Fc variants with binding to cancellation of fcgamrar and c1q

Abstract

The present disclosure provides, among other things, Fc variants with significantly reduced ADCC, ADCP, and CDC functions. As described herein, the disclosure is based in part on identifying novel combinations of mutations that eliminate binding to all of Fc [gamma] RI, Fc [gamma] RIIa, Fc [gamma] RIIb, Fc [gamma] RIIIa, Fc [gamma] RIIIb, and C1q.

Description

Background Art

[0001] The immune response is a mechanism by which the body defends itself from invasion by foreign substances leading to infection or disease. This mechanism is based on the ability of antibodies produced or administered to a host to bind to antigens through their variable regions. Once an antigen is bound by an antibody, the antigen is targeted for destruction, usually mediated in part by the constant region or Fc domain of the antibody.

[0002] For example, an activity of the Fc domain of an antibody is to bind complement proteins, which can help to lyse target antigens, such as cellular pathogens. Another activity of the Fc region is to bind to Fc receptors (FcR) on the surface of immune cells or so-called effector cells, which have the ability to trigger 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 responses are most important for the efficacy of antibodies, and in other cases, the response causes undesirable side effects. An example of effector-mediated side effects is the release of inflammatory cytokines that cause acute febrile reactions. Another example is the long-term absence of cells carrying antigens.

[0003] Effector functions of antibodies can be avoided by using antibody fragments lacking the Fc region (e.g., Fab, Fab'2, or single-chain antibodies (sFv)), however these fragments have a reduced half-life, 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

[0004] In addition, the present disclosure provides, among other things, Fc variants with significantly reduced ADCC, ADCP and CDC functions. As described herein, the present disclosure is based in part on identifying novel combinations of mutations that abolish binding to all of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb and C1q, while retaining its ability to bind to FcRn.

[0005] In one aspect, the present 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 substituted with Gly, wherein the residues are numbered according to the EU index.

[0006] In some embodiments, the Fc variants further comprise one or more amino acid substitutions at 234, 237, 329, 330, or 331. In some embodiments, the Fc variants further comprise an amino acid substitution at 234. In some embodiments, the Fc variants further comprise amino acid substitutions at 234 and 237. In some embodiments, the Fc variants further comprise amino acid substitutions at 234, 330, and 331. In some embodiments, the Fc variants further comprise amino acid substitutions at 234, 237, 330, and 331. In some embodiments, the Fc variants further comprise amino acid substitutions at 237. In some embodiments, the Fc variants further comprise amino acid substitutions at 330 and 331. In some embodiments, the Fc variants further comprise amino acid substitutions at 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, wherein the residues are numbered according to the EU index.

[0008] In some embodiments, the Fc variant further comprises amino acid substitutions at 235 and 237. In some embodiments, the Fc variant further comprises amino acid substitutions at 235, 237, 330, and 331. In some embodiments, the Fc variant further comprises amino acid substitutions at 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, wherein the residues are numbered according to the EU index.

[0010] In one aspect, the present 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 of 234V, L235E and D265G, wherein the 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 includes amino acid substitutions of S228P, F234V, L235E, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and includes amino acid substitutions of 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 amino acid substitutions of 234F, L235E and D265G, wherein the 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 amino acid substitutions of L234F, L235E, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and comprises amino acid substitutions of L234F, L235E, and D265G.

[0014] In one aspect, the present 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 of 234F, L235E, G237A and D265G, wherein the 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 includes amino acid substitutions of L234F, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and includes amino acid substitutions of 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 amino acid substitutions of 234V, L235E, G237A and D265G, wherein the 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 includes amino acid substitutions of L234V, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and includes amino acid substitutions of 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 amino acid substitutions of 234F, L235E, D265D, A330S and P331S, wherein the 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 includes amino acid substitutions of L234F, L235E, D265D, A330S, and P331S. In some embodiments, the Fc variant is an IgG1 Fc region and includes amino acid substitutions of 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 amino acid substitutions of 234V, L235A, G237A and D265G, wherein the residues are numbered according to the EU index.

[0021] In some embodiments, Fc variants are IgG1 Fc regions. In some embodiments, Fc variants include amino acid substitutions of L234V, L235A, G237A and D265G. In some embodiments, Fc variants are IgG1 Fc regions, and include amino acid substitutions of 234F, L234V, L235A, G237A and D265G. In some embodiments, Fc variants are IgG4 Fc regions. In some embodiments, Fc variants include amino acid substitutions of S228P, L234V, L235A, G237A and D265G. In some embodiments, Fc variants are IgG4 Fc regions, and include amino acid substitutions of S228P, L234V, L235A, G237A and D265G.

[0022] In one aspect, the present 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 of 234V, L235A, G237A, D265G, A330S and P331S, wherein the 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 includes amino acid substitutions of L234V, L235A, G237A, D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG1 Fc region and includes amino acid substitutions of L234V, L235A, G237A, D265G, A330S, and P331S.

[0024] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions of L235E and D265G, wherein the 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 includes amino acid substitutions of S228P, L235E, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and includes amino acid substitutions of S228P, L235E, and D265G.

[0026] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions of L235E, G237A and D265G, wherein the 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 includes amino acid substitutions of S228P, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and includes amino acid substitutions of S228P, L235E, G237A, and D265G.

[0028] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions of L235E, G237A and P329G, wherein the 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 includes amino acid substitutions of S228P, L235E, G237A, and P329G. In some embodiments, the Fc variant is an IgG4 Fc region and includes amino acid substitutions of S228P, L235E, G237A, and P329G.

[0030] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions of L235E, G237A and L328R, wherein the 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 includes amino acid substitutions of S228P, L235E, G237A, and L328R. In some embodiments, the Fc variant is an IgG4 Fc region and includes amino acid substitutions of S228P, L235E, G237A, and L328R.

[0032] In one aspect, the present invention provides, inter alia, 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 the 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 includes amino acid substitutions of D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG2 Fc region and includes amino acid substitutions of D265G, A330S, and P331S.

[0034] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions of 235E, D265G, A330S and P331S, wherein the 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 includes amino acid substitutions of A235E, D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG2 Fc region and includes amino acid substitutions of 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 amino acid substitutions of 235E, D265G and P329G, wherein the 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 includes amino acid substitutions of A235E, D265G, and P329G. In some embodiments, the Fc variant is an IgG2 Fc region and includes amino acid substitutions of 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, among other things, 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 for treating a disease or condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of an isolated polypeptide comprising an Fc variant. In some embodiments, the disease or condition is ANCA-associated vasculitis. In some embodiments, the disease or condition is C3 glomerulopathy (C3G). In some embodiments, the disease or condition is an autoimmune disease. In some embodiments, the disease or condition is wet age-related macular degeneration (wAMD). In some embodiments, the disease or condition is passive Heimann nephritis (NHP). In some embodiments, the disease or condition is collagen antibody-induced arthritis (CAIA). BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1A -D is a series of exemplary graphs showing the binding of Abl and Ab2 engineered with various Fc variants, respectively, to FcγRI, FcγRIIa, FcγRIIb and C1q as determined by the assay described in Example 2.

[0042] Figure 2A is an exemplary bar graph and table showing the expression levels of Abl engineered with wild-type IgGl, wild-type IgG4, vFclO, and vFcl7. Figure 2B are exemplary graphs and tables showing the Protein A binding properties of Abl engineered with wild-type IgGl, wild-type IgG4, and vFcl7.

[0043] Figure 3A is an exemplary graph showing the binding of Ab1 engineered with wild-type IgG1, wild-type IgG4, vFc10 or vFc17 to FcγRI, thereby demonstrating that the Fc variants of the present invention significantly reduce the binding of Fc to FcγRI compared to wild-type IgG1 and IgG4. Figure 3B is an exemplary graph showing the binding of Ab1 engineered with wild-type IgG1, wild-type IgG4, vFc10 or vFc17 to FcγRIIa and FcγRIIb, thereby demonstrating that the Fc variants of the present invention significantly reduce the binding of Fc to FcγRIIa and FcγRIIb compared to wild-type IgG1 and IgG4. Figure 3C are exemplary graphs showing binding of Abl engineered with wild-type IgG1, wild-type IgG4, vFclO, or vFcl7 to FcγRIIIa and FcγRIIIb. Figure 3D are exemplary graphs showing binding of Abl engineered with wild-type IgGl, wild-type IgG4, vFclO, or vFcl7 to C1q.

[0044] Figure 4Ais an exemplary graph showing the fold induction of ADCC by Abl engineered with wild-type IgG1, wild-type IgG4, vFclO, or vFcl7. Figure 4B is an exemplary graph showing the fold induction of ADCP by Abl engineered with wild-type IgGl, wild-type IgG4, vFclO, or vFcl7. Figure 4C is an exemplary graph showing the fold induction of CDC by Abl engineered with wild-type IgGl, wild-type IgG4, vFclO, or vFcl7.

[0045] definition

[0046] 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 to (immunoreacts with) an antigen. "Binding" or "immunoreacting 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 dAbs (domain antibodies), single chains, Fab, Fab', F(ab')2 fragments, scFv and Fab expression libraries. Antibodies can be complete antibodies, or immunoglobulins, or antibody fragments.

[0047] Fc domain: As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl end of the heavy chain. However, the C-terminal lysine (Lys447) in 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 referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0048] 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. Especially in bispecific or biparatopic molecules, this produces functional monovalent antibodies with unknown specificity and therefore potentially reduced therapeutic efficacy. Mutations can be introduced into the Fc domain to inhibit Fab arm exchange. It is well known that the S228P mutation prevents IgG4 FAEs from reaching undetectable levels in vitro and in vivo.

[0049] Humanized antibody: The term "humanized antibody" includes non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof, which contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementarity determining regions (CDRs) are replaced with residues from CDRs of non-human species (e.g., mouse, rat, rabbit, hamster) having 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).

[0050] Monoclonal antibody: The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, 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 for a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method.

[0051] Multispecific antibody: As used herein, the term "multispecific antibody" refers to a binding molecule, antibody, or antigen-binding fragment thereof, that is capable of specifically binding to two or more different epitopes on the same or different targets.

[0052] Biparatopic antibody: As used herein, the term "biparatopic antibody" refers to a multispecific antibody capable of binding to two different non-overlapping epitopes on the same target antigen molecule.

[0053] K i Live K d : As used herein, the term "K d ” refers to the dissociation constant for a particular antibody-antigen interaction as known in the art, and will be applied for the subject compositions as a parameter for the binding affinity of the targeting moiety to its cognate ligand.

[0054] IC50: As used herein, the term "IC50" refers to the concentration required to inhibit half of the maximal biological response of a ligand agonist, and is generally determined by a competition binding assay.

[0055] 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 toxin that induces a response halfway between 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.

[0056] Linker: As used herein, the term "linker" refers to a molecule or group of molecules (e.g., a monomer or polymer) that connects two molecules and is generally used to place the two molecules in a preferred configuration. Many strategies can be used to covalently link molecules together. These strategies include, but are not limited to, polypeptide connections between the N-terminus and the C-terminus of a protein or protein domain, connections through disulfide bonds, and connections through chemical cross-linking agents. In one aspect of this embodiment, the linker is a peptide bond produced by recombinant technology or peptide synthesis. The linker may contain amino acid residues that provide flexibility. Therefore, the linker peptide may mainly include the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length sufficient to connect the two molecules in a manner that allows the two molecules to present the correct configuration relative to each other, so that it retains the desired activity. Suitable lengths for this purpose include at least one and no more than 30 amino acid residues. In one embodiment, the length of the linker is about 1 to 30 amino acids. In another embodiment, the length of the linker is about 1 to 15 amino acids. In addition, the amino acid residues selected to be included in the linker peptide should exhibit characteristics that do not significantly interfere with the activity of the polypeptide.

[0057] scFv: As used herein, the term "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, connected to a short linker peptide of ten to about 25 amino acids.

[0058] Fab: As used herein, the term "Fab" refers to an antibody fragment that comprises a portion of an intact antibody, including the antigen binding or variable region thereof.

[0059] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.

[0060] In vivo: As used herein, the term "in vivo" refers to events that occur in multicellular organisms 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).

[0061] 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). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, which refers to a human who goes to a healthcare provider for diagnosis or treatment of a disease. The term "subject" may be used interchangeably herein with "individual" or "patient". A subject may be suffering from or susceptible to a disease or condition, but may or may not show symptoms of the disease or condition.

[0062] Dysfunction: As used herein, the term "dysfunction" refers to an abnormal function. Dysfunction of a molecule (e.g., a protein) can be caused by an increase or decrease in an activity associated with such molecule. Dysfunction of a molecule can be caused by defects associated with the molecule itself or with other molecules that directly or indirectly interact with or regulate the molecule.

[0063] Derivative: As used herein, the term "derivative" when used in connection with an antibody or C5aR1 antibody refers to a portion of an original molecule that has some sequence that retains at least some of the functions and / or properties of the original molecule.

[0064] Identity: As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as known in the art, which compares the sequences of these molecules. Determine the relationship by performing. In the art, "identity" also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, and in some cases more than one nucleotide sequence or more than one. It can be determined by the match between amino acid sequence strings. "Identity" means the identity between the gap alignment (if any) solved by a specific mathematical model or computer program (i.e., "algorithm") and the smaller sequence of two or more sequences. Measure the consistency match percentage.

[0065] Similarity or resemblance: As used herein, the term "similarity" used in the art with respect to related concepts, but in contrast to "identity" and "similarity", refers to both identity and conservative substitution matches. Indicating relevance, including if two polypeptide sequences have, for example, 10 identical amino acids out of 20 amino acids, and the rest are non-conservative substitutions, then the percentage of identity and percentage of similarity are both 50%. In the same example, if there are 5 or more conservative substitutions, the percentage of identity is still 50%, but the percentage of similarity is 75%. Therefore, if there are conservative substitutions, the percentage of similarity between the two polypeptides is higher than the percentage of identity between the two polypeptides.

[0066] Treatment: As used herein, the term "treat / treatment / treating" refers to any method used to partially or completely alleviate, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment can be administered to subjects who do not show signs of the disease and / or only show early signs of the disease in order to reduce the risk of developing lesions associated with the disease.

[0067] Vector: The term "vector" refers to a polynucleotide (usually DNA) used to artificially carry foreign genetic material to another cell where the foreign genetic material can be replicated or expressed. Non-limiting exemplary vectors include plasmids, viral vectors, cosmids, and artificial chromosomes. These vectors can be derived from a variety of sources, including bacterial and viral sources. A non-limiting exemplary viral source of plasmids is adeno-associated virus.

[0068] Various aspects of the present disclosure are described in detail in the following sections. The use of sections is not intended to limit the present disclosure. Each section may apply to any aspect of the present disclosure. In this application, unless otherwise stated, the use of "or" means "and / or". As used herein, unless the context clearly dictates otherwise, the singular forms "a / an" and "the" include both singular and plural referents. DETAILED DESCRIPTION

[0069] The Fc region of an antibody controls antibody cytotoxic activity and may affect the serum half-life of the antibody. However, in a therapeutic setting, the cytotoxic effector function of an antibody is generally undesirable and can generate safety issues and undesirable side effects by activating host immune defenses. In cases where activation is otherwise harmful, Fc engineering is required to silence the IgG Fc domain so that it cannot bind to the Fc-γ receptor.

[0070] This disclosure describes a new class of Fc variants that are particularly effective in silencing IgG Fc domains. Notably, the combined Fc mutations of the invention are novel and reduce binding to Fc-γ receptors and C1q, thereby effectively reducing undesirable ADCC, ADCP and CDC effector functions.

[0071] Fc region and its effector functions

[0072] The Fc region of an antibody interacts with many Fc receptors and ligands, thereby conferring a series of important functional capabilities known as effector functions. For IgG, the Fc region includes Ig domains Cγ2 and Cγ3 and the N-terminal hinge leading to Cγ2. The important family of Fc receptors of IgG categories is Fcγ receptors (FcγR). These receptors mediate the communication between antibodies and the cell arm (cellular arm) of the immune system (Raghavan et al., 1996, "Annual Review of Cell and Developmental Biology (Annu Rev Cell Dev Biol)" 12: 181-220; Ravetch et al., 2001, "Annual Review of Immunology (Annu Rev Immunol)" 19: 275-290). In humans, this family of proteins 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 usually have an extracellular domain, a transmembrane region and an intracellular domain that can mediate some signal transduction events in the cell that mediate the combination with Fc. These receptors are expressed in various immune cells, and the immune cells include monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells and γδT cells. The formation of Fc / FcγR complexes recruits these effector cells to the site of antigen binding, thereby typically causing intracellular signal transduction events and important subsequent immune responses, such as the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis and cytotoxic attacks. The ability to mediate cytotoxicity and engulf effector functions is the potential mechanism of antibody destruction targeted cells.The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγRs recognize bound antibodies on target cells and subsequently cause lysis of the target cells is called antibody-dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annual Review of Cell and Developmental Biology 12: 181-220; Ghetie et al., 2000, Annual Review of Immunology 18: 739-766; Ravetch et al., 2001, Annual Review of Immunology 19: 275-290, the documents are incorporated by reference). The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγRs recognize bound antibodies on target cells and subsequently cause phagocytosis of the target cells is called antibody-dependent cell-mediated cellular phagocytosis (ADCP). Many structures have been solved for the extracellular domains of human FcγRs, 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 the hinge preceding it. This interaction is well characterized structurally (Sondermann et al., 2001, J. Biol. Chem. 309:737-749, incorporated by reference), and several structures of human Fc bound to the extracellular domain of human FcγRIIIb have been 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).

[0073] Overlapping but separate sites on Fc serve as the interface for the complement protein C1q. In the same way that 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 is able to bind six antibodies, but binding to two IgGs is sufficient to activate the complement cascade. Similar to the Fc interaction with FcγRs, different IgG subclasses have different affinities for C1q, with IgG1 and IgG3 generally binding significantly better to FcγRs than IgG2 and IgG4.

[0074] The site on Fc between the Cγ2 and Cγ3 domains mediates interaction with the neonatal receptor FcRn, whose binding recycles endocytosed antibodies from endosomes back into the bloodstream (Raghavan et al., 1996, Annual Review of Cell and Developmental Biology 12: 181-220; Gheti et al., 2000, Annual Review of Immunology 18: 739-766, which are incorporated by reference). This process, coupled with the exclusion of kidney filtration due to the large size of the full-length molecule, produces a favorable antibody serum half-life ranging from one to three weeks. The binding of Fc to FcRn also plays a key role in antibody transport. The binding site of FcRn on Fc is also the site where bacterial proteins A and G bind. The tight binding of these proteins is often used as a method for purifying antibodies by using protein A or protein G affinity chromatography during protein purification. Therefore, 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) and of Fc in complex 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) have provided insights into the interaction of Fc with these proteins.

[0075] The present invention relates to optimized Fc variants for various contexts. As outlined above, current antibody therapies face various problems. The present invention provides a promising method for enhancing the therapeutic efficacy of antibodies by eliminating their ability to mediate cytotoxic effector functions, such as ADCC, ADCP and CDC.

[0076] Fc variants of the present invention

[0077] The Fc variants of the present invention can be used in various Fc polypeptides. Fc polypeptides comprising Fc variants of the present invention are referred to herein as "Fc polypeptides of the present invention". Fc polypeptides of the present invention include polypeptides comprising Fc variants of the present invention in the context of larger polypeptides such as antibodies or Fc fusions. That is, the Fc polypeptides of the present invention include antibodies and Fc fusions comprising Fc variants of the present invention. As used herein, "antibodies of the present invention" means antibodies comprising Fc variants of the present invention. As used herein, "Fc fusions of the present invention" refers to Fc fusions comprising Fc variants of the present invention. The Fc polypeptides of the present invention also include polypeptides that include little or no additional polypeptide sequences in addition to 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 can be fused with one or more fusion partners or conjugated partners to provide desired functional properties.

[0078] The parent Fc polypeptides described herein can be derived from a wide range of sources and can be substantially encoded by one or more Fc genes from any organism, including but not limited to humans, rodents, including but not limited to 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), macaques (old world monkeys) and the most popular superfamily of humans, including gibbons, small apes and great apes. The parent Fc polypeptides of the present invention can be substantially encoded by immunoglobulin genes belonging to any one 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 present invention include sequences belonging to the human IgG class of antibodies. For example, the parent Fc polypeptide can be a parent antibody, such as a human IgG1 antibody, a human IgA antibody, or a mouse IgG2a or IgG2b antibody. The parent antibody can be non-human, chimeric, humanized, or fully human as described in detail below. The parent Fc polypeptide can be modified or engineered in some way, such as a parent antibody can be affinity matured, or can have an engineered glycoform, all of which are described more fully below. Alternatively, the parent Fc polypeptide can be an Fc fusion, such as an Fc fusion in which the fusion partner targets a cell surface receptor. Alternatively, the parent Fc polypeptide can be an isolated Fc region, which hardly includes or does not include other polypeptide sequences outside the Fc region. The parent Fc polypeptide can be a naturally occurring Fc region, or can be an existing engineered variant of the Fc polypeptide. Importantly, the parent Fc polypeptide includes an Fc region, and then the Fc region can be mutated to produce an Fc variant.

[0079] The Fc variants of the present invention can be used in a wide range of products. In one embodiment, the Fc variants of the present invention are therapeutic agents, diagnostic agents or research reagents, preferably therapeutic agents. Alternatively, the Fc variants of the present invention can be used for agricultural or industrial purposes. The antibodies of the present invention can be used in monoclonal or polyclonal antibody compositions. The Fc variants of the present invention can be agonists, antagonists, neutralizers, inhibitors or stimulants. In a preferred embodiment, the Fc variants of the present invention are used to kill target cells carrying target antigens, such as cancer cells. In an alternative embodiment, the Fc variants of the present invention are used to block, antagonize or excite target antigens. In an alternatively preferred embodiment, the Fc variants of the present invention are used to block, antagonize or excite target antigens and kill target cells carrying target antigens.

[0080] Optimized features

[0081] The present invention provides Fc variants optimized for many treatment-related properties. Relative to the parent Fc polypeptide, the Fc variant includes one or more amino acid modifications, wherein the amino acid modifications provide one or more optimized properties. The amino acid sequence of the Fc variant of the present invention is different from its parent Fc polypeptide in that at least one amino acid modification is present. Therefore, compared with the parent, the Fc variant of the present invention has at least one amino acid modification. Alternatively, compared with the parent, the Fc variant of the present invention can have more than one amino acid modification, for example, compared with the parent, there are about one to fifty amino acid modifications, about one to ten amino acid modifications, or about one to about five amino acid modifications. Therefore, 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 with the parent Fc variant sequence, preferably at least about 90% homology, and most preferably at least about 95% homology.

[0082] The Fc variants of the present invention can be optimized for various properties. Fc variants that are engineered or predicted to show one or more optimized properties are referred to herein as "optimized Fc variants". The properties that can be optimized include, but are not limited to, enhancing or reducing affinity for FcγR. In some embodiments, the Fc variants of the present invention are optimized to have reduced or ablated affinity for human FcγR, including, but not limited to, FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIb. These embodiments are expected to provide Fc polypeptides with enhanced therapeutic properties of people, such as reduced effector functions and reduced toxicity. In other embodiments, the Fc variants of the present invention provide enhanced affinity for one or more FcγRs, but provide reduced affinity for 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, the Fc variants of the invention may have enhanced binding to FcγRIIa and FcγRI, but reduced binding to FcγRIIb. In yet another embodiment, the Fc variants of the invention may have enhanced affinity for FcγRIIb, but reduced affinity for one or more activating FcγRs.

[0083] In some embodiments, the Fc variants have reduced or ablated affinity for FcγRI. In some embodiments, the Fc variants have reduced or ablated affinity for FcγRIIa. In some embodiments, the Fc variants have reduced or ablated affinity for FcγRIIb. In some embodiments, the Fc variants have reduced or ablated affinity for FcγRIIc. In some embodiments, the Fc variants have reduced or ablated affinity for FcγRIIIa. In some embodiments, the Fc variants have reduced or ablated affinity for FcγRIIIb. In some embodiments, the Fc variants have reduced or ablated 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 ablated affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb and C1q. In some embodiments, the Fc variant has reduced or ablated affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q, and retains binding to FcRn.

[0084] The Fc variants of the present invention can also be optimized to enhance the functionality and / or solution properties of the deglycosylated form. In a preferred embodiment, the deglycosylated Fc variants of the present invention bind to Fc ligands with reduced affinity than the deglycosylated form of the parent Fc variant. The Fc ligands include but are not limited to FcγR, C1q, FcRn and proteins A and G, and can be from any source, including but not limited to humans, mice, rats, rabbits or monkeys, preferably humans. In an alternatively preferred embodiment, the Fc variant is optimized to be more stable and / or more soluble than the deglycosylated form of the parent Fc variant.

[0085] Engineered Fc mutations

[0086] In one aspect, the present 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 the residues are numbered according to the EU index.

[0087] In one aspect, the present 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, wherein the residues are numbered according to the EU index.

[0088] In one aspect, the present 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 the residues are numbered according to the EU index.

[0089] In one aspect, the present 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 the residues are numbered according to the EU index.

[0090] In one aspect, the present 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 the residues are numbered according to the EU index.

[0091] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG1 Fc region, the Fc variant comprising the amino acid substitutions L234V / L235A / G237A / D265G / A330S / P331S, wherein the residues are numbered according to the EU index.

[0092] In one aspect, the present 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 the residues are numbered according to the EU index.

[0093] In one aspect, the present 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 the residues are numbered according to the EU index.

[0094] In one aspect, the present 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 the residues are numbered according to the EU index.

[0095] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG4 Fc region, the Fc variant comprising the amino acid substitutions F234V / L235A / G237A / D265G / S228P, wherein the residues are numbered according to the EU index.

[0096] In one aspect, the present 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 the residues are numbered according to the EU index.

[0097] In one aspect, the present 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 the residues are numbered according to the EU index.

[0098] In one aspect, the present 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 the residues are numbered according to the EU index.

[0099] In one aspect, the present 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 the residues are numbered according to the EU index.

[0100] In one aspect, the present 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, wherein the residues are numbered according to the EU index.

[0101] In one aspect, the present 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, wherein the residues are numbered according to the EU index.

[0102] In one aspect, the present 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 substituted with Gly, wherein the residues are numbered according to the EU index.

[0103] In some embodiments, the Fc variants further comprise one or more amino acid substitutions at 234, 237, 329, 330, or 331. In some embodiments, the Fc variants further comprise an amino acid substitution at 234. In some embodiments, the Fc variants further comprise amino acid substitutions at 234 and 237. In some embodiments, the Fc variants further comprise amino acid substitutions at 234, 330, and 331. In some embodiments, the Fc variants further comprise amino acid substitutions at 234, 237, 330, and 331. In some embodiments, the Fc variants further comprise amino acid substitutions at 237. In some embodiments, the Fc variants further comprise amino acid substitutions at 330 and 331. In some embodiments, the Fc variants further comprise amino acid substitutions at 329.

[0104] 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, wherein the residues are numbered according to the EU index.

[0105] In some embodiments, the Fc variant further comprises amino acid substitutions at 235 and 237. In some embodiments, the Fc variant further comprises amino acid substitutions at 235, 237, 330, and 331. In some embodiments, the Fc variant further comprises amino acid substitutions at 235.

[0106] 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, wherein the residues are numbered according to the EU index.

[0107] In one aspect, the present 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 of 234V, L235E and D265G, wherein the residues are numbered according to the EU index.

[0108] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant includes amino acid substitutions of S228P, F234V, L235E, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and includes amino acid substitutions of S228P, F234V, L235E, and D265G.

[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 amino acid substitutions of 234F, L235E and D265G, wherein the 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 amino acid substitutions of L234F, L235E, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and comprises amino acid substitutions of L234F, L235E, and D265G.

[0111] In one aspect, the present 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 of 234F, L235E, G237A and D265G, wherein the 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 includes amino acid substitutions of L234F, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and includes amino acid substitutions of L234F, L235E, G237A, and D265G.

[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 amino acid substitutions of 234V, L235E, G237A and D265G, wherein the residues are numbered according to the EU index.

[0114] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant includes amino acid substitutions of L234V, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG1 Fc region and includes amino acid substitutions of L234V, L235E, G237A, 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 amino acid substitutions of 234F, L235E, D265D, A330S and P331S, wherein the residues are numbered according to the EU index.

[0116] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant includes amino acid substitutions of L234F, L235E, D265D, A330S, and P331S. In some embodiments, the Fc variant is an IgG1 Fc region and includes amino acid substitutions of L234F, L235E, D265D, A330S, and P331S.

[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 amino acid substitutions of 234V, L235A, G237A and D265G, wherein the residues are numbered according to the EU index.

[0118] In some embodiments, Fc variants are IgG1 Fc regions. In some embodiments, Fc variants include amino acid substitutions of L234V, L235A, G237A and D265G. In some embodiments, Fc variants are IgG1 Fc regions, and include amino acid substitutions of 234F, L234V, L235A, G237A and D265G. In some embodiments, Fc variants are IgG4 Fc regions. In some embodiments, Fc variants include amino acid substitutions of S228P, L234V, L235A, G237A and D265G. In some embodiments, Fc variants are IgG4 Fc regions, and include amino acid substitutions of S228P, L234V, L235A, G237A and D265G.

[0119] In one aspect, the present 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 of 234V, L235A, G237A, D265G, A330S and P331S, wherein the residues are numbered according to the EU index.

[0120] In some embodiments, the Fc variant is an IgG1 Fc region. In some embodiments, the Fc variant includes amino acid substitutions of L234V, L235A, G237A, D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG1 Fc region and includes amino acid substitutions of L234V, L235A, G237A, D265G, A330S, and P331S.

[0121] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions of L235E and D265G, wherein the residues are numbered according to the EU index.

[0122] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant includes amino acid substitutions of S228P, L235E, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and includes amino acid substitutions of S228P, L235E, and D265G.

[0123] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions of L235E, G237A and D265G, wherein the residues are numbered according to the EU index.

[0124] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant includes amino acid substitutions of S228P, L235E, G237A, and D265G. In some embodiments, the Fc variant is an IgG4 Fc region and includes amino acid substitutions of S228P, L235E, G237A, and D265G.

[0125] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions of L235E, G237A and P329G, wherein the residues are numbered according to the EU index.

[0126] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant includes amino acid substitutions of S228P, L235E, G237A, and P329G. In some embodiments, the Fc variant is an IgG4 Fc region and includes amino acid substitutions of S228P, L235E, G237A, and P329G.

[0127] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions of L235E, G237A and L328R, wherein the residues are numbered according to the EU index.

[0128] In some embodiments, the Fc variant is an IgG4 Fc region. In some embodiments, the Fc variant includes amino acid substitutions of S228P, L235E, G237A, and L328R. In some embodiments, the Fc variant is an IgG4 Fc region and includes amino acid substitutions of S228P, L235E, G237A, and L328R.

[0129] In one aspect, the present invention provides, inter alia, 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 the residues are numbered according to the EU index.

[0130] In some embodiments, the Fc variant is an IgG2 Fc region. In some embodiments, the Fc variant includes amino acid substitutions of D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG2 Fc region and includes amino acid substitutions of D265G, A330S, and P331S.

[0131] In one aspect, the present invention provides, inter alia, an isolated polypeptide comprising an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising the amino acid substitutions of 235E, D265G, A330S and P331S, wherein the residues are numbered according to the EU index.

[0132] In some embodiments, the Fc variant is an IgG2 Fc region. In some embodiments, the Fc variant includes amino acid substitutions of A235E, D265G, A330S, and P331S. In some embodiments, the Fc variant is an IgG2 Fc region and includes amino acid substitutions of A235E, D265G, A330S, and P331S.

[0133] 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 amino acid substitutions of 235E, D265G and P329G, wherein the residues are numbered according to the EU index.

[0134] In some embodiments, the Fc variant is an IgG2 Fc region. In some embodiments, the Fc variant includes amino acid substitutions of A235E, D265G, and P329G. In some embodiments, the Fc variant is an IgG2 Fc region and includes amino acid substitutions of A235E, D265G, and P329G.

[0135] Design of antibodies with Fc variants

[0136] The Fc variants of the present invention can be antibodies, referred to herein as "antibodies of the present invention". The antibodies of the present invention can include immunoglobulin sequences substantially encoded by immunoglobulin genes belonging to any one of the antibody classes, including but not limited to IgG (including human subclass IgG1, IgG2, IgG3 or IgG4), IgA (including human subclass IgA1 and IgA2), IgD, IgE, IgG and IgM classes of antibodies. Most preferably, the antibodies of the present invention include sequences belonging to the human IgG class of antibodies. The antibodies of the present invention can be non-human, chimeric, humanized or fully human. As will be appreciated by those skilled in the art, these different types of antibodies reflect the degree of "humanity" in humans or the potential level of immunogenicity. For a description of these concepts, see Clark et al., 2000 and references cited therein (Clark, 2000, "Immunol Today" 21: 397-402, which are incorporated by reference). Chimeric antibodies include variable regions of non-human antibodies operably linked to the constant regions of human antibodies, such as VH and VL domains of mouse or rat origin. The non-human variable regions may be derived from any organism as described above, preferably mammals, and most preferably rodents or primates. In one embodiment, the antibodies of the present invention include monkey variable domains, such as Newman et al., 1992, Biotechnology 10: 1455-1460 U.S. Patent No. 5,658,570 and U.S. Patent No. 5,750,105, the document being incorporated by reference). In a preferred embodiment, the variable region is derived from a non-human source, but its immunogenicity has been reduced using protein engineering. In a preferred embodiment, the antibodies of the present invention are humanized (Tsurushita and Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), which is incorporated by reference). As used herein, a "humanized" antibody means an antibody comprising a human framework region (FR) and one or more complementary determining regions (CDRs) from a non-human (usually mouse or rat) antibody. The non-human antibody providing the CDR is referred to as the "donor", and the human immunoglobulin providing the framework is referred to as the "acceptor". Humanization mainly relies on the grafting of donor CDRs onto the acceptor (human) VL and VH frameworks (Winter U.S. Pat. No. 5,225,539, which is incorporated by reference). This strategy is called "CDR grafting"."Back mutation" of selected acceptor framework residues to corresponding donor residues is often required to restore the 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, which are incorporated by reference). A large number of other methods for humanization are known in the art (Tsurushita and Vasquez, 2004, Humanization of Monoclonal Antibodies, B Cell Molecular Biology, 533-545, Elsevier Science (USA), which is incorporated by reference), and any of such methods can be used in the present invention to modify 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 will typically include a human Fc region.

[0137] 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, for example, comprising a plurality of immunoglobulin variable region sequences, wherein the first immunoglobulin variable region sequence in the plurality of immunoglobulin variable region sequences has binding specificity to the first epitope and the second immunoglobulin variable region sequence of the plurality of immunoglobulin variable region sequences has binding specificity to the second epitope. In one embodiment, the first epitope and the second epitope are on the same antigen, for example, on the same protein (or subunit of a multimeric protein). Bispecific or biparatopic antibodies have specificity for no more than two antigens or epitopes. Bispecific or biparatopic antibody molecules are generally characterized by a first immunoglobulin variable region sequence having binding specificity to the first epitope and a second immunoglobulin variable region sequence having binding specificity to the second epitope. In one embodiment, a bispecific or biparatopic antibody molecule comprises a half antibody or fragment thereof having binding specificity to the first epitope and a half antibody or fragment thereof having binding specificity to the second epitope. In one embodiment, the first epitope and the second epitope are on the same antigen, such as the same protein (or subunit of a multimeric protein). In some embodiments, the biparatopic antibodies provided in the present disclosure include Fab-Fc and a single chain variable fragment (scFv), wherein the Fc is connected to the scFv via a linker.

[0138] In some embodiments, the biparatopic antibody is a bispecific antibody with a two-arm single-chain Fab-Fc design that includes "knobs-in-hole" (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 mutation includes a T366Y mutation in one CH3 domain that can be used to create a knob, while 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, while 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, while 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 the knob-hole technology (e.g., comprising the hole mutations: Y349C, T366S, L368A, Y407V; the knob mutations: S354C, T366W).

[0139] In some embodiments, the biparatopic antibody comprises the antibody formats described in Table 2.

[0140] Table 2. Format of biparatopic antibodies

[0141] (Site II) Fab-IgG-scFv (Site I) (Site II) Fab-IgG-Linker-VL (Site I)-Linker-VH (Site I) (Site II) Fab-IgG-Linker-VH (Site I)-Linker-VL (Site I) (Site I) Fab-IgG-scFv (Site II)

[0142] In one embodiment, the biparatopic antibody molecule comprises two heavy chain variable regions and two light chain variable regions. In one embodiment, the anti-C5aR1 antibody molecule comprises Fab, F(ab')2, Fv, Fd or a single chain Fv fragment (scFv).

[0143] In some embodiments, the Fc domain used in the present application includes or is derived from IgG, IgM, IgE, Fc part. In addition to the KiH mutation described above, the Fc domain includes a S228P mutation. In some embodiments, S228P enhances the homogeneity of the antibody. In some embodiments, the Fc domain includes 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 includes an IgG4 Fc domain. In some embodiments, the Fc domain is derived from or includes an IgG4 Fc domain with a S228P mutation. In some embodiments, the Fc domain is derived from or includes an IgG1 Fc domain. In some embodiments, the Fc domain is derived from or includes an IgG1 Fc domain with a S228P mutation.

[0144] In some exemplary embodiments, monospecific antibodies and biparatopic antibodies may be modified or mutated to enhance the thermal stability of the antibody. The thermal stability of the antibody can be assessed by determining the aggregation onset temperature. One way to increase the stability of the antibody is to increase the thermal transition midpoint (Tm) as measured by differential scanning calorimetry (DSC). In general, the protein Tm is related to its stability and is negatively correlated with its susceptibility to unfolding and denaturation in solution and the degradation process that depends on the unfolding trend of the protein. Many studies have found a correlation between the grade of physical stability of formulations measured as thermal stability by DSC 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 have shown that the Fab Tm is involved in the long-term physical stability of the corresponding mAb.

[0145] In some exemplary embodiments, strategically introduced disulfide bonds can stabilize monomeric and multi-subunit proteins, playing a role in enhancing the thermal stability of antibodies.

[0146] 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.

[0147] In some embodiments, strategic introduction of salt bridges occurring between amino acid side chains with opposite positive or negative full electron charges, ie, (at neutral pH) Glu or Asp versus Arg or Lys, enhances the stability of proteins, particularly antibodies.

[0148] In some exemplary embodiments, the monospecific antibody or biparatopic antibody comprises one or more modifications that enhance thermal stability. In some embodiments, the modification that enhances thermal stability is the introduction of a cysteine ​​residue.

[0149] In some embodiments, exemplary biparatopic antibodies have a Tm greater than 65° C. In some embodiments, exemplary biparatopic antibodies have a Tm greater than 60° C., and exemplary biparatopic antibodies have a Tm greater than 55° C. In some embodiments, exemplary biparatopic antibodies have a Tm greater than 50° C.

[0150] In some embodiments, a peptide linker is used to connect a scFv or a single-chain antibody to the Fc domain of a Fab. Several examples of suitable linkers include a single glycine (G) residue; a glycine 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 peptide GGGGS, peptide GGGGSGGGGS, peptide GGGGSGGGGSGGGGS, peptide GGGGSGGGGSGGGGSGGGGS, peptide GGSGSSGSGG, QRIEG, and peptide GQPKAAP. Other suitable linkers include single Ser and Val residues; dipeptides RTQP, SS, TK, SL, TKGPS, TVAAP, QPKAA. The examples listed above are not intended to limit the scope of the present disclosure in any way, and have been shown to be suitable for binding proteins including 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. For further description of linker sequences, see, for example, WO2012135345.

[0151] The identity and sequence of amino acid residues in the linker can vary depending on the type of secondary structural elements required to be achieved in the linker. For example, glycine, serine and alanine are most preferably used for linkers with maximum flexibility. If a more rigid and extended linker is desired, some combination of glycine, proline, threonine and serine is applicable. Depending on the desired properties, any amino acid residue can be considered as a linker combined with other amino acid residues, as needed, to construct a larger peptide linker.

[0152] Target

[0153] The Fc variants of the present invention can target almost any antigen, including but not limited to proteins, subunits, domains, motifs and / or epitopes belonging to the following target list: 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, neurilemmal, anti-Id, ASPAR TIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B lymphocyte stimulating 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-2BMP-2a, BMP-3 osteogenic factor, BMP-4BMP-2b, BMP- 5. BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, 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) C1 0, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin 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, CCL28, 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, C D25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), 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, Clostridium botulinum toxin, Clostridium perfringens 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, DNA enzyme, 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, Fractal Chemokine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6. 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-α1, GFR-α2, GFR-α3, GITR, glucagon, Glut 4, 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), HepB 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), HIVgp120, HIVIIIB gp120 V3 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)-α, INF-β, INF-γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1,Integrin α2, Integrin α3, Integrin α4, Integrin α4 / β1, Integrin α4 / β7, Integrin α5 (αV), Integrin α5 / β1, Integrin α5 / β3, Integrin α6, Integrin β1, Integrin β2, Interferon γ, 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), Potential TGF-1, Potential TGF-1bpi, LBP, LDGF, LECT2, Lefty, Lewis Y antigen (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, metalloproteinases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, M MP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mud), MUC18, Muellerian-inhibitin substance), Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, neurotrophin-4 or neurotrophin-6, neurotrophin, neuron growth factor (NGF), NGFR, NGF-β, 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, RSV Fgp, 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 receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testis P LAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β pan-specific, TGF-βR1 (ALK-5), TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, 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 (TWEAKR FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF R1CD120a, p55-60),TNFRSF1B (TNFRIICD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40ACT35, 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 titin, 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, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, Lewis Y-related carbohydrate expressing tumor-associated antigen, 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 integrin, von Willebrands 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 receptors for hormones and growth factors.

[0154] In some embodiments, antibodies comprising Fc variants specifically bind to complement factors. In some embodiments, antibodies comprising Fc variants specifically bind to SARS-CoV-2. In some embodiments, antibodies comprising Fc variants specifically bind to amyloid beta fibrils. In some embodiments, antibodies comprising Fc variants specifically bind to LAG-3. In some embodiments, antibodies comprising Fc variants specifically bind to CD3. In some embodiments, antibodies comprising Fc variants specifically bind to VEGF. In some embodiments, antibodies comprising Fc variants specifically bind to Ang-2. In some embodiments, antibodies comprising Fc variants specifically bind to PD-1. In some embodiments, antibodies comprising Fc variants specifically bind to EGFR. In some embodiments, antibodies comprising Fc variants specifically bind to IFNAR1. In some embodiments, antibodies comprising Fc variants specifically bind to CD19. In some embodiments, antibodies comprising Fc variants specifically bind to IL-17A. In some embodiments, antibodies comprising Fc variants specifically bind to IL-17B. In some embodiments, antibodies comprising Fc variants specifically bind to IL-13. In some embodiments, antibodies comprising Fc variants specifically bind to angiopoietin-like 3. In some embodiments, antibodies comprising Fc variants specifically bind to nerve growth factor. In some embodiments, antibodies comprising Fc variants specifically bind to Ebola virus. In some embodiments, antibodies comprising Fc variants specifically bind to HER2. In some embodiments, antibodies comprising Fc variants specifically bind to GD2. In some embodiments, antibodies comprising Fc variants specifically bind to BCMA. In some embodiments, antibodies comprising Fc variants specifically bind to IL-6R. In some embodiments, antibodies comprising Fc variants specifically bind to TROP-2. In some embodiments, antibodies comprising Fc variants specifically bind to IGF-1R. In some embodiments, antibodies comprising Fc variants specifically bind to CD38. In some embodiments, antibodies comprising Fc variants specifically bind to adhesion protein-4. In some embodiments, antibodies comprising Fc variants specifically bind to P-selectin. In some embodiments, antibodies comprising Fc variants specifically bind to CD79b. In some embodiments, antibodies comprising Fc variants specifically bind to sclerostin. In some embodiments, antibodies comprising Fc variants specifically bind to IFNγ. In some embodiments, antibodies comprising Fc variants specifically bind to CCR4. In some embodiments, antibodies comprising Fc variants specifically bind to CGRP receptors. In some embodiments, antibodies comprising Fc variants specifically bind to receptors for complement factors. In some embodiments, antibodies comprising Fc variants specifically bind to C5a receptor 1.In some embodiments, antibodies comprising Fc variants specifically bind to C5a. In some embodiments, antibodies comprising Fc variants specifically bind to C3. In some embodiments, antibodies comprising Fc variants specifically bind to C3a. In some embodiments, antibodies comprising Fc variants specifically bind to C3b. In some embodiments, antibodies comprising Fc variants specifically bind to C3 receptor. In some embodiments, antibodies comprising Fc variants specifically bind to C10.

[0155] Carrier

[0156] Further provided herein are vectors comprising a nucleotide sequence encoding an isolated polypeptide comprising an Fc variant, as described herein.

[0157] In one embodiment, the vector includes a nucleic acid described herein. For example, the vector may include a first nucleic acid and a second nucleic acid, wherein the first nucleic acid and the second nucleic acid respectively encode a heavy chain variable region and a light chain variable region of one or more antibody molecules selected from the antibody molecules disclosed herein.

[0158] 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 having one or more substitutions, e.g., conservative substitutions).

[0159] Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). A variety of vector systems can be used. For example, one type of vector 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 type of vector utilizes RNA elements derived from RNA viruses, such as Semliki Forestvirus, Eastern Equine Encephalitis virus, and Flaviviruses.

[0160] In addition, can select the cell that DNA is stably integrated into its chromosome by introducing one or more markers that allow to select transfected host cells.Marker can provide the original trophotype of for example auxotrophic host, biocide resistance (for example, antibiotic) or resistance to heavy metal (for example copper) etc.Selectable marker gene can be directly connected to DNA sequence to be expressed or introduced into the same cell by cotransformation.The most preferred synthesis of mRNA may also need other elements.These elements can comprise splicing signal and transcription promoter, enhancer and termination signal.

[0161] Once an expression vector or DNA sequence containing a construct is prepared for expression, the expression vector can be transfected or introduced into an appropriate host cell. A variety of techniques can be used to achieve this, such as plasmid fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. In the case of plasmid fusion, cells are grown in culture medium and screened for appropriate activity.

[0162] The methods and conditions for culturing the resulting transfected cells and recovering the produced antibody molecules are known to those of ordinary skill in the art and can be modified or optimized based on the description herein, depending on the specific expression vector and mammalian host cell employed.

[0163] Therapeutic Uses

[0164] Isolated polypeptides comprising Fc variants (e.g., antibodies) of the invention can be used to treat 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 virus infection, HER2+ breast cancer, multiple myeloma, breast cancer, thyroid eye disease, sickle cell disease, HIV infection, gastric cancer, anthrax infection, osteoporosis, Crohn's disease, ANCA-associated vasculitis, lupus, rheumatoid arthritis, inflammatory bowel disease, C3 glomerulopathy (C3G), C3 glomerulonephritis (C3GN), dense deposition disease (DDD), suppuration Hidradenitis (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, pustular gangrene, psoriasis, paroxysmal nocturnal hemoglobinuria with extravascular hemolysis, acute kidney injury (AKI), chronic kidney disease (CKD), geographic atrophy (GA), autoimmune hemolytic anemia (AIHA), and age-related macular degeneration (AMD).

[0165] In some embodiments, the present invention provides a method of treating a disease or condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of an isolated polypeptide comprising an Fc variant. In some embodiments, the disease or condition is ANCA-associated vasculitis. In some embodiments, the disease or condition is C3 glomerulopathy (C3G).

[0166] In some embodiments, the Fc variants of the present invention can be engineered into antibodies that specifically bind to an antigen. In some embodiments, the Fc variants of the present invention can be engineered into antibodies listed in Table 3.

[0167] Table 3. Commercially available therapeutic antibodies

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] Examples

[0178] Other features, objectives and advantages of the present disclosure are apparent in the following examples. However, it should be understood that the examples, although indicating embodiments of the present disclosure, are given only in an illustrative and non-limiting manner. Various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art from the examples.

[0179] Example 1. Generation of new Fc mutations

[0180] Fc-γ receptors recognize the Fc region of IgG antibodies and bind to it. This combination regulates immune response by triggering effector functions. In some disease indications, therapeutic antibodies interact with Fc-γ receptors and enhance natural Fc-γ receptor activation may be beneficial, but for other indications, this may be harmful. In the case of other activations that are harmful, Fc engineering is required to silence the IgG Fc domain so that it cannot bind to the Fc-γ receptor. In this example, new Fc variants are engineered, which are particularly effective in silencing the IgG Fc domain. It is worth noting that the combination of Fc mutations of the present invention is new and effective in eliminating affinity for Fc-γ receptors and C1q. Fc variants of the present invention are shown in Table 1.

[0181] Table 1. Novel Fc variants

[0182] Fc mutation Subtype VfC07 L234F / L235E / D265G IgG1 VxF L234F / L235E / G237A / D265G IgG1 VFc09 L234V / L235E / G237A / D265G IgG1 VFc10 L234F / L235E / D265G / A330S / P331S IgG1 VFc11 L234V / L235A / G237A / D265G IgG1 VFc12 L234V / L235A / G237A / D265G / A330S / P331S IgG1 VFc13 D265G IgG1 VFc16 L235E / D265G IgG4-S228P VFc17 F234V / L235E / D265G IgG4-S228P VFc18 F234V / L235A / G237A / D265G IgG4-S228P VFc19 L235E / G237A / D265G IgG4-S228P VFc20 L235E / G237A / P329G IgG4-S228P VFc21 L235E / G237A / L328R IgG4-S228P VFc22 D265G / A330S / P331S IgG2 VFc23 A235E / D265G / A330S / P331S IgG2 VFc24 A235E / D265G / P329G IgG2

[0183] Example 2. Fc variants show significantly reduced Fcγ receptor and C1q binding

[0184] This example demonstrates that the novel Fc variants shown in Table 1 successfully abrogate Fc-gamma receptor and CIq binding. The Fc variants were engineered into antibodies "Abl" and "Ab2," each of which includes a different variable domain and targets a different antigen.

[0185] Method for measuring binding between Fc-γ receptor and C1q

[0186] This protocol describes a method that can be used to determine the degree of binding of Fc-silent antibodies to Fc-γ receptors or C1q using biolayer interferometry (BLI) or ELISA on an Octet Red 384 system. FcγRI, FcγRIIa H167, FcγRIIb, FcγRIIIaV176 and FcγRIIIb binding are evaluated using NiNTA of a HIS1K biosensor. In both procedures, the Fc-γ receptor is first loaded onto the biosensor, and then the biosensor is blocked with casein or BSA to prevent nonspecific binding of antibodies to any unbound ligand on the biosensor. Next, the biosensor is introduced into the antibody, and then returned to the blocking buffer for a short dissociation phase. The resulting binding sensorgram is used to quantitatively evaluate the intensity of each measured interaction, and the maximum equilibrium binding response is used as a quantitative readout, wherein the maximum equilibrium binding response is proportional to the binding affinity. According to methods known in the art, the binding to C1q is measured in a similar manner.

[0187] result

[0188] like Figure 1A As shown in Figure 2-C, all Fc variants resulted in significantly reduced binding to FcγRI, FcγRIIa and FcγRIIb compared to their counterpart wild-type IgG isotypes. Notably, for both Abl and Ab2, the Fc region of any of vFc07-vFc12 and vFc16-vFc24 did not bind to FcγRI, FcγRIIa and FcγRIIb. It is also noteworthy that a single mutation, D265G, was able to significantly reduce FcγRI, FcγRIIa and FcγRIIb binding (compare Figure 1A -C vFc13 relative to IgG1 WT). Additionally, all Fc variants resulted in significantly reduced binding to C1q compared to their counterpart wild-type IgG isotype ( Figure 1D ), regardless of the variable region (Ab1 or Ab2).

[0189] Example 3. Additional validation of two Fc variants in reducing Fcγ receptor and C1q binding

[0190] This embodiment verifies that the Fc variants of the present invention successfully eliminate the binding of Fc-γ receptors and C1q. In this particular example, 2 Fc variants, vFc10 and vFc17, were used in the experiment. vFc10 includes L234F / L235E / D265G / A330S / P331S mutations in the IgG1 isotype, and vFc17 includes F234V / L235E / D265G mutations in the IgG4-S228P isotype. (The S228P mutation reduces Fab arm exchange by stabilizing the disulfide in the core hinge of the IgG molecule.) These two Fc variants are each engineered into antibody "Ab1". The combination of the engineered antibody and FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb and C1q is tested by the method described in Example 2.

[0191] The major drawback of antibodies is their demanding production requirements (Garber, 2001, Nat Biotechnol 19:184-185; Dove, 2002, Nat Biotechnol 20:777-779, which are 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 Abl are expressed in culture according to methods known in the art, and expression yields are measured. As described in Figure 2A As shown in, both antibodies with vFc10 or vFc17 Fc variants produced high yields, with yields significantly higher than wild-type IgG1 antibodies, and yields comparable to or higher than wild-type IgG4 antibodies. As will be appreciated by those of ordinary skill in the art, Protein A interacts with the Fc portion of immunoglobulins. Therefore, Octet experiments were performed to check that the mutations introduced into the Fc variants did not alter the Protein A binding properties. Figure 2B The Fc variants were shown to display similar binding to wild-type IgG1 or IgG4, indicating that the mutations did not alter the Protein A binding properties.

[0192] like Figure 3A As shown, when paired with antibodies, both vFc10 and vFc17 abolished FcγRI binding to near baseline levels. Similarly, both vFc10 and vFc17 abolished FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb binding to near baseline levels ( Figure 3B-3C ). IgG4 has a short hinge and low Fab arm flexibility, which partially prevents it from binding to C1q. Figure 3D It is shown that neither vFclO nor vFcl7 induce C1q binding when paired with Abl.

[0193] In summary, the data in this example show that both Fc variants tested showed abrogated binding to all of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q when paired with the variable region of an antibody.

[0194] Example 4. Fc variants show significantly reduced ADCC, ADCP and CDC induction

[0195] Antibodies of the IgG subclass are bifunctional molecules, having a F(ab) domain, which is variable in sequence and responsible for antigen binding, and an Fc domain, which is constant in sequence and responsible for mediating a range of antibody effector functions. These functions are mainly triggered by interaction with complement component C1q or with the FcγR family, which is mainly expressed on the surface of leukocytes. Fcγ receptors (FcγRs) trigger cell-mediated cytotoxic effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0196] Antibody-dependent cellular cytotoxicity (ADCC) is the Fc-dependent effector function of IgG, which is important for antiviral immunity and anti-tumor therapy. NK cell-mediated ADCC is mainly triggered by IgG-Fc receptor (FcγR) IIIa. Phagocytes including monocytes, macrophages, neutrophils, eosinophils and dendritic cells (DC) express FcγRI, FcγRII and FcαRI, which can mediate immune complex uptake. ADCP causes immune complexes to be cleared from infected hosts by transporting complexes to lysosomes for degradation and antigen processing to present on major histocompatibility complex (MHC) molecules on the cell surface. Interestingly, some viruses have used this mechanism to infect phagocytes by escaping from lysosomal degradation (described below in "antibody-dependent infection enhancement").

[0197] This example shows that the Fc variants of the present invention that are capable of abolishing binding to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb and C1q have reduced ADCC, ADCP and CDC functions.

[0198] In this particular example, 2 Fc variants, vFc10 and vFc17, were used in the experiment. vFc10 includes L234F / L235E / D265G / A330S / P331S mutations in the IgG1 isotype, and vFc17 includes F234V / L235E / D265G mutations in the IgG4-S228P isotype. The two Fc variants were each engineered into antibody "Ab1". The amount of ADCC, ADCP and CDC induction was measured according to the antibody concentration.

[0199] Figure 4A It is shown that VFc10 and VFc17 maintain low ADCC compared to wild-type IgG1 antibody. Similarly, VFc10 and VFc17 maintain low ADCP and CDC ( Figure 4B and Figure 4C ).

[0200] In summary, the data in this Example show that the Fc variants of the present invention abolish binding to all FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb and C1q, and effectively reduce ADCC, ADCP and CDC functions.

[0201] Equivalents and scope

[0202] 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 present disclosure is not intended to be limited to the above description, but rather is set forth in the following claims.

Claims

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

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

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

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

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

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

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

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

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

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

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

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

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

15. The isolated polypeptide according to any one of the preceding claims, wherein the Fc variant is an IgG1 Fc region, an IgG2 Fc region or an IgG4 Fc region.

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

17. An antibody comprising the isolated polypeptide according to any one of claims 1-16.

18. A nucleic acid encoding the isolated polypeptide according to any one of the preceding claims.

19. A cell comprising the nucleic acid according to claim 18.

20. A method for preparing an isolated polypeptide according to any one of claims 1 to 16, the method comprising: culturing a host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof; and culturing the cell under conditions allowing production of the isolated polypeptide.

21. Use of an isolated polypeptide according to any one of claims 1 to 16 or an antibody according to claim 17 in the preparation of a medicament for treating a disease or disorder.

22. An isolated polypeptide according to any one of claims 1 to 16 or an antibody according to claim 17 for use in treating a disease or disorder.

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