Immunoglobulin fc-region variants comprising stability-enhancing mutations

Abstract

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

Description

Technical Field

[0001] This disclosure relates to the field of immunoglobulins, and in particular to immunoglobulin Fc variants containing stability-enhancing mutations. Background Technology

[0002] Immunoglobulin-based drugs are becoming an increasingly important treatment approach, and monoclonal antibodies have been identified as a major treatment for various diseases over the past 25 years.

[0003] Numerous studies have focused on engineering immunoglobulins to improve various functions. For example, the Fc region of antibodies has been modified to improve pharmacokinetics, enhance or reduce antibody-dependent 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 antibody properties, including thermal stability.

[0004] Work on improving the stability of engineered antibodies includes introducing mutations that provide novel disulfide bonds (Gong et al., 2009, J Biol Chem, 284(21):14203-14210; Jacobsen et al., 2017, J Biol Chem, 292(5):1865-1875) and combinations of introducing point mutations (International Patent Application Publication No. WO 2012 / 032080). Methods for improving the stability of the Fc region of an antibody by introducing various amino acid substitutions into the loop region of the Fc region are also described (US Patent Application Publication No. 2015 / 0210763).

[0005] This background information is provided so that the applicant may recognize known information as potentially relevant to this disclosure. It is not to be acknowledged, nor should it be construed, that any of the foregoing information constitutes prior art to the claimed invention. Summary of the Invention

[0006] This document describes immunoglobulin Fc region variants containing stability-enhancing mutations. One aspect of this disclosure relates to an Fc variant comprising one or more stability-enhancing amino acid mutations selected from: a mutation at position 250, wherein the mutation is a substitution of the amino acid at position 250 by Ala, Ile, or Val; a mutation at position 287, wherein the mutation is a substitution of the amino acid at position 287 by Phe, His, Met, Trp, or Tyr; a mutation at position 308, wherein the mutation is a substitution of the amino acid at position 308 by Ile; a mutation at position 309, wherein the mutation is a substitution of the amino acid at position 309 by Gln or Thr; a mutation at position 428, wherein the mutation is a substitution of the amino acid at position 428 by Phe; and a pair of mutations at positions 242 and 336, wherein both mutations are substitutions by Cys, wherein the CH2 domain melting temperature (Tm) of the Fc variant is increased compared to the parental Fc without the one or more stability-enhancing amino acid mutations.

[0007] Another aspect of this disclosure relates to an Fc variant comprising one to three stability-enhancing amino acid mutations, said mutations comprising: (a) one or more mutations selected from: a mutation at position 287, said mutation being a substitution of Phe, His, Met, Trp, or Tyr; a mutation at position 308, said mutation being a substitution of Ile; and a mutation at position 309, said mutation being a substitution of Gln or Thr; or (b) two or more mutations selected from: a mutation at position 250, said mutation being a substitution of Ala, Ile, or Val; a mutation at position 287, said mutation being a substitution of Phe, His, Met, Trp, or Tyr; a mutation at position 308, said mutation being a substitution of Ile; a mutation at position 309, said mutation being a substitution of Gln or Thr; a mutation at position 428, said mutation being a substitution of Ile; a mutation at position 309, said mutation being a substitution of Gln or Thr; a mutation at position 428, said mutation being a substitution of Ile; a mutation at position 309, said mutation being a substitution of Ile ... The substitution is a Phe substitution; and a pair of mutations at positions 242 and 336, both of which are Cys substitutions; or (c) three or more mutations, including: a pair of mutations at positions 242 and 336, both of which are Cys substitutions, and a mutation selected from: a mutation at position 250, which is a substitution of Ala, Ile, or Val; a mutation at position 287, which is a substitution of Phe, His, Met, Trp, or Tyr; a mutation at position 308, which is a substitution of Ile; a mutation at position 309, which is a substitution of Gln or Thr; and a mutation at position 428, which is a substitution of Phe, wherein the CH2 domain melting temperature (Tm) of the Fc variant is increased compared to the parent Fc that does not contain the one or more stability-enhancing amino acid mutations.

[0008] Another aspect of this disclosure relates to a polypeptide comprising an Fc variant as described herein and one or more protein moieties fused or covalently linked to the Fc variant.

[0009] Another aspect of this disclosure relates to a polynucleotide or polynucleotide group that encodes an Fc variant as described herein.

[0010] Another aspect of this disclosure relates to a polynucleotide or polynucleotide group that encodes a polypeptide comprising an Fc variant as described herein and one or more protein portions fused or covalently linked to the Fc variant.

[0011] Another aspect of this disclosure relates to a vector or group of vectors comprising one or more polynucleotides encoding an Fc variant as described herein.

[0012] Another aspect of this disclosure relates to a vector or group of vectors comprising one or more polynucleotides encoding a polypeptide comprising an Fc variant as described herein and one or more protein portions fused or covalently linked to the Fc variant.

[0013] Another aspect of this disclosure relates to a host cell containing one or more polynucleotides that encode the Fc variant as described herein.

[0014] Another aspect of this disclosure relates to a host cell comprising one or more polynucleotides encoding a polypeptide comprising an Fc variant as described herein and one or more protein portions fused or covalently linked to the Fc variant.

[0015] Another aspect of this disclosure relates to a method for preparing an Fc variant as described herein, the method comprising transfecting a host cell with one or more polynucleotides encoding the Fc variant, and culturing the host cell under conditions suitable for expressing the Fc variant.

[0016] Another aspect of this disclosure relates to a method for preparing a polypeptide comprising an Fc variant as described herein and one or more protein 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.

[0017] Another aspect of this disclosure relates to a pharmaceutical composition comprising the Fc variant as described herein.

[0018] Another aspect of this disclosure relates to a pharmaceutical composition comprising a polypeptide including an Fc variant as described herein and one or more protein portions fused or covalently linked to the Fc variant.

[0019] Another aspect of this disclosure relates to a method for increasing the melting temperature (Tm) of the CH2 domain of an Fc, comprising introducing one or more stability-enhancing amino acid mutations into a parental Fc to provide an Fc variant with an increased CH2 domain Tm compared to the parental Fc, the mutations being selected from: 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 Ile; 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, wherein both mutations are substitutions with Cys.

[0020] Another aspect of this disclosure relates to a method for increasing the melting temperature (Tm) of the CH2 domain of an Fc, comprising introducing one to three stability-enhancing amino acid mutations into a parental Fc to provide an Fc variant with an increased CH2 domain Tm compared to the parental Fc, the mutations comprising: (a) one or more mutations selected from: a mutation at position 287, the mutation being a substitution of Phe, His, Met, Trp, or Tyr; a mutation at position 308, the mutation being a substitution of Ile; and a mutation at position 309, the mutation being a substitution of Gln or Thr; or (b) two or more mutations selected from: a mutation at position 250, the mutation being a substitution of Ala, Ile, or Val; a mutation at position 287, the mutation being a substitution of Phe, His, Met, Trp, or Tyr; a mutation at position 308, the mutation being a substitution of Gln or Thr; or (c) two or more mutations selected from: a mutation at position 250, the mutation being a substitution of Ala, Ile, or Val; a mutation at position 287, the mutation being a substitution of Phe, His, Met, Trp, or Tyr; a mutation at position 308, the mutation being a substitution of Gln or Thr; or (d ... The mutation is a substitution made with Ile; a mutation at position 309, which is a substitution made with Gln or Thr; a mutation at position 428, which is a substitution made with Phe; and a pair of mutations at positions 242 and 336, both of which are substitutions made with Cys; or (c) three or more mutations, including: a pair of mutations at positions 242 and 336, both of which are substitutions made with Cys, and mutations selected from: a mutation at position 250, which is a substitution made with Ala, Ile, or Val; a mutation at position 287, which is a substitution made with Phe, His, Met, Trp, or Tyr; a mutation at position 308, which is a substitution made with Ile; a mutation at position 309, which is a substitution made with Gln or Thr; and a mutation at position 428, which is a substitution made with Phe. Attached Figure Description

[0021] Figure 1 Provided are (A) the sequence of the IgG1 Fc region [SEQ ID NO:1] and (B) the structural diagram of the IgG1 Fc region (PDB ID:4BSV), which illustrates the locations of exemplary stability-enhancing designs T250V, A287F, and M428F.

[0022] Figure 2 The improved CH2 domain melting temperature (Tm) is shown by introducing exemplary stability-enhancing mutations into various Fc scaffolds: (A) scaffold 3, which contains an asymmetric mutation that promotes heterodimer Fc formation; (B) scaffold 6, which contains an N297A mutation; and (C) scaffold 7, which contains an S239D / I332E mutation. Scaffold 1 is a homodimer IgG1 Fc.

[0023] Figure 3 The sequence alignments are shown as follows: (A) sequence alignments of the CH2 domains of IgA, IgD, and IgG with the CH3 domains of IgE and IgM; and (B) sequence alignments of the CH3 domains of IgA, IgD, and IgG with the CH4 domains of IgE and IgM. Positions equivalent to IgG1 T250, A287, and M428 are outlined.

[0024] Figure 4 The correlation between aggregation induced by incubation at 40°C for 2 weeks under acidic or neutral conditions and the thermostability of the CH2 domain of antibody variants with and without the stability-enhancing mutation T250V / A287F is shown in (A) standard scale x-axis, incubation under weakly acidic conditions, (B) standard scale x-axis, incubation under neutral conditions, (C) logarithmic scale x-axis, incubation under weakly acidic conditions, and (D) logarithmic scale x-axis, incubation under neutral conditions. Variants showing small aggregation changes or not showing negative aggregation changes in the study are omitted. Parental sequences (unstabilized) are represented by circles, and stabilized variants are represented by squares. Each unstabilized variant and its corresponding stabilized variant are connected by an arrow. Detailed Implementation

[0025] This article describes Fc variants that contain one or more amino acid mutations that increase the stability of the Fc variants compared to parental Fc variants that do not contain one or more amino acid mutations. These mutations are referred to herein as “stability-enhancing amino acid mutations” or “stability-enhancing mutations”.

[0026] In some implementations, the Fc variant contains one or more stability-enhancing amino acid mutations selected from:

[0027] A mutation at position 250, wherein the mutation is the substitution of amino acid at position 250 by Ala, Ile, or Val;

[0028] A mutation at position 287, wherein the mutation is the substitution of the amino acid at position 287 by Phe, His, Met, Trp, or Tyr;

[0029] A mutation at position 308, wherein the mutation is the substitution of amino acid 1e at position 308;

[0030] A mutation at position 309, wherein the mutation is the substitution of amino acid at position 309 by Gln or Thr;

[0031] A mutation at position 428, wherein the mutation is the substitution of amino acid 428 with Phe; and

[0032] The mutations at positions 242 and 336, both of which involved substitutions using Cys.

[0033] Some embodiments of this disclosure relate to peptides that comprise Fc variants as described herein. Examples of such peptides include, but are not limited to, antibodies, antibody fragments, and Fc fusion proteins. Peptides comprising Fc variants as described herein can be used as therapeutic agents, diagnostic agents, or research tools.

[0034] Some embodiments of this disclosure relate to polynucleotides encoding the Fc variants described herein and polynucleotides encoding polypeptides containing the Fc variants, as well as host cells containing the polynucleotides and methods for preparing Fc variants or polypeptides containing the Fc variants using the polynucleotides and host cells.

[0035] Some embodiments of this disclosure relate to methods for stabilizing an Fc (parental Fc) by introducing one or more stability-enhancing mutations as described herein into the Fc. Some embodiments of this disclosure relate to methods for increasing the CH2 domain melting temperature (Tm) of an Fc (parental Fc) by introducing one or more stability-enhancing mutations as described herein into the Fc. The parental Fc can be a wild-type Fc, or it can itself be a variant Fc that already contains one or more amino acid mutations, for example, that improve the function of the Fc region.

[0036] definition

[0037] 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.

[0038] As used herein, the term “about” refers to a change of approximately + / - 10% from a given value. It should be understood that such a change is always included in any given value provided herein, unless specifically mentioned otherwise.

[0039] When used in conjunction with the term “containing” in this text, the word “a or an” can mean “one”, but it is also consistent with the meaning of “one or more”, “at least one” and “one or more”.

[0040] As used herein, the terms “comprising,” “having,” “including,” and “containing,” and their grammatical variations, are inclusive or open-ended and do not exclude additional, unlisted elements and / or method steps. When used herein in conjunction with a composition, use, or method, the term “consistently comprising” indicates that additional elements and / or method steps may be present, but these additions do not materially affect how the said composition, method, or use functions. When used herein in conjunction 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 comprising certain elements and / or steps may, in some embodiments, consist substantially of those elements and / or steps, and in other embodiments, consist of those elements and / or steps, whether or not these embodiments are specifically mentioned.

[0041] As used herein, the term "isolated" in relation to materials means that the material has been removed from its initial environment (e.g., its natural environment if it is naturally occurring). For example, naturally occurring polynucleotides or polypeptides present in living animals are not isolated, but the same polynucleotides or polypeptides separated from some or all of the coexisting materials in the natural system are isolated. Such polynucleotides may be part of a carrier, and / or such polynucleotides or polypeptides may be part of a composition and are still isolated because such carriers or compositions are not part of their natural environment.

[0042] As used interchangeably herein, the terms "Fc region" and "Fc" refer to the C-terminal region of the immunoglobulin heavy chain. For example, the Fc region sequence of the human IgG heavy chain is typically defined as extending from position 239 of the heavy chain to the C-terminus. The "Fc polypeptide" of a dimer Fc refers to one of the two polypeptides forming the dimer Fc domain, i.e., a polypeptide containing the C-terminal constant region of the immunoglobulin heavy chain capable of stable self-association. The Fc region typically contains both a CH2 domain and a CH3 domain. In some embodiments, the Fc region may also be considered to encompass the hinge region.

[0043] The "CH2 domain" of the human IgG Fc region is typically defined as extending from position 239 to 340. The "CH3 domain" is typically defined as containing the amino acid residues at the C-terminus of the CH2 domain in the Fc region, i.e., from position 341 to 447. The "hinge region" of human IgG1 is typically defined as extending from position 216 to 238 (Burton, 1985, Molec. Immunol., 22:161-206). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by aligning the first and last cysteine ​​residues that form the inter-heavy chain disulfide bond.

[0044] Unless otherwise stated herein, the amino acid residues in the Fc region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0045] Naturally occurring amino acids are always identified by the conventional three-letter or one-letter abbreviations shown in Table A below, as is generally accepted in the art and recommended by the IUPAC-IUB Committee in its biochemical nomenclature.

[0046] Table A: Amino Acid Codes

[0047]

[0048] It should be understood that a positive statement of a feature in one embodiment serves as the basis for excluding said feature in alternative embodiments. In particular, when a list of options is provided for a given embodiment or claim, it should be understood that one or more options may be removed from said list, and a shortened list may form alternative embodiments, whether or not such alternative embodiments are specifically mentioned.

[0049] It is anticipated that any implementation of the Fc variant disclosed herein can be implemented with respect to any of the methods, uses or compositions disclosed herein, and vice versa.

[0050] Fc variant

[0051] The Fc variants disclosed herein contain one or more amino acid mutations (“stability-enhancing mutations”) that increase the stability of the Fc variant compared to the parental Fc. This increased stability can result in increased thermal stability of the CH2 domain, decreased likelihood of aggregation, increased serum half-life, improved manufacturability, or a combination thereof. 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 parental Fc.

[0052] In some embodiments, the Fc variant contains one or more stability-enhancing mutations that increase the thermal stability (Tm) of the CH2 domain and also reduce Fc region aggregation compared to the parental Fc. In some embodiments, the Fc variant contains one or more stability-enhancing mutations that increase the thermal stability (Tm) of the CH2 domain and reduce Fc region aggregation at low pH levels compared to the parental Fc. In this context, "low pH" refers to a pH between approximately 4.0 and 7.5.

[0053] In some embodiments, compared to the parental Fc, the Fc variant contains one or more stability-enhancing mutations that increase the thermal stability (Tm) of the CH2 domain and reduce the aggregation of the Fc region under weakly acidic conditions, wherein weakly acidic conditions include a pH below neutral. In some embodiments, weakly acidic conditions include a pH between about 4.0 and 7.0. In some embodiments, weakly acidic conditions include a pH between about 4.0 and 6.5.

[0054] The Fc variant may contain one stability-enhancing mutation, or it may contain more than one stability-enhancing mutation. In some embodiments, the Fc variant contains between one and five stability-enhancing mutations. In some embodiments, the Fc variant contains between one and four stability-enhancing mutations. In some embodiments, the Fc variant contains between one and three stability-enhancing mutations. In some embodiments, the Fc variant contains 1, 2, or 3 stability-enhancing mutations.

[0055] In some embodiments, the CH2 domain Tm of the Fc variant is increased by at least 0.5 °C compared to the parental 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 parental 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 parental Fc.

[0056] In some embodiments, the Tm of the CH2 domain of the Fc variant is increased by about 0.5°C to about 6.5°C compared to the parental Fc. In some embodiments, the Tm of the CH2 domain of the Fc variant is increased by about 0.5°C to about 9.0°C compared to the parental Fc. In some embodiments, the Tm of the CH2 domain 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 parental Fc. In some embodiments, the Tm of the CH2 domain 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 parental Fc.

[0057] In some implementations, the CH2 domain Tm is measured by DSC or DSF.

[0058] The parental Fc can be a wild-type Fc, or it can be a variant Fc that already contains one or more amino acid mutations, such as those that improve the function of the Fc region. In some embodiments, the parental Fc can be an Fc containing one or more amino acid mutations that functionally enhance the Fc region. In some embodiments, the parental Fc can 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 parental Fc can 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.

[0059] Examples of amino acid mutations that functionally enhance the Fc region but reduce the thermal stability of the CH2 domain compared to wild-type Fc include, but are not limited to: mutations that promote heterodimer Fc formation (such as mortising or electrostatic orientation mutations, as described by Atwell et al., 1997, J Biol Chem, 270:26-35 and Gunasekaran et al., 2010, J Biol Chem, 270:26-35). Chem, 285(25):19637-19646 (described); mutations that produce glycosylated Fc (such as the N297A mutation, described in 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 (such as the S239D / I332E or S239D / A330L / I332E mutations that increase affinity for FcγRIIIa, as Lazar et al., 2006, PNAS, 103(11):4005-4010 and Oganesyan et al., 2008, Molec, 2008). As described in Immunol, 45(7):1872-1882, or the E233D / G237D / P238D / H268D / P271G / A330R mutations that increase selectivity for FcγRIIb, as described in Mimoto et al., 2013, Protein Eng. Des. Sel., 26:589-598. Other examples of amino acid mutations that functionally enhance the Fc region but reduce the thermal stability of the CH2 domain compared to the wild-type or parental Fc are described in the embodiments herein.

[0060] The parental Fc incorporating the stability-enhancing mutation can be an IgG Fc, IgA Fc, IgD Fc, IgE Fc, or IgM Fc. Although the amino acid numbering used herein refers to IgG Fc, those skilled in the art can readily determine the equivalent location of the mutation in other Ig Fc sequences using sequence alignment with one of the many sequence alignment tools known in the art. Therefore, references to stability-enhancing mutations at specific locations in the Fc region herein are intended to cover the specified locations in IgG Fc, and the corresponding locations in the IgA, IgD, IgE, or IgM Fc regions. Sequence alignments of the CH2 domains of IgA, IgD, and IgG with the CH3 domains of IgE and IgM, and sequence alignments of the CH3 domains of IgA, IgD, and IgG with the CH4 domains of IgE and IgM are shown in [illustration / example]. Figure 3 In A and 3B.

[0061] In some embodiments, the Fc variant is based on IgG, IgA, IgD, IgE, or IgM Fc. In some embodiments, the Fc variant is based on human IgG, IgA, IgD, IgE, or IgM Fc. In some embodiments, the Fc variant is based on IgG or IgA Fc. In some embodiments, the Fc variant is based on human IgG Fc. In some embodiments, the Fc variant is based on human IgG Fc.

[0062] In some embodiments, the Fc variant is based on IgG Fc, which may be IgG1, IgG2, IgG3, or IgG4 Fc. In some embodiments, the Fc variant is based on human IgG1, IgG2, IgG3, or IgG4 Fc. Sequence alignment of the CH2 and CH3 domains of human IgG1, IgG2, IgG3, and IgG4 is provided. Figure 3 In A and 3B. In some embodiments, the Fc variant is based on IgG1 Fc. In some embodiments, the Fc variant is based on human IgG1 Fc.

[0063] Stability-enhancing mutations

[0064] As described in this paper, computer simulations and bioinformatics methods were used to identify locations within the Fc region where mutations could enhance Fc stability. These methods identified the mutations shown in Table 1 as stability-enhancing mutations.

[0065] Table 1: Stability-enhancing mutations

[0066] 250 T250A, T250I, T250V 287 A287F, A287H, A287M, A287W, A287Y 308 V308I 309 L309Q, L309T 428 M428F 240 and 332 V240C_I332C 242 and 336 L242C_I336C

[0067] Specifically, computer simulation methods identified the following amino acid mutations as those that increase Fc stability when introduced as single mutations:

[0068] Mutations at position 287 selected from A287F, A287H, A287M, A287W, and A287Y; and

[0069] Mutant M428F,

[0070] Furthermore, bioinformatics methods identified the following amino acid mutations as those that increase Fc stability when introduced as single mutations:

[0071] Mutations selected from position 250 of T250A, T250I, and T250V;

[0072] Mutant V308I; and

[0073] The mutation at position 309 was selected from L309Q and L309T.

[0074] Furthermore, the mutations L242C_I336C, V240C_I332C, and V263C_V302C each introduce additional disulfide bonds into the Fc region, demonstrating an increase in Fc stability in the absence of any other stability-enhancing mutations.

[0075] The combinations of stability-enhancing mutations shown in Table 1 also demonstrate a further increase in the stability of the Fc variant.

[0076] Therefore, in some embodiments, the Fc variant includes one or more stability-enhancing mutations, wherein at least one mutation is selected from the mutations shown in Table 1. In the same embodiment, the Fc variant includes one or more stability-enhancing mutations selected from the mutations shown in Table 1.

[0077] In some implementations, the Fc variant contains one or more stability-enhancing mutations, wherein at least one mutation is selected from:

[0078] A mutation at position 287, wherein the mutation is the substitution of the amino acid at position 287 by Phe, His, Met, Trp, or Tyr;

[0079] A mutation at position 308, wherein the mutation is the substitution of amino acid 1e at position 308; and

[0080] A mutation at position 309, wherein the mutation is the substitution of an amino acid at position 309 with Gln or Thr.

[0081] In some implementations, the Fc variant contains a single stability-enhancing mutation. In some implementations, the Fc variant contains a single stability-enhancing mutation selected from the following:

[0082] A mutation at position 287, wherein the mutation is the substitution of the amino acid at position 287 by Phe, His, Met, Trp, or Tyr;

[0083] A mutation at position 308, wherein the mutation is the substitution of amino acid 1e at position 308; and

[0084] A mutation at position 309, wherein the mutation is the substitution of an amino acid at position 309 with Gln or Thr.

[0085] In some embodiments, the mutation at position 287 in the Fc variant is the substitution of amino acid Phe at position 287. In some embodiments, the mutation at position 309 in the Fc variant is the substitution of amino acid Gln at position 309.

[0086] In some embodiments, the Fc variant includes a stability-enhancing mutant pair, each mutation introducing a cysteine ​​residue to allow the formation of a novel disulfide bond in the Fc region. In some embodiments, the stability-enhancing mutant pair is selected from 242C_336C, 240C_332C, and 263C_302C. In some embodiments, the stability-enhancing mutant pair is either 242C_336C or 240C_332C. In some embodiments, the stability-enhancing mutant pair is 242C_336C.

[0087] In some implementations, the Fc variant contains two or more stability-enhancing mutations. In some implementations, the Fc variant contains two or more stability-enhancing mutations selected from the following:

[0088] A mutation at position 250, wherein the mutation is the substitution of amino acid at position 250 by Ala, Ile, or Val;

[0089] A mutation at position 287, wherein the mutation is the substitution of the amino acid at position 287 by Phe, His, Met, Trp, or Tyr;

[0090] A mutation at position 308, wherein the mutation is the substitution of amino acid 1e at position 308;

[0091] A mutation at position 309, wherein the mutation is the substitution of amino acid at position 309 by Gln or Thr;

[0092] A mutation at position 428, wherein the mutation is the substitution of amino acid 428 with Phe; and

[0093] A pair of mutations at positions 242 and 336, where each mutation is a substitution made by Cys.

[0094] In some implementations, the Fc variant contains two stability-enhancing mutations. In some implementations, the Fc variant contains:

[0095] The following two stability-enhancing mutations are selected: mutations at positions 250, 287, 308, 309, and 428, wherein the mutation at position 250 is replaced by Ala, Ile, or Val; the mutation at position 287 is replaced by Phe, His, Met, Trp, or Tyr; the mutation at position 308 is replaced by Ile; the mutation at position 309 is replaced by Gln or Thr; and the mutation at position 428 is replaced by Phe; or

[0096] A pair of mutations at positions 242 and 336, where each mutation is a substitution made by Cys.

[0097] In some implementations, the Fc variant includes:

[0098] The mutations at positions 250 and 287, wherein the mutation at position 250 is a substitution made by Ala, Ile, or Val, and the mutation at position 287 is a substitution made by Phe, His, Met, Trp, or Tyr.

[0099] The mutations at positions 250 and 308, where the 250-position mutation is a substitution made by Ala, Ile, or Val, and the 308-position mutation is a substitution made by Ile.

[0100] The mutations at positions 250 and 309, where the mutation at position 250 is a substitution made by Ala, Ile, or Val, and the mutation at position 309 is a substitution made by Gln or Thr.

[0101] The mutations at positions 250 and 428 are used, where the 250-position mutation is a substitution made with Ala, Ile, or Val, and the 428-position mutation is a substitution made with Phe.

[0102] The mutations at positions 287 and 308 are used, where the mutation at position 287 is a substitution made with Phe, His, Met, Trp or Tyr, and the mutation at position 308 is a substitution made with Ile.

[0103] The mutations at positions 287 and 309 are used, where the mutation at position 287 is a substitution made by Phe, His, Met, Trp or Tyr, and the mutation at position 309 is a substitution made by Gln or Thr.

[0104] The mutations at positions 287 and 428 are used, where the 287-position mutation is a substitution made with Phe, His, Met, Trp, or Tyr, and the 428-position mutation is a substitution made with Phe.

[0105] The mutations at positions 308 and 309 are as follows: the mutation at position 308 is a substitution made with Ile, and the mutation at position 309 is a substitution made with Gln or Thr.

[0106] The mutations at positions 308 and 428 are as follows: the mutation at position 308 is a substitution made with Ile, and the mutation at position 428 is a substitution made with Phe.

[0107] The mutations at positions 309 and 428, where the 309-position mutation is a substitution made with Gln or Thr, and the 428-position mutation is a substitution made with Phe; or

[0108] A pair of mutations at positions 242 and 336, where each mutation is a substitution made by Cys.

[0109] In some implementations, the Fc variant includes:

[0110] The mutations at positions 250 and 287, wherein the mutation at position 250 is a substitution made by Ala, Ile, or Val, and the mutation at position 287 is a substitution made by Phe, His, Met, Trp, or Tyr.

[0111] The mutations at positions 250 and 309, where the mutation at position 250 is a substitution made by Ala, Ile, or Val, and the mutation at position 309 is a substitution made by Gln or Thr.

[0112] The mutations at positions 250 and 428 are used, where the 250-position mutation is a substitution made with Ala, Ile, or Val, and the 428-position mutation is a substitution made with Phe.

[0113] Mutations at positions 287 and 428, wherein the 287-position mutation is a substitution made with Phe, His, Met, Trp, or Tyr, and the 428-position mutation is a substitution made with Phe; or

[0114] A pair of mutations at positions 242 and 336, where each mutation is a substitution made by Cys.

[0115] In some embodiments, the 250-position mutation in the Fc variant is replaced by Val. In some embodiments, the 287-position mutation in the Fc variant is replaced by Phe. In some embodiments, the 309-position mutation in the Fc variant is replaced by Gln.

[0116] In some implementations, the Fc variants include 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.

[0117] In some implementations, the Fc variant includes stability-enhancing mutations 250V / 287F, 250V / 309Q, 250V / 428F, 287F / 428F, or 242C_336C.

[0118] In some implementations, the Fc variants contain stability-enhancing mutations 250V / 287F, 250V / 309Q, 250V / 428F, or 287F / 428F.

[0119] In some implementations, the stability-enhancing mutations included in the Fc variants are selected from: 250V, 287F, 308I, 309Q, 428F, 242C_336C, 287F / 428F, 250V / 287F, 250V / 309Q, 250V / 428F, and 242C_336C / 308I.

[0120] In some implementations, the stability-enhancing mutations included in the Fc variants are selected from: 287F, 308I, 309Q, 242C_336C, 287F / 428F, 250V / 287F, 250V / 309Q, 250V / 428F, and 242C_336C / 308I.

[0121] In some implementations, the Fc variant contains three or more stability-enhancing mutations. In some implementations, the Fc variant contains three or more stability-enhancing mutations selected from the following:

[0122] The mutation at position 250 is a substitution made with Ala, Ile, or Val; the mutation at position 287 is a substitution made with Phe, His, Met, Trp, or Tyr; the mutation at position 308 is a substitution made with Ile; the mutation at position 309 is a substitution made with Gln or Thr; the mutation at position 428 is a substitution made with Phe; and the pair of mutations at positions 242 and 336 are both substitutions made with Cys.

[0123] In some implementations, the Fc variant contains three stability-enhancing mutations. In some implementations, the Fc variant contains three stability-enhancing mutations selected from the following:

[0124] The mutation at position 250 is a substitution made with Ala, Ile, or Val; the mutation at position 287 is a substitution made with Phe, His, Met, Trp, or Tyr; the mutation at position 308 is a substitution made with Ile; the mutation at position 309 is a substitution made with Gln or Thr; the mutation at position 428 is a substitution made with Phe; and the pair of mutations at positions 242 and 336 are both substitutions made with Cys.

[0125] In some implementations, the Fc variant includes:

[0126] A pair of mutations at positions 242 and 336, both of which are substitutions performed using Cys; and

[0127] The mutations are selected from the following: a mutation at position 250, which is a substitution of Ala, Ile, or Val; a mutation at position 287, which is a substitution of Phe, His, Met, Trp, or Tyr; a mutation at position 308, which is a substitution of Ile; a mutation at position 309, which is a substitution of Gln or Thr; and a mutation at position 428, which is a substitution of Phe.

[0128] In some implementations, the Fc variant contains one to three stability-enhancing mutations. In some implementations, the Fc variant contains:

[0129] One or more mutations, selected from: a mutation at position 287, wherein the mutation is a substitution made by Phe, His, Met, Trp, or Tyr; a mutation at position 308, wherein the mutation is a substitution made by Ile; and a mutation at position 309, wherein the mutation is a substitution made by Gln or Thr; or

[0130] Two or more mutations selected from: a 250-position mutation, the substitution being made with Ala, Ile, or Val; a 287-position mutation, the substitution being made with Phe, His, Met, Trp, or Tyr; a 308-position mutation, the substitution being made with Ile; a 309-position mutation, the substitution being made with Gln or Thr; a 428-position mutation, the substitution being made with Phe; and a pair of mutations at positions 242 and 336, both of which are substitutions being made with Cys; or

[0131] Three or more mutations, comprising: a 242-bit mutation and a 336-bit mutation, both of which are substitutions using Cys, and a mutation selected from: a 250-bit mutation, the substitution of which is Ala, Ile, or Val; a 287-bit mutation, the substitution of which is Phe, His, Met, Trp, or Tyr; a 308-bit mutation, the substitution of which is Ile; a 309-bit mutation, the substitution of which is Gln or Thr; and a 428-bit mutation, the substitution of which is Phe.

[0132] In some embodiments, the Fc variant is an IgG Fc variant. In some embodiments, the IgG Fc variant includes one or more stability-enhancing mutations. In some embodiments, the IgG Fc variant includes one or more stability-enhancing mutations, wherein at least one mutation is selected from:

[0133] Mutations at position 287 selected from A287F, A287H, A287M, A287W, and A287Y;

[0134] Mutant V308I; and

[0135] The mutation at position 309 was selected from L309Q and L309T.

[0136] In some embodiments, the IgG Fc variant contains a single stability-enhancing mutation. In some embodiments, the IgG Fc variant contains a single stability-enhancing mutation selected from the following:

[0137] Mutations at position 287 selected from A287F, A287H, A287M, A287W, and A287Y;

[0138] Mutant V308I; and

[0139] The mutation at position 309 was selected from L309Q and L309T.

[0140] In some implementations, the mutation at position 287 in the IgG Fc variant is A287F. In some implementations, the mutation at position 309 in the IgG Fc variant is L309Q.

[0141] In some embodiments, the Fc variant includes a stability-enhancing mutation pair, each mutation introducing a cysteine ​​residue that allows the formation of a new disulfide bond in the Fc region. In some embodiments, the stability-enhancing mutation pair is selected from L242C_I336C, V240C_I332C, and V263C_V302C. In some embodiments, the stability-enhancing mutation pair is either L242C_I336C or V240C_I332C. In some embodiments, the stability-enhancing mutation pair is L242C_I336C.

[0142] In some embodiments, the IgG Fc variant contains two or more stability-enhancing mutations. In some embodiments, the IgG Fc variant contains two or more stability-enhancing mutations selected from the following:

[0143] Mutations selected from position 250 of T250A, T250I, and T250V;

[0144] Mutations at position 287 selected from A287F, A287H, A287M, A287W, and A287Y;

[0145] Mutant V308I;

[0146] A mutation at position 309 selected from L309Q and L309T;

[0147] Mutant M428F, and

[0148] Mutations L242C and I336C.

[0149] In some embodiments, the IgG Fc variant contains two stability-enhancing mutations. In some embodiments, the IgG Fc variant contains:

[0150] The mutations are selected from the following two stability-enhancing mutations: the mutation at position 250, the mutation at position 287, the mutation at position 308, the mutation at position 309, and the mutation at position 428. Specifically, 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...

[0151] Mutations L242C and I336C.

[0152] In some implementations, the IgG Fc variant comprises:

[0153] Mutations at position 250 selected from T250A, T250I, and T250V, and mutations at position 287 selected from A287F, A287H, A287M, A287W, and A287Y;

[0154] Mutations at position 250 selected from T250A, T250I, and T250V, as well as mutation V308I;

[0155] Mutations at position 250 selected from T250A, T250I, and T250V, and mutations at position 309 selected from L309Q and L309T;

[0156] Mutations at position 250 selected from T250A, T250I, and T250V, as well as mutation M428F;

[0157] Mutations at position 287 selected from A287F, A287H, A287M, A287W, and A287Y, as well as mutation V308I;

[0158] Mutations at position 287 selected from A287F, A287H, A287M, A287W, and A287Y, and mutations at position 309 selected from L309Q and L309T;

[0159] Mutations at position 287 selected from A287F, A287H, A287M, A287W, and A287Y, as well as mutation M428F;

[0160] Mutation V308I and mutations at position 309 selected from L309Q and L309T;

[0161] Mutations V308I and M428F;

[0162] Mutations selected from L309Q and L309T at position 309, and the mutation M428F; or

[0163] Mutations L242C and I336C.

[0164] In some implementations, the IgG Fc variant comprises:

[0165] Mutations at position 250 selected from T250A, T250I, and T250V, and mutations at position 287 selected from A287F, A287H, A287M, A287W, and A287Y;

[0166] Mutations at position 250 selected from T250A, T250I, and T250V, and mutations at position 309 selected from L309Q and L309T;

[0167] Mutations at position 250 selected from T250A, T250I, and T250V, as well as mutation M428F;

[0168] Mutations at position 287 selected from A287F, A287H, A287M, A287W, and A287Y, as well as mutation M428F; or

[0169] Mutations L242C and I336C.

[0170] In some embodiments, the mutation at position 250 in the IgG Fc variant is T250V. In some embodiments, the mutation at position 287 in the IgG Fc variant is A287F. In some embodiments, the mutation at position 309 in the IgG Fc variant is L309Q.

[0171] In some implementations, the IgG Fc variant includes 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.

[0172] In some implementations, the IgG Fc variant contains stability-enhancing mutations T250V / A287F, T250V / L309Q, T250V / M428F, A287F / M428F, or L242C_I336C.

[0173] In some implementations, the IgG Fc variant includes stability-enhancing mutations T250V / A287F, T250V / L309Q, T250V / M428F, or A287F / M428F.

[0174] In some implementations, the stability-enhancing mutations included in the Fc variants are selected from: T250V, A287F, V308I, L309Q, M428F, L242C_I336C, A287F / M428F, T250V / A287F, T250V / L309Q, T250V / M428F, and L242C_I336C / V308I.

[0175] In some implementations, the stability-enhancing mutations included in the Fc variants are selected from: A287F, V308I, L309Q, L242C_I336C, A287F / M428F, T250V / A287F, T250V / L309Q, T250V / M428F, and L242C_I336C / V308I.

[0176] In some embodiments, the IgG Fc variant contains three or more stability-enhancing mutations. In some embodiments, the IgG Fc variant contains three or more stability-enhancing mutations selected from the following:

[0177] 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 V308I; the mutation at position 309 is selected from L309Q and L309T; the mutation M428F; and the mutations L242C and I336C.

[0178] In some implementations, the IgG Fc variant contains three stability-enhancing mutations. In some implementations, the IgG Fc variant contains three stability-enhancing mutations selected from the following:

[0179] 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 V308I; the mutation at position 309 is selected from L309Q and L309T; the mutation M428F; and the mutations L242C and I336C.

[0180] In some implementations, the IgG Fc variant includes mutations L242C and I336C, as well as 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 V308I; a mutation at position 309, selected from L309Q and L309T; and a mutation M428F.

[0181] In some embodiments, the IgG Fc variant contains one to three stability-enhancing mutations. In some embodiments, the IgG Fc variant contains:

[0182] One or more mutations, said mutations being selected from: a 287-bit mutation selected from A287F, A287H, A287M, A287W, and A287Y; mutation V308I; and a 309-bit mutation selected from L309Q and L309T; or

[0183] Two or more mutations, said mutations being selected from: a 250-bit mutation selected from T250A, T250I, and T250V; a 287-bit mutation selected from A287F, A287H, A287M, A287W, and A287Y; mutation V308I; a 309-bit mutation selected from L309Q and L309T; mutation M428F; and mutations L242C and I336C; or

[0184] Three or more mutations, including: mutations L242C and I336C, and mutations selected from: a 250-bit mutation selected from T250A, T250I, and T250V; a 287-bit mutation selected from A287F, A287H, A287M, A287W, and A287Y; mutation V308I; a 309-bit mutation selected from L309Q and L309T; and mutation M428F.

[0185] Certain stability-enhancing mutations are known in the art. For example, introducing additional disulfide bonds by including the 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 U.S. Patent Application Publication No. 2015 / 0210763. Certain embodiments of this disclosure contemplate Fc variants comprising one or more of the stability-enhancing mutations disclosed herein combined with one or more previously shown mutations that increase the stability of the Fc region.

[0186] method

[0187] Some embodiments of this disclosure relate to a method for stabilizing the Fc region (parental Fc) by introducing one or more stability-enhancing mutations, as described herein, into the parental Fc to provide an Fc variant.

[0188] Some embodiments of this disclosure relate to a method of increasing the CH2 domain melting temperature (Tm) of the Fc region (parental Fc) by introducing one or more stability-enhancing mutations described herein into the parental Fc to provide an Fc variant that increases the CH2 domain Tm by at least 0.5 °C compared to the parental Fc.

[0189] Some embodiments of this disclosure relate to a method for increasing the CH2 domain Tm of a parental Fc, the method comprising introducing one or more stability-enhancing mutations as described herein into the Fc to provide an Fc variant wherein the CH2 domain Tm of the Fc variant is at least 0.5 °C higher than that of the parental Fc.

[0190] The parental Fc can be a wild-type Fc, or it can be a variant Fc that already contains one or more amino acid mutations, such as those that improve the function of the Fc region. In some embodiments, the parental Fc may contain one or more amino acid mutations that improve the function of the Fc region but also reduce the Tm of the CH2 domain.

[0191] Some embodiments of this disclosure relate to a method of increasing the CH2 domain Tm below that of a parental Fc corresponding to the wild-type Fc, the method comprising introducing one or more stability-enhancing mutations as described herein into the Fc to provide an Fc variant wherein the CH2 domain Tm of the Fc variant is at least 0.5°C higher than that of the parental Fc.

[0192] 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.

[0193] In some embodiments, the method provides an Fc variant with 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.

[0194] In some embodiments, the method further includes measuring the CH2 domain Tm of the Fc variant. In some embodiments, the method further includes measuring the CH2 domain Tm of the Fc variant by DSC or DSF.

[0195] In some embodiments, the method includes introducing a stability-enhancing mutation between one and five as described herein into the parental Fc. In some embodiments, the method includes introducing a stability-enhancing mutation between one and four as described herein into the parental Fc. In some embodiments, the method includes introducing a stability-enhancing mutation between one and three as described herein into the parental Fc. In some embodiments, the method includes introducing one, two, or three stability-enhancing mutations into the parental Fc.

[0196] In some implementations, the method includes introducing one or more stability-enhancing mutations selected from the following parental Fc:

[0197] A mutation at position 250, wherein the mutation is the substitution of amino acid at position 250 by Ala, Ile, or Val;

[0198] A mutation at position 287, wherein the mutation is the substitution of the amino acid at position 287 by Phe, His, Met, Trp, or Tyr;

[0199] A mutation at position 308, wherein the mutation is the substitution of amino acid 1e at position 308;

[0200] A mutation at position 309, wherein the mutation is the substitution of amino acid at position 309 by Gln or Thr;

[0201] A mutation at position 428, wherein the mutation is the substitution of amino acid 428 with Phe; and

[0202] The mutations at positions 242 and 336, both of which involved substitutions using Cys.

[0203] In some embodiments, the method includes introducing a single stability-enhancing amino acid mutation selected from the following: a mutation at position 287, wherein the mutation is a substitution made by Phe, His, Met, Trp, or Tyr; a mutation at position 308, wherein the mutation is a substitution made by Ile; and a mutation at position 309, wherein the mutation is a substitution made by Gln or Thr.

[0204] In some embodiments, the method includes introducing two or more stability-enhancing amino acid mutations selected from the following: a mutation at position 250, wherein the mutation is a substitution of Ala, Ile, or Val; a mutation at position 287, wherein the mutation is a substitution of Phe, His, Met, Trp, or Tyr; a mutation at position 308, wherein the mutation is a substitution of Ile; a mutation at position 309, wherein the mutation is a substitution of Gln or Thr; a mutation at position 428, wherein the mutation is a substitution of Phe; and mutations at positions 242 and 336, both of which are substitutions of Cys.

[0205] In some embodiments, the method includes introducing three or more stability-enhancing amino acid mutations into the parental Fc, the 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 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.

[0206] Some implementations involve a method for increasing the CH2 domain Tm of the Fc region (parental Fc), the method comprising introducing one to three stability-enhancing amino acid mutations into the parental Fc to provide an Fc variant with an increased CH2 domain Tm compared to the parental Fc region, wherein one to three stability-enhancing mutations include:

[0207] (a) One or more mutations selected from: a mutation at position 287, wherein the mutation is a substitution made by Phe, His, Met, Trp, or Tyr; a mutation at position 308, wherein the mutation is a substitution made by Ile; a mutation at position 309, wherein the mutation is a substitution made by Gln or Thr; or

[0208] (b) Two or more mutations selected from: a 250-bit mutation, the mutation being a substitution made with Ala, Ile, or Val; a 287-bit mutation, the mutation being a substitution made with Phe, His, Met, Trp, or Tyr; a 308-bit mutation, the mutation being a substitution made with Ile; a 309-bit mutation, the mutation being a substitution made with Gln or Thr; a 428-bit mutation, the mutation being a substitution made with Phe; and mutations at bits 242 and 336, both of which are substitutions made with Cys; or

[0209] (c) Three or more mutations, comprising: a 242-bit mutation and a 336-bit mutation, both of which are substitutions using Cys, and a mutation selected from: a 250-bit mutation, the substitution of which is Ala, Ile, or Val; a 287-bit mutation, the substitution of which is Phe, His, Met, Trp, or Tyr; a 308-bit mutation, the substitution of which is Ile; a 309-bit mutation, the substitution of which is Gln or Thr; and a 428-bit mutation, the substitution of which is Phe.

[0210] In some embodiments, the method includes introducing 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 into the parental Fc. In some embodiments, the method includes introducing the stability-enhancing mutations 250V / 287F, 250V / 309Q, 250V / 428F, 287F / 428F, or 242C_336C into the parental Fc. In some implementations, the method includes introducing stability-enhancing mutations 250V / 287F, 250V / 309Q, 250V / 428F, or 287F / 428F into the parental Fc.

[0211] In some embodiments, the method includes introducing a stability-enhancing mutation selected from the following parental Fc: 250V, 287F, 308I, 309Q, 428F, 242C_336C, 287F / 428F, 250V / 287F, 250V / 309Q, 250V / 428F, and 242C_336C / 308I. In some embodiments, the method includes introducing a stability-enhancing mutation selected from the following parental Fc: 287F, 308I, 309Q, 242C_336C, 287F / 428F, 250V / 287F, 250V / 309Q, 250V / 428F, and 242C_336C / 308I.

[0212] In some embodiments, the parental Fc is an IgG, IgA, IgD, IgE, or IgM Fc, such as human IgG, IgA, IgD, IgE, or IgM Fc. In some embodiments, the parental Fc is an IgG or IgA Fc, such as human IgG or IgA Fc. In some embodiments, the parental Fc is an IgG Fc, such as human IgG Fc.

[0213] In some embodiments, the parental Fc is IgG1, IgG2, IgG3, or IgG4 Fc, such as human IgG1, IgG2, IgG3, or IgG4 Fc. In some embodiments, the parental Fc is IgG1 Fc, such as human IgG1 Fc.

[0214] In some embodiments, the method provides Fc variants with a CH2 domain Tm increased by at least 0.5 °C compared to the parental Fc and exhibiting reduced Fc region aggregation compared to the parental 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 parental Fc and exhibit reduced Fc region aggregation at low pH compared to the parental 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 parental Fc and exhibit reduced aggregation under weakly acidic conditions compared to the parental Fc.

[0215] Measurement

[0216] Compared to the parental Fc, the Fc variants of this disclosure exhibit increased stability. This increased stability can lead to increased thermal stability of the CH2 domain, reduced aggregation, increased serum half-life, improved manufacturability, or a combination thereof.

[0217] In some embodiments, the thermal stability of the Fc variant is increased compared to the parent Fc, as determined by the CH2 domain melting temperature (Tm). The CH2 domain Tm of the Fc variant and the parent Fc can be measured using standard techniques such as circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorescence (DSF). In some embodiments, the stability of the Fc variant is increased compared to the parent Fc, as determined by the CH2 domain Tm, wherein the CH2 domain Tm is measured by DSC or DSF.

[0218] In some embodiments, when a single mutation is introduced into Fc, the stability-enhancing mutation results in an increase in the Tm of the CH2 domain of the Fc variant by at least 0.5 °C compared to the parental Fc. In some embodiments, when a single mutation is introduced into Fc, the stability-enhancing mutation results in an increase in the Tm of the CH2 domain of the Fc variant 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 parental Fc. In some embodiments, when a single mutation is introduced into Fc, the stability-enhancing mutation results in an increase in the Tm of the CH2 domain of the Fc variant by about 0.5 °C to about 6.5 °C compared to the parental Fc.

[0219] In some embodiments, when Fc is introduced in combination with two or more mutations, the stability-enhancing mutation results in an increase in the Tm of the CH2 domain of the Fc variant by at least 0.5 °C compared to the parental Fc. In some embodiments, when Fc is introduced in combination with two or more mutations, the stability-enhancing mutation results in an increase in the Tm of the CH2 domain of the Fc variant 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 parental Fc. In some embodiments, when Fc is introduced in combination with two or more mutations, the stability-enhancing mutation results in an increase in the Tm of the CH2 domain of the Fc variant 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 parental Fc. In some embodiments, when Fc is introduced in combination with two or more mutations, the stability-enhancing mutation results in an increase in the Tm of the CH2 domain of the Fc variant by about 0.5 °C to about 9.0 °C compared to the parental Fc. In some embodiments, when Fc is introduced in combination with two or more mutations, the stability-enhancing mutation results in an increase in the Tm of the CH2 domain 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 parental Fc. In some embodiments, when Fc is introduced in combination with two or more mutations, the stability-enhancing mutation results in an increase in the Tm of the CH2 domain 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 parental Fc.

[0220] In some embodiments, increased stability of the Fc variant leads to reduced aggregation of the Fc variant compared to the parental Fc and / or an increased serum half-life. Aggregation and serum half-life can be measured using various standard techniques known in the art. For example, the aggregation of the Fc variant and the parental Fc can be assessed by size exclusion chromatography (SEC) or dynamic light scattering (DLS). The serum half-life of the Fc variant and the parental Fc can be assessed, for example, by pharmacokinetic studies in animal models.

[0221] In some embodiments, as determined by the CH2 domain melting temperature (Tm), the thermal stability of the Fc variant is increased compared to the parent Fc, and it also exhibits reduced aggregation. In some embodiments, the thermal stability (Tm) of the Fc variant is increased compared to the parent Fc, and it also exhibits reduced aggregation at low pH. In some embodiments, the thermal stability (Tm) of the Fc variant is increased compared to the parent Fc, and it also exhibits reduced aggregation under weakly acidic conditions.

[0222] Optional standard techniques can be used for further characterization of Fc variants. For example, purity, FcR binding, FcRn binding, aggregation, and / or C1q binding of Fc variants can be assessed. For instance, purity and aggregation can be assessed by liquid chromatography-mass spectrometry (LC-MS) and size exclusion chromatography (SEC), respectively. FcR and FcRn binding can be assessed by, for example, surface plasmon resonance (SPR), SPR imaging (SPRi), biofilm interference (BLI), ELISA, and kinetic exclusion assays. Or based on Meso Scale Discovery TM (MSD TM C1q binding can be measured using methods such as (see, for example, Current Protocols in Immunology: Ligand-Receptor Interactions in the Immune System, J. Coligan et al., eds., 2018 & updates, Wiley Inc., Hoboken, NJ; and Yang et al., 2016, Analytical Biochem, 508:78-96). C1q binding can be assessed by, for example, ELISA or SPR.

[0223] In some implementations, the Fc variant is an IgG Fc variant and its FcγR binding and / or FcRn binding can be evaluated. Typically, binding affinity is expressed as the dissociation constant (Kc) of the Fc variant binding to FcγR or FcRn. D() indicates. In some embodiments where the Fc variant is an IgG Fc variant, the Fc variant retains substantially the same binding to each Fcγ receptor as the parental 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 parental Fc. In this context, "substantially the same binding" means that compared to the parental Fc, K D The change is 3 times or less.

[0224] polypeptide

[0225] Some embodiments of this disclosure relate to peptides that comprise Fc variants as described herein. Typically, a peptide comprises one or more other protein moieties fused to or covalently linked to an Fc variant (e.g., via a linker). For example, a peptide may be an Fc fusion protein or an antibody or antibody fragment. Examples of protein moieties that may be fused to or linked to an Fc variant include, but are not limited to, antigen-binding domains, ligands, receptors, receptor fragments, cytokines, and antigens.

[0226] When a polypeptide comprises more than one other protein moiety, these moieties may be the same or they may be different. One or more other protein moieties may be fused to the N-terminus, C-terminus, or both of the N-terminus and C-terminus of one or both of the Fc polypeptides. In some embodiments, the polypeptide comprises one or more other protein moieties fused to the N-terminus of one or both of the Fc polypeptides. In some embodiments, the polypeptide comprises one other protein moieties fused to the N-terminus of one of the Fc polypeptides. In some embodiments, the polypeptide comprises two other protein 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 other protein moieties comprised of the polypeptide may be tandemly linked.

[0227] In some embodiments, the polypeptide comprises an Fc variant fused to one or more protein portions that serve as 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, such as 2, 3, 4, 5, 6, 7, or 8 antigen-binding domains. When the 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.

[0228] In some embodiments, the polypeptide comprises an Fc variant fused to one or more protein moieties that serve as an antigen-binding domain and one or more other protein moieties. Examples of other protein moieties in this context include, but are not limited to, receptors, receptor fragments (such as extracellular portions), ligands, and cytokines.

[0229] In some embodiments, the peptide may be an antibody or an antibody fragment, wherein at least one of one or more protein moieties is an antigen-binding domain. For example, the antigen-binding domain may be a Fab fragment, an Fv fragment, a single-chain Fv fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the peptide may be a monospecific antibody. In some embodiments, the peptide may be a monospecific antibody containing one antigen-binding domain. In some embodiments, the peptide may be a monospecific antibody containing two antigen-binding domains. In some embodiments, the peptide may be a monospecific antibody containing more than two antigen-binding domains. In some embodiments, the peptide may be a bispecific or multispecific antibody containing an Fc variant and two or more antigen-binding domains, wherein the two or more antigen-binding domains bind to different antigens.

[0230] In some implementations, the polypeptide may be a therapeutic or diagnostic antibody or antibody fragment, wherein at least one of one or more protein portions thereof is an antigen-binding domain.

[0231] In some implementations, the peptide comprises an Fc variant and one or more antigen-binding domains that bind to tumor-associated antigens or tumor-specific antigens.

[0232] Preparation of Fc variants

[0233] The Fc variants and peptides containing Fc variants as described herein can be prepared using standard recombination methods. Recombinant generation of Fc variants and peptides typically involves: synthesizing one or more polynucleotides encoding the Fc variant or peptide; cloning one or more polynucleotides into one or more suitable vectors; and introducing the vectors into suitable host cells to express the Fc variant or peptide. Recombinant generation of proteins is well known in the art and can be achieved using standard methods described, for example, in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., *Current Protocols in Molecular Biology*, (1987 and updated), John Wiley & Sons, New York, NY; and Harlow and Lane, *Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990).

[0234] Some embodiments of this disclosure therefore relate to isolated polynucleotides or polynucleotide sequences encoding Fc variants as described herein or encoding polypeptides comprising Fc variants as described herein. In this context, the polynucleotides may encode all or part of the Fc variants or polypeptides.

[0235] The terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used interchangeably herein and refer to nucleotides of any length, deoxyribonucleotides, or ribonucleotides, or their polymeric forms. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, isolated DNA, isolated RNA, nucleic acid probes, and primers.

[0236] A polynucleotide that “encodes” a given polypeptide is a polynucleotide 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 the start codon at the 5' (amino) end and the translation stop codon at the 3' (carboxyl) end. The transcription termination sequence can be located at the 3' end of the coding sequence.

[0237] One or more polynucleotides encoding an Fc variant or polypeptide can be inserted into one or more suitable expression vectors, either directly or after one or more subcloning steps, using standard ligation techniques. Examples of suitable vectors include, but are not limited to, plasmids, phages, phages, bacteriophages, baculoviruses, retroviruses, or DNA viruses. Vectors are typically chosen to function in the specific host cells to be used, i.e., the vector is compatible with host cell mechanisms, allowing for the amplification and / or expression of the polynucleotide. At this point, selecting an appropriate combination of vector and host cell is entirely within the ordinary skill of those skilled in the art.

[0238] Some embodiments of this disclosure therefore relate to vectors (such as expression vectors) that contain one or more polynucleotides encoding Fc variants or polypeptides containing Fc variants. The polynucleotide may be contained in a single vector or in more than one vector. In some embodiments, the polynucleotide is contained in a polycistronic vector.

[0239] Typically, expression vectors contain one or more regulatory elements for plasmid maintenance and for cloning and expressing exogenous polynucleotide sequences. Examples of such regulatory elements include promoters, enhancer sequences, origins of replication, transcription termination sequences, donor and acceptor splicing sites, leader sequences for polypeptide secretion, ribosome binding sites, polyadenylation sequences, multi-connector regions for inserting polynucleotides encoding the polypeptide to be expressed, and optional markers.

[0240] 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., from a combination of regulatory elements from more than one source), or synthetic. Therefore, the source of a regulatory element can be any prokaryote or eukaryote, provided that the sequence functions in the host cell mechanism in use and can be activated by the host cell mechanism in use.

[0241] Optionally, the vector may also contain a "tag" coding sequence. The tag coding sequence is a nucleic acid sequence located at the 5' or 3' end of the coding sequence encoding the heteropeptide sequence, such as polyHis (e.g., 6xHis). HA (hemagglutinin influenza virus), myc, metal affinity, avidin / streptavidin, glutathione S-transferase (GST), or biotin tags. This tag typically remains fused to the expressed peptide and can be used as a means of affinity purification or detection of the peptide. Optionally, the tag can subsequently be removed from the purified peptide by various means, such as cleavage using certain peptidases.

[0242] Various expression vectors are readily available from commercial sources. Alternatively, when commercially available vectors containing all desired regulatory elements are unavailable, commercially available vectors can be used as starting vectors to construct expression vectors. When one or more desired regulatory elements are not yet present in a vector, they can be obtained individually and ligated into the vector. Methods and sources for obtaining various regulatory elements are well known to those skilled in the art.

[0243] After constructing an expression vector containing a multinucleotide encoding an Fc variant or polypeptide, the vector can be inserted into a suitable host cell for amplification and / or protein expression. The conversion of the expression vector into selected host cells can be accomplished by well-known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, liposome transfection, DEAE-glucan-mediated transfection, and other known techniques. The method chosen will vary in part depending on the type of host cell to be used. These and other suitable methods are well known to those skilled in the art (see, for example, Sambrook et al., ibid.).

[0244] When cultured under appropriate conditions, the host cell expresses the polypeptide encoded by the vector, and the polypeptide can subsequently be collected from the culture medium (if the host cell secretes the polypeptide) or directly from the host cell that produced it (if the polypeptide is not secreted). The host cell can be a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a yeast, fungus, plant, or mammalian cell). Appropriate host cells can be easily selected by a technician, taking into account various factors such as the desired expression level, the polypeptide modifications required or necessary for activity (e.g., glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

[0245] Some embodiments of this disclosure therefore relate to host cells that contain polynucleotides encoding Fc variants or polypeptides containing Fc variants, or one or more vectors containing polynucleotides. In some embodiments, the host cell is a eukaryotic cell.

[0246] For example, eukaryotic microorganisms such as filamentous fungi or yeasts can be used as host cells, including fungal and yeast strains whose glycosylation pathways have been “humanized” (see, for example, Gerngross, (2004), Nat. Biotech., 22:1409-1414, and Li et al., (2006), Nat. Biotech., 24:210-215). Plant cells can also be used as host cells (see, for example, descriptions of PLANTIBODIES). TM The technology is referenced in U.S. patents 5,959,177; 6,040,498; 6,420,548; 7,125,978 and 6,417,429.

[0247] In some implementations, the eukaryotic host cell is a mammalian cell. Various mammalian cell lines can be used as host cells. Examples of useful mammalian host cell lines include, but are not limited to, monkey kidney CV1 line transformed from SV40 (COS-7), human embryonic kidney line 293 (e.g., HEK293 cells described in Graham et al., (1977), J. Gen Virol., 36:59), hamster kidney cells (BHK), mouse sertoli cells (e.g., TM4 cells described in Mather, (1980), Biol. Reprod., 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HeLa), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumors (MMT 060562), and TRI cells (e.g., Mather et al., 1982, Annals). (described in NYAcad. Sci., 383:44-68), MRC5 cells, FS4 cells, and Chinese hamster ovary (CHO) cells (including DHFR cells described in Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA, 77:4216). - CHO cells) and myeloma cell lines (such as Y0, NSO, and Sp2 / 0). See also Yazaki and Wu, 2003, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKCLo, ed., Humana Press, Totowa, NJ).

[0248] Some embodiments of this disclosure relate to methods for preparing Fc variants as described herein or polypeptides containing Fc variants as 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 containing polynucleotides), and culturing the host cell under conditions suitable for expressing the encoded Fc variant or polypeptide.

[0249] Typically, after expression, the Fc variant or peptide is isolated from the host cell and can optionally be purified. Methods for isolating and purifying expressed proteins are well known in the art. For example, standard purification methods include, for instance, chromatographic techniques such as ion-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, size exclusion chromatography, gel filtration chromatography, or reversed-phase chromatography, which can be performed using systems such as FPLC, MPLC, and HPLC at atmospheric, medium, or high pressure. Other purification methods include electrophoresis, immunology, precipitation, dialysis, and chromatographic focusing techniques. Ultrafiltration and dialysis techniques combined with protein concentration may also be useful.

[0250] Various natural proteins that bind to the Fc region of antibodies are known in the art, 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 typically be achieved using specific fusion partners or affinity tags as described above. For example, if GST fusion is used, the antibody can be purified using glutathione resin; if a His tag is used, Ni... +2 Antibodies can be purified by affinity chromatography, or, if using a FLAG tag, by immobilized anti-flag antibodies. Examples of useful purification techniques are described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), and Protein Purification: Principles and Practice, 3rd Edition, Scopes, Springer-Verlag, NY (1994).

[0251] Pharmaceutical Composition

[0252] Certain embodiments of this disclosure relate to the therapeutic use of Fc variants or peptides comprising Fc variants. For therapeutic use, the Fc variants and peptides may be provided in the form of a composition comprising the Fc variant or peptide and a pharmaceutically acceptable carrier or diluent. The composition may be prepared using known and readily available ingredients through known procedures and may be formulated for administration to a subject via, for example, oral (including, for example, buccal or sublingual), topical, parenteral, rectal, or vaginal routes, or by inhalation or spray. As used herein, the term “parenteral” includes injection or infusion via subcutaneous, intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal, or intrathecal routes.

[0253] The composition is typically formulated in a form suitable for administration to a subject via a chosen route (e.g., as a syrup, elixir, tablet, lozenge, tablet, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granules, emulsion, injection, or solution). The composition may be provided as a unit-dose formulation.

[0254] Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used. Examples of such carriers include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives, such as octadecyl dimethyl benzyl ammonium chloride, hexamethyl diammonium chloride, benzalkonium chloride, benzyl chloride, phenol, butanol, benzyl alcohol, alkyl esters of parabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 amino acids) peptides; proteins, Examples of such substances include 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium ions; metal complexes such as Zn-protein complexes; and nonionic surfactants such as polyethylene glycol (PEG).

[0255] In some embodiments, the composition may be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions can be formulated using suitable dispersants or wetting agents and / or suspending agents known in the art. The sterile injectable solution or suspension may contain Fc variants or peptides in a non-toxic, parenteral acceptable diluent or solvent. Acceptable diluents and solvents that can be used include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. Furthermore, sterile non-volatile oils can be used as solvents or suspension media. For this purpose, a variety of mild non-volatile oils can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid can be used to prepare the injection. Adjuvants known in the art, such as local anesthetics, preservatives, and / or buffers, may also be included in the injectable solution or suspension.

[0256] Other pharmaceutical compositions and methods for preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0257] How to use

[0258] Some embodiments of this disclosure relate to the use of Fc variants or peptides containing Fc variants as therapeutic agents, diagnostic agents, or research tools. Some embodiments relate to the therapeutic use of Fc variants and peptides containing Fc variants.

[0259] Peptides comprising an Fc variant as described herein and one or more antigen-binding domains (e.g., antibodies or antibody fragments) are particularly useful as diagnostic and therapeutic agents. Therefore, some embodiments relate to methods of using peptides comprising an Fc variant and one or more antigen-binding domains in diagnosing a disease or condition in a subject. Some embodiments relate to methods of using peptides comprising an Fc variant and one or more antigen-binding domains in treating a disease or condition in a subject in need.

[0260] The disease or condition to be diagnosed or treated will depend on one or more antigens targeted by the antigen-binding domain. Examples of diseases and conditions that can be diagnosed or treated include, but are not limited to, inflammatory diseases and conditions, autoimmune diseases and conditions, and proliferative diseases and conditions, such as various cancers.

[0261] The following examples are provided for illustrative purposes and are not intended to limit the scope of the invention in any way.

[0262] Example

[0263] General methods

[0264] Preparation of variants

[0265] Variants and controls were prepared using standard methods via site-directed mutagenesis and / or restriction / ligation. The final DNA was subcloned into the vector pTT5 (see U.S. Patent No. 9,353,382). All scaffolds used for variant preparation were based on IgG1 Fc. The sequence of the IgG1 Fc region was provided in... Figure 1 In A, the C-terminal lysine residue is omitted from the Fc sequence in some clones.

[0266] The following brackets were used:

[0267] Bracket 1: A full-size antibody (FSA) based on trastuzumab, which has a homodimer IgG1 Fc, SEQ ID NO:1.

[0268] Support 2: A single-arm antibody scaffold (OAA) containing trastuzumab Fab and heterodimer IgG1 Fc, which contains the following mutations:

[0269] Chain A: T350V_L351Y_F405A_Y407V

[0270] Chain B: T350V_T366L_K392L_T394W

[0271] Support 3: A full-size antibody (FSA) based on trastuzumab, which contains the same heterodimer Fc as scaffold 2.

[0272] Bracket 4 : A full-size antibody (FSA) based on 4G7 anti-CD19 antibody (Meeker et al., 1984, Hybridoma, 3:305-320; US Patent No. 8,524,867), which contains the same heterodimer Fc as scaffold 2.

[0273] Bracket 5 A full-size antibody (FSA) based on CP-870,893 anti-CD40 antibodies (Gladue et al., 2011, Cancer Immunol Immunother, 60:1009-1017) containing the same heterodimer Fc as scaffold 2. The variable domain sequence is obtained from international patent application publication number WO 2013 / 132044.

[0274] Bracket 6 : A full-size trastuzumab-based antibody (FSA) containing the N297A mutation (Leabman et al., 2013, mAb, 5(6): 896-903), which leads to glycosylation of Fc and elimination of binding to all FcγRs.

[0275] Bracket 7 : A full-size trastuzumab-based antibody (FSA) containing S239D and I332E mutations (Lazar et al., 2006, PNAS, 103:4005-4010), which results in increased FcγRIIIa binding.

[0276] Bracket 8A single-arm antibody scaffold (OAA) comprising trastuzumab Fab and heterodimer IgG1 Fc, containing the following mutations that result in increased FcγRIIb selectivity:

[0277] Chain A: CH2: G236N_G237A

[0278] CH3: T350V_L351Y_F405A_Y407V

[0279] Chain B: CH2: G236D_G237F_S239D_S267V_H268D_“Template 1”

[0280] CH3: T350V_T366L_K392L_T394W

[0281] "Template 1" means replacing the amino acid residues at positions 325-331 with the following sequence: STWFDGGYAT[SEQ IDNO:2].

[0282] Bracket 9 A single-arm antibody scaffold (OAA) comprising trastuzumab Fab and heterodimer IgG1 Fc, containing the following mutations that result in increased FcγRIIb selectivity:

[0283] Chain A: CH2: L234F_G236N_H268Q_A327G_A330K_P331S

[0284] CH3: T350V_L351Y_F405A_Y407V

[0285] Chain B: CH2: G236D_S239D_V266L_S267A_H268D

[0286] CH3: T350V_T366L_K392L_T394W

[0287] Expression - Solution 1

[0288] Expression was performed in 200 mL of CHO 3E7 cells. CHO cells were in the exponential growth phase (1.5 to 2 million cells / mL) and expressed with aqueous 1 mg / mL 25 kDa polyethyleneimine (PEI). proPolyplus Transfection SA, Illkirch, France) transfected cells at a PEI:DNA ratio of 2.5:1 (Delafosse et al., 2016, J. Biotechnol., 227:103-111). To determine the optimal concentration range for heterodimer formation, the optimal DNA ratio (e.g., HC-A / HC-B / LC ratio = 30:30:40%) for heavy chain A (HC-A), light chain (LC), and heavy chain B (HC-B) was used for transfection. When expressing homodimers, a ratio of 50:50% was used for HC:LC. Transfected cells were harvested after 5–6 days, and the culture medium was collected after centrifugation at 4000 rpm and clarified using a 0.45 μm filter.

[0289] Load the clarified culture medium into MabSelect TM Sure TM (GE Healthcare, Baie-d'Urfé, QC, Canada) Protein A column was used, and the sample was washed with 10 column volumes of PBS buffer at pH 7.2. The antibody was eluted with 10 column volumes of citrate buffer at pH 3.6, and the combined fractions contained antibody neutralized with TRIS at pH 11. The sample was then buffer-exchanged in PBS at pH 7.4 and stored at -80°C.

[0290] Expression - Option 2

[0291] Expression was performed using HEK 293-6E cells (NRC, Canada) in small-scale (1 mL) or large-scale (30 mL or more) assays.

[0292] For 1 mL-scale expression, the cationic lipid 293Fectin was used. TM (Life Technologies, Paisley, UK) Pre-complexed DNA was transfected into HEK 293-6E cells at a concentration of 1 μg DNA / mL during the exponential growth phase (1.5 to 2 million cells / mL). Heavy and light chain DNA were mixed at a ratio of 47.5:52.5%, and the DNA was combined with 293Fectin. TM The final concentration of the compound was 11.7 μg / mL DNA and 1.65% (v / v) 293 Fectin. TMThe mixture was then incubated at ambient temperature for 30 min before being added to the cells. For optimal heterodimer formation, the ratio of HC-A to HC-B DNA in the transfection mixture was 50:50%, or a slight variation thereof. Cells were cultured for 5–6 days in humidified shakers in 96-well deep-well plates sealed with a breathable seal at 37°C and 5% CO2. The culture medium was collected after centrifugation at 1600 x g.

[0293] For large-scale expression, HEK 293-6E cells were transfected with DNA pre-complexed with Gemini cationic lipids (Camilleri et al., 2000, Chem. Commun., 1253-1254) at a concentration of 1 μg DNA / mL cells in the exponential growth phase (1.5 to 2 million cells / mL). Heavy and light chain DNA were mixed at a 50:50 ratio and complexed with Gemini to a final concentration of 10 μg / mL DNA, 40 μg / mL Gemini. This mixture was then incubated at ambient temperature for 15–30 min before being added to the cells. The HC-A and HC-B DNA ratio of the transfection mixture was as described above. Cells were cultured at 37°C and 5% CO2 in appropriately sized Erlenmeyer flasks or BioReactor tubes in a humidified shaker for up to 10 days. The culture medium was then collected after centrifugation at 2750 x g and clarified using a 0.22 μm filter.

[0294] Load the clarified culture medium into MabSelect TM Sure TM (GE Healthcare, Little Chalfont, UK) Protein A column was washed with 3-10 column volumes of Tris-acetate buffer (pH 7.5), followed by 2-5 column volumes of acetic acid (pH 2.6). The elution fraction was neutralized with TRIS. Size exclusion chromatography (using PBS run buffer) was then performed using Superdex. TM Further purification and / or cation exchange (Resource) was performed on a 200-column (GE Healthcare, Little Chalfont, UK) column. TM The S column (GE Healthcare, Little Chalfont, UK) was used for the selected sample. The antibody buffer purified from protein A was exchanged for PBS.

[0295] Preparation of Fcγ and FcRn receptors

[0296] Option 1

[0297] As previously described, FcγRIIaH, IIaR, IIb, IIIaF, and IIIaV are produced in HEK293-6E cells, while FcγRIa is produced in CHO-3E7 cells (Dorian-Thibaudeau et al., 2014, J. Immunol. Methods, 408:24-34). Human FcRn is also expressed in HEK293-6E cells via co-transfection with a 1:1 ratio of TEV-cleavable C-terminal His tag to the extracellular domain of the α subunit (p51) of β2-microglobulin. Following purification as described by Dorion-Thibaudeau et al. (ibid.), the C-terminal His tag was removed by TEV cleavage.

[0298] Option 2

[0299] Soluble FcγRI extracellular domains with a C-terminal 6xHis tag were purchased from the R&D system (catalog number 1257-Fc). Soluble FcγRI IaH, IIaR, IIb, IIIaF, and IIIaV extracellular domains were generated in HEK293-6E cells with a C-terminal 10xHis tag. Cells were transfected at 1 μg DNA / mL at the exponential growth phase (1.5 to 2 million cells / mL) using DNA pre-complexed with Gemini cationic lipids (Camilleri et al., 2000, Chem. Commun., 1253-1254.). Cells were cultured for up to 7 days in appropriately sized Erlenmeyer flasks in a humidified shaker at 37°C and 5% CO2. Harvest time was determined by the time when cell viability dropped below 50%. The culture medium was then collected after centrifugation at 2750 x g and clarified using a 0.22 μm filter.

[0300] The clarified culture medium buffer was exchanged for a loading buffer containing 25 mM imidazole at pH 7.7 via dialysis or tangential flow filtration and applied to a Ni Sepharose 6 column (GE Healthcare, Little Chalfont, UK). Elution was then performed by increasing the imidazole concentration in the buffer to 300 mM. The eluted proteins were concentrated by dialysis and buffer-exchanged in PBS, and then subjected to size exclusion chromatography (MSC). Further purification was performed using a 75 column (GE Healthcare, Little Chalfont, UK).

[0301] Soluble human FcRn extracellular domains were expressed in HEK 293-6E cells via co-transfection at a 1:1 ratio of α-subunits containing a C-terminal 6xHis tag with β2 microglobulin, and expressed as described otherwise for FcγR. The pH of clarifying medium was adjusted to pH 5.3 with citrate and then loaded onto an IgG Sepharose column (GE Healthcare, Little Chalfont, UK). Bound proteins were eluted with HEPES buffer at pH 7.7. The eluted proteins were concentrated by dialysis and buffer-exchanged in PBS, and then subjected to size exclusion chromatography (MSC). Further purification was performed using a 75 column (GE Healthcare, Little Chalfont, UK).

[0302] Fcγ receptor binding (surface plasmon resonance (SPR))

[0303] Option 1

[0304] Using ProteOn at 25°C TM XPR36 was used as the run buffer in PBS (pH 7.4) containing 150 mM NaCl, 3.4 mM EDTA, and 0.05% Tween 20. The affinity of FcγR for antibody Fc was measured via SPR. For the trastuzumab variant, recombinant HER2 was immobilized on the GLM sensor chip using a standard amine conjugation kit via the BioRad Amine Conjugation Kit. Briefly, the GLM sensor chip was activated with NHS / EDC, followed by infusion of 4.0 μg / mL HER2 in 10 mM NaOAc (pH 4.5) until approximately 3000 resonance units (RUs) were immobilized. The remaining active groups were then quenched with ethanolamine. The wild-type trastuzumab variant was indirectly captured onto its SPR surface by purifying the antibody for 240 seconds via ligand-directed injection of 40 μg / mL solution at 25 μL / min, resulting in approximately 500 RUs on the surface. After injecting buffer to establish a stable baseline in the analyte orientation, the analyte was injected at 50 μL / min for 120 s, followed by a dissociation phase of 180 s to obtain a set of binding sensing maps. In addition to 30 nM of FcγR1a, five concentrations of a 3-fold dilution series of FcγR were used, with the highest nominal concentration of all receptors at 10 μM, and buffer was included for dual reference. ProteOn was used. TM The Equilibrium Fit model in Manager v3.1.0 determines the resulting Kd (affinity) value from the aligned and referenced sensor maps, and reports the value as the average of two or three independent runs.

[0305] Option 2

[0306] Use Biacore at 25℃ TM 4000 (GE Healthcare, Little Chalfont, UK) was used as the run buffer in PBSTE (PBS containing 0.05% Tween-20 and 3.4 mM EDTA), and the affinity of FcγR for antibody Fc was measured by SPR. For anti-HER2 antibodies, amine conjugation (EDC / NHS Chemistry) was used to immobilize the CM5 chip (GE Healthcare, Little Chalfont, UK) with recombinant HER2 extracellular domains (Merck, Darmstadt, Germany, or ThermoFisher Scientific, Loughborough, UK). Briefly, the CM5 sensor chip was activated with NHS / EDC and then 10.0 μg / mL of HER2 was injected in 10 mM NaOAc (pH 4.5). Immobilization levels were maintained in the range of 1000–4000 RU. Any remaining active groups were then quenched with ethanolamine. Antibodies were first captured on the immobilized surface of the chip by infusion at a flow rate of 10 μl / min at points and flow cells for 35 s, leaving point 3 blank for reference subtraction. Receptors were diluted in PBSTE buffer to a defined concentration range, depending on their expected affinity. Six concentrations were used for each analyte, including zero. Analyte contact time was optimized based on the receptors used and their expected kinetics. For example, for FcγRIIB and FcγRIIaR, the contact time was 18 s at 30 μl / min. The chip surface was regenerated after infusion of 87 mM phosphate at each analyte concentration. The chip was prepared with 3 x 18 s infusions of 87 mM phosphate prior to testing. Double reference subtraction (reference point 3 and 0 receptor concentration) was performed, and binding reactions were normalized by antibody capture levels. Samples were analyzed using either kinetic and / or steady-state (equilibrium) fitting models.

[0307] FcRn bonding (surface plasmon resonance (SPR))

[0308] Option 1

[0309] Using ProteOn at 25°C TMXPR36, using HBS-EP+ (10 mM HEPES, 150 mM NaCl, 0.003% M EDTA, and 0.05% (v / v) surfactant P20 (Teknova, Hollister, USA)) as a run buffer at pH 7.4 or pH 6.0, was used to measure the affinity of FcRn for the antibody variant Fc via SPR. Protein L (ThermoScientific, Loughborough, UK) was immobilized on a GLM sensor chip using a standard amine conjugation kit from GE Healthcare. Briefly, the GLM sensor chip was activated with NHS / EDC, followed by injection of 50 μg / mL protein L in 10 mM NaOAc (pH 4.5) until approximately 3000 resonance units (RUs) were immobilized, and then the remaining active groups were quenched with ethanolamine. Antibody variants were indirectly captured onto their SPR surfaces by injecting a solution of 50 μg / mL purified antibody at 30 μL / min in the ligand direction for 120 s. After injecting buffer to establish a stable baseline in the analyte direction, the analyte was injected at 40 μL / min for 300 s, followed by a dissociation phase of 600 s to obtain a set of binding sensory maps. Five concentrations of FcγR in a 4-fold dilution series, with the highest nominal concentration being 2048 nM, were used, and buffer was included for dual reference. ProteOn was used. TM The Equilibrium Fit model in Manager v3.1.0 determines the resulting Kd (affinity) value from the aligned and referenced sensor maps.

[0310] Option 2

[0311] Use Biacore at 25℃ TMThe T200 (GE Healthcare, Little Chalfont, UK) was used with HBS-EP+ at pH 7.4 or MES at pH 6.0 as the run buffer to measure the affinity of FcRn for the antibody variant Fc by SPR. Samples were captured on an immobilized protein LCM5 chip (GE Healthcare), but the 4G7 anti-CD19 antibody failed to be captured. The antibody was first captured on the immobilized surface of the chip by instillation at a flow rate of 5 μl / min at approximately 15 μg / ml for 60 s at both the spot and flow cell sites. Receptors were diluted to the defined concentration range in HBS-EP+ buffer at pH 7.4 or MES buffer at pH 6.0. At pH 7.4, three concentrations (4096, 512, and 0 nM) were used for each analyte, and at pH 6.0, four concentrations (512, 64, 8, and 0 nM) were used for each analyte. The chip surface was regenerated after instillation of 10 mM glycine at pH 1.5 for each analyte concentration. Use Biacore TM The T200 evaluation results were obtained using the V2 software and a 1:1 combined dynamic model analysis.

[0312] Option 3

[0313] The affinity of FcRn was measured by SPR using an IBIS MX96 (IBIS Technologies, Enschede, The Netherlands) at 25°C with HBS-EP+ at pH 7.4 or MES at pH 6.0 as the run buffer. Samples were diluted in acetate buffer at pH 4.5 and then captured using a continuous flow micro-dispenser (Carterra, Salt Lake City, USA). G Sensor chips (Carterra, Salt Lake City, USA) were used. Receptors were diluted to defined concentration ranges in HBS-EP+ buffer at pH 7.4 or MES buffer at pH 6.0. Three concentrations (4096, 512, and 0 nM) were used for each analyte at pH 7.4, and four concentrations (512, 64, 8, and 0 nM) were used for each analyte at pH 6.0. The chip surface was regenerated after infusion of 10 mM glycine at pH 2.0 for each analyte concentration. Results were analyzed using Scrubber V2 (BioLogic Software, Campbell, Australia) and a kinetic fitting model.

[0314] Option 4

[0315] Use BiacoreTM The T200 (GE Healthcare) surface plasmon resonance spectrometer was used to screen antibodies for FcRn binding. The experiment was conducted at 25°C using a pH 6 surface plasmon resonance spectrometer with a binding rate of 0.05%. Run buffer containing PBS with 20 and 3.4 mM EDTA was used. Biotinylated FcRn (generated by Scheme 1 above) was captured onto a CM-5 sensor chip having neutral avidin (Thermo Fisher, Waltham MA) previously immobilized on blank and capture surfaces using standard amine coupling. Antibody dilution was then passed through the FcRn and control surfaces. Using the immobilization wizard in Biacore control software, 25 μg / mL neutral avidin in 10 mM sodium acetate buffer at pH 4.5 was added to each NHS / EDC activation surface until 2000 RU was reached. To obtain the FcRn surface, biotinylated FcRn was placed in a solution containing 0.05% PBS. Diluted to 2 μg / mL in PBS with 20 and 3.4 mM EDTA (pH 7.4) and injected onto the capture surface at a flow rate of 25 μg / mL for 110 s 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 25 μL / min using a 3-fold dilution in run buffer at pH 6, with 90 s for association and 180 s for dissociation. The FcRn surface was regenerated between different antibody variants after 30 s of injection into pH 7.4 buffer at 30 μL / min. Sensing maps were generated using a dual-reference blank control surface and fitted using an affinity binding model to generate the KD value for each antibody-FcRn interaction.

[0316] Differential scanning calorimetry

[0317] Option 1

[0318] 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). Five buffer blank injections were performed at the start of each DSC run to stabilize the baseline, and one buffer injection was performed before each antibody injection for reference. Each sample was scanned from 20°C to 100°C at a rate of 60°C / h, with low feedback, 8-s filtration, 5-min preTstat, and 70 psi nitrogen pressure. The resulting thermograms were referenced and analyzed using Origin 7 software (OriginLab Corporation, Northampton, MA).

[0319] Option 2

[0320] The antibody construct was evaluated using the same method as described for Scheme 1, except that an antibody concentration of 0.1–1.0 mg / ml was used, with a concentration of 0.4 mg / ml or greater being preferred.

[0321] Differential scanning fluorescence method

[0322] Option 1

[0323] Add 20 μL of purified sample (between 0.2 and 1.0 mg / mL) to 10 μL In Orange (Invitrogen, Paisley, UK), the stock solution was diluted from 5000x to 20x with reverse osmosis (RO) water and placed in a clear 96-well PCR plate. The samples were incubated at 40°C for 5 min, and then processed using BioRad CFX Connect. TM The RT-PCR machine (BioRad, Watford, UK) measures at a rate of 15°C / h between 40-95°C. Orange fluorescence emission. (Using Bio-Rad CFX Manager) TM Version 3.1 analyzes each peak and derives the temperature of protein unfolding events, then correlates them with the unfolding of known domains within the protein.

[0324] Option 2

[0325] 10 μL of purified sample (between 0.2 and 1.0 mg / mL) was loaded into a Prometheus NT.Plex nanoDSF standard-grade glass capillary (PR-AC002, NanoTemper Technologies, London, UK) and analyzed at a rate of 60 °C / h between 20 and 95 °C. Peaks were analyzed using PR.stability Analysis software version 1.02 to determine the temperature of protein unfolding events, which was then correlated with the unfolding of known domains within the protein.

[0326] Size exclusion chromatography

[0327] Using an Agilent 1100 HPLC system (Agilent, Stockport, UK), 10 μL of purified sample (in the concentration range between 0.2 and 2 mg / mL) was injected into Supelco. On a G3000 SWXL size size exclusion column (Tosoh, Reading, UK), a mobile phase of 400 mM sodium phosphate and 200 mM NaCl at pH 6.8 was flowed at a constant rate of 1.0 mL / min for 15 minutes per sample. A diode array detector was connected to the post-column flowline and recorded the UV / vis absorbance at 210 and 280 nm. The resulting traces were integrated using Chemstation software (Agilent, Stockport, UK) and subsequently analyzed using ChromView. TM The software performs analysis. Sample purity is recorded by classifying the peak area percentage by comparing the percentage of total peak area with molecular weights higher than the main peak and the percentage of total peak area with molecular weights lower than the main peak.

[0328] Liquid chromatography-mass spectrometry

[0329] Mass spectrometry was used to identify the samples. To remove N-linked glycosylation, 10 μl of 80 μg / mL PNG enzyme F in 5% glycerol was added to 50 μL of antibody sample (concentration range between 0.2 and 2 mg / mL), and the mixture was incubated overnight at 30°C. 5 μL of 0.5 M DTT was added to each sample prior to MS analysis. Using an Agilent 1200 HPLC system (Agilent Technologies, Stockport, UK), 3 to 5 μg of sample was injected into a reversed-phase guard column washed with 0.1% formate and 5% acetonitrile, followed by elution with 0.1% formate and 90% acetonitrile at a flow rate of 0.5 mL / min. The eluent was directed to a 6224 Precision Mass TOF LC / MS mass spectrometer (Agilent Technologies), controlled by MassHunter software (Agilent Technologies) running on a PC. Sample mass was determined using MassHunter deconvolution charge envelope.

[0330] C1q combination

[0331] The binding of the antibody construct to human C1q was assessed by ELISA. The antibody construct was coated onto a 96-well flat-bottom Nunc plate by adding 100 μl of 10 μg / ml test antibody in PBS to each well. The plates (Invitrogen, Paisley, UK) were placed on the wells. The plates were sealed and incubated at 4°C for 16 hours. The plates were then dipped in 300 μl of solution containing 0.05% (volume / volume). Wash three times with PBS at 20°C. Then block the plate surface by adding 200 μl of 1% (w / v) bovine serum albumin to each well. Incubate the plate at ambient temperature for 1 h, then wash as before. Dilute recombinant human C1q (C1740, Sigma Aldrich, Gillingham, UK) to the final assay concentration in 50 mM carbonate / bicarbonate buffer (C3041, Sigma Aldrich), and add 100 μl to each well. Incubate the sample at ambient temperature for 2 h, then wash the plate as before. Then add 100 μl of sheep anti-human C1q-HRP (Ab46191, AbCam, Cambridge, UK) diluted to 2 μg / ml with PBS to each well. Incubate the sample at ambient temperature for 1 h, then wash the plate as before. For detection, add 100 μl of Sureblue to each well. TM TMB (52-00-01, Seracare, Milford, USA) was used, and the samples were incubated with stirring at ambient temperature for 20 min. The reaction was stopped by adding 100 μl of 1M HCl to each well. Then, M5e was used... A plate reader (Molecular Devices, Wokingham, UK) measured the absorbance of each sample well at 450 nm. For each antibody variant, seven C1q concentrations ranging from 2 μg / ml to 6 ng / ml, plus a C1q-free control, were tested in duplicate in a semi-logarithmic step. 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 concentrations. C1q concentrations exceeding a threshold absorbance were extrapolated from the fitted curves.

[0332] Example 1: Stability mutations identified by computer simulation prediction tools

[0333] Using the three-dimensional structure of FcγRIIb that binds to IgG1 Fc, single mutations were computer-simulated at each position within the CH2 domain and at specific positions within the CH3 domain within the two shells of the CH2 domain. The first shell residues were residues that directly interacted with the CH2 domain, and the second shell residues were residues that interacted with at least one first shell residue. At each position, all possible amino acids except proline or cysteine ​​were substituted. Mutations that enhanced the thermal stability of Fc were identified and termed stability-enhancing mutations.

[0334] Stability-enhancing mutations were initially investigated in the context of Fc variants selective for FcγRIIb. Since the binding of FcγRIIb to IgG Fc results in an asymmetric complex, only mutations that computer-simulated enhance the stability of both Fc strands were selected for testing to ensure compatibility with FcγRIIb-selective variants and other antibody therapies.

[0335] All models generated by computer simulations were analyzed using a variety of molecular modeling tools, including computer-simulated mutagenesis and packaging modeling using in-house software tools. All models were scored and ranked based on numerous factors, including knowledge-based and physics-based folding and complex formation potential, as well as the combined energy contributions of electrostatics, solvent screening, Lennard-Jones interactions, and hydrophobic interactions.

[0336] Further filtering of the results selects the optimal stability-enhancing mutations with the following characteristics:

[0337] • No exposure to solvents (<30% solvent-accessible surface area (SASA) is advantageous, <50% is acceptable)

[0338] • Away from the FcγR interface

[0339] It is unknown whether this will have a negative impact on FcRn binding.

[0340] It is unknown whether this will have a negative impact on C1q binding.

[0341] • It does not affect the N-glycosylation of Fc at N297.

[0342] The above procedure identified mutations A287F, T289W, A339W, A339Q, A378W, and M428F as potential stability-enhancing mutations. Six variants of trastuzumab (scaffold 1) were constructed, each containing one of the identified mutations, as described in the general methodology. Expression, aggregation, thermostability, and binding affinity for FcγRIIa, FcγRIIb, and FcRn were assessed for each variant as described in the general methodology. Specifically, aggregation was assessed by SEC analysis; thermostability was assessed by DSC (Program 2) and DSF (Program 1); FcγRIIa and FcγRIIb binding was assessed by Biacore binding (Program 2), and FcRn binding was assessed by Protocol 1. The results are shown in Tables 1.1 and 1.2.

[0343] Table 1.1: Characteristics of mutations identified by computer simulation prediction tools

[0344]

[0345] 1The relative SASA is calculated based on the average solvent-accessible volume of both chains and the area of ​​each residue. The output is relative to the fully exposed residue.

[0346] 2 ΔT m Indicates T m Mutant-T m Differences between wild-type (v16588, WT trastuzumab)

[0347] 3 The data for variant 19305 were generated at a flow rate of 0.5 ml / min instead of 1.0 ml / min. Therefore, the retention time was very consistent with the column retention compared to other variants.

[0348] Table 1.2: Characteristics of mutations identified by computer simulation prediction tools

[0349]

[0350] 1 The variation relative to the wild type is represented by K for FcγRIIb and FcγRIIa. D wt / K D mut.

[0351] 2 NB = No binding.

[0352] 3 ND = Not determined.

[0353] Mutations A287F and M428F were selected for further evaluation based on the following criteria:

[0354] For single-point mutations, T is obtained through DSC. m Increase >2℃

[0355] • Regarding the binding of FcγRIIb and FcγRIIa, wild-type characteristics were preserved (WT value +30%).

[0356] • Retain the binding with FcRn (relative to WT, <K D (2 times difference)

[0357] • Analysis of the SEC revealed a monomer content >95%.

[0358] The stability achieved by the A287F mutation is energy-favorable and likely stems from the generation of stacked PP interactions at the W277 position and the burying of hydrogen bonds between W277 and S304. Therefore, substitution mutations at these positions with amino acids possessing similar aromaticity and hydrophobicity are predicted to increase stability, and are thus stability-enhancing mutations. These include mutations A287Y, A287W, and A287H, as well as A287M. The latter mutation is predicted to bury hydrogen bonds, but may offer lower stability due to the loss of π-π stacking interactions.

[0359] Figure 1 B shows the positions of A287 and M428 in the IgG Fc region.

[0360] Example 2: Stable mutations identified through bioinformatics analysis

[0361] Sequences from multiple organisms and IgG subtypes (59 non-redundant sequences extracted from Uniprot) were aligned with ClustalX (Larkin et al., 2007, Bioinformatics, 23:2947-2948) to distinguish residues conserved for folding or function from residues that can be substituted. Non-surface exposed sites with some sequence identity in all IgGs were identified along with commonly found substituted residues present in other species or subtypes. Additional filtering based on relative SASA at selected sites was included to reduce the risk of immunogenicity.

[0362] The filtering results are used to select the optimal stability-enhancing mutations with the following characteristics:

[0363] • No exposure to solvents (<30% solvent-accessible surface area (SASA) is advantageous, <50% is acceptable)

[0364] • Partially conserved (sequence identity of <85% for all CH2 IgGs is favorable, <95% is acceptable)

[0365] • Away from the FcγR interface

[0366] It is unknown whether this will have a negative impact on FcRn binding.

[0367] It is unknown whether this will have a negative impact on C1q binding.

[0368] • It does not affect the N-glycosylation of Fc at N297.

[0369] Amino acid substitutions present in mouse IgG2a are more favorable than in other species because the CH2 domain T in mouse IgG2a... m (T m = (approximately 80℃) compared to the CH2 domain Tm(T in human IgG1)m =72℃) High. The stability of the CH2 domain in other species was not considered.

[0370] The above methods 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 the general methods, each containing one of the identified mutations. Expression, post-purification aggregation, thermostability, and binding affinity for FcγRIIb, FcγRIIa, and FcRn of each variant were evaluated in mammalian cells as described in Example 1. The results are shown in Tables 2.1 and 2.2.

[0371] Table 2.1: Characteristics of mutations identified by bioinformatics analysis

[0372]

[0373] 1 The relative SASA is calculated based on the average solvent-accessible volume of both chains and the area of ​​each residue. The output is relative to the fully exposed residue.

[0374] 2 ΔT m Indicates T m Mutant-T m Differences between wild-type (v16588, WT trastuzumab)

[0375] 3 ND = Not measured

[0376] Table 2.2: Characteristics of mutations identified through bioinformatics analysis

[0377]

[0378] 1 The variation relative to the wild type is represented by K for FcγRIIb and FcγRIIa. D wt / K D mut.

[0379] 2 NB = No binding.

[0380] 3 ND = Not determined.

[0381] 4 Due to poor antibody capture, reliable data for v19315 could not be obtained.

[0382] Mutations T250V, L309Q, and V308I were selected for further evaluation based on the following criteria:

[0383] For single-point mutations, T is obtained through DSC. m Increase >2℃

[0384] • Regarding the binding of FcγRIIb and FcγRIIa, wild-type characteristics were preserved (WT value ± 30%).

[0385] • Retain the binding with FcRn (relative to WT, <K D (2 times difference)

[0386] • Analysis of the SEC revealed a monomer content >95%.

[0387] Due to similar amino acid properties in terms of size and hydrophobicity, substitution mutations at these positions (such as T250I, T250A, or L309T) are predicted to increase stability. Small differences in amino acid size (V vs. I or A) and side chain branching (Cβ-branched vs. unbranched residues) may lead to minor variations in stabilizing effects.

[0388] Figure 1 B shows the location of the T250 site in the IgG Fc region.

[0389] Example 3: Stability mutation involving non-natural disulfide bonds

[0390] Using the three-dimensional structure of FcγRIIb bound to IgG1 Fc, the distances between all Cα and Cβ atom pairs in the CH2 domain were calculated and averaged across the two chains of Fc to determine sites where disulfide bonds could be introduced. Filtering was performed based on the maximum distance between Cα-Cα pairs. The maximum distance between Cβ and Cβ pairs

[0391] Because the binding of FcγRIIb to IgG Fc results in an asymmetric complex, only mutations that improve the stability of both Fc strands, as demonstrated by computer simulations, were selected for testing. This selection biases stability mutations towards those compatible with variants that are selective for both FcγRIIb and unbound Fc.

[0392] Computer simulation models were created for all possible artificial disulfide bonds, and the energy of these models was minimized to determine those with the lowest energies. The models were visually examined and scored based on the relative solvent accessible surface area (SASA) of cysteine ​​residues in both chains, the root mean square deviation (RMSD) of the main chain and side chains relative to the wild-type structure, improvements based on knowledge and physics of affinity and stability potential, stereoconflict, and disulfide strain energy (DSE) scores (Katz and Kossiakoff, 1986, J Biol Chem, 261(33):15480-15485).

[0393] The filtering results are used to select the optimal stability-enhancing mutations with the following characteristics:

[0394] • No exposure to solvents (<30% is advantageous compared to SASA, <50% is acceptable)

[0395] • Away from the FcγR interface

[0396] It is unknown whether this will have a negative impact on FcRn binding.

[0397] It is unknown whether this will have a negative impact on C1q binding.

[0398] • Does not affect N-glycosylation of Fc at N297

[0399] • Low RMSD of main chain and side chain (main chain) And the side chain )

[0400] • Equivalent or enhanced knowledge- and physical stability potential

[0401] • Low DSE score (<30 (DSE) is favorable, <50 is acceptable).

[0402] The above process identified the mutations D249C-P257C, F275C-S304C, V263C-V302C, L242C-I336C, T289C-S304C, F243C-T260C, V266C-Y300C, V240C-I332C, and W277C-V284C as potential stability-enhancing mutations. Thirteen variants of trastuzumab (scaffold 1) were constructed as described in the general methodology, each containing one of the identified mutation pairs or a previously reported mutation pair in the literature to enhance stability by introducing 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). Expression, aggregation, thermostability, and binding affinity to FcγRIIb, FcγRIIa, and FcRn were evaluated for each variant in mammalian cells as described in Example 1. Affinity to the FcRn receptor was also evaluated in a subset of successful variants. The results are shown in Tables 3.1 and 3.2.

[0403] Table 3.1: Characteristics of mutations introducing non-natural disulfide bonds

[0404]

[0405]

[0406] 1 The relative SASA is calculated based on the average solvent-accessible volume of both chains and the area of ​​each residue. The output is relative to the fully exposed residue.

[0407] 2 ΔT m Indicates T m Mutant-T m Differences between wild-type (v16588, WT trastuzumab)

[0408] 3 The DSC plot of v19326 is atypical, leading to a T value for the CH2 domain. m The determination is unclear.

[0409] Table 3.2: Characteristics of mutations introducing non-natural disulfide bonds

[0410]

[0411] 1 The variation relative to the wild type is represented by K for FcγRIIb and FcγRIIa.D wt / K D mut.

[0412] 2 NB = No binding.

[0413] 3 ND indicates that the data was not measured.

[0414] 4 The purity curve of v19326 is atypical, and this leads to K D The determination is unclear.

[0415] 5 LB ＝ Low binding.

[0416] Of the disulfide bonds identified, only L242C-I336C were selected for further evaluation based on the following criteria:

[0417] For single mutation pairs, T values ​​obtained through DSC m Increase >2℃

[0418] • Regarding the binding of FcγRIIb and FcγRIIa, wild-type characteristics were preserved (WT value ± 30%).

[0419] • Analysis of the SEC revealed a monomer content >95%.

[0420] Mutations known in the art for L242C-K334C, A287C-L306C, and V259C-L306C meet the following criteria:

[0421] For single mutation pairs, T values ​​obtained through DSC m Increase >2℃

[0422] • Regarding the binding of FcγRIIb and FcγRIIa, wild-type characteristics were preserved (WT value ± 30%).

[0423] The V240C-I332C disulfide bond increases Tm but partially eliminates FcγR binding. It is expected that such disulfide bonds will still be available for binding elimination in certain cases where it is necessary or can be mitigated by including other mutations that promote binding to one or more FcγRs.

[0424] Example 4: Stability of FcγRIIB-selective Fc variants

[0425] Six optimal individual mutations (A287F, M428F, T250V, L309Q, L242C_I336C, and V308I) identified in trastuzumab homodimers as described in Examples 1-3 were transplanted into two different heterodimeric trastuzumab FcγRIIb selective variants (scaffold 8 and scaffold 9) to assess their compatibility with other CH2 domain mutations. Furthermore, 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 assess whether a stable increase could be achieved through additive or synergistic effects.

[0426] Twenty-four trastuzumab variants in single-arm antibody form were constructed as described in the general methodology. Each variant included one of two sets of FcγRIIb selective enhancement mutations (scaffold 8 or scaffold 9; see Table 4.1) and stability enhancement mutations as shown in Tables 4.2 to 4.4. Expression, aggregation, thermostability, and binding affinity to FcγRIIb, FcγRIIa, and FcγRI were evaluated for each variant as described in Example 1. The results are shown in Tables 4.2 to 4.4.

[0427] Table 4.1: Parental variants used to assess the compatibility of stability and selective enhancement mutations

[0428]

[0429] *Template 1 indicates that the sequence between positions 325 and 331 should be replaced with the following sequence: STWFDGGYAT[SEQ ID NO:1]

[0430] Based on the analysis of the SEC curve, a first-layer filtration was applied after purification. The area under the chromatogram for all present signals was integrated and converted to the percentage of each species present in the variant sample. The percentage of high molecular weight (HMW) species observed in the analysis of the SEC curve indicates the abundance of full-size antibodies formed by each variant when expressed at a single DNA ratio. Variants with less than 20% HMW species when expressed 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 in further characterization. Low molecular weight (LMW) species indicate the presence of mismatched Fc homodimers, which do not interfere with the determination of Tm or binding affinity for any FcγR.

[0431] Table 4.2: Compatibility of stability and selective enhancement mutations: expression and aggregation

[0432]

[0433]

[0434] 1 The percentages of HMW, heterodimer, LMW, and monomer retention time were all correlated with the graphs observed for each variant in the SEC analysis, indicating their relative abundance. The HMW percentage corresponds to mismatched full-size antibodies, the heterodimer percentage corresponds to heterodimer single-arm antibodies, and the LMW percentage corresponds to mismatched homodimer Fc or half-antibodies.

[0435] 2 Bracket 8

[0436] 3 Bracket 9

[0437] Table 4.3: Compatibility of Stability and Selectivity-Enhancing Mutations: Thermal Stability

[0438]

[0439]

[0440] 1 The report describes the transformation of the CH2 domain observed via DSF.

[0441] 2 ΔT m Indicates T m Mutant-T m Differences between the parents (v27923 or v27924).

[0442] 3 Theoretical ΔT m This refers to the additive stabilizing effect based on point mutations in the corresponding parent variants.

[0443] 4 Bracket 8

[0444] 5 Bracket 9

[0445] 6 N / A indicates that data was not collected due to low sample purity.

[0446] Table 4.4: Compatibility of stability and selectivity-enhancing mutations: binding affinity to FcγRIIb, FcγRIIa, and FcγRI

[0447]

[0448]

[0449] 1 Bracket 8

[0450] 2 Bracket 9

[0451] 3 N / A indicates that data was not collected due to low sample purity.

[0452] Mutations are evaluated based on the following criteria:

[0453] For single-point mutations, T is obtained through DSF. m Increase >1℃, and the additive effect is minimal when combined.

[0454] • In terms of binding of FcγRIIb, FcγRIIa, and FcγRI, it retains characteristics of the wild type (<2-fold difference compared to the parental variant).

[0455] • Analysis showed that the heterodimer content of SEC was >75%.

[0456] Based on the above, the most effective single mutations are A287F (+3.5-4℃), T250V (+5.5℃), L309Q (+2-2.5℃), and M428F (+2℃). V308I, as a single mutation, also provides a small increase in Tm (+0.5 to 1.0℃).

[0457] Stability-enhancing designs with additive or synergistic contributions include A287F / M428F (+6.5-7℃), A287F / T250V (+9.0-9.5℃), M428F / T250V (+8.5℃), and T250V / L309Q (+8.5-9.0℃). The combinations of A287F / M428F, M428 / T250V, and T250V / L309Q produce a Tstability slightly higher than the additive effect. m The increase is due to the addition of A287F / T250V. The combination of L242C_I336C / V308I also provides a slight increase in Tm compared to the L242C_I336C mutation alone.

[0458] Example 5: Stability of other full-size antibody testing systems

[0459] Three stability-enhancing designs were each combined with three FcγRIIb-selective enhancement designs and transferred to three different full-size antibody systems to evaluate 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 the general methodology. The three FcγRIIb-selective enhancement designs are shown in Table 5.1, and the three selected stability-enhancing designs are shown in Tables 5.2-5.5.

[0460] Table 5.1: Parental variants used to assess stability and selective enhancement mutation compatibility

[0461]

[0462] 1 "Template 1.1" means replacing the amino acid residues at positions 325-331 with the following sequence: STWFIGGYAT[SEQ IDNO:3].

[0463] 2 "Template 7.1" means replacing the amino acid residues at positions 325-331 with the following sequence: GLDHRGKGYV [SEQ ID NO:4].

[0464] Expression, post-purification aggregation, and thermostability of each variant in mammalian cells were evaluated as described in Example 1. Binding affinities of the trastuzumab-based variants to FcγRI, FcγRIIb, FcγRIIa, and FcγRIIIa were evaluated as described in Example 1. C1q binding of the trastuzumab-based variants was evaluated as described in the general methodology. Thermostability was assessed by DSF of the various antibodies and DSC of the trastuzumab-based variants. The results are shown in Tables 5.2 to 5.6.

[0465] SEC plots were collected and analyzed as described in the general methodology, with the following modifications made to full-size antibody species. The area under the chromatogram for all present signals was integrated and converted to a percentage for each species. The percentage of high molecular weight (HMW) species observed in the aSEC plot indicates the amount of aggregates formed by each variant after the introduction of stability-enhancing mutations and is compared to the parental variant in each antibody system. Variants with parental-like characteristics (+ / - 5% HMW species) are preferred among multiple antibodies. Lower molecular weight (LMW) species indicate the presence of mismatched heterodimeric heavy chains, which leads to half-antibodies, and this is due to the use of an unoptimized DNA ratio during expression. The results are shown in Table 5.2.

[0466] Purity was assessed by LC-MS as described in the general methods to confirm that the monomeric inclusions were predominantly the desired heterodimeric species. The cumulative percentages of half-body A and half-body B indicate the amount of mismatched homodimers present in each sample for a given ratio of DNA used for expression. Since the stability-enhancing mutation is symmetric, the binding of the CH2 domains Tm, FcRn, and C1q is unaffected by the presence of mismatched species. However, high levels of mismatched homodimers will affect the FcγRIIb selectivity observed by SPR. Most samples showed less than 10% mismatched homodimers. See Table 5.3.

[0467] Table 5.2: Purity of Heterodimers (SEC Analysis)

[0468]

[0469]

[0470] 1 The HMW percentage, monomer percentage, LMW percentage, and monomer retention time are all correlated with the graphs observed for each variant in the SEC analysis, and indicate their relative abundance. The HMW percentage corresponds to aggregates, the monomer percentage corresponds to heterodimers and mismatched homodimers, and the LMW percentage corresponds to hemimers.

[0471] 2 Heterodimer trastuzumab-stent 3.

[0472] Table 5.3: Heterodimer Purity (LCMS)

[0473]

[0474]

[0475] 1 Heterodimer trastuzumab-stent 3.

[0476] Table 5.4: Thermal Stability

[0477]

[0478]

[0479] 1 ΔT m T represents the value of each design evaluated by DSC or DSF. m Mutant-T m Differences between parents (v29689, v29715 or v29724).

[0480] 2 Heterodimer trastuzumab-stent 3.

[0481] 3 ND = Not measured

[0482] Table 5.5: Trastuzumab-based variants 1 FcγR binding

[0483]

[0484] 1 No variants showed any detectable binding to FcγRIIIaV and FcγRIIIaF via SPR.

[0485] 2 The variation relative to the wild type is represented by K for FcγRIIb and FcγRIIa. D wt / K D mut.

[0486] 3 Heterodimer trastuzumab-stent 3.

[0487] 4 NB = No binding observed.

[0488] Table 5.6: C1q binding of trastuzumab-based variants

[0489]

[0490] 1 Heterodimer trastuzumab-stent 3.

[0491] 2 C1q binding affinity is calculated from the C1q concentration at which the binding signal exceeds 0.5 absorbance, as extrapolated from curve fitting.

[0492] 3 C1q binding affinity, as calculated from absorbance measured at 2 μg / ml.

[0493] 2 NB = No binding detected.

[0494] All three stability-enhanced designs tested (A287F / T250V, M428F / T250V, and A287F / M428F) successfully increased thermal stability by 6-10°C while maintaining parent-sample characteristics in all other aspects evaluated. Specifically, the designs met the following criteria:

[0495] • Of all three antibodies, the T-cell antibody obtained via DSF m Increase by >5℃.

[0496] • In terms of binding to FcγRI, FcγRIIaH, FcγRIIaR, FcγRIIb, FcγRIIIa and C1q, wild-type characteristics are retained (<2-fold difference compared to parental variants).

[0497] • The heterodimer content measured by LC-MS was equal to or better than that of the parent variant.

[0498] • The monomer content is similar to or better than that of the parental variants obtained through aSEC.

[0499] Example 6: FcRn binding of a full-size antibody with stability-enhancing design

[0500] As described in Example 5, three stability-enhancing designs were each combined with three FcγRIIb-selective enhancement designs and transferred to three different full-size antibody systems (trastuzumab, anti-CD19 antibody, and anti-CD40 antibody; scaffolds 3-5). The FcRn binding of the resulting variants was evaluated as described in the general methods (Scheme 2 for scaffolds 3 / 5 and Scheme 3 for scaffolds 3-5). The results are shown in Table 6.1.

[0501] Table 6.1: FcRn binding of antibodies designed with enhanced stability and FcγRIIb selectivity.

[0502]

[0503]

[0504] 1 Heterodimer trastuzumab – stent 3.

[0505] 2 K is determined through scheme 2 in the general method. D The fold change relative to the parent variant is denoted as K. D wt / K D mut

[0506] 3 K is determined through scheme 3 in the general method. D The fold change relative to the parent variant is denoted as K. D wt / K D mut

[0507] 4 ND ＝ Not detected because the antibody did not bind to protein L.

[0508] 5 v29724 exhibits a non-standard form when evaluated using scheme 3 (general method). The combination evaluated using scheme 2 (general method) is comparable to other variants.

[0509] For all antibodies tested, the stability-enhancing design did not alter FcRn binding (K) compared to the corresponding parental variants. D (3 times that of the parental variant), indicating that the design is transferable between different antibodies.

[0510] Example 7: Compatibility of stability-enhancing designs with other mutations that lead to stability loss

[0511] To further evaluate the transferability and compatibility of the three best-performing stability-enhancing designs (A287F / T250V, M428F / T250V, and A287F / M428F), these designs were each combined with the following three groups of CH2 or CH3 mutations (scaffolds 3, 6, and 7; see general approach):

[0512] • Scaffold 3: An asymmetric mutation in the CH3 domain that promotes the formation of heterodimer Fc.

[0513] • Scaffold 6: The N297A mutation generates non-glycosylated antibodies and eliminates the effector function of the antibody. Compared with the wild-type antibody, the introduction of the N297A mutation reduces the thermal stability of the variant antibody by 10°C.

[0514] • Scaffold 7: The S239D / I332E mutation increases the antibody's affinity for the FcγRIIIa receptor. Compared to the wild-type antibody, the introduction of the S239D / I332E mutation reduces the thermostability of the variant antibody by 20°C.

[0515] As described in the general methodology, the corresponding parental variants and mutants were cloned into trastuzumab scaffolds. Expression of each variant in mammalian cells (Protocol 1), thermostability, binding affinity for FcγRI, FcγRIIb, FcγRIIa, and FcγRIIIa (Protocol 1), and FcRn binding (Protocol 4) were assessed as described in the general methodology. Thermostability was assessed by DSC (Protocol 1). Results are shown in Tables 7.1 to 7.4 and Figure 2 A through C.

[0516] Table 7.1: Effects of stability-enhancing mutations on the binding of FcγRI and FcγRIII in different antibody scaffolds

[0517]

[0518] 1 The fold change relative to the parent variant is denoted as K. D wt / K D mut

[0519] 2 NB = No binding detected.

[0520] Table 7.2: Effect of stability-enhancing mutations on FcγRII binding in different antibody scaffolds

[0521]

[0522] 1 The fold change relative to the parent variant is denoted as K. D wt / K D mut

[0523] 2 NB = No binding detected.

[0524] Table 7.3: Effect of stability-enhancing mutations on FcRn binding in different antibody scaffolds

[0525]

[0526] 1 The fold change relative to the parent variant is denoted as K. D wt / K D mut

[0527] Table 7.3: Effect of stability-enhancing mutations on thermal stability (Tm) in different antibody scaffolds

[0528]

[0529] 1 The CH2 transition overlaps with the Fab and CH3 transitions, and cannot be accurately measured.

[0530] All three stability-enhanced designs tested (A287F / T250V, M428F / T250V, and A287F / M428F) successfully improved the thermal stability of each tested stent by 6–10°C, while maintaining parent-sample characteristics in all other aspects evaluated. Specifically, all three designs met the following criteria:

[0531] • Of all three types of stents, the T-cell stent via DSC m Increase by >5℃.

[0532] • In terms of binding to FcγRI, FcγRIIaH, FcγRIIaR, FcγRIIb, FcγRIIIa and FcRn, wild-type characteristics are retained (<2-fold difference compared to parental variants).

[0533] Therefore, the stability enhancement design is compatible with mutations in the CH2 and CH3 domains that alter antibody function and biological properties and can stabilize these mutations.

[0534] The aforementioned CH2 or CH3 mutation groups (taking scaffolds 3, 6, and 7 as examples) are examples of mutations currently included in therapeutic molecules under clinical evaluation (Saxena et al., 2016, Front Immunol, 7:580). Other CH2 and / or CH3 mutations affecting antibody function and stability, such as club-and-mortar structures (Ridgeway et al., 1996, Protein Eng., 9:617-621), electrostatic shifting (Gunasekaran et al., 2010, JBC, 285, 19637-19646), or other mutations known in the art, are also expected to be compatible with and stabilized by stability-enhancing mutations.

[0535] Example 8: Stability-enhanced design for compatibility with other immunoglobulin classes

[0536] Evaluate the sequence and topology of the constant domains of IgG, IgA, IgD, IgE, and IgM to determine whether the most effective stability-enhancing designs identified above can be transferred to other immunoglobulin (Ig) classes and / or subtypes.

[0537] Different Ig classes have different functions and biological activities, but share a common topological structure. IgG, IgA, IgD, IgE, and IgM are all composed of heavy and light chains. The constant regions of IgG, IgA, and IgD contain CH1, CH2, and CH3 domains, and they share a common Ig fold, indicating that mutations that increase IgG stability can be transferred to IgA and IgD classes. IgE and IgM differ from other Ig classes and are composed 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 [link to relevant documentation]). Figure 3 A review of the structures of IgG, IgA, and IgM Ig domains (PDB IDs: 2QEJ, 2WAH, and 6KXS, respectively) obtained from the Protein Database (PDB) revealed that, structurally, these domains exhibit similar folding, suggesting that mutations increasing IgG stability can be transferred to both IgM and IgE classes.

[0538] Further analysis of the specific interactions of each of the T250V, A287F, and M428F mutations within the CH2 and CH3 domains provides further support for the proposal that stability-enhancing mutations should be transferable to other Ig classes.

[0539] Residue T250 is located in the helical region of IgG1 near the FcRn binding site of the CH2 domain and spatially near the CH3 domain. Threonine residues at this position are conserved in all IgG isoforms and are substituted by similarly polar residues (serine) in IgM and charged residues (aspartic acid) in IgA, IgD, and IgE (see [link to relevant documentation]). Figure 3 A). Structural analysis of this region in IgA, IgM, and IgG showed that the helices in IgA and IgM were less buried compared to IgG1 (PDBID: 2WAH) (PDBID: 2QEJ and 6KXS). Therefore, mutating the charged or polar residues at the T250 position in other Ig classes to smaller hydrophobic residues could improve the stacking of the first helix (246-254) against the second helix (309-316) and the connection of the CH2-CH3 domains, resulting in a more compact structure with an increased burial interface. This, in turn, can stabilize the CH2 domain to prevent thermal denaturation. Therefore, the stability-enhancing mutation T250V is expected to effectively improve the stability of the CH2 domain in IgA, IgD, and IgG antibodies, as well as the CH3 domain in IgE and IgM antibodies.

[0540] Residue A287 is located in the exposed β-chain region outside the Ig fold of the CH2 domain of IgG1. The alanine residue at position 287 is conserved in all IgG isoforms and in IgA, but is substituted by residues such as valine, histidine, and threonine in IgD, IgE, and IgM. However, regardless of the different residues present at this position, the local environment and folding are similar in all Ig classes where the structure is available. As described in Example 1, the stabilization via the mutant A287F in IgG1 is energy-advantageous and likely stems from the generation of stacked π-π interactions with the W277 position and the burying of hydrogen bonds between the W277 and S304 positions. Residue W277 is conserved in all Ig classes, while residue S304 is conserved in all Igs except IgM (see Example 1). Figure 3 A and Figure 3 B). Therefore, it is also expected that the A287F mutation will effectively improve the stability of the CH2 domain of IgA, IgD and IgG antibodies and the CH3 domain of IgE and IgM antibodies.

[0541] Residue M428 is located in the exposed β-chain region of the CH3 domain of IgG1 and is spatially situated at the interface with the CH2 domain. The methionine at position 428 is conserved in all IgG isoforms, while other Ig classes contain smaller residues at this position, such as glycine, valine, alanine, or serine (see [link to relevant documentation]). Figure 3Introducing a larger residue (such as phenylalanine) at this location may bury the hydrophobic side chain on the helix of the CH2 domain. The resulting increased burial surface at the CH2-CH3 domain junction reduces the flexibility of the CH2 domain and increases its stability. The local structural environment in IgA and IgM is similar to that in IgG in this region, and the introduction of large aromatic residues (such as phenylalanine, tyrosine, or tryptophan) is expected to form additional stacking π-π interactions with the surrounding aromatics. Therefore, the mutant M428F is also predicted to increase the stability of the CH2 domain of IgA, IgD, and IgG antibodies, as well as the CH3 domain of IgE and IgM antibodies.

[0542] As demonstrated in Examples 4-7, the stability-enhancing mutations of T250V, A287F, and M428F are pairwise compatible and can be combined to produce additive stabilizing effects. Similar compatibility is expected for other Ig classes for these stability-enhancing designs (A287F / T250V, M428F / T250V, and A287F / M428F).

[0543] Example 9: The effect of abrupt changes in stability under stress on aggregation tendency

[0544] To assess whether the increased thermostability achieved through the incorporation of stability-enhancing mutations could reduce antibody aggregation tendency, fifteen antibody variants with or without the T250V / A287F stability-enhancing design and a range of thermostability Fc regions were prepared. The resulting antibody variants were then subjected to stress experiments, where changes in their aggregate ratios were evaluated after incubation at 40°C for 2 weeks under neutral or weakly acidic conditions.

[0545] Each antibody variant was based on scaffold 3 and contained various combinations of mutations in the CH2 domain as shown in Table 9.1. The corresponding parental variants and stable mutants were cloned into scaffold 3 as described in the general methods. Expression of each variant in mammalian cells (Scheme 2), aggregation by size exclusion chromatography, and thermostability by DSF (Scheme 2) were evaluated. The results are shown in Tables 9.1 and 9.2.

[0546] Subsequently, each variant was normalized to 10 mg / ml and dialyzed into acetate- or phosphate-based buffers for testing under weakly acidic or neutral conditions, respectively, and incubated at 4°C or 40°C for 2 weeks. The aggregate, monomer, and fragment ratios of each variant were then assessed by size exclusion chromatography, comparing samples at 4°C and 40°C. The results are shown in Table 9.3 and... Figure 4 .

[0547]

[0548]

[0549] Table 9.2: Thermal stability of parent and stabilized variants

[0550]

[0551] Table 9.3: Changes in the aggregation tendency of parents and stabilized variants after stress under neutral and acidic conditions.

[0552]

[0553] 1 Changes in total HMW species after incubation at 40°C in a specified buffer for 2 weeks.

[0554] 2 ΔHMW species [stabilized] - ΔHMW species [unstabilized]

[0555] As shown in Table 9.2, the incorporation of the T250V / A287F stability-enhancing mutation successfully increased the thermal stability of fifteen variants by 7.7 °C to 10.6 °C. Therefore, these stability-enhancing mutations can increase CH2 stability, regardless of the initial stability of the antibody construct.

[0556] The results in Table 9.3 show that after 2 weeks of incubation at 40°C under acidic conditions, the three variants with the lowest CH2 Tm (variants v31186, v32210, and v32242) exhibited high levels of aggregation observed by SEC analysis. When the stability-enhancing T250V / A287F mutation was added to these variants, the Tm of the variants increased, and the least amount of HMW species was detected after the stress experiment. Figure 4 A strong and approximate exponential correlation can also be observed between CH2 Tm and the aggregation observed in these variants under weakly acidic conditions. For variants with an initial (unstabilized) CH2 Tm above 60 °C, very small changes (<2%) in high molecular weight species were observed. The small changes in high molecular weight species observed after incubation under neutral conditions appear to be independent of CH2 Tm.

[0557] Overall, these results suggest that the incorporation of stable mutations (and specifically T250V / A287F) can generally reduce the tendency to aggregate under weakly acidic conditions.

[0558] All disclosures of patents, patent applications, publications and database entries mentioned in this specification are hereby expressly incorporated in their entirety by reference, as if each such individual patent, patent application, publication and database entry were expressly and individually incorporated by reference.

[0559] It is intended that modifications of the specific embodiments described herein, which would be obvious to those skilled in the art, be included within the scope of the following claims.

Claims

1. An Fc variant comprising a stability-enhancing amino acid mutation, said stability-enhancing amino acid mutation comprising a mutation at position 287, wherein said mutation is the substitution of amino acid at position 287 with Phe, and: A mutation at position 250, wherein the mutation is the substitution of the amino acid at position 250 by Val; or A mutation at position 428, wherein the amino acid at position 428 is replaced by Phe. The Fc variants described herein are based on parental IgG Fc that do not contain the stability-enhancing amino acid mutations. The CH2 domain melting temperature (Tm) of the Fc variant is increased compared to the parental IgG Fc without the stability-enhancing amino acid mutation, and The amino acid numbers are based on the EU index.

2. The Fc variant according to claim 1, wherein the stability-enhancing amino acid mutation contained in the Fc variant is: 287F / 428F.

3. The Fc variant of claim 1, wherein the stability-enhancing amino acid mutation contained in the Fc variant is: 250V / 287F.

4. The Fc variant according to any one of claims 1 to 3, wherein the parental IgG Fc is IgG1 Fc.

5. The Fc variant according to any one of claims 1 to 3, wherein the parental IgG Fc is human IgG1 Fc.

6. The Fc variant according to any one of claims 1 to 3, wherein the parental IgG Fc comprises one or more amino acid mutations that improve the function of the Fc region.

7. The Fc variant according to any one of claims 1 to 3, wherein the parental IgG Fc comprises one or more amino acid mutations that improve the function of the Fc region and reduce the Tm of the CH2 domain of the corresponding wild-type Fc.

8. The Fc variant according to any one of claims 1 to 3, wherein the CH2 domain Tm of the Fc variant is increased by at least 2.0°C or at least 3.0°C compared to the parental IgG Fc.

9. The Fc variant according to any one of claims 1 to 3, wherein the CH2 domain Tm of the Fc variant is increased by 3.0°C to 9.0°C compared to the parental IgG Fc.

10. The Fc variant according to any one of claims 1 to 3, wherein the CH2 domain Tm of the Fc variant is increased by 3.0°C to 10.5°C compared to the parental IgG Fc.

11. The Fc variant according to any one of claims 1 to 3, wherein the Fc variant exhibits reduced aggregation under weakly acidic conditions compared to the parental IgG Fc region.

12. A polypeptide comprising an Fc variant according to any one of claims 1 to 11 and one or more protein moieties fused or covalently linked to the Fc variant, wherein the one or more protein moieties comprise an antigen-binding domain, a ligand, a receptor, a receptor fragment, a cytokine, or an antigen.

13. The polypeptide of claim 12, wherein at least one of the one or more protein portions is an antigen-binding domain.

14. The polypeptide of claim 13, wherein the antigen-binding domain is Fab or scFv.

15. The polypeptide of claim 13, wherein the polypeptide is an antibody or an antigen-binding antibody fragment.

16. The polypeptide of claim 15, wherein the polypeptide is a therapeutic antibody or antibody fragment.

17. A polynucleotide or polynucleotide group, said polynucleotide encoding an Fc variant according to any one of claims 1 to 11.

18. A polynucleotide or polynucleotide group, said polynucleotide encoding a polypeptide according to any one of claims 12 to 16.

19. A vector or group of vectors, said vector comprising one or more polynucleotides encoding an Fc variant according to any one of claims 1 to 11 or a polypeptide according to any one of claims 12 to 16.

20. A host cell comprising one or more polynucleotides, said polynucleotide encoding an Fc variant according to any one of claims 1 to 11 or a polypeptide according to any one of claims 12 to 16.

21. A method for preparing an Fc variant according to any one of claims 1 to 11, comprising transfecting a host cell with one or more polynucleotides encoding the Fc variant, and culturing the host cell under conditions suitable for expressing the Fc variant.

22. A method for preparing a polypeptide according to any one of claims 12 to 16, comprising transfecting a host cell with one or more polynucleotides encoding the polypeptide, and culturing the host cell under conditions suitable for expressing the polypeptide.

23. A pharmaceutical composition comprising an Fc variant according to any one of claims 1 to 11 or a polypeptide according to any one of claims 12 to 16.

24. A method for increasing the melting temperature (Tm) of the CH2 domain of an Fc, comprising introducing a stability-enhancing amino acid mutation into a parental Fc to provide an Fc variant with an increased CH2 domain Tm compared to the parental Fc, the stability-enhancing amino acid mutation comprising a mutation at position 287, wherein the mutation is the substitution of amino acid Phe at position 287, and: A mutation at position 250, wherein the mutation is the substitution of the amino acid at position 250 by Val; or A mutation at position 428, wherein the amino acid at position 428 is replaced by Phe. The parental Fc is IgG Fc, and The amino acid numbers are based on the EU index.

25. The method of claim 24, wherein the method includes introducing the mutation at bit 287 and the mutation at bit 250, the mutation at bit 250 being a substitution performed using Val.

26. The method of claim 24, wherein the method includes introducing the 287-bit mutation and the 428-bit mutation, the 428-bit mutation being a substitution performed using Phe.

27. The method according to any one of claims 24 to 26, wherein the CH2 domain Tm of the Fc variant is increased by at least 2.0°C or at least 3.0°C compared to the parent Fc.

28. The method according to any one of claims 24 to 26, wherein the CH2 domain Tm of the Fc variant is increased by 3.0°C to 9.0°C compared to the parent Fc.

29. The method according to any one of claims 24 to 26, wherein the CH2 domain Tm of the Fc variant is increased by 3.0°C to 10.5°C compared to the parent Fc.

30. The method according to any one of claims 24 to 26, wherein introducing the stability-enhancing amino acid mutation into the parental Fc provides an Fc variant that exhibits reduced aggregation under weakly acidic conditions compared to the parental Fc region.

31. The method of claim 30, wherein the stability-enhancing amino acid mutations include 250V and 287F.

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