FC variants with abrogated binding to FCGammar and C1Q

Abstract

The present disclosure provides, inter alia, Fc variants with significantly reduced ADCC, ADCP, and CDC functions. As described herein, the present disclosure is based in part on the identification of novel combinations of mutations that abolish binding to all of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q.

Description

[Background technology]

[0001] The immune response is the mechanism by which the body defends itself against foreign agents that invade the body and cause infection or disease. This mechanism is based on the ability of antibodies, produced or administered to a host, to bind antigens through their variable regions. Once an antigen is bound by an antibody, it becomes a target for destruction, often mediated in part by the antibody's constant region or Fc domain.

[0002] For example, one activity of the Fc domain of an antibody is to bind to a target antigen, e.g., a complement protein that can assist in the lysis of a cellular pathogen. Another activity of the Fc region is to bind to Fc receptors (FcRs) on the surface of immune cells, or so-called effector cells, that have the ability to induce other immune effects. These immune effects include, for example, the release of immune activators, the regulation of antibody production, endocytosis, phagocytosis, and cell killing. In some clinical applications, these reactions are important for the efficacy of the antibody, while in other cases they induce undesirable side effects. One example of an effector-mediated side effect is the release of inflammatory cytokines that cause an acute febrile reaction. Another example is the long-term deletion of antigen-bearing cells.

[0003] Antibody effector functions can be avoided by using antibody fragments that lack the Fc region, such as, for example, Fab, Fab'2, or single-chain antibodies (sFv), but these fragments have shortened half-lives, only one antigen-binding site instead of two (e.g., in the case of Fab antibody fragments and single-chain antibodies (sFv)), and are more difficult to purify. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides, inter alia, Fc variants with significantly reduced ADCC, ADCP and CDC functions. As described herein, the present disclosure is based in part on the identification of novel combinations of mutations that abolish binding to all FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q, while maintaining its ability to bind to FcRn.

[0005] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising amino acid substitutions at positions 235 and 265, wherein the amino acid at position 265 is replaced by Gly, and wherein residues are numbered according to the EU index.

[0006] In some embodiments, the Fc variant further comprises one or more amino acid substitutions at positions 234, 237, 329, 330 or 331. In some embodiments, the Fc variant further comprises an amino acid substitution at position 234. In some embodiments, the Fc variant further comprises an amino acid substitution at positions 234 and 237. In some embodiments, the Fc variant further comprises an amino acid substitution at positions 234, 330 and 331. In some embodiments, the Fc variant further comprises an amino acid substitution at position 234, 237, 330 and 331. In some embodiments, the Fc variant further comprises an amino acid substitution at position 237. In some embodiments, the Fc variant further comprises an amino acid substitution at positions 330 and 331. In some embodiments, the Fc variant further comprises an amino acid substitution at position 329.

[0007] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising amino acid substitutions at positions 234 and 265, wherein the amino acid at position 234 is substituted with Val, and wherein residues are numbered according to the EU index.

[0008] In some embodiments, the Fc variant further comprises amino acid substitutions at positions 235 and 237. In some embodiments, the Fc variant further comprises amino acid substitutions at positions 235, 237, 330 and 331. In some embodiments, the Fc variant further comprises an amino acid substitution at position 235.

[0009] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG4 Fc region, said Fc variant comprising amino acid substitutions at positions F234, L235 and D265, where residues are numbered according to the EU index.

[0010] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234V, L235E and D265G, wherein residues are numbered according to the EU index.

[0011] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, F234V, L235E, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, F234V, L235E, and D265G.

[0012] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234F, L235E and D265G, wherein residues are numbered according to the EU index.

[0013] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234F, L235E, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: L234F, L235E, and D265G.

[0014] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234F, L235E, G237A and D265G, wherein residues are numbered according to the EU index.

[0015] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234F, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: L234F, L235E, G237A, and D265G.

[0016] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234V, L235E, G237A and D265G, wherein residues are numbered according to the EU index.

[0017] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234V, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: L234V, L235E, G237A, and D265G.

[0018] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234F, L235E, D265D, A330S and P331S, where residues are numbered according to the EU index.

[0019] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234F, L235E, D265D, A330S, and P331S. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: L234F, L235E, D265D, A330S, and P331S.

[0020] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234V, L235A, G237A and D265G, wherein residues are numbered according to the EU index.

[0021] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234V, L235A, G237A, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: 234F, L234V, L235A, G237A, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, L234V, L235A, G237A, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, L234V, L235A, G237A, and D265G.

[0022] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234V, L235A, G237A, D265G, A330S and P331S, where residues are numbered according to the EU index.

[0023] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234V, L235A, G237A, D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: L234V, L235A, G237A, D265G, A330S, and P331S.

[0024] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: L235E and D265G, wherein residues are numbered according to the EU index.

[0025] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, L235E, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, L235E, and D265G.

[0026] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: L235E, G237A and D265G, wherein residues are numbered according to the EU index.

[0027] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, L235E, G237A, and D265G.

[0028] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: L235E, G237A and P329G, wherein residues are numbered according to the EU index.

[0029] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, L235E, G237A, and P329G. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, L235E, G237A, and P329G.

[0030] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: L235E, G237A and L328R, wherein residues are numbered according to the EU index.

[0031] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, L235E, G237A, and L328R. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, L235E, G237A, and L328R.

[0032] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: D265G, A330S and P331S, wherein residues are numbered according to the EU index.

[0033] In some embodiments, the Fc variant is an IgG2 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG2 Fc region and comprises the following amino acid substitutions: D265G, A330S, and P331S.

[0034] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 235E, D265G, A330S and P331S, wherein residues are numbered according to the EU index.

[0035] In some embodiments, the Fc variant is an IgG2 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: A235E, D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG2 Fc region and comprises the following amino acid substitutions: A235E, D265G, A330S, and P331S.

[0036] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 235E, D265G and P329G, wherein residues are numbered according to the EU index.

[0037] In some embodiments, the Fc variant is an IgG2 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: A235E, D265G, and P329G. In some embodiments, the Fc variant is an IgG2 Fc region and comprises the following amino acid substitutions: A235E, D265G, and P329G.

[0038] In one aspect, the invention provides, inter alia, a nucleic acid encoding an isolated polypeptide comprising an Fc variant of the invention.

[0039] In one aspect, the invention provides, inter alia, a cell comprising a nucleic acid encoding an isolated polypeptide comprising an Fc variant of the invention.

[0040] In one aspect, the present invention provides a method of treating a disease or disorder, the method comprising administering a therapeutically effective amount of an isolated polypeptide comprising an Fc variant to a subject in need thereof. In some embodiments, the disease or disorder is ANCA-associated vasculitis. In some embodiments, the disease or disorder is C3 nephropathy (C3G). In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the disease or disorder is wet age-related macular degeneration (wAMD). In some embodiments, the disease or disorder is passive Heymann nephritis (NHP). In some embodiments, the disease or disorder is collagen antibody-induced arthritis (CAIA). [Brief description of the drawings]

[0041] [Figure 1A] 1A-D are a series of exemplary graphs showing binding of Ab1 and Ab2 engineered with various Fc variants to FcγRI, FcγRIIa, FcγRIIb, and C1q, respectively, as determined by the assays described in Example 2. [Figure 1B] 1A-D are a series of exemplary graphs showing binding of Ab1 and Ab2 engineered with various Fc variants to FcγRI, FcγRIIa, FcγRIIb, and C1q, respectively, as determined by the assays described in Example 2. [Figure 1C] 1A-D are a series of exemplary graphs showing binding of Ab1 and Ab2 engineered with various Fc variants to FcγRI, FcγRIIa, FcγRIIb, and C1q, respectively, as determined by the assays described in Example 2. [Figure 1D] 1A-D are a series of exemplary graphs showing binding of Ab1 and Ab2 engineered with various Fc variants to FcγRI, FcγRIIa, FcγRIIb, and C1q, respectively, as determined by the assays described in Example 2. [Figure 2A] FIG. 2A is an exemplary bar graph and table showing expression levels of wild-type IgG1, wild-type IgG4, vFc10, and vFc17 engineered Ab1. [Figure 2B] FIG. 2B is an exemplary graph and table showing the Protein A binding properties of wild-type IgG1, wild-type IgG4, and vFc17 engineered Ab1. [Figure 3A] FIG. 3A is an exemplary graph showing binding of wild type IgG1, wild type IgG4, vFc10 or vFc17 engineered Ab1 to FcγRI, demonstrating that the Fc variants of the invention have significantly reduced Fc binding to FcγRI compared to wild type IgG1 and IgG4. [Figure 3B] FIG. 3B is an exemplary graph showing binding of wild type IgG1, wild type IgG4, vFc10 or vFc17 engineered Ab1 to FcγRIIa and FcγRIIb, demonstrating that the Fc variants of the invention have significantly reduced Fc binding to FcγRIIa and FcγRIIb compared to wild type IgG1 and IgG4. [Figure 3C] FIG. 3C is an exemplary graph showing binding of wild-type IgG1, wild-type IgG4, vFc10 or vFc17 engineered Ab1 to FcγRIIIa and FcγRIIIb. [Figure 3D] FIG. 3D is an exemplary graph showing binding of wild-type IgG1, wild-type IgG4, vFc10 or vFc17 engineered Ab1 to C1q. [Figure 4A] FIG. 4A is an exemplary graph showing the fold induction of ADCC by wild-type IgG1, wild-type IgG4, vFc10, or vFc17 engineered Ab1. [Figure 4B] FIG. 4B is an exemplary graph showing fold induction of ADCP by wild-type IgG1, wild-type IgG4, vFc10 or vFc17 engineered Ab1. [Figure 4C] FIG. 4C is an exemplary graph showing fold induction of CDC by wild-type IgG1, wild-type IgG4, vFc10 or vFc17 engineered Ab1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] definition Antibody: As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that binds (immunoreacts with) an antigen. "Binds" or "immunoreacts with" means that the antibody reacts with one or more desired antigenic determinants. Antibodies include antibody fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibodies), single chain, Fab, Fab', F(ab')2 fragments, scFv, and Fab expression libraries. Antibodies may be whole antibodies, or immunoglobulins, or antibody fragments.

[0043] Fc domain: As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0044] Fab arm exchange: The term "Fab arm exchange" refers to the phenomenon that IgG4 antibodies can exchange "half molecules", an activity referred to herein as Fab arm exchange. Particularly in bispecific or biparatopic molecules, this can result in functionally monovalent antibodies with unknown specificity and therefore reduced therapeutic efficacy. To inhibit Fab arm exchange, mutations can be introduced into the Fc domain. It is known that the S228P mutation can prevent IgG4 FAE to undetectable levels both in vitro and in vivo.

[0045] Humanized antibody: The term "humanized antibody" includes non-human (e.g., mouse) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., mouse) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from complementary determining regions (CDRs) are replaced by residues from CDRs of non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capacity (Jones et al., Nature 321:522-525, 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988).

[0046] Monoclonal antibody: The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0047] Multispecific antibody: As used herein, the term "multispecific antibody" refers to a binding molecule, antibody, or antigen-binding fragment thereof, that has the ability to specifically bind to two or more different epitopes on the same or different targets.

[0048] Biparatopic antibody: As used herein, the term "biparatopic antibody" refers to a multispecific antibody that has the ability to bind to two different, non-overlapping epitopes on the same target antigen molecule.

[0049] K i or K d As used herein, the term "K d " as used herein refers to the dissociation constant of a particular antibody-antigen interaction known in the art and will apply to a subject composition as a parameter of the binding affinity of a targeting moiety to its cognate ligand.

[0050] IC50: As used herein, the term "IC50" refers to the concentration required to inhibit half of the maximal biological response of a ligand agonist, typically determined by competitive binding assays.

[0051] EC50: As used herein, the term "EC50" refers to the half-maximal effective concentration. The term EC50 refers to the concentration of a drug, antibody, or toxicant that induces a response halfway between the baseline and maximum after a specified exposure time. More simply, EC50 can be defined as the concentration required to obtain 50% of the desired effect.

[0052] Linker: As used herein, the term "linker" refers to a molecule or group of molecules (such as a monomer or polymer) that connects two molecules and often serves to place the two molecules in a preferred configuration. Several strategies may be used to covalently link molecules to one another. These include, but are not limited to, a polypeptide bond between the N-terminus and C-terminus of a protein or protein domain, linkage via a disulfide bond, and linkage via a chemical cross-linking reagent. In one aspect of this embodiment, the linker is a peptide bond, generated by recombinant technology or peptide synthesis. The linker may contain amino acid residues that provide flexibility. Thus, the linker peptide may contain primarily the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a suitable length to link two molecules such that they adopt the correct conformation relative to one another such that they retain the desired activity. Suitable lengths for this purpose include at least one and no more than 30 amino acid residues. In one embodiment, the linker is about 1-30 amino acids in length. In another embodiment, the linker is about 1-15 amino acids in length. Furthermore, the amino acid residues selected for inclusion in the linker peptide should exhibit properties that do not significantly interfere with the activity of the polypeptide.

[0053] scFv: As used herein, the term "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins linked to a short linker peptide of 10 to about 25 amino acids.

[0054] Fab: As used herein, the term "Fab" refers to an antibody fragment that contains a portion of an intact antibody, including its antigen-binding region or its variable region.

[0055] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism, but in an artificial environment, such as a test tube or reaction vessel, cell culture, etc.

[0056] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0057] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. A subject can be a patient, which refers to a person who visits a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject can be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0058] Dysfunction: As used herein, the term "dysfunction" refers to abnormal function. Dysfunction of a molecule (e.g., a protein) may be caused by an increase or decrease in activity associated with such a molecule. Dysfunction of a molecule may be caused by a defect associated with the molecule itself, or by a defect associated with other molecules that directly or indirectly interact with or regulate the molecule.

[0059] Derivative: As used herein, the term "derivative" when used in relation to an antibody or C5aR1 antibody, refers to a moiety having part of the sequence of the original molecule that retains at least some of the functions and / or properties of the original molecule.

[0060] Identity: As used herein, the term "identity" refers to the relationship between sequences of two or more polypeptide molecules or two or more nucleic acid molecules known in the art, comparing the sequences of these molecules. The relationship is determined by performing. In the art, "identity" also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, and in some cases means multiple nucleotide sequences or multiples. It can be determined by the match between amino acid sequence strings. "Identity" means between gap alignments (if any) and smaller sequences of two or more sequences that are addressed by a particular mathematical model or computer program (i.e., "algorithm"). The percent match of identity is measured.

[0061] Similarity or similarity: As used herein, the term "similarity" is used in the art with respect to related concepts, but in contrast to "identity", "similarity" refers to both identity and conservative substitution matching. If two polypeptide sequences have, for example, 10 identical amino acids out of 20 amino acids, and the rest are all non-conservative substitutions, the percentage of identity and the percentage of similarity are both included at 50%, indicating relatedness. In the same example, if there are five more conservative substitutions, the percentage of identity remains 50%, but the percentage of similarity is 75%. Thus, when there are conservative substitutions, the percentage of similarity between two polypeptides is higher than the percentage of identity between these two polypeptides.

[0062] Treatment: As used herein, the terms "treat", "treatment", or "treating" refer to any method used to partially or completely alleviate, ameliorate, mitigate, inhibit, prevent, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease and / or who show only early signs of the disease, with the intent of reducing the risk of developing pathology associated with the disease.

[0063] Vector: The term "vector" refers to a polynucleotide (usually DNA) used to artificially carry exogenous genetic material into another cell where it can be replicated or expressed. Non-limiting exemplary vectors include plasmids, viral vectors, cosmids, and artificial chromosomes. Such vectors can be derived from a variety of sources, including bacterial and viral sources. A non-limiting exemplary viral source of plasmids is the adeno-associated virus.

[0064] Various aspects of the present disclosure are described in detail in the following sections. The use of the sections is not intended to limit the present disclosure. Each section may be applied to any aspect of the present disclosure. In this application, the use of "or" means "and / or" unless otherwise stated. As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise. Detailed Description

[0065] The Fc region of an antibody can control the antibody cytotoxic activity and affect the serum half-life of the antibody. However, in therapeutic situations, the cytotoxic effector function of an antibody is often undesirable and can cause safety concerns and undesirable side effects by activating the host immune defense. If additional activation is harmful, Fc engineering is necessary to silence the IgG Fc domain so that it cannot bind to the Fc-gamma receptor.

[0066] This disclosure describes a new class of Fc variants that are particularly effective at silencing IgG Fc domains. Notably, the combinatorial Fc mutations of the present invention are novel and reduce binding to Fc gamma receptors and C1q, effectively reducing undesirable ADCC, ADCP, and CDC effector functions. The Fc region and its effector functions

[0067] The Fc region of an antibody interacts with several Fc receptors and ligands, conferring a number of important functional capabilities called effector functions. For IgG, the Fc region includes Ig domains Cγ2 and Cγ3, as well as the N-terminal hinge leading to Cγ2. An important family of Fc receptors for the IgG class are the Fc gamma receptors (FcγRs). These receptors mediate communication between the antibody and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans, this protein family includes FcγRI (CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, incorporated by reference). These receptors typically have an extracellular domain that mediates binding to Fc, a transmembrane region, and an intracellular domain that may mediate some signaling events within the cell. These receptors are expressed on a variety of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and γδ T cells. Formation of the Fc / FcγR complex recruits these effector cells to the site of bound antigen, typically resulting in intracellular signaling events and important subsequent immune responses such as release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy target cells.The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize the bound antibody on target cells and then cause the lysis of target cells is called antibody-dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290, incorporated by reference). The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize the bound antibody on target cells and then cause the phagocytosis of target cells is called antibody-dependent cell-mediated phagocytosis (ADCP). Several structures of the extracellular domains of human FcγRs have been solved, including FcγRIIa (pdb accession code 1H9V) (Sondermann et al., 2001, J Mol Biol 309:737-749) (pdb accession code 1FCG) (Maxwell et al., 1999, Nat Struct Biol 6:437-442), FcγRIIb (pdb accession code 2FCB) (Sondermann et al., 1999, Embo J 18:1095-1103), and FcγRIIIb (pdb accession code 1E4J) (Sondermann et al., 2000, Nature 406:267-273, incorporated by reference). All FcγRs bind to the same region on Fc, at the N-terminus of the Cγ2 domain and preceding hinge.This interaction has been structurally well characterized (Sondermann et al., 2001, J Mol Biol 309:737-749, incorporated by reference), with several structures of human Fc bound to the extracellular domain of human FcγRIIIb being solved (pdb accession code 1E4K) (Sondermann et al., 2000, Nature 406:267-273) (pdb accession codes 1IIS and 1IIX) (Radaev et al., 2001, J Biol Chem 276:16469-16477, incorporated by reference), as well as the structure of the human IgE Fd / FcεRIa complex (pdb accession code 1F6A) (Garman et al., 2000, Nature 406:259-266, incorporated by reference).

[0068] An overlapping but distinct site on Fc serves as an interface for the complement protein C1q. Just as Fc / FcγR binding mediates ADCC, Fc / C1q binding mediates complement-dependent cytotoxicity (CDC). C1q forms a complex with the serine proteases C1r and C1s to form the C1 complex. C1q can bind six antibodies, but binding to two IgGs is sufficient to activate the complement cascade. Similar to the Fc interaction with FcγR, different IgG subclasses have different affinities for C1q, with IgG1 and IgG3 typically binding substantially better to FcγR than IgG2 and IgG4.

[0069] The site on Fc between the Cγ2 and Cγ3 domains mediates interaction with the neonatal receptor FcRn, whose binding recycles internalized antibodies from endosomes back into the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766, incorporated by reference). This process, coupled with exclusion of renal filtration due to the large size of the full-length molecule, results in favorable antibody serum half-lives in the range of 1-3 weeks. Binding of Fc to FcRn also plays an important role in antibody transport. The binding site of FcRn on Fc is also the site to which bacterial proteins A and G bind. Tight binding by these proteins is typically exploited as a means to purify antibodies by employing protein A or protein G affinity chromatography during protein purification. Hence, the fidelity of this region on Fc is important for both the clinical properties of antibodies and their purification. Available structures of the rat Fc / FcRn complex (Martin et al., 2001, Mol Cell 7:867-877, incorporated by reference), as well as complexes of Fc with proteins A and G (Deisenhofer, 1981, Biochemistry 20:2361-2370; Sauer-Eriksson et al., 1995, Structure 3:265-278; Tashiro et al., 1995, Curr Opin Struct Biol 5:471-481, incorporated by reference), provide insight into the interactions of Fc with these proteins.

[0070] The present invention is directed to an optimized Fc variant that is useful in various situations.As outlined above, current antibody therapy suffers from various problems.The present invention provides a promising means for enhancing the therapeutic efficacy of antibody through the abolishment of the ability to mediate cytotoxic effector functions such as ADCC, ADCP and CDC. Fc variants of the present invention

[0071] The Fc variants of the present invention may find use in various Fc polypeptides. The Fc polypeptides comprising the Fc variants of the present invention are referred to herein as "Fc polypeptides of the present invention". The Fc polypeptides of the present invention include polypeptides comprising the Fc variants of the present invention in the context of a larger polypeptide, such as an antibody or Fc fusion. That is, the Fc polypeptides of the present invention include antibodies and Fc fusions comprising the Fc variants of the present invention. As used herein, "antibodies of the present invention" refers to antibodies comprising the Fc variants of the present invention. As used herein, "Fc fusions of the present invention" refers to Fc fusions comprising the Fc variants of the present invention. The Fc polypeptides of the present invention also include polypeptides that contain little or no additional polypeptide sequence other than the Fc region, referred to as isolated Fc. The Fc polypeptides of the present invention also include fragments of the Fc region. As described below, any of the aforementioned Fc polypeptides of the present invention may be fused to one or more fusion or conjugate partners to provide desired functional properties.

[0072] The parent Fc polypeptides described herein may be derived from a wide range of sources and may be substantially encoded by one or more Fc genes from any organism, including but not limited to rodents, including but not limited to humans, mice and rats, lagomorphs such as rabbits and hares, camelids such as camels, llamas and dromedaries, and non-human primates, including but not limited to prosimians, platyrrhines (New World monkeys), Cercopithecinae (Old World monkeys), and Hominoidea, including gibbons, lesser apes and greater apes, with humans being the most preferred. The parent Fc polypeptides of the invention may be substantially encoded by immunoglobulin genes belonging to any of the antibody classes, including but not limited to sequences belonging to the IgG (including human subclasses IgG1, IgG2, IgG3, or IgG4), IgA (including human subclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM classes of antibodies. The parent Fc polypeptides of the invention include sequences belonging to the human IgG class of antibodies. For example, the parent Fc polypeptide may be a parent antibody, such as a human IgG1 antibody, a human IgA antibody, or a mouse IgG2a or IgG2b antibody. The parent antibody may be non-human, chimeric, humanized, or fully human, as described in detail below. The parent Fc polypeptide may be modified or engineered in some way, for example, the parent antibody may be affinity matured or have engineered glycoforms, all of which are described more fully below. Alternatively, the parent Fc polypeptide may be an Fc fusion, for example, an Fc fusion in which the fusion partner targets a cell surface receptor. Alternatively, the parent Fc polypeptide may be an isolated Fc region that contains little or no other polypeptide sequence outside the Fc region. The parent Fc polypeptide may be a naturally occurring Fc region or an existing engineered variant of an Fc polypeptide. What is important is that the parent Fc polypeptide contains an Fc region, which can then be mutated to generate an Fc variant.

[0073] The Fc variants of the present invention may find use in a wide range of products. In one embodiment, the Fc variants of the present invention are therapeutic, diagnostic, or research reagents, preferably therapeutic. Alternatively, the Fc variants of the present invention may be used in agricultural or industrial applications. The antibodies of the present invention may find use in antibody compositions that are monoclonal or polyclonal. The Fc variants of the present invention may be agonistic, antagonistic, neutralizing, inhibiting, or stimulating. In a preferred embodiment, the Fc variants of the present invention are used to kill target cells bearing the target antigen, e.g., cancer cells. In an alternative embodiment, the Fc variants of the present invention are used to block, antagonize, or stimulate the target antigen. In an alternative preferred embodiment, the Fc variants of the present invention are used to block, antagonize, or stimulate the target antigen and kill target cells bearing the target antigen. Optimized Properties

[0074] The present invention provides Fc variants optimized for several therapeutically relevant properties. The Fc variants comprise one or more amino acid modifications relative to a parent Fc polypeptide, said amino acid modification(s) providing one or more optimized properties. The Fc variants of the present invention differ in amino acid sequence from their parent Fc polypeptide by at least one amino acid modification. Thus, the Fc variants of the present invention have at least one amino acid modification compared to the parent. Alternatively, the Fc variants of the present invention may have multiple amino acid modifications compared to the parent, for example, about 1-50 amino acid modifications, about 1-10 amino acid modifications, or about 1-5 amino acid modifications compared to the parent. Thus, the sequence of the Fc variant and the sequence of the parent Fc polypeptide are substantially homologous. For example, the variant Fc variant sequence herein will have about 80% homology, preferably at least about 90% homology, most preferably at least about 95% homology with the parent Fc variant sequence.

[0075] The Fc variants of the present invention may be optimized for various properties. Fc variants engineered or predicted to exhibit one or more optimized properties are referred to herein as "optimized Fc variants". Properties that may be optimized include, but are not limited to, enhanced or reduced affinity to FcγR. In some embodiments, the Fc variants of the present invention are optimized to have reduced or ablated affinity to human FcγRs, including, but not limited to, FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. These embodiments are expected to provide Fc polypeptides with enhanced therapeutic properties in humans, such as reduced effector function and reduced toxicity. In other embodiments, the Fc variants of the present invention provide enhanced affinity to one or more FcγRs, but reduced affinity to one or more other FcγRs. For example, the Fc variants of the present invention may have enhanced binding to FcγRIIIa, but reduced binding to FcγRIIb. Alternatively, an Fc variant of the invention may have enhanced binding to FcγRIIa and FcγRI, but reduced binding to FcγRIIb, hi yet another embodiment, an Fc variant of the invention may have enhanced affinity for FcγRIIb, but reduced affinity for one or more activating FcγR.

[0076] In some embodiments, the Fc variants have reduced or truncated affinity for FcγRI. In some embodiments, the Fc variants have reduced or truncated affinity for FcγRIIa. In some embodiments, the Fc variants have reduced or truncated affinity for FcγRIIb. In some embodiments, the Fc variants have reduced or truncated affinity for FcγRIIc. In some embodiments, the Fc variants have reduced or truncated affinity for FcγRIIIa. In some embodiments, the Fc variants have reduced or truncated affinity for FcγRIIIb. In some embodiments, the Fc variants have reduced or truncated affinity for C1q. In some embodiments, the Fc variants have enhanced affinity for FcRn. In some embodiments, the Fc variants maintain affinity for FcRn. In some embodiments, the Fc variants have reduced or truncated affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q, hi some embodiments, the Fc variants have reduced or truncated affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q and retain binding to FcRn.

[0077] The Fc variants of the invention may also be optimized for enhanced function and / or solution properties in the aglycosylated form. In a preferred embodiment, the aglycosylated Fc variants of the invention bind Fc ligands with reduced affinity than the aglycosylated form of the parent Fc variant. Said Fc ligands may be from any source, including but not limited to FcγR, C1q, FcRn, and proteins A and G, including but not limited to human, mouse, rat, rabbit, or monkey, preferably human. Alternatively, in a preferred embodiment, the Fc variants are optimized to be more stable and / or soluble than the aglycosylated form of the parent Fc variant. Engineered Fc mutations

[0078] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG1 Fc region, said Fc variant comprising the amino acid substitutions L234F / L235E / D265G, wherein residues are numbered according to the EU index.

[0079] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG1 Fc region, said Fc variant comprising the amino acid substitutions L234F / L235E / G237A / D265G, where residues are numbered according to the EU index.

[0080] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG1 Fc region, said Fc variant comprising the amino acid substitutions L234V / L235E / G237A / D265G, wherein residues are numbered according to the EU index.

[0081] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG1 Fc region, said Fc variant comprising the amino acid substitutions L234F / L235E / D265G / A330S / P331S, wherein residues are numbered according to the EU index.

[0082] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG1 Fc region, said Fc variant comprising the amino acid substitutions L234V / L235A / G237A / D265G, wherein residues are numbered according to the EU index.

[0083] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG1 Fc region, said Fc variant comprising the amino acid substitutions L234V / L235A / G237A / D265G / A330S / P331S, where residues are numbered according to the EU index.

[0084] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG1 Fc region, said Fc variant comprising the amino acid substitution D265G, wherein residues are numbered according to the EU index.

[0085] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG4 Fc region, said Fc variant comprising the amino acid substitutions L235E / D265G / S228P, wherein residues are numbered according to the EU index.

[0086] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG4 Fc region, said Fc variant comprising the amino acid substitutions F234V / L235E / D265G / S228P, wherein residues are numbered according to the EU index.

[0087] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG4 Fc region, said Fc variant comprising the amino acid substitutions F234V / L235A / G237A / D265G / S228P, where residues are numbered according to the EU index.

[0088] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG4 Fc region, said Fc variant comprising the amino acid substitutions L235E / G237A / D265G / S228P, wherein residues are numbered according to the EU index.

[0089] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG4 Fc region, said Fc variant comprising the amino acid substitutions L235E / G237A / P329G / S228P, wherein residues are numbered according to the EU index.

[0090] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG4 Fc region, said Fc variant comprising the amino acid substitutions L235E / G237A / L328R / S228P, wherein residues are numbered according to the EU index.

[0091] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG2 Fc region, said Fc variant comprising the amino acid substitutions D265G / A330S / P331S, wherein residues are numbered according to the EU index.

[0092] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG2 Fc region, said Fc variant comprising the amino acid substitutions A235E / D265G / A330S / P331S, where residues are numbered according to the EU index.

[0093] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG2 Fc region, said Fc variant comprising the amino acid substitutions A235E / D265G / P329G, where residues are numbered according to the EU index.

[0094] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising amino acid substitutions at positions 235 and 265, wherein the amino acid at position 265 is replaced by Gly, and wherein residues are numbered according to the EU index.

[0095] In some embodiments, the Fc variant further comprises one or more amino acid substitutions at positions 234, 237, 329, 330 or 331. In some embodiments, the Fc variant further comprises an amino acid substitution at position 234. In some embodiments, the Fc variant further comprises an amino acid substitution at positions 234 and 237. In some embodiments, the Fc variant further comprises an amino acid substitution at positions 234, 330 and 331. In some embodiments, the Fc variant further comprises an amino acid substitution at position 234, 237, 330 and 331. In some embodiments, the Fc variant further comprises an amino acid substitution at position 237. In some embodiments, the Fc variant further comprises an amino acid substitution at positions 330 and 331. In some embodiments, the Fc variant further comprises an amino acid substitution at position 329.

[0096] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising amino acid substitutions at positions 234 and 265, wherein the amino acid at position 234 is substituted with Val, and wherein residues are numbered according to the EU index.

[0097] In some embodiments, the Fc variant further comprises amino acid substitutions at positions 235 and 237. In some embodiments, the Fc variant further comprises amino acid substitutions at positions 235, 237, 330 and 331. In some embodiments, the Fc variant further comprises an amino acid substitution at position 235.

[0098] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG4 Fc region, said Fc variant comprising amino acid substitutions at positions F234, L235 and D265, where residues are numbered according to the EU index.

[0099] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234V, L235E and D265G, wherein residues are numbered according to the EU index.

[0100] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, F234V, L235E, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, F234V, L235E, and D265G.

[0101] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234F, L235E and D265G, wherein residues are numbered according to the EU index.

[0102] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234F, L235E, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: L234F, L235E, and D265G.

[0103] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234F, L235E, G237A and D265G, wherein residues are numbered according to the EU index.

[0104] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234F, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: L234F, L235E, G237A, and D265G.

[0105] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234V, L235E, G237A and D265G, wherein residues are numbered according to the EU index.

[0106] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234V, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: L234V, L235E, G237A, and D265G.

[0107] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234F, L235E, D265D, A330S and P331S, where residues are numbered according to the EU index.

[0108] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234F, L235E, D265D, A330S, and P331S. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: L234F, L235E, D265D, A330S, and P331S.

[0109] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234V, L235A, G237A and D265G, wherein residues are numbered according to the EU index.

[0110] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234V, L235A, G237A, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: 234F, L234V, L235A, G237A, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, L234V, L235A, G237A, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, L234V, L235A, G237A, and D265G.

[0111] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234V, L235A, G237A, D265G, A330S and P331S, where residues are numbered according to the EU index.

[0112] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: L234V, L235A, G237A, D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG1 Fc region and comprises the following amino acid substitutions: L234V, L235A, G237A, D265G, A330S, and P331S.

[0113] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: L235E and D265G, wherein residues are numbered according to the EU index.

[0114] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, L235E, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, L235E, and D265G.

[0115] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: L235E, G237A and D265G, wherein residues are numbered according to the EU index.

[0116] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, L235E, G237A, and D265G.

[0117] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: L235E, G237A and P329G, wherein residues are numbered according to the EU index.

[0118] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, L235E, G237A, and P329G. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, L235E, G237A, and P329G.

[0119] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: L235E, G237A and L328R, wherein residues are numbered according to the EU index.

[0120] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: S228P, L235E, G237A, and L328R. In some embodiments, the Fc variant is an IgG4 Fc region and comprises the following amino acid substitutions: S228P, L235E, G237A, and L328R.

[0121] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: D265G, A330S and P331S, wherein residues are numbered according to the EU index.

[0122] In some embodiments, the Fc variant is an IgG2 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG2 Fc region and comprises the following amino acid substitutions: D265G, A330S, and P331S.

[0123] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 235E, D265G, A330S and P331S, wherein residues are numbered according to the EU index.

[0124] In some embodiments, the Fc variant is an IgG2 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: A235E, D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG2 Fc region and comprises the following amino acid substitutions: A235E, D265G, A330S, and P331S.

[0125] In one aspect, the invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 235E, D265G and P329G, wherein residues are numbered according to the EU index. In some embodiments, the Fc variant is an IgG2 Fc region. In some embodiments, the Fc variant comprises the following amino acid substitutions: A235E, D265G, and P329G. In some embodiments, the Fc variant is an IgG2 Fc region and comprises the following amino acid substitutions: A235E, D265G, and P329G. Designing antibodies with Fc variants

[0126] The Fc variants of the present invention may be antibodies, and are referred to herein as "antibodies of the present invention". Antibodies of the present invention may comprise immunoglobulin sequences substantially encoded by immunoglobulin genes belonging to any of the antibody classes, including but not limited to the IgG (including human subclasses IgG1, IgG2, IgG3, or IgG4), IgA (including human subclasses IgA1 and IgA2), IgD, IgE, IgG, and IgM classes of antibodies. Most preferably, antibodies of the present invention comprise sequences belonging to the human IgG class of antibodies. Antibodies of the present invention may be non-human, chimeric, humanized, or fully human. As will be appreciated by those skilled in the art, these different types of antibodies reflect degrees of "humanness", or potential levels of human immunogenicity. For an explanation of these concepts, see Clark et al., 2000 and references cited therein (Clark, 2000, Immunol Today 21:397-402, incorporated by reference). A chimeric antibody comprises a variable region of a non-human antibody, e.g., a VH domain and a VL domain of mouse or rat origin, operably linked to a constant region of a human antibody. The non-human variable region may be derived from any of the organisms mentioned above, preferably a mammal, most preferably a rodent or a primate. In one embodiment, the antibody of the invention comprises a monkey variable domain, e.g., as described in Newman et al., 1992, Biotechnology 10:1455-1460, U.S. Pat. No. 5,658,570, and U.S. Pat. No. 5,750,105 (incorporated by reference). In a preferred embodiment, the variable region is derived from a non-human source, but its immunogenicity is reduced using protein engineering. In a preferred embodiment, the antibody of the invention is humanized (Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), incorporated by reference).As used herein, a "humanized" antibody refers to an antibody that comprises a human framework region (FR) and one or more complementarity determining regions (CDRs) derived from a non-human (usually mouse or rat) antibody. The non-human antibody that provides the CDRs is called the "donor" and the human immunoglobulin that provides the framework is called the "acceptor". Humanization mainly relies on the grafting of donor CDRs into the acceptor (human) VL and VH frameworks (Winter U.S. Patent No. 5,225,539, incorporated by reference). This strategy is called "CDR grafting". "Backmutation" of selected acceptor framework residues to the corresponding donor residues is often required to restore affinity lost in the initial grafted construct (U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213, incorporated by reference). Many other methods for humanization are known in the art (Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), incorporated by reference), and any of these methods may find use in the present invention to engineer Fc variants to reduce immunogenicity. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus typically will comprise a human Fc region.

[0127] In some embodiments, the antibody comprising the Fc variant is a monospecific antibody. In some embodiments, the antibody comprising the Fc variant is a multispecific antibody. In some embodiments, the antibody comprising the Fc variant is a bispecific antibody. In some embodiments, the antibody comprising the Fc variant is a multiparatopic antibody, e.g., comprising a plurality of immunoglobulin variable region sequences, a first immunoglobulin variable region sequence of the plurality has binding specificity for a first epitope, and a second immunoglobulin variable region sequence of the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). A bispecific or biparatopic antibody has specificity for no more than two antigens or epitopes. A bispecific or biparatopic antibody molecule is typically characterized by a first immunoglobulin variable region sequence having binding specificity for a first epitope, and a second immunoglobulin variable region sequence having binding specificity for a second epitope. In one embodiment, a bispecific or biparatopic antibody molecule comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the biparatopic antibody presented in the present disclosure comprises a Fab-Fc and a single chain variable fragment (scFv), and the Fc is linked to the scFv via a linker.

[0128] In some embodiments, the biparatopic antibody is a bispecific antibody with a two-arm single chain Fab-Fc design and includes "knobs-in-holes" (KiH) mutations in the CH3 domain to assemble two half antibodies (a common Fc heterodimer and unique VH-CH and VL-CL domains). In some embodiments, the KiH mutations include that a T366Y mutation in one CH3 domain can be used to create a knob and a Y407T mutation in the other CH3 domain can be used to create a hole. In some embodiments, a F405A mutation in one CH3 domain can be used to create a knob and a T394W mutation in the other CH3 domain can be used to create a hole. In some embodiments, a T366W mutation in one CH3 domain can be used to create a knob and a Y407A mutation in the other CH3 domain can be used to create a hole. In some embodiments, biparatopic scFv-Fc molecules can be generated using knobs-and-holes technology (e.g., including hole mutations: Y349C, T366S, L368A, Y407V; knob mutations: S354C, T366W).

[0129] In some embodiments, the biparatopic antibody comprises an antibody format described in Table 2. Table 2. Biparatopic antibody formats [Table 2]

[0130] In one embodiment, a biparatopic antibody molecule comprises two heavy chain variable regions and two light chain variable regions. In one embodiment, the anti-C5aR1 antibody molecule comprises a Fab, F(ab')2, Fv, Fd, or single chain Fv fragment (scFv).

[0131] In some embodiments, the Fc domain used in the present application comprises or is derived from an IgG, IgM, IgE, Fc portion. In addition to the KiH mutations described above, the Fc domain comprises an S228P mutation. In some embodiments, S228P enhances the homogeneity of the antibody. In some embodiments, the Fc domain comprises or is derived from an IgG Fc domain. In some embodiments, the IgG Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the Fc domain is derived from or comprises an IgG4 Fc domain. In some embodiments, the Fc domain is derived from or comprises an IgG4 Fc domain with an S228P mutation. In some embodiments, the Fc domain is derived from or comprises an IgG1 Fc domain. In some embodiments, the Fc domain is derived from or comprises an IgG1 Fc domain with an S228P mutation.

[0132] In some exemplary embodiments, monospecific and biparatopic antibodies can be modified or mutated to enhance the thermal stability of the antibody. The thermal stability of an antibody can be evaluated by determining the aggregation onset temperature. One way to increase the stability of an antibody is to increase the thermal transition midpoint (Tm) measured by differential scanning calorimetry (DSC). In general, protein Tm correlates with its stability and inversely correlates with its susceptibility to unfolding and denaturation in solution, as well as the degradation process, which depends on the unfolding tendency of the protein. Several studies have found a correlation between the ranking of formulation physical stability as measured by DSC as thermal stability and physical stability measured by other methods (Maa et al. (1996) Int. J. Pharm. 140:155-68; Remmele et al. (1997) Pharm. Res. 15:200-8; Gupta et al. (2003) AAPS Pharm Sci. 5E8:2003; Bedu-Addo et al. (2004) Pharm. Res. 21:1353-61; Zhang et al. (2004) J. Pharm. Sci. 93:3076-89). Formulation studies suggest that the Fab Tm influences the long-term physical stability of the corresponding mAb.

[0133] In some exemplary embodiments, the strategic introduction of disulfide bonds can stabilize monomeric and multi-subunit proteins and can play a role in enhancing the thermal stability of antibodies.

[0134] In some exemplary embodiments, strategic introduction of π-stacking interactions with aromatic amino acids (AA), such as tryptophan (TRP), tyrosine (TYR), phenylalanine (PHE), and histidine (HIS), plays a role in enhancing the thermal stability of antibodies.

[0135] In some embodiments, the strategic introduction of salt bridges between amino acid side chains with opposite positive or negative overall electronic charge, i.e., Glu or Asp versus Arg or Lys (at neutral pH), enhances the stability of proteins, particularly antibodies.

[0136] In some exemplary embodiments, the monospecific or biparatopic antibody comprises one or more thermostability-enhancing modifications, hi some embodiments, the thermostability-enhancing modification is the introduction of a cysteine ​​residue.

[0137] In some embodiments, the Tm of an exemplary biparatopic antibody is greater than 65° C. In some embodiments, the Tm of an exemplary biparatopic antibody is greater than 60° C. In some embodiments, the Tm of an exemplary biparatopic antibody is greater than 55° C. In some embodiments, the Tm of an exemplary biparatopic antibody is greater than 50° C.

[0138] In some embodiments, a peptide linker is used to link the scFv antibody or single chain antibody to the Fc domain of the Fab. Some examples of suitable linkers include a single glycine (G) residue, a diglycine peptide (GG), a tripeptide (GGG), a peptide with four glycine residues (GGGG), a peptide with five glycine residues (GGGGG), a peptide with six glycine residues (GGGGGG), a peptide with seven glycine residues (GGGGGGG), a peptide with eight glycine residues (GGGGGGGG). Other combinations of amino acid residues may be used, such as the peptide GGGGS, the peptide GGGGSGGGGS, the peptide GGGGSGGGGSGGGGGS, the peptide GGGGSGGGGSGGGGSGGGGS, the peptide GGSGSSGSGG, QRIEG, and the peptide GQPKAAP. Other suitable linkers include a single Ser residue, and a Val residue, the dipeptides RTQP, SS, TK, SL, TKGPS, TVAAP, QPKAA. The above-listed examples are not intended to limit the scope of the present disclosure in any way, and it has been shown that linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline are suitable for binding proteins.For further description of linker sequences, see, for example, WO2012135345.

[0139] The identity and sequence of the amino acid residues in the linker can vary depending on the type of secondary structure elements that need to be achieved within the linker.For example, glycine, serine, and alanine are best for linkers with maximum flexibility.Some combinations of glycine, proline, threonine, and serine are useful when a more rigid and extended linker is required.Depending on the desired properties, any amino acid residue can be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as needed. target

[0140] Virtually any antigen may be targeted by the Fc variants of the present invention, including but not limited to proteins, subunits, domains, motifs, and / or epitopes belonging to the following list of targets: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (0P-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C3b, C4, C5, C5a, C5a receptor 1 (C5aR1) C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI,カテプシンD, カテプシンE, カテプシンH, カテプシンL, カテプシンO、カテプシンS、カテプシンV、カテプシンX / Z / P、CBL、 CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CC L28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5 , CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67タンパクquality), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, cytomegalovirus toxin, cytomegalovirus toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5,CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(1-3)-IGF-1 (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZ D9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIlb / 111a), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, Tegrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpi, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, Meta protease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mud), MUC18, Müllerian inhibitory factor, Mug, MuSK, NAIP, NAP, NCAD , N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF , PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV)F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-beta R1 (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymic Ck-1, Thyroid Stimulating Hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFα, TNF-R1, TNF-RII, TNFRSF10A (TRAIL R1Apo-2, DR4), TNFRSF10B(TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C(TRAIL R3DcRl, LIT, TRID), TNFRSF10D(TRAIL R4 DcR2, TRUNDD), TNFRSF11A(RANK ODF R, TRANCE R), TNFRSF11B(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF R1CD120a, p55~60), TNFRSF1B(TNF RIICD120b, p75~80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB) CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2TNFRH2), TNFRST23(DcTRAIL R1TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (Light HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1),VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigens, VLA, VLA-1, VLA-4, VNR integrins, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and hormone and growth factor receptors.

[0141] In some embodiments, an antibody comprising an Fc variant specifically binds to a complement factor. In some embodiments, an antibody comprising an Fc variant specifically binds to SARS-CoV-2. In some embodiments, an antibody comprising an Fc variant specifically binds to amyloid beta protofibrils. In some embodiments, an antibody comprising an Fc variant specifically binds to LAG-3. In some embodiments, an antibody comprising an Fc variant specifically binds to CD3. In some embodiments, an antibody comprising an Fc variant specifically binds to VEGF. In some embodiments, an antibody comprising an Fc variant specifically binds to Ang-2. In some embodiments, an antibody comprising an Fc variant specifically binds to PD-1. In some embodiments, an antibody comprising an Fc variant specifically binds to EGFR. In some embodiments, an antibody comprising an Fc variant specifically binds to IFNAR1. In some embodiments, an antibody comprising an Fc variant specifically binds to CD19. In some embodiments, an antibody comprising an Fc variant specifically binds to IL-17A. In some embodiments, an antibody comprising an Fc variant specifically binds to IL-17B. In some embodiments, an antibody comprising an Fc variant specifically binds to IL-13. In some embodiments, an antibody comprising an Fc variant specifically binds to angiopoietin-like 3. In some embodiments, an antibody comprising an Fc variant specifically binds to nerve growth factor. In some embodiments, an antibody comprising an Fc variant specifically binds to Ebola virus. In some embodiments, an antibody comprising an Fc variant specifically binds to HER2. In some embodiments, an antibody comprising an Fc variant specifically binds to GD2. In some embodiments, an antibody comprising an Fc variant specifically binds to BCMA. In some embodiments, an antibody comprising an Fc variant specifically binds to IL-6R. In some embodiments, an antibody comprising an Fc variant specifically binds to TROP-2. In some embodiments, an antibody comprising an Fc variant specifically binds to IGF-1R. In some embodiments, an antibody comprising an Fc variant specifically binds to CD38. In some embodiments, an antibody comprising an Fc variant specifically binds to Nectin-4.In some embodiments, an antibody comprising an Fc variant specifically binds to P-selectin. In some embodiments, an antibody comprising an Fc variant specifically binds to CD79b. In some embodiments, an antibody comprising an Fc variant specifically binds to sclerostin. In some embodiments, an antibody comprising an Fc variant specifically binds to IFN gamma. In some embodiments, an antibody comprising an Fc variant specifically binds to CCR4. In some embodiments, an antibody comprising an Fc variant specifically binds to a CGRP receptor. In some embodiments, an antibody comprising an Fc variant specifically binds to a receptor for a complement factor. In some embodiments, an antibody comprising an Fc variant specifically binds to C5a receptor 1. In some embodiments, an antibody comprising an Fc variant specifically binds to C5a. In some embodiments, an antibody comprising an Fc variant specifically binds to C3. In some embodiments, an antibody comprising an Fc variant specifically binds to C3a. In some embodiments, an antibody comprising an Fc variant specifically binds to C3b. In some embodiments, an antibody comprising an Fc variant specifically binds to the C3 receptor. In some embodiments, an antibody comprising an Fc variant specifically binds C10. vector

[0142] Further provided herein is a vector comprising a nucleotide sequence encoding an isolated polypeptide comprising an Fc variant, as described herein.

[0143] In one embodiment, the vector comprises a nucleic acid as described herein. For example, the vector may comprise a first and a second nucleic acid encoding a heavy chain variable region and a light chain variable region, respectively, of an antibody molecule selected from one or more of the antibody molecules disclosed herein.

[0144] In certain embodiments, the vector comprises a nucleotide sequence encoding an Fc variant region, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and / or a sequence having one or more substitutions, e.g., conservative substitutions).

[0145] Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). Numerous vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV or MOMLV) or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, Eastern equine encephalitis virus, and flavivirus.

[0146] Furthermore, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can provide, for example, prototropy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or can be introduced into the same cell by cotransformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0147] When the expression vector or DNA sequence containing construct is prepared for expression, the expression vector can be transfected or introduced into suitable host cells.To achieve this, various techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques.In the case of protoplast fusion, cells are grown in culture medium and screened for suitable activity.

[0148] Methods and conditions for culturing the resulting transfected cells and recovering the antibody molecules produced are known to those skilled in the art and may be modified or optimized based on the present description, depending on the particular expression vector and mammalian host cell used. therapeutic use

[0149] Isolated polypeptides comprising the Fc variants (e.g., antibodies) of the invention can be used for the treatment of a variety of diseases, including, but not limited to, melanoma, wAMD, DME, esophageal squamous cell carcinoma, type 1 diabetes, non-small cell lung cancer, asthma, CNS, cervical cancer, cold agglutinin disease, systemic lupus erythematosus, psoriasis, atopic dermatitis, endometrial cancer, bladder cancer, Ebola infection, HER2+ breast cancer, multiple myeloma, breast cancer, thyroid eye disease, sickle cell disease, HIV infection, and the like. Infection, gastric cancer, anthrax infection, bone loss, Crohn's disease, ANCA-associated vasculitis, lupus, rheumatoid arthritis, inflammatory bowel disease, C3 glomerulopathy (C3G), C3 glomerulonephritis (C3GN), membranoproliferative glomerulonephritis type II (DDD), hidradenitis suppurativa (HS), atypical hemolytic uremic syndrome, lupus nephritis, IgA nephropathy, myasthenia gravis, macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, neuropathic pain, COVID-19 infection, allergic asthma, chronic obstructive pulmonary disease, Bullous pemphigoid, pyoderma gangrenosum, psoriasis, paroxysmal nocturnal hemoglobinuria with extravascular hemolysis, acute kidney injury (AKI), chronic kidney disease (CKD), geographic atrophy (GA), autoimmune hemolytic anemia (AIHA), age-related macular degeneration (AMD).

[0150] In some embodiments, the present invention provides a method for treating a disease or disorder, comprising administering a therapeutically effective amount of an isolated polypeptide comprising an Fc variant to a subject in need thereof. In some embodiments, the disease or disorder is ANCA-associated vasculitis. In some embodiments, the disease or disorder is C3 nephropathy (C3G).

[0151] In some embodiments, the Fc variants of the invention may be engineered into antibodies that specifically bind to an antigen. In some embodiments, the Fc variants of the invention may be engineered into the antibodies listed in Table 3. Table 3. Commercially available therapeutic antibodies [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] EXAMPLES

[0152] Other features, objects, and advantages of the present disclosure will become apparent from the following examples. However, it should be understood that the examples, while showing embodiments of the present disclosure, are given by way of illustration only and are not limiting. Various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art from the examples. Example 1. Generation of new Fc mutations

[0153] Fc gamma receptors recognize and bind the Fc region of IgG antibodies. This binding regulates immune responses by triggering effector functions. In some disease indications, it may be beneficial for therapeutic antibodies to interact with Fc gamma receptors and enhance innate Fc gamma receptor activation, while in other indications it may be detrimental. If additional activation is detrimental, Fc engineering is necessary to silence the IgG Fc domain so that it cannot bind to the Fc-gamma receptor. In this example, new Fc variants were engineered that are particularly effective at silencing the IgG Fc domain. Notably, the Fc mutation combinations of the present invention are new and effective at abolishing affinity for Fc gamma receptors and C1q. The Fc variants of the present invention are shown in Table 1. Table 1. Novel Fc variants [Table 1] Example 2. Fc variants showed significantly reduced Fcγ receptor and C1q binding

[0154] This example confirms that the new Fc variants shown in Table 1 successfully abrogate Fc gamma receptor and C1q binding. The Fc variants were engineered into antibodies "Ab1" and "Ab2," each of which contains a distinct variable domain and targets a different antigen. Methods for determining Fc gamma receptor and C1q binding

[0155] This protocol describes methods that can be used to determine the extent to which Fc-silenced antibodies bind to Fc gamma receptors or C1q using biolayer interferometry (BLI) or ELISA on an Octet Red 384 system. FcγRI, FcγRIIa H167, FcγRIIb, FcγRIIIa V176, and FcγRIIIb binding were assessed using NiNTA on either HIS1K biosensors. In both procedures, Fc gamma receptors were first loaded onto the biosensor, which was then blocked with either casein or BSA to prevent nonspecific binding of the antibody to any unbound ligands on the biosensor. The biosensor was then introduced to the antibody and then returned to blocking buffer for a short dissociation step. The resulting binding sensorgrams were used to qualitatively assess the strength of each measured interaction, while the maximum equilibrium binding response was used as a quantitative readout, which was proportional to the binding affinity. Binding to C1q was measured in a similar manner, according to methods known in the art. result

[0156] As shown in Figure 1A-C, all Fc variants significantly reduced binding to FcγRI, FcγRIIa, and FcγRIIb compared to their corresponding wild-type IgG isotypes. Notably, for both Ab1 and Ab2, none of the Fc regions of vFc07-vFc12 and vFc16-vFc24 bound to FcγRI, FcγRIIa, and FcγRIIb. It was also notable that the single mutation D265G was able to significantly reduce FcγRI, FcγRIIa, and FcγRIIb binding (compare vFc13 and IgG1 WT in Figure 1A-C). In addition, all Fc variants, regardless of the variable region (Ab1 or Ab2), significantly reduced binding to C1q compared to their corresponding wild-type IgG isotypes (Figure 1D). Example 3. Further validation of two Fc variants in reducing Fcγ receptor and C1q binding

[0157] This example verifies that the Fc variants of the present invention successfully abolished Fc gamma receptor and C1q binding. In this particular example, two Fc variants, vFc10 and vFc17, were used in the experiment. vFc10 contains L234F / L235E / D265G / A330S / P331S mutations in the IgG1 isotype, and vFc17 contains F234V / L235E / D265G mutations in the IgG4-S228P isotype. (The S228P mutation reduces Fab arm exchange by stabilizing disulfides in the core hinge of the IgG molecule.) These two Fc variants were each engineered into antibody "Ab1". The engineered antibodies were tested for binding to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q by the methods described in Example 2.

[0158] A major drawback of antibodies is their stringent production requirements (Garber, 2001, Nat Biotechnol 19:184-185; Dove, 2002, Nat Biotechnol 20:777-779, incorporated by reference). Therefore, it is important that engineered antibodies are not limited by expression and purification yields. Wild-type IgG1, wild-type IgG4, vFc10, or vFc17 Fc regions engineered into antibody Ab1 were expressed in culture medium according to methods known in the art, and expression yields were measured. As shown in Figure 2A, both antibodies with vFc10 or vFc17 Fc variants produced high yields, significantly higher than wild-type IgG1 antibody, and comparable or higher than wild-type IgG4 antibody. As one skilled in the art would understand, protein A interacts with the Fc portion of immunoglobulins. Therefore, Octet experiments were performed to confirm that the mutations introduced into the Fc variants do not alter the Protein A binding properties. Figure 2B shows that the Fc variants exhibit similar binding to wild-type IgG1 or IgG4, demonstrating that the mutations do not alter the Protein A binding properties.

[0159] As shown in Figure 3A, both vFc10 and vFc17 abolished FcγRI binding to near baseline levels when paired with Ab1. Similarly, both vFc10 and vFc17 abolished FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb binding to near baseline levels (Figures 3B-3C). IgG4 has a short hinge and low Fab arm flexibility that partially shields it from binding to C1q. Figure 3D shows that neither vFc10 nor vFc17 induced C1q binding when paired with Ab1.

[0160] Overall, the data in this example show that the two Fc variants tested, when paired with antibody variable regions, exhibited abrogation of binding to all FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q. Example 4. Fc variants showed significantly reduced ADCC, ADCP, and CDC induction

[0161] Antibodies of the IgG subclass are bifunctional molecules with an F(ab) domain, which is variable in sequence and responsible for binding antigens, and an Fc domain, which is constant in sequence and responsible for mediating various antibody effector functions. These functions are primarily triggered by interaction with the complement component C1q or with a family of FcγRs expressed primarily on the surface of leukocytes. Fc gamma receptors (FcγRs) trigger cell-mediated cytotoxic effector functions such as antibody-dependent cellular cytotoxicity (ADCC), phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0162] Antibody-dependent cellular cytotoxicity (ADCC) is an Fc-dependent effector function of IgG that is important for antiviral immunotherapy and antitumor therapy. NK cell-mediated ADCC is primarily triggered through IgG-Fc receptor (FcγR)IIIa. Phagocytes, including monocytes, macrophages, neutrophils, eosinophils, and dendritic cells (DCs), express FcγRI, FcγRII, and FcαRI, all of which can mediate the uptake of immune complexes. ADCP results in the clearance of immune complexes from the infected host by transporting the complexes to lysosomes for degradation and antigen processing for presentation on major histocompatibility complex (MHC) molecules on the cell surface. Interestingly, some viruses exploit this mechanism to infect phagocytes by escaping lysosomal degradation (described below in "Antibody-Dependent Enhancement of Infection").

[0163] This example shows that Fc variants of the invention that were able to abolish binding to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q have reduced ADCC, ADCP, and CDC functions.

[0164] In this particular example, two Fc variants, vFc10 and vFc17, were used in the experiment. vFc10 contains L234F / L235E / D265G / A330S / P331S mutations in the IgG1 isotype, and vFc17 contains F234V / L235E / D265G mutations in the IgG4-S228P isotype. These two Fc variants were each engineered into antibody "Ab1". The amount of ADCC, ADCP, and CDC induction was measured versus antibody concentration.

[0165] Figure 4A shows that VFc10 and VFc17 maintained low ADCC compared to wild-type IgG1 antibody. Similarly, VFc10 and VFc17 maintained low ADCP and CDC compared to wild-type IgG1 antibody (Figure 4B and Figure 4C).

[0166] Overall, the data in this example demonstrate that the Fc variants of the invention abolished binding to all FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q, effectively reducing ADCC, ADCP, and CDC function. Equivalents and Scope

[0167] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. The scope of the disclosure is not intended to be limited to the above description, but rather is as set forth in the following claims.

Claims

1. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising an amino acid substitution of D265G and one or more amino acid substitutions at positions 234, 235, 237, 329, 330, or 331, wherein the residues are numbered according to the EU index.

2. 1. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising amino acid substitutions 234V, 235E and D265G, wherein residues are numbered according to the EU index.

3. 3. The isolated polypeptide of claim 2, wherein the Fc variant is an IgG4 Fc region and comprises the amino acid substitutions S228P, F234V, L235E, and D265G.

4. 1. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising amino acid substitutions 234F, 235E and D265G, wherein residues are numbered according to the EU index.

5. 1. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 234V, 235A, G237A, and D265G, wherein residues are numbered according to the EU index.

6. 1. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising amino acid substitutions 235E and D265G, wherein residues are numbered according to the EU index.

7. 1. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions 235E, G237A and D265G, wherein residues are numbered according to the EU index.

8. 1. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions 235E, G237A and P329G, wherein residues are numbered according to the EU index.

9. 9. The isolated polypeptide of claim 8, wherein the Fc variant comprises the amino acid substitutions S228P, L235E, G237A and P329G.

10. 1. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions 235E, G237A and L328R, wherein residues are numbered according to the EU index.

11. 11. The isolated polypeptide of claim 10, wherein the Fc variant comprises the amino acid substitutions S228P, L235E, G237A and L328R.

12. 1. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions D265G, A330S and P331S, wherein residues are numbered according to the EU index.

13. 1. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 235E, D265G, A330S, and P331S, wherein residues are numbered according to the EU index.

14. 1. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the following amino acid substitutions: 235E, D265G, and P329G, wherein residues are numbered according to the EU index.

15. The isolated polypeptide of claim 1, wherein the Fc variant is an IgG1, IgG2, or IgG4 Fc region.

16. An isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising a D265G amino acid substitution, wherein residues are numbered according to the EU index, and wherein said Fc variant has reduced affinity for human FcγR compared to said wild-type human IgG Fc region.

17. An antibody comprising an isolated polypeptide described in any one of claims 1 to 14 and 16.

18. A nucleic acid encoding the isolated polypeptide of any one of claims 1 to 14 and 16.

19. A cell comprising the nucleic acid of claim 18.

20. 17. A method of making an isolated polypeptide of any one of claims 1 to 14 and 16, comprising culturing a host cell comprising nucleic acid encoding an antibody or antigen-binding fragment thereof, and culturing the cell under conditions that allow production of the isolated polypeptide.

21. Use of an isolated polypeptide according to any one of claims 1 to 14 and 16, or an antibody comprising an isolated polypeptide according to any one of claims 1 to 14 and 16, in the manufacture of a medicament for treating a disease or disorder.

22. A composition for use in the treatment of a disease or disorder, comprising an isolated polypeptide described in any one of claims 1 to 14 and 16, or an antibody comprising an isolated polypeptide described in any one of claims 1 to 14 and 16.

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