1875; Gong, et al., 2009, J Biol Chem, 284(21):14203-14210; Gong, et al., 2011, J Biol Chem, 286(31):27288-27293). Each variant was evaluated for expression in mammalian cells, aggregation, thermostability, and binding affinity for FcγRIIb, FcγRIIa, and FcRn as described in Example 1. Affinity for the FcRn receptor was also evaluated in a subset of successful variants. The results are shown in Tables 3.1 and 3.2.

[0217] [Table 3.1]

[0218] [Table 3.2]

[0219] Of the identified disulfide bonds, only L242C-I336C was selected for further evaluation based on the following criteria: T by DSC above 2°C for a single pair of mutations m Increase in Retains wild-type-like properties for FcγRIIb and FcγRIIa binding (WT value ±30%) • Monomer content by analytical SEC greater than 95%.

[0220] Mutation Pairs The mutation pairs known in the art, L242C-K334C, A287C-L306C and V259C-L306C, met the following criteria: T by DSC above 2°C for a single pair of mutations m Increase in Retains wild-type-like properties for FcγRIIb and FcγRIIa binding (WT value ±30%)

[0221] The disulfide bond V240C-I332C improved T but partially inhibited FcγR binding, and it is believed that this disulfide bond may still be useful in certain situations where inhibited binding is desired or can be alleviated by including other mutations that enhance binding to one or more FcγRs.

[0222] Example 4: Stabilization of FcγRIIB-selective Fc variants The six best individual mutations identified in the trastuzumab homodimer described in Examples 1-3 (A287F, M428F, T250V, L309Q, L242C_I336C, and V308I) were transferred to two different heterodimeric trastuzumab FcγRIIb-selective variants (scaffolds 8 and 9) to evaluate their compatibility with other CH2 domain mutations. Additionally, six combinations of two or three stability-enhancing mutations (A287F / M428F, A287F / T250V, M428F / T250V, A287F / M428F / T250V, T250V / L309Q, and L242C_I336C / V308I) were tested to evaluate whether they conferred increased stability through additive or synergistic effects.

[0223] Twenty-four variants of trastuzumab in a panel-armed antibody format were constructed as described in General Methods. Each variant contained one of two sets of FcγRIIb-selectivity-enhancing mutations (scaffold 8 or scaffold 9, see Table 4.1) along with the stability-enhancing mutations shown in Tables 4.2-4.4. Each variant was evaluated for expression, aggregation, thermal stability, and binding affinity for FcγRIIb, FcγRIIa, and FcγRI as described in Example 1. The results are shown in Tables 4.2-4.4.

[0224] [Table 4.1]

[0225] First-layer filtering was performed after purification based on analytical SEC profiles. The area under the curve of the chromatogram was integrated for all signals present and converted to the percentage of each species present in the variant sample. The percentage of high molecular weight (HMW) species observed in the analytical SEC profile indicates the abundance of full-size antibody formed for each variant using a single DNA ratio for expression. Variants with less than 20% HMW species upon expression at a single DNA ratio were considered successful. Only three variants had more than 20% HMW species (see Table 4.2) and were not included for further characterization. Low molecular weight (LMW) species indicate the presence of mismatched Fc homodimers, which do not interfere with Tm determination or binding affinity for either FcγR.

[0226] [Table 4.2]

[0227] [Table 4.3]

[0228] [Table 4.4]

[0229] Mutations were evaluated based on the following criteria: T with DSF>1°C for a single point mutation m Increased and minimal additive effects when combined Retention of wild-type-like properties for FcγRIIb, FcγRIIa, and FcγRI binding (less than 2-fold difference compared to the parental variant) • Heterodimer content greater than 75% by analytical SEC.

[0230] Based on the above, the most effective single mutations were A287F (+3.5-4°C), T250V (+5.5°C), L309Q (+2-2.5°C), and M428F (+2°C). V308I as a single mutation also resulted in a slight increase in Tm (+0.5-1.0°C).

[0231] Stability-enhanced designs with additive or synergistic contributions include A287F / M428F (+6.5-7°C), A287F / T250V (+9.0-9.5°C), M428F / T250V (+8.5°C), and T250V / L309Q (+8.5-9.0°C). The combinations A287F / M428F, M428 / T250V, and T250V / L309Q have slightly higher T than additive effects. m The combination L242C_I336C / V308I also resulted in a slight increase in Tm compared to the L242C_I336C mutation alone.

[0232] Example 5: Stabilization of additional full-size antibody test systems Three of the stability-enhanced designs were each combined with three FcγRIIb selectivity-enhanced designs and transferred into three different full-size antibody systems to assess the transferability of the designs between antibodies. The designs were cloned into heterodimeric trastuzumab, anti-CD19, and anti-CD40 antibodies (scaffolds 3-5) as described in General Methods. The three FcγRIIb selectivity-enhanced designs are shown in Table 5.1, and the three selected stability-enhanced designs are shown in Tables 5.2-5.5.

[0233] [Table 5.1]

[0234] Each variant was evaluated for aggregation and thermal stability after expression and purification in mammalian cells as described in Example 1. Binding affinity for FcγRI, FcγRIIb, FcγRIIa, and FcγRIIIa for trastuzumab-based variants was evaluated as described in Example 1. C1q binding for trastuzumab-based variants was evaluated as described in General Methods. Thermal stability was evaluated by DSF across multiple antibodies and by DSC for trastuzumab-based variants. Results are shown in Tables 5.2-5.6.

[0235] Analytical SEC profiles were collected as described in General Methods, with the following modifications for full-size antibody species: The area under the curve of the chromatogram was integrated for all signals present and converted to a percentage of each species. The percentage of high molecular weight (HMW) species observed in the aSEC profile indicates the amount of aggregates formed for each variant upon introduction of stability-enhancing mutations and was compared to the parental variant across each antibody series. Variants with parent-like properties (+ / - 5% HMW species) across multiple antibodies were preferred. Low molecular weight (LMW) species indicate the presence of mismatched heterodimeric heavy chains resulting in half antibodies and are due to the use of non-optimized DNA ratios during expression. The results are shown in Table 5.2.

[0236] Purity by LC-MS was assessed as described in the general methods to confirm that the monomer content was predominantly the desired heterodimeric species. The cumulative percentage of half A and half B indicates the amount of mismatched homodimers present in each sample for a given DNA ratio used for expression. Because the stability-enhancing mutations are symmetric, the CH2 domain Tm, FcRn, and C1q binding are unaffected by the presence of mismatched species. However, a high content of mismatched homodimers would affect the FcγRIIb selectivity observed by SPR. Most samples showed less than 10% mismatched homodimers. See Table 5.3.

[0237] [Table 5.2]

[0238] [Table 5.3]

[0239] [Table 5.4]

[0240] [Table 5.5]

[0241] [Table 5.6]

[0242] All three stability-enhanced designs tested (A287F / T250V, M428F / T250V, and A287F / M428F) successfully increased thermal stability by 6-10°C while maintaining parent-like properties in all other aspects evaluated. Specifically, the designs met the following criteria: T of DSF>5°C across all three antibodies m Increase in. • Retention of wild-type-like properties with respect to FcγRI, FcγRIIaH, FcγRIIaR, FcγRIIb, FcγRIIIa and C1q binding (less than 2-fold difference from the parental variant). ●Heterodimer content equal to or greater than that of the parent variant by LC-MS. Monomer content similar to or better than the parent variant by aSEC.

[0243] Example 6: FcRn binding of full-size antibodies containing stability-enhancing designs Three of the stability-enhanced designs were each combined with three FcγRIIb selectivity-enhanced designs and transferred to three different full-size antibody systems (trastuzumab, anti-CD19 antibody, and anti-CD40 antibody; scaffolds 3-5) as described in Example 5. The resulting variants were evaluated for FcRn binding as described in the general method (protocol 2 for scaffolds 3 / 5 and protocol 3 for scaffolds 3-5). The results are shown in Table 6.1.

[0244] [Table 6.1]

[0245] For all antibodies tested, the stability enhancement design was performed using the respective parent variants (K D <3-fold), demonstrating that the design is transferable across antibodies with different FcRn binding.

[0246] Example 7: Compatibility of stability-enhancing designs with other mutations that cause stability loss To further evaluate the portability and suitability of three of the best stability-enhancing designs (A287F / T250V, M428F / T250V, and A287F / M428F), these designs were each combined with the following three sets of CH2 or CH3 mutations (scaffolds 3, 6, and 7; see General Methods): ●Scaffold 3: Asymmetric mutations in the CH3 domain to promote heterodimeric Fc formation. Scaffold 6: The N297A mutation produces an aglycosylated antibody and inhibits the antibody's effector function. The introduction of the N297A mutation reduces the thermal stability of the variant antibody by 10°C compared to the wild-type antibody. ● Scaffold 7: S239D / I332E mutations that increase the affinity of the antibody for the FcγRIIIa receptor. Introduction of the S239D / I332E mutations reduces the thermal stability of the variant antibody by 20°C compared to the wild-type antibody.

[0247] Each parental variant and mutant was cloned into the trastuzumab backbone as described in General Methods. Each variant was evaluated for expression in mammalian cells (Protocol 1), thermal stability, binding affinity for FcγRI, FcγRIIb, FcγRIIa, and FcγRIIIa (Protocol 1), and FcRn binding (Protocol 4) as described in General Methods. Thermal stability was assessed by DSC (Protocol 1). Results are shown in Tables 7.1-7.4 and Figures 2A-C.

[0248] [Table 7.1]

[0249] [Table 7.2]

[0250] [Table 7.3]

[0251] [Table 7.3]

[0252] All three stability-enhanced designs tested (A287F / T250V, M428F / T250V, and A287F / M428F) successfully increased the thermal stability of each of the scaffolds tested by 6-10°C while maintaining parent-like properties in all other aspects evaluated. Specifically, all three designs met the following criteria: DSC T > 5°C across all three frameworks m Increase in • Retention of wild-type-like properties with respect to FcγRI, FcγRIIaH, FcγRIIaR, FcγRIIb, FcγRIIIa and FcRn binding (less than 2-fold difference from the parental variant).

[0253] Thus, the stability-enhancing design can accommodate and stabilize mutations in the CH2 and CH3 domains that alter the functional and biological properties of the antibody.

[0254] The above set of CH2 or CH3 mutations (exemplified by scaffolds 3, 6, and 7) are examples of mutations contained in therapeutic molecules currently being clinically evaluated (Saxena et al., 2016, Front Immunol, 7:580). Other CH2 and / or CH3 mutations that affect antibody function and stability, such as knobs-into-holes (Ridgeway et al., 1996, Protein Eng., 9:617-621), electrostatic steering (Gunasekaran et al., 2010, JBC, 285, 19637-19646), or others known in the art, are also expected to be compatible with and stabilized by the stability-enhancing mutations.

[0255] Example 8: Compatibility of Stability-Enhanced Design with Other Immunoglobulin Classes The sequences and topologies of IgG, IgA, IgD, IgE, and IgM constant domains were evaluated to determine whether the most effective stability-enhancing designs identified above could be transferred to other immunoglobulin (Ig) classes and / or subtypes.

[0256] The various Ig classes have different functions and biological activities but share a common topology. IgG, IgA, IgD, IgE, and IgM all consist of heavy and light chains. IgG, IgA, and IgD constant regions contain CH1, CH2, and CH3 domains that share a common Ig fold, suggesting that mutations that increase IgG stability may be transferable to the IgA and IgD classes. Unlike other Ig classes, IgE and IgM consist of CH1, CH2, CH3, and CH4 domains. Based on sequence identity, the CH3 and CH4 domains of IgM and IgE can be considered equivalent to the CH2 and CH3 domains of other Ig classes (see Figures 3A and 3B). A review of the structures of IgG, IgA, and IgM Ig domains obtained from the Protein Data Bank (PDB) (PDB IDs: 2QEJ, 2WAH, and 6KXS, respectively) shows that, from a structural point of view, these domains have a similar fold, suggesting that mutations that increase the stability of IgG may be transferable to the IgM and IgE classes.

[0257] Further analysis of the specific interactions of each of the T250V, A287F, and M428F mutations within the CH2 and CH3 domains further supported the proposition that stability-enhancing mutations should be transferable to other Ig classes.

[0258] Residue T250 is located within a helical region of IgG1, close to the FcRn-binding site of the CH2 domain and spatially close to the CH3 domain. A threonine residue is conserved across all IgG subtypes at this position and is replaced by a similar polar residue (serine) in IgM and a charged residue (aspartic acid) in IgA, IgD, and IgE (see Figure 3A). Structural analysis of this region in IgA, IgM, and IgG showed that the helices in IgA and IgM are less buried (PDB IDs: 2QEJ and 6KXS) than in IgG1 (PDB ID: 2WAH). Therefore, mutation of a charged or polar residue at the position corresponding to T250 in other Ig classes to a smaller, hydrophobic residue may improve the packing of the first helix (246-254) relative to the second helix (309-316) and the junction of the CH2-CH3 domains, resulting in a more compact structure with an increased buried interface. This, in turn, may stabilize the CH2 domain against thermal denaturation. Therefore, the stability-enhancing mutation T250V is predicted to be effective in increasing the stability of the CH2 domains of IgA, IgD, and IgG antibodies, and the CH3 domains of IgE and IgM antibodies.

[0259] Residue A287 is located in the exposed β-strand region outside the Ig fold of the CH2 domain of IgG1. An alanine residue is conserved at position 287 across all IgG subtypes and IgA, but is replaced by residues such as valine, histidine, and threonine in IgD, IgE, and IgM. However, despite the different residues present at this position, the local environment and fold are similar across all Ig classes for which structures are available. As described in Example 1, stabilization by the A287F mutation in IgG1 likely results from energetically favorable, stacked π-π interactions with W277 and buried hydrogen bonds between W277 and S304. Residue W277 is conserved across all Ig classes, and residue S304 is conserved across all Igs except IgM (see Figures 3A and 3B). Therefore, the A287F mutation is also predicted to be effective in increasing the stability of the CH2 domains of IgA, IgD and IgG antibodies, and the CH3 domains of IgE and IgM antibodies.

[0260] Residue M428 is located in the exposed β-strand region of the CH3 domain of IgG1, spatially at the interface with the CH2 domain. While the methionine at position 428 is conserved across all IgG subtypes, other Ig classes contain smaller residues at this position, such as glycine, valine, alanine, or serine (see Figures 3A and 3B). The introduction of a larger residue, such as phenylalanine, at this position likely buries its hydrophobic side chain against the helix from the CH2 domain, increasing the buried surface at the CH2-CH3 domain junction, reducing flexibility and increasing the stability of the CH2 domain. The local structural environment in IgA and IgM is similar to that of IgG in this region, and the introduction of a larger aromatic residue, such as phenylalanine, tyrosine, or tryptophan, is expected to form more stacked π-π interactions with surrounding aromatic residues. Therefore, the mutation M428F is also predicted to increase the stability of the CH2 domains of IgA, IgD, and IgG antibodies, and the CH3 domains of IgE and IgM antibodies.

[0261] As shown in Examples 4-7, the stability-enhancing mutations T250V, A287F, and M428F are pairwise compatible and can be combined to provide additive stabilizing effects. Similar compatibility is expected for these stability-enhancing designs (A287F / T250V, M428F / T250V, and A287F / M428F) across other Ig classes.

[0262] Example 9: Effect of stability mutations on aggregation tendency under stress conditions To assess whether the increased thermal stability achieved by incorporating stability-enhancing mutations could reduce the aggregation tendency of antibodies, 15 antibodies with variant Fc regions with some degree of thermal stability were prepared, with or without the T250V / A287F stability-enhancing design. The resulting antibody variants were then subjected to stress experiments and evaluated for changes in aggregation rate after two weeks of incubation at 40°C under neutral or slightly acidic conditions.

[0263] Each antibody variant was based on scaffold 3 and contained various combinations of mutations in the CH2 domain, as shown in Table 9.1. Each parental variant and stability mutant was cloned into scaffold 3 as described in General Methods. Each variant was evaluated for expression in mammalian cells (Protocol 2), aggregation by size exclusion chromatography, and thermal stability by DSF (Protocol 2). The results are shown in Tables 9.1 and 9.2.

[0264] Each variant was then normalized to 10 mg / ml and dialyzed into either acetate buffer or phosphate-based buffer to be tested under weakly acidic or neutral conditions, respectively, and incubated at either 4°C or 40°C for 2 weeks. The variants were then assessed for the percentage of aggregates, monomers, and fragments by size exclusion chromatography, and the 4°C and 40°C samples were compared. The results are shown in Table 9.3 and Figure 4.

[0265] [Table 9.1] TIFF0007766049000027.tif23168 1 Mimoto, et al., 2013, Prot. Eng. Des. Sel., 26:589-598 2 Chu, et al., 2008, Mol. Immunol., 45:3926-3933 3 Lazar, et al., 2006, PNAS, 103:4005-4010 4 "Template 1.1" shows the substitution of amino acid residues 325-331 of chain B with the sequence: STWFIGGYAT [SEQ ID NO: 3]. 5 "Template 1.2" shows the substitution of amino acid residues 325-331 of chain B with the sequence: STWFDKGYAT [SEQ ID NO: 5]. 6 "Template 7.1" shows the substitution of amino acid residues 325-331 of chain B with the sequence: GLDHRGKGYV [SEQ ID NO: 4].

[0266] [Table 9.2]

[0267] [Table 9.3]

[0268] As can be seen from Table 9.2, incorporation of the T250V / A287F stability-enhancing mutations successfully increased the thermal stability of 15 variants by between 7.7°C and 10.6°C. Thus, these stability-enhancing mutations can increase CH2 stability independent of the starting stability of the antibody construct.

[0269] The results in Table 9.3 show that after two weeks of incubation at 40°C under acidic conditions, three of the variants with the lowest CH2 Tm (variants v31186, v32210, and v32242) exhibited high levels of observed aggregation by analytical SEC. Addition of the stability-enhancing T250V / A287F mutations to these variants increased the variant's Tm, and minimal amounts of HMW species were detected after stress experiments. Figure 4 also shows a strong, nearly exponential correlation between CH2 Tm and observed aggregation under mildly acidic conditions for these variants. For variants with initial (unstabilized) CH2 Tm above 60°C, little change in HMW species (less than 2%) was observed. The small changes in HMW species observed after incubation under neutral conditions appear to be unrelated to CH2 Tm.

[0270] Overall, these results indicate that in general, the incorporation of stabilizing mutations, specifically T250V / A287F, can reduce aggregation propensity under mildly acidic conditions.

[0271] The disclosures of all patents, patent applications, publications, and database entries referenced in this specification are specifically incorporated by reference in their entireties herein to the same extent as if each individual patent, patent application, publication, and database entry was specifically and individually indicated to be incorporated by reference.

[0272] Modifications of the specific embodiments described herein which may be obvious to those skilled in the art are intended to be included within the scope of the following claims.

Claims

1. An Fc variant comprising two or more stability-enhancing amino acid mutations, wherein the stability-enhancing amino acid mutations are: a mutation at position 287, wherein said mutation is a substitution of the amino acid at position 287 with Phe; and a mutation at position 250, wherein the mutation is a substitution of the amino acid at position 250 with Ala, He, or Val; and A mutation at position 428, wherein the mutation is a substitution of the amino acid at position 428 with Phe. one or more additional mutations selected from Including, the Fc variant is based on a parent IgG Fc that does not comprise the two or more stability-enhancing amino acid mutations and that has a reduced CH2 domain melting temperature (Tm) compared to the corresponding wild-type IgG Fc; the Fc variant has an increased CH2 domain Tm compared to a parent IgG Fc that does not contain the two or more stability-enhancing amino acid mutations; and Amino acid numbering is according to the EU index, The Fc variant.

2. The Fc variant of claim 1, wherein the additional mutation comprises a mutation at position 250 that is a substitution with Ala, Ile, or Val.

3. The Fc variant of claim 2, wherein the mutation at position 250 is a substitution with Val.

4. An Fc variant described in any one of claims 1 to 3, wherein the additional mutation is a mutation at position 428, which is a substitution of the amino acid at position 428 with Phe.

5. The Fc variant of claim 1, wherein the stability-enhancing mutations contained in the Fc variant are selected from 287F / 428F, 250V / 287F, and 250V / 287F / 428F.

6. The Fc variant of claim 1, wherein the stability-enhancing mutations contained in the Fc variant are 287F / 428F or 250V / 287F.

7. 7. The Fc variant of any one of claims 1 to 6, wherein the parent IgG Fc is an IgG1 Fc.

8. 8. The Fc variant of claim 7, wherein the parent IgG Fc is a human IgG1 Fc.

9. The Fc variant of any one of claims 1 to 8, wherein the parent Fc comprises one or more amino acid mutations that improve the function of the Fc region.

10. 10. The Fc variant of any one of claims 1 to 9, wherein the CH2 domain Tm of said Fc variant is increased by at least 3.0°C compared to the parent IgG Fc.

11. The Fc variant of any one of claims 1 to 9, wherein the CH2 domain Tm of the Fc variant is increased by 3.0°C to 9.0°C compared to the parent IgG Fc.

12. The Fc variant of any one of claims 1 to 9, wherein the CH2 domain Tm of the Fc variant is increased by 3.0°C to 10.5°C compared to the parent IgG Fc.

13. The Fc variant of any one of claims 1 to 12, wherein the Fc variant exhibits reduced aggregation under weakly acidic conditions compared to the parent IgG Fc.

14. The two or more stability-enhancing amino acid mutations are a mutation at position 308, wherein the mutation is a substitution of the amino acid at position 308 with He; A mutation at position 309, wherein the mutation is a substitution of the amino acid at position 309 with Gln or Thr; and A pair of mutations at positions 242 and 336, both of which are substitutions with Cys.

14. The Fc variant of any one of claims 1 to 13, further comprising one or more mutations selected from:

15. 15. A polypeptide comprising an Fc variant according to any one of claims 1 to 14 and one or more proteinaceous moieties fused or covalently linked to said Fc variant, wherein said one or more proteinaceous moieties comprise an antigen-binding domain, a ligand, a receptor, a receptor fragment, a cytokine, or an antigen.

16. 16. The polypeptide of claim 15, wherein at least one of the one or more proteinaceous moieties is an antigen-binding domain.

17. The polypeptide of claim 16, wherein the antigen-binding domain is a Fab or scFv.

18. The polypeptide of any one of claims 15 to 17, which is an antibody or an antigen-binding antibody fragment.

19. 19. The polypeptide of claim 18, which is a therapeutic antibody or antibody fragment.

20. A polynucleotide or a set of polynucleotides encoding the Fc variant of any one of claims 1 to 14, or the polypeptide of any one of claims 15 to 19.

21. A vector or a set of vectors comprising one or more polynucleotides encoding an Fc variant according to any one of claims 1 to 14, or a polypeptide according to any one of claims 15 to 19.

22. A host cell comprising one or more polynucleotides encoding an Fc variant according to any one of claims 1 to 14 or a polypeptide according to any one of claims 15 to 19.

23. 20. A method for preparing an Fc variant according to any one of claims 1 to 14, or a polypeptide according to any one of claims 15 to 19, comprising transfecting a host cell with one or more polynucleotides encoding said Fc variant or polypeptide, and culturing said host cell under conditions suitable for expression of said Fc variant or polypeptide.

24. A pharmaceutical composition comprising an Fc variant according to any one of claims 1 to 14 or a polypeptide according to any one of claims 15 to 19.

25. 1. A method for increasing the CH2 domain melting temperature (Tm) of an Fc, the method comprising introducing two or more stability-enhancing amino acid mutations into a parent Fc to provide an Fc variant having an increased CH2 domain Tm compared to said parent Fc, wherein the stability-enhancing amino acid mutations are: a mutation at position 287, wherein said mutation is a substitution of the amino acid at position 287 with Phe; and a mutation at position 250, wherein the mutation is a substitution of the amino acid at position 250 with Ala, He, or Val; and A mutation at position 428, wherein the mutation is a substitution of the amino acid at position 428 with Phe. one or more additional mutations selected from Including, the parent Fc is an IgG Fc with a reduced CH2 domain Tm compared to the corresponding wild-type IgG Fc; Amino acid numbering is according to the EU index, The method.

26. The method of claim 25, wherein the Fc variant comprises a mutation at position 250 that is a substitution with Ala, Ile, or Val.

27. ​​The method described in claim 26, wherein the mutation at position 250 is a substitution with Val.

28. The method of claim 25, wherein the Fc variant comprises a mutation at position 428 that is a substitution with Phe.

29. 29. The method of any one of claims 25-28, wherein the CH2 domain Tm of the Fc variant is increased by at least 3.0°C compared to the parent Fc.

30. A method described in any one of claims 25 to 28, wherein the CH2 domain Tm of the Fc variant is increased by 3.0°C to 9.0°C compared to the parent Fc.

31. A method described in any one of claims 25 to 28, wherein the CH2 domain Tm of the Fc variant is increased by 3.0°C to 10.5°C compared to the parent Fc.

32. The method of any one of claims 25 to 31, wherein the introduction of the stability-enhancing amino acid mutations into the parent Fc provides an Fc variant that exhibits reduced aggregation under weakly acidic conditions compared to the parent Fc.

33. 33. The method of claim 32, wherein the stability-enhancing amino acid mutations include 250V and 287F.

Images