Fc variants and preparation thereof

Abstract

To provide novel Fc variants having modified binding affinity to FcRn, preferably higher binding affinity to FcRn.SOLUTION: The problem is solved by an Fc variant that binds to human FcRn with a higher affinity as compared with wild type human Fc protein, where the Fc variant comprises an amino acid sequence of SEQ ID NO:22 with a combination of substituted amino acids at specific EU positions of wild type Fc protein.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to Fc variant proteins and their preparation. The Fc variants have altered binding affinity to FcRn. The Fc variants prepared according to the present invention can be used to prepare FcRn antagonist compositions or to prepare Fc variant-containing drugs or molecules with altered effector functions. [Background technology]

[0002] The neonatal Fc receptor (FcRn) is structurally homologous to major histocompatibility complex (MHC) class I heterodimeric molecules, consisting of a type I transmembrane heavy chain noncovalently associated with a soluble light chain, β2-microglobulin (β2m). β2m is essential for the proper folding, trafficking, and function of FcRn and other MHC class I homologs. The FcRn heavy chain contains three soluble extracellular domains (α1, α2, and α3), a single transmembrane helix, and a cytoplasmic tail. Unlike MHC class I molecules, FcRn does not directly present antigens to T cells due to point mutations on its surface that disrupt peptide binding. The ability of FcRn to protect IgG from intracellular catabolism is the result of a specific pH-dependent interaction with the Fc portion of IgG. IgG binds to FcRn in a strictly pH-dependent manner at acidic pH (<6.5) but not at neutral pH (>7), mediated by electrostatic interactions between adjustable histidine residues within the CH2-CH3 domain of the IgG Fc and acidic residues on the α2-domain of FcRn. Binding is further stabilized by a series of hydrophobic interactions and hydrogen bonds between the Fc and residues within the FcRn α2 domain and the N-terminus of the β2m light chain. The homodimeric nature of IgG allows one IgG molecule to simultaneously bind to two FcRn molecules, increasing the avidity of the interaction, resulting in a high affinity interaction between FcRn and IgG at pH 6. The 2:1 interaction between FcRn and IgG is important for efficient binding, recycling, and transcytosis in FcRn-expressing cells, ultimately maintaining the long-term serum persistence of IgG, thereby eliminating the need for repeated production of protective antibody molecules by B cells and providing efficient immunity against infection. Unfortunately, this same interaction is also involved in the pathogenesis of IgG molecules reactive against self-antigens, leading to many autoimmune disorders. Mice lacking FcRn are protected from IgG-mediated autoimmune diseases, suggesting that FcRn contributes, at least in part, to autoimmunity by maintaining pathogenic IgG in the circulation longer. Because FcRn contributes to the serum persistence of IgG, therapies that block IgG-FcRn interactions represent a potential modality for the treatment of IgG-mediated autoimmunity.High-dose intravenous immunoglobulin (IVIg) therapy has been approved by the FDA for the treatment of IgG-mediated autoimmune diseases, providing partial therapeutic benefit by saturating FcRn receptors and thereby enhancing the catabolism of pathogenic IgG. However, IVIg therapy faces several challenges. For example, because IVIg is a blood-derived product, it is susceptible to contamination with human pathogens such as HIV, HBV, and HCV. Therefore, the treatment of chronic disorders such as autoimmune diseases poses significant risks to recipients. Furthermore, IVIg therapy is expensive because it requires IgG extraction from the plasma of many donors. Furthermore, scalability is a concern due to its dependency on human donors. Therefore, novel recombinant approaches are now needed to provide safe, scalable, and cost-effective alternative treatment options. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent No. 8,067,232 [Patent Document 2] U.S. Patent No. 7,083,970 [Patent Document 3] U.S. Patent No. 7,524,647 [Patent Document 4] International Publication No. 2007 / 017903 [Patent Document 5] International Publication No. 2012 / 046255 Summary of the Invention [Means for solving the problem]

[0004] Monoclonal antibody-based antagonists have been developed to inhibit endogenous IgG-FcRn interactions. One approach is the use of monoclonal antibodies against FcRn that act via the classical antibody:antigen binding mechanism. Examples of such antibodies are M281 and UCB7665, which bind to human FcRn and inhibit IgG binding to FcRn, thereby promoting the clearance of endogenous IgG. M281 has been shown to produce a dose-dependent mean reduction in endogenous IgG levels ranging from 25 to 80%. Furthermore, a single dose of M281 at 30 mg / kg or 60 mg / kg maintained serum IgG levels below 50% of baseline for 18 and 27 days, respectively (1). In healthy subjects, serum IgG levels were reduced by up to 50% with a single IV dose of UCB7665 at 7 mg / kg. The decline in serum IgG concentrations observed in this study persisted for several weeks, with the maximum decline achieved by days 7–10, followed by a gradual return to baseline by day 57 ( 2 ).

[0005] Another antibody engineering approach to lowering serum IgG levels involves engineering the Fc domain of IgG to have high affinity for FcRn at both pH 6 and pH 7.4. Such molecules, developed using "Abdegs" (i.e., antibodies that promote IgG degradation) technology, have been shown to be effective in treating mouse models of arthritis at doses 25–50 times lower than IVIg, suggesting that potent IgG-FcRn-based antagonists of the IgG-FcRn interaction represent an alternative therapeutic intervention in autoimmunity. This molecule, Efgartigimod, is currently in clinical development by arGEN-X, a Netherlands-based biotechnology company.

[0006] More recently, synthetic peptides that compete with IgG for binding to FcRn have been identified from phage libraries. A chemically optimized peptide dimer (SYN1436) that binds to FcRn with subnanomolar affinity at pH 6 and pH 7.4 was effective in increasing the clearance of exogenous IgG in mice and endogenous IgG in monkeys, but has not been evaluated in therapeutic models of IgG-mediated autoimmunity. Alternatively, additional molecules that block IgG-FcRn interactions by binding at the CH2-CH3 domain interface instead of Fc itself include a 13-amino acid cyclic peptide (FcIII) selected by phage display, a computationally designed IgG-Fc binding protein (FcBP6.1), and the endogenous Fc receptor TRIM21 (3). Thus, the present invention provides FcRn binders that address various unmet needs, such as downregulating FcRn activity, extending the circulating half-life of various drugs, and targeting drugs and / or vaccines to specific cell types. The FcRn binders of the present invention can be used to treat a variety of diseases.

[0007] Another class of molecules targets immune complex (IC) formation and immune complex-mediated FcγR-mediated activation. This class of molecules includes recombinant Fc multimers such as Pf-06755347 (GL-2045) and CSL730 (M230). These molecules are structurally complex and can be highly heterogeneous. These molecules are being developed to treat autoimmune diseases. Therefore, molecules targeting different mechanisms are currently in early clinical trials and are being developed as alternatives to intravenous immunoglobulin (IVIg) and subcutaneous immunoglobulin (SCIg) to avoid the reliance on human plasma supplies and the high doses of up to 2 g / kg body weight required for treatment. In this context, it would be advantageous to have a single drug that combines the effects of multiple mechanisms of action, such as FcRn blockade, inhibition of FcγR activation, and / or inhibition of complete complement activation.

[0008] In one aspect, the present invention provides Fc variant molecules that are FcRn binders as well as FcγR binders, and that can simultaneously address various needs, such as downregulation of FcRn activity, extension of the circulating half-life of various drugs, IC inhibition, inhibition of cytokine release mediated by IC formation, and inhibition of phagocytosis mediated by FcγR activation. Preferably, the Fc variant molecules prepared according to the present invention provide one or more of the above-mentioned desired effects at a lower dose compared to the dose of IVIg.

[0009] The present invention provides novel Fc variants with altered binding affinity to FcRn, preferably higher binding affinity to FcRn. In one aspect, the Fc variants of the present invention have altered binding affinity to FcγR, preferably higher binding affinity to FcγR. Preferably, the Fc variants can be used to develop pharmaceutical compositions with FcRn antagonist function or extended circulating half-life, or to target specific cells and / or tissues. The present invention also provides methods for producing novel Fc variants. The Fc variants of the present invention can be further used in the preparation of drugs for treating diseases in which FcRn activity is deleterious, or for extending the circulating half-life of drugs, or for targeting drugs to certain cells or tissues. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows a map of the vector used for library construction to generate the Fc variants of the present invention. [Figure 2] FIG. 1 shows maps of vectors used to produce the Fc monomers and Fc dimers of the present invention. [Figure 3] FIG. 1 shows a map of a vector used to generate a monoclonal antibody comprising an Fc monomer of the present invention. [Figure 4] FIG. 1 shows the results of experiments to determine the effect of Fc variants (Fc dimers) and IVIg on total IgG serum levels in wild-type mice. [Figure 5] FIG. 1 shows the results of experiments to determine the effect of Fc variants (Fc dimers) and IVIg on serum albumin levels in wild-type mice. [Figure 6] FIG. 1 shows the results of experiments to determine the effect of Fc variants on ADCC activity. [Figure 7] FIG. 1 shows the results of experiments to determine the effect of Fc variants on platelet counts in a mouse model of acute immune thrombocytopenia (ITP). DETAILED DESCRIPTION OF THE INVENTION

[0011] definition The term "defucosylated" as used herein refers to glycosylated proteins having N-linked glycans lacking a core fucose molecule as described in U.S. Pat. No. 8,067,232, the entire contents of which are incorporated herein by reference.

[0012] The term "amino acid modification" as used herein is an amino acid substitution, insertion, and / or deletion in a polypeptide sequence.

[0013] The term "amino acid substitution" or "substitution" as used herein refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. For example, the substitution T307N refers to a variant polypeptide, in this case an Fc variant, in which the threonine at position 307 has been replaced with an asparagine.

[0014] The term "antigen-binding molecule" according to the present invention refers to a protein capable of binding to a target antigen and comprising an "FcRn-binding domain." The FcRn-binding domain according to the present invention is present in an Fc protein, preferably in an Fc variant of the present invention. Preferred "antigen-binding molecules" according to the present invention may include antibodies or peptibodies or fusion proteins comprising the Fc variants of the present invention.

[0015] The term "antibody" as used herein includes whole antibodies and any antigen-binding fragments (i.e., "antigen-binding portions"). An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain contains a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated as V) and a heavy chain constant region (Fc). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The light chain constant region is composed of C L It consists of one domain: V H Area and V L The regions can be further subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with regions of high conservation called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells such as NK cells, T cells, macrophages, and dendritic cells) and the first component (C1q) of the classical complement system.

[0016] The term "operably linked" is intended to mean that a gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of such gene.

[0017] "K a The term "K" refers to the binding rate of the interaction between two molecules. d The term "K" is the dissociation rate of the interaction between two molecules. D The term "affinity rate constant" is K d Ka This can be measured using surface plasma resonance techniques well known in the art.

[0018] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. The term "bispecific antibody" refers to a homogeneous antibody population involved in the highly specific recognition and binding of two different antigenic determinants or epitopes. The term "recombinant antibody" as used herein includes all antibodies prepared, expressed, generated, or isolated by recombinant means. However, in certain embodiments, such recombinant antibodies can be obtained by in vitro mutagenesis, thus allowing for the V of the recombinant antibodies described herein to be further refined. H Area and V L The amino acid sequence of the region is one that may not naturally occur in vivo within the human antibody germline repertoire. As used herein, the term "effector function" refers to a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. The term also refers to physiological events such as the circulating half-life of a drug or the targeting of a drug to a specific cell or tissue type.

[0019] The term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" as used herein refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibody on target cells and subsequently cause lysis of the target cells.

[0020] The term "ADCP" or "antibody-dependent cell-mediated phagocytosis" as used herein refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibody on target cells and subsequently cause phagocytosis of the target cells.

[0021] The term "effector cells," as used herein, refers to cells that express one or more Fc receptors and mediate one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and γδ T cells, and can be from any organism, including, but not limited to, human, mouse, rat, rabbit, or monkey.

[0022] The term "Fc" fragment reflects its ability to readily crystallize. The crystal structure of the Fc region of human IgG has been determined (4). In human IgG molecules, the Fc region is generated by papain cleavage from the N-terminus to Cys226. The Fc region is central to antibody effector function.

[0023] The term "Fc protein" as used herein means the portion of a single immunoglobulin heavy chain beginning at the hinge region immediately upstream of the papain cleavage site and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes the hinge (e.g., upper, middle, and / or lower hinge regions), CH2 domain, and at least a portion of the CH3 domain.

[0024] As used herein, the term "Fc variant-containing protein" refers to any molecule comprising an Fc variant of the present invention. Preferably, Fc variant-containing proteins include antibodies, fusion proteins, and peptibodies. Antibodies, fusion proteins, and peptibodies as referred to herein include antibodies, fusion proteins, and peptibodies that are approved or in clinical or preclinical trials.

[0025] The term "Fc variant-containing molecule" as used herein means any molecule comprising an Fc variant of the invention. It may be a fusion product in which an Fc variant of the invention is linked or conjugated to a drug, which may be a small peptide or receptor or toxin or any chemical molecule.

[0026] As used herein, the term "Fc variant protein" or "Fc protein variant" or "Fc variant" refers to an Fc protein that differs from that of a wild-type Fc protein by at least one amino acid modification. An Fc variant can refer to the Fc variant itself, a composition comprising the Fc variant, or the amino acid sequence encoding it. Preferably, an Fc variant has at least one amino acid modification compared to the parent protein, e.g., about 1 to about 10 amino acid modifications, preferably about 1 to about 5 amino acid modifications, compared to the parent.

[0027] The term "EU position" as used herein refers to an amino acid position in the EU numbering system for the Fc region as set out in reference 5.

[0028] The term "CH1 domain" as used herein means the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain, extending from about EU position 118 to EU position 215. The CH1 domain is the V domain of an immunoglobulin heavy chain molecule. H It is adjacent to the amino terminus of the domain and hinge region and does not form part of the Fc region of an immunoglobulin heavy chain.

[0029] As used herein, the term "hinge region" refers to the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper hinge domain, the middle hinge domain, and the lower hinge domain (6). The Fc variants of the present invention may comprise all or part of the hinge region.

[0030] The term "CH2 domain" as used herein means the portion of a heavy chain immunoglobulin molecule extending from about EU positions 231-340.

[0031] The term "CH3 domain" as used herein means the portion of a heavy chain immunoglobulin molecule extending from the N-terminus of the CH2 domain to about 110 residues, e.g., from about positions 341 to 446 (EU numbering system).

[0032] As used herein, the term "Fc gamma receptor" or "FcγR" refers to a member of a family of proteins that bind to the Fc region of an IgG antibody and are encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), including the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb1 and FcγRIIb2), and FcγRIIc; FcγRIII (CD16) (7), including the isoforms FcγRIIIa (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIIbNA1 and FcγRIIIbNA2), as well as any unidentified human FcγR or FcγR isoforms or allotypes. FcγRs can be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγR or FcγR isoform or allotype.

[0033] As used herein, the term "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn can be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. As is known in the art, a functional FcRn protein often comprises two polypeptides, referred to as a heavy chain and a light chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. As used herein, unless otherwise specified, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and beta-2-microglobulin.

[0034] The term "wild-type Fc" as used herein refers to an unmodified Fc polypeptide that is later modified to produce a variant. A wild-type Fc may be a naturally occurring polypeptide or a recombinant version of a naturally occurring polypeptide. A wild-type Fc may refer to the unmodified Fc polypeptide itself, a composition comprising the unmodified Fc polypeptide, or the amino acid sequence encoding it.

[0035] The term "position" as used herein refers to a location in the sequence of a protein. Positions may be numbered sequentially or according to established formats, such as the EU index, such as Kabat. For example, position 305 is a position in human antibody IgG1.

[0036] The terms "patient" and "subject" are used interchangeably and are used in their conventional sense to refer to an organism suffering from or susceptible to a condition that can be prevented or treated by administering a composition of the invention, including both human and non-human animals. Examples of subjects include, but are not limited to, humans, chimpanzees, and other ape and monkey species; livestock, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats, and guinea pigs; and birds, including poultry, wild birds, and game birds, such as chickens, turkeys, and other pheasants, ducks, and geese. The terms do not denote a particular age. Thus, adults, juveniles, and newborns are included.

[0037] The term "residue" as used herein refers to a position in a protein and its associated amino acid identity. For example, threonine 307 (also referred to as Thr307, or T307) is a residue in the human antibody IgG1.

[0038] The term "immunoconjugate" or "conjugate" as used herein means a compound or derivative thereof linked to a cell-binding agent (i.e., an antibody or Fc variant described herein) and is defined by the general formula: ALD, where A is the cell-binding agent or antibody or Fc variant of the invention, L is a linker, and D is a drug (toxin or pharmaceutical). Immunoconjugates can also be defined by the general formula ALD in reverse order.

[0039] The term "linker" refers to any chemical moiety capable of linking a compound, typically a drug such as a maytansinoid or auristatin, to a cell-binding agent, such as anti-FcRn or a fragment thereof, in a stable, covalent manner. The linker may be sensitive to or substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions under which the compound or antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. Linkers also include charged linkers and hydrophilic forms thereof, as described herein and known in the art.

[0040] The term "antagonist" refers to an agent that inhibits or reduces the biological activity of an antigen to which it binds, such as FcRn. In certain embodiments, an antagonist substantially or completely inhibits the biological activity of an antigen, such as FcRn. Desirably, the biological activity is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

[0041] The term "treatment" or "therapy," as used herein, means any treatment of a disease in a mammal, particularly a human, including (a) preventing the disease from occurring in a subject susceptible to or at risk of developing the disease, but not yet diagnosed with the disease; (b) suppressing the disease, i.e., arresting the onset of the disease; and (c) palliating the disease, i.e., causing regression of the disease.

[0042] As used herein, the term "wild-type" or "WT" refers to an amino acid or nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid or nucleotide sequence that has not been intentionally modified.

[0043] The term "non-naturally encoded amino acid" refers to an amino acid that is not one of the common amino acids, or pyrrolysine, pyrroline-carboxy-lysine, or selenocysteine. Other terms that may be used synonymously with the term "non-naturally encoded amino acid" are "non-natural amino acid," "unnatural amino acid," "non-naturally occurring amino acid," and various hyphenated and non-hyphenated versions thereof. The term "non-naturally encoded amino acid" also includes, but is not limited to, amino acids that arise by modification (e.g., post-translational modification) of naturally encoded amino acids (including, but not limited to, the 20 common amino acids, or pyrrolysine, pyrroline-carboxy-lysine, and selenocysteine), but that are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0044] [Table 1]

[0045] Other abbreviations used in this patent application: %:percentage ℃: Celsius temperature μg: microgram μL: microliter A: Adenine ADCC: Antibody-dependent cytotoxicity C: Cytosine CDC: Complement-dependent cytotoxicity CFU: colony forming unit CHO: Chinese hamster ovary DNA: deoxyribonucleic acid ELISA: Enzyme-linked immunosorbent assay EC 50: 50% of the maximum effective concentration of the drug EC 75 : 75% of the maximum effective concentration of the drug Fc: crystallizable fragment FcGRT: Fc fragment of IgG receptor and transporter FcRn: neonatal Fc receptor FcγRs: Fc gamma receptors G: Guanine h: time H2SO4: sulfuric acid HRP: horseradish peroxidase IC: immune complex IgG: immunoglobulin IPTG: Isopropyl β-D-1-thiogalactopyranoside ITP: Acute immune thrombocytopenia IVIg: intravenous immunoglobulin K a / k assoc : Coupling constant K d / k dissoc :Dissociation constant K D :equilibrium dissociation constant M: mole mg: milligram MgCl2: Magnesium chloride min:minutes mL: milliliter mM: millimolar MOI: Multiplicity of infection NaCl: Sodium chloride NaHCO3: Sodium bicarbonate ng: nanogram nm: nanometer OD: optical density OPD: o-phenylenediamine dihydrochloride PBS: Phosphate buffered saline PBST: Phosphate buffered saline containing 0.05% Tween PEG: polyethylene glycol PFU: plaque forming unit rFcRn: recombinant neonatal Fc receptor rhFcRn: recombinant human neonatal Fc receptor rpm: revolutions per minute s: second SCIg: Subcutaneous immunoglobulin SEQ / seq: Sequence SPR: surface plasmon resonance T: Thymine YT medium: Yeast extract-tryptone medium

[0046] Embodiments of the invention The present disclosure relates to novel Fc variant proteins that can be used for therapeutic purposes.

[0047] In one embodiment, the Fc variant proteins or Fc variant-containing proteins or molecules of the invention bind with higher affinity to human FcRn compared to wild-type Fc proteins. -8 M or less, more preferably 10 -10 K below M D K D The value is a measure of the binding affinity of the antibody for its target antigen.

[0048] In one embodiment, an Fc variant according to the invention has an altered (increased or decreased) affinity for an Fc gamma receptor compared to the affinity of a wild-type IgG1 Fc region for said Fc gamma receptor, hi certain embodiments, an Fc variant has an increased affinity for FcγRIIIa (CD16a) compared to the affinity of a wild-type IgG1 Fc region for said Fc gamma receptor.

[0049] In one embodiment, the Fc variants of the invention have a 10 to 15 affinity for FcγRIIIa (CD16a) including allotypes V158 and F158. -7 K below M DIn one embodiment, the Fc variants of the invention inhibit immune complex-mediated FcγR activation as assessed by inhibition of ADCC. In one embodiment, the Fc variants of the invention inhibit FcγR-mediated phagocytosis. In one embodiment, the Fc variants of the invention inhibit FcγR-mediated cytokine release, such as IL-6 or IL-8.

[0050] In one embodiment, the amino acid sequence of the Fc variant is that of an IgG1, IgG2, IgG3, IgG4 or IgG2 / IgG4 isotype, preferably an IgG1 isotype.

[0051] In another embodiment, one or more Fc variants of the invention have altered, reduced, or no effector function. In one embodiment, an Fc variant of the invention or an Fc variant-containing protein has a reduced potential to cause ADCC and CDC safety issues. In a preferred embodiment, an Fc variant of the invention has no CDC activity. In a preferred embodiment, an Fc variant of the invention has reduced ADCC activity compared to the ADCC activity of wild-type Fc or the ADCC activity of IVIg. In one embodiment, an Fc variant of the invention has reduced or no ADCP activity. In one embodiment, an Fc variant of the invention does not interfere with the binding properties of FcRn to albumin.

[0052] In one embodiment, the Fc variants of the invention cross-react with FcRn from species other than human.

[0053] In one embodiment, the Fc variants of the invention have higher binding specificity for human FcRn.

[0054] In one embodiment, the Fc variants or Fc variant-containing proteins or Fc variant-containing molecules of the present invention have an extended half-life in a subject. In certain embodiments, polyethylene glycol or human serum albumin can be linked to the Fc variants of the present invention to further extend the half-life of the Fc variants. In alternative embodiments, the Fc variants of the present invention can include mutations to extend their half-life in a subject. In another alternative embodiment, the Fc variants of the present invention can be expressed in a multimeric form, which can extend their half-life by increasing the molecular size and affinity through the higher avidity of the Fc variant. As used herein, the term "multimeric form" refers to a form of a protein that contains more than one unit of protein, preferably an Fc protein as used herein. For example, it can be a dimer, trimer, tetramer, pentamer, hexamer, etc. For example, a monomeric Fc protein has two binding sites for FcRn and FcγR. Similarly, the Fc dimer according to the present invention has four binding sites for FcRn as well as FcγRs.

[0055] In another embodiment, the Fc variants of the invention are capable of binding to monkey FcRn and may facilitate drug development by providing relevant animal pharmacology and toxicology models.

[0056] In certain embodiments, the Fc variant comprises a variant Fc region comprising a defucosylated N-linked glycan at EU position 297.

[0057] In one embodiment, the Fc variant comprises a variant Fc region that comprises increased sialylation compared to wild-type IgG.

[0058] In one embodiment, the invention provides a composition comprising an Fc variant protein or Fc variant-containing protein or molecule and an acceptable carrier.

[0059] In another embodiment, the Fc variant proteins or Fc variant-containing proteins of the present invention may be used in the treatment of diseases such as infectious diseases, various cancers, autoimmune disorders, and the like.

[0060] In certain embodiments, the Fc variant or Fc variant-containing protein or Fc variant-containing molecule comprises an amino acid sequence selected from the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 347. The sequences comprising SEQ ID NOs: 1 to 347 are set forth in Table 3 herein.

[0061] In another embodiment, the Fc variants of the invention can be used to generate full-length antibodies that have an Fc variant of the invention in place of a conventional Fc region. The antibody can be a modified version of an already approved or discovered antibody, where the modified version of an existing antibody has an Fc variant of the invention. It can also be a novel antibody developed against any target.

[0062] In a particular embodiment, the Fc variants of the invention may be used to manufacture drugs with extended half-lives, where the Fc variants of the invention are fused, linked, or conjugated to drugs that originally have a short half-life in circulation.

[0063] In another embodiment, the Fc variants of the invention can be used to extend the half-life and reduce off-target toxicity of ADCs by increasing recycling via the FcRn receptor.

[0064] In one embodiment, the Fc variants of the present invention may be fused to any drug to improve its half-life and in-vivo stability.

[0065] In one embodiment, the Fc variants of the present invention can be used to replace albumin in albumin fusion drugs to improve pharmacokinetics.

[0066] In one embodiment, mutations in the Fc variants of the invention can be used to improve the affinity of the intact antibody for FcRn at pH 6 and increase its circulating half-life by reducing catabolism.

[0067] In one embodiment, antibody molecules containing Fc variants may be used as scavengers to remove pathogenic proteins or toxins, such as TNFalpha, VEGF, etc., from the circulation.

[0068] In one embodiment, antibody molecules or proteins containing Fc variants can be used as trappers to capture desired proteins, peptides, carbohydrates or drugs from the environment and internalize them into FcRn-expressing target cells.

[0069] In one embodiment, the Fc variants of the present invention can be used to develop monomeric Fc fusion proteins fused to any therapeutic peptide, retaining FcRn binding activity and improving tissue penetration.

[0070] In one embodiment, the Fc variants of the invention can be fused with PEG to further improve their circulating half-life.

[0071] In one embodiment, the Fc variants of the present invention may be used to deliver drugs via a non-invasive route, such as pulmonary administration.

[0072] In one embodiment, the Fc variants of the invention block the IgG binding site on FcRn, thereby speeding up the clearance of endogenous IgG by competing with FcRn.

[0073] In one embodiment, the Fc variants of the invention may improve the potency / effectiveness of a drug independent of pharmacokinetic half-life.

[0074] In one embodiment, the Fc variants of the present invention fused to an immunogen / antigen can improve the delivery of the antigen to APCs expressing FcRn, improving the immune response.

[0075] In one embodiment, the Fc variants of the present invention may be used to orally deliver drugs, including nanoparticles, to various organs across the intestinal epithelium.

[0076] In one embodiment, the Fc variants of the present invention fused to peptides / immunogens may be used for intranasal immunization as a general delivery route for subunit vaccines against many mucosal pathogens.

[0077] In one embodiment, the Fc variants of the present invention fused to any broadly neutralizing antibody may be used to extend the half-life of such antibodies and may also be used to enhance mucosal localization to confer immune protection against viral entry into the gastrointestinal or cervicovaginal tract.

[0078] Detailed Description of the Invention In one embodiment, the Fc variant proteins or Fc variant-containing proteins or molecules of the invention bind to human FcRn with high affinity. The Fc variants of the invention have a lower K for binding to FcRn at pH 6.0 than the native Fc. D The Fc variants of the present invention have an affinity of 10 for FcRn at pH 6.0. -8 M or less, more preferably 10 -10 K below M D K D The value is a measure of the binding affinity of the antibody for its target antigen.

[0079] Amino acid sequences of Fc variants Fc variants of the invention comprise an amino acid substitution in the CH2 or CH3 domain, or in the CH2 and CH3 domains of the Fc protein. More preferably, Fc variants of the invention have an amino acid substitution at one or more residues selected from EU250, EU251, EU252, EU253, EU254, EU255, EU256, EU257, EU258, EU259, EU307, EU308, EU309, EU310, EU311, EU375, EU377, EU379, EU380, EU381, EU432, EU433, EU434 and EU435. Preferred replacement amino acids at positions EU250, EU251, EU252, EU253, EU307, EU308, EU309, EU375, EU377, EU379, EU380, EU381, EU432, EU433, EU434, EU435, EU436, ​​and EU437 are shown in Table 2.

[0080] [Table 2A]

[0081] [Table 2B]

[0082] In a preferred embodiment, the amino acid substitutions in the Fc variants according to the invention are T250Q, T250R, T250A, T250H, T250V, T250K, L251H, L251A, L251E, L251Q, L251R, L251Y, L251C, L251V, M252L, M252V, M252Q, M252Y, M252H, M 252F, I253P, I253S, I253Q, I253R, I253T, I253N, I253Y, S254H, R255Q, T256Q, P257Q, E25 8Y, E258W, V259R, V259G, T307N, T307Y, T307Q, V308P, V308D, V308T, V308S, L309Y, L309S , L309T, L309Q, H310P, H310V, Q311H, Q311N, S375R, I377L, V379I, E380Q, W381Q, L432S, L 432C, L432Q, L432Y, L432P, L432W, L432F, L432A, L432M, H433R, H433T, H433Q, H433P, H43 3K, H433F, H433S, H433N, H433M, N434W, N434Y, N434H, N434F, N434D, N434G, N434I, N434L, N434T, N434Q, H435Q, H435K, H435P, H435N, H435D, Y436F, T437N and suitable combinations thereof.

[0083] The Fc variants of the present invention may contain substitutions at more than one position of the wild-type Fc, referred to herein as "combinations" of substitutions. The amino acid sequence of the wild-type Fc is referred to as SEQ ID NO: 348. Preferred combinations of amino acid substitutions for Fc variants are shown in Table 3. Amino acid substitutions present in a combination are separated by a " / " symbol.

[0084] [Table 3A]

[0085] [Table 3B]

[0086]

Table 3C

[0087]

Table 3D

[0088] Table 3E

[0089]

Table 3F

[0090]

Table 3G

[0091]

Table 3H

[0092]

Table 3I

[0093]

Table 3J

[0094]

Table 3K

[0095]

Table 3L

[0096]

Table 3M

[0097]

Table 3N

[0098] In a preferred embodiment, the combination of substituted amino acids at specific EU positions in an Fc variant according to the invention is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77 7, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 1, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO: No. 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236 6, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267,SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273, SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308 , SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346 and SEQ ID NO: 347.

[0099] In a preferred embodiment, the combination of substituted amino acids at specific EU positions in an Fc variant according to the invention is selected from the sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49.

[0100] In a preferred embodiment, the combination of substituted amino acids at specific EU positions in an Fc variant according to the invention is selected from the sequences set forth in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:20 or SEQ ID NO:22.

[0101] In a more preferred embodiment, the amino acid sequence of the Fc variant of the invention is selected from SEQ ID NO: 22a, SEQ ID NO: 22b, SEQ ID NO: 22c, SEQ ID NO: 22d, SEQ ID NO: 22e and SEQ ID NO: 22f and SEQ ID NO: 22g.

[0102] The residues of the Fc variants described herein, their respective amino acid substitutions, and combinations of amino acid substitutions for specific residues may be present in Fc proteins of the IgG1, IgG2, IgG3, IgG4, or IgG2 / G4 isotype, preferably the IgG1 isotype. Fc variant constructs of the IgG2, IgG4, or IgG2 / G4 isotype may include a single amino acid substitution (i.e., S228P) in the hinge region of the Fc variant to mitigate disruption of the disulfide bond between the two Fc chains (8). The Fc variants prepared according to the present invention may optionally be modified to alter their functionality, for example, to remove residual effector functions such as ADCC and CDC activity. Preferably, the Fc variants of the present invention do not have CDC activity. Preferably, the Fc variants of the present invention have reduced ADCC activity compared to the ADCC activity of the wild-type Fc or the ADCC activity of IVIg. Fc variants according to the present invention have altered (increased or decreased) affinity for Fc gamma receptors compared to the affinity of the wild-type IgG1 Fc region for said Fc gamma receptors. In one embodiment, the Fc variants have increased affinity for FcγRIIIa (CD16a), FcγRIIa (CD32a) and FcγRI (CD64) compared to the affinity of the wild-type IgG1 Fc region for said Fc gamma receptors. In one embodiment, the Fc variants of the present invention have increased affinity for FcγRIIIa (CD16a), FcγRIIa (CD32a) and FcγRI (CD64), including allotypes V158 and F158, over 10 -7 K below M D K DThe AF value is a measure of the binding affinity of the antibody to its target antigen. In one embodiment, the Fc variants of the invention inhibit immune complex-mediated FcγR activation as assessed by inhibition of ADCC. In one embodiment, the Fc variants of the invention inhibit FcγR-mediated phagocytosis. In one embodiment, the Fc variants of the invention inhibit FcγR-mediated cytokine release, such as IL-6 or IL-8. The invention provides Fc variants that can bind to FcRn and FcγR with high affinity. Thus, the Fc variants of the invention possess the combined effects of multiple mechanisms of action in a single drug, such as FcRn blockade and inhibition of FcγR activation, resulting in the potent efficacy of the Fc variants of the invention.

[0103] This combined effect is illustrated in the Examples herein using one of the non-limiting Fc variants of the present invention, which has the substitutions of SEQ ID NO: 22 as set forth in Table 3. The amino acid sequences of the non-limiting Fc variants of the present invention are shown in Table 4 below. A specific variant of SEQ ID NO: 22, SEQ ID NO: 22b, was used in different experiments described in the Examples herein.

[0104] [Table 4]

[0105] In one aspect, the Fc variants of the invention may comprise unnatural amino acids at one or more positions. The introduction of unnatural amino acids into peptides is well known to those skilled in the art. Methods for producing and incorporating non-naturally occurring amino acids into proteins are known (U.S. Patent Nos. 7,083,970 and 7,524,647). In one aspect, the Fc variants of the invention have increased FcRn binding and extended half-life, and altered, reduced, or no ADCC and / or CDC activity. The ADCC and / or CDC activity of the Fc variants of the invention is compared to that of a wild-type Fc protein or IVIg. In one embodiment, the Fc variants of the invention have an amino acid substitution selected from P329G and / or M428L and N434S mutations.

[0106] Preparation of Fc variants The Fc variants of the present invention are prepared using phage display library methods, following the generation of a mutant library using site-directed mutagenesis methods described in the Examples herein.

[0107] Nucleic acid molecules, vectors, and host cells encoding Fc variants In one embodiment, the present invention provides nucleic acid molecules encoding Fc variants or Fc variant-containing proteins, as well as expression vectors comprising such nucleic acids, and host cells comprising such nucleic acid molecules encoding Fc variants derived from expression vectors. Suitable vectors for recombinantly producing the Fc variants or Fc variant-containing proteins of the present invention are known to those skilled in the art. Examples of such known vectors are described in patent publications WO 2007 / 017903 and WO 2012 / 046255, which are incorporated herein by reference. Host cells according to the present invention are prokaryotic or eukaryotic cells; preferably, the host cells are mammalian cells, more preferably CHO cells.

[0108] Pharmaceutical Composition Pharmaceutical compositions can be developed in which one or a combination of the Fc variants or Fc variant-containing proteins or Fc variant-containing molecules of the invention are formulated together with a pharmaceutically acceptable carrier. Such compositions can include one or a combination (e.g., two or more different) of the Fc variants or Fc variant-containing proteins, or immunoconjugates or bispecific molecules of the invention. For example, a pharmaceutical composition of the invention can include a combination of an Fc variant or Fc variant-containing protein and an antibody (or immunoconjugate or bispecific) that binds to different epitopes on the target antigen.

[0109] Therapeutic Use Fc variants or Fc variant-containing proteins or molecules may be used to treat diseases, including therapeutic methods requiring binding of drugs to FcRn.

[0110] In one embodiment of the present invention, Fc variants or Fc variant-containing proteins may be used to inhibit FcRn in vivo in subjects, including but not limited to humans, suffering from diseases such as autoimmune diseases and inflammation. In certain embodiments, the Fc variant or Fc variant-containing protein or Fc variant-containing molecule is an Fc variant or Fc variant-containing molecule that is a member of the Fc variant family of Fc variants, including autoimmune hemolytic anemia, pernicious anemia, idiopathic thrombocytopenic purpura, Goodpasture's syndrome, bullous pemphigoid, pemphigus vulgaris, Hashimoto's thyroiditis, insulin-dependent diabetes mellitus (IDDM), Graves' disease, myasthenia gravis, CIDP, Diabetes, antisera (β-thalassemia), hematopoietic organ drugs, ophthalmic drugs, bone diseases, nervous system genetic diseases, age-related macular degeneration, diabetic retinopathy, macular diseases, non-small cell lung cancer treatment, hereditary eye diseases, hemophilia B, drugs for treating circulatory system diseases (coagulation factor IX deficiency), type 2 diabetes, immunosuppressants, rheumatoid arthritis, treatment of transplant rejection, brain tumors, breast cancer, colon cancer, diabetic retinopathy, digestive system cancer, endocrine system cancer, female reproductive system cancer, stomach cancer, urinary system cancer, macular diseases, melanoma, multiple myeloma, myelodysplastic syndrome treatment, myeloid leukemia treatment, non-Hodgkin's lymphoma treatment, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer treatment, kidney cancer treatment, retinopathy, aplastic anemia The present invention is used to treat diseases selected from the group consisting of chronic anemia, analgesics, hematological genetic diseases, multisystem genetic diseases, osteoarthritis, scleroderma, treatment of autoimmune diseases, gout, urticaria, treatment of ankylosing spondylitis, asthma treatment, dermatological therapeutic agents, idiopathic inflammatory myopathy, immunosuppressants, inflammatory bowel disease, interstitial lung disease, treatment of multiple myeloma, multiple sclerosis, nephrosis, psoriatic arthritis, systemic lupus erythematosus, acute alcoholic hepatitis treatment, Alzheimer's dementia, antiallergic / antiasthmatic agents, antiarthritis drugs, analgesics, breast cancer therapeutic agents, cancer-related diseases, dermatological therapeutic agents, heart failure therapeutic agents, lymphoma therapeutic agents, nephritis, neurological therapeutic agents (other), respiratory diseases, and therapeutic agents for congenital metabolic disorders. The present invention will now be described in detail with reference to the following non-limiting examples, which should not be construed as limiting the scope of the present invention in any way. [Example]

[0111] The following examples are presented to provide those of skill in the art with disclosure and description of how the methods and Fc variants claimed herein are performed. They are intended purely for illustrative purposes and are not intended to limit the scope of the present disclosure. Other Fc variants of the present invention can be developed using the methods described in the provided examples, with some modifications. Such modifications will be known to those of skill in the art.

[0112] Example 1 Preparation of phage display libraries (a) Construction of wild-type Fc plasmid To generate the library, a human IgG1 Fc region containing a partial sequence of the hinge of the CH2 and CH3 domains of the heavy chain (EU221-447) was chemically synthesized with EcoRI and BamHI overhangs and cloned into the pMA / pMK vector.

[0113] The Fc gene (SEQ ID NO:348) was isolated from these constructs after digestion with EcoRI and BamHI. The amino sequence of the cloned Fc gene is provided herein as SEQ ID NO:348.

[0114] [ka]

[0115] The pSEX81 (catalog no. PR3005, Progen) phagemid vector was modified by excising the pIII gene of the phagemid vector, digesting it with EcoRI and BamHI, and ligating the linearized vector with the digested Fc gene. The resulting modified vector is referred to herein as pSEX83. The vector map of pSEX83 is shown in Figure 1. The digested Fc and pSEX83 vector fragments were analyzed on an agarose gel and purified from the gel using a QIAquick Gel Extraction Kit (Qiagen catalog no. 28706). The ligation products of both the Fc (insert) and pSEX83 (vector) were transformed into electrocompetent E. coli TG1 cells (catalog no. 60502-1, Lucigen). The transformed cells were plated on 2xYT agar plates containing ampicillin (100 μg / mL). Clones were analyzed by EcoRI and BamHI restriction digestion and DNA sequencing using the Sanger method.

[0116] (b) Preparation of secondary libraries for affinity maturation Two different strategies were taken to prepare the Fc variant phage display library.

[0117] Strategy 1: PCR primer-based site-directed mutagenesis Four different regions in the CH2-CH3 domain of the Fc gene (EU250-259, EU307-311, EU375-381, EU432-437) were targeted for random mutagenesis and preparation of phage display libraries.

[0118] Mutations in selected regions were introduced using PCR with primer pairs (RK173 / RK174, RK175 / RK176, RK177 / RK178, RK179 / RK180, RK181 / RK182, and RK183 / RK184) containing a SapI restriction site (Table 4). Each primer pair was designed to amplify the entire plasmid in linear form, resulting in a circular plasmid containing the mutations introduced by the primer pair after restriction digestion with SapI and subsequent ligation with T4 DNA ligase. Each pair was used to generate a separate library. DNA from one library was also used to generate libraries for mutagenizing other regions.

[0119] Briefly, 100 ng of template DNA of the Fc gene of the pSEX83 vector was used for amplification using the primer pair listed in Table 5. After amplifying the template using randomized primers, the product was digested with SapI to generate sticky ends. After PCR purification, the digested products were ligated overnight at 16°C. The ligated DNA was then transformed into freshly prepared electrocompetent cells (TG1). The transformed cells were cultured to produce phages. Phage production was performed separately for each library as previously described (Brockmann et al., 2011).

[0120] [Table 5]

[0121] Strategy 2: Kunkel mutagenesis and sRCA Kunkel mutagenesis was performed by sRCA according to reference 9. Small cultures of CJ236 cells were infected with phages carrying the Fc gene plasmid. The infected cells were grown in 2xYT containing uridine (6 μg / mL) and carbenicillin antibiotic (100 μg / mL). The phages were superinfected with VCS M13 helper phage (Agilent Technologies, USA) and purified by precipitation with PEG6000 (4%) and NaCl (500 mM). Single-stranded uridylated DNA (ss(U)DNA) was extracted from the purified phages using the M13 purification kit from the EZNA® M13 DNA Mini Kit (OMEGA bio-tek, USA) according to the manufacturer's instructions.

[0122] This ss(U) DNA was used as a template in Kunkel mutagenesis, and various primers with random mutations were used to generate libraries. Primers targeting different regions of Fc (Table 6) were separately hybridized to the ss(U) DNA template and extended by Kunkel mutagenesis. The products were treated with UDG and selectively amplified by RCA (9). The RCA products were digested with Hind III, circularized using T4 DNA ligase, and transformed into SS320 cells. Phages (secondary libraries) were generated separately for each mutagenic primer by superinfection with helper phage as described below.

[0123] [Table 6]

[0124] Example 2 Production and purification of mutant Fc phages from PCR and Kunkel libraries Cells from the glycerol stock of the library described above were cultured in a flask containing 2xYT medium containing carbenicillin or ampicillin (100 μL / mL) at an initial concentration of 0.06 OD 600 The culture was incubated at 37°C with shaking at 250 rpm until OD600 The cultures were grown until a maximum β-reactive index (MOI) of 0.4–0.6 was reached. Then, helper phage, either VCSM13 (Agilent, Cat. No. 200251) or M13KO7 (GE Healthcare, Cat. No. 27152401), was added to the cultures at a multiplicity of infection (MOI) of 20. The cultures were first incubated at 37°C for 40 minutes without shaking, followed by an additional 40 minutes with shaking at 37°C. Kanamycin was then added to the cultures, and the cultures were grown overnight at 26°C and 150 rpm. The overnight phage cultures were centrifuged at 4000 g, and the cell pellet was discarded. A PEG (20%) / NaCl (2.5 M) solution was added to the supernatant at a 1:5 ratio to precipitate the phage. The resuspended solution was incubated on ice for 20 minutes, followed by centrifugation at 14,000 g for 15 minutes at 4°C. The supernatant was discarded and the pellet was resuspended in 1 mL of sterile PBS containing 0.01% sodium azide. The phages were stored at 4°C until further use.

[0125] Example 3 Biopanning of high-affinity Fc variant expressing phage The Fc variant library (1x10) prepared in Example 2 12PFU) were screened for high-affinity Fc binders to the FcRn receptor. The first round of panning against the FcRn / FcGRT antigen (Sino biological, catalog no. CT071-H27H) at pH 6.0 was performed using antigen-immobilized immunotubes (Quidel, USA) prepared by overnight incubation at 4°C with a 5 μg / mL FcRn protein solution in carbonate buffer (0.1 M, pH 9.6). The immunotubes were washed three times with PBS and then incubated with phage in PBS for 2 hours at 25°C with constant rotation. The tubes were washed 10 times with 4 mL of PBS containing Tween 20 (0.1%), followed by 10 washes with PBS. Bound phage were eluted with glycine-HCl pH 2.1 (0.1 M). The eluted phages were rescued by infection of TG1 E. coli cells, plated, and phage were produced as described in Example 3.

[0126] The second and third rounds of panning were performed using the output phage (1x10) from the first round of panning. 11 CFU) and output phage from the second round of panning (1x10 10 Panning was performed on immunotubes coated with FcRn antigen, with the resulting phage (CFU) used as input phage for the second and third rounds of panning, respectively. Phages from the third round of panning were infected into TG1 cells as described above, and phagemid DNA was isolated for cloning of the Fc variants into appropriate expression vectors.

[0127] Example 4 Screening of individual Fc variant clones as soluble Fc proteins Cloning into expression vectors and protein production The Fc variant genes from the enriched library generated after the third round of panning were cloned into the expression vector pOPE101 (carbenicillin-resistant) (Progen, Germany) or its improved version, pOPE102 (kanamycin-resistant), so that individual Fc variant clones could be produced as HIS-tagged fusion products. Both the vector DNA (pOPE101 or pOPE102) and phagemid DNA were digested with restriction enzymes (EcoRI and BamHI), and the vector gene and Fc variant gene, respectively, were isolated. The restriction-digested vector and Fc gene were ligated and transformed into TG1 electrocompetent cells. The transformed cells were plated on 2xYT agar plates containing carbenicillin antibiotic. Individual clones from the 2xYT agar plates were picked and cultured overnight at 32°C and 200 rpm in 15 mL tubes containing 5 mL of 2xYT medium containing carbenicillin or ampicillin (100 μL / mL). The next day, the culture was reinoculated into fresh 2xYT medium containing carbenicillin (100 μg / mL) and glucose (0.1%) at a volume ratio of 1:200. 600 The culture was grown until its RI reached approximately 0.6-0.8. Then, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture and grown overnight at 30°C and 200 rpm. The overnight culture was spun at 10,800 g for 15 minutes, and the supernatant was removed. The cell pellet was resuspended in 1 / 20 the original culture volume of 1x PBS buffer, pH 7.4, containing 2 mg / mL lysozyme, 0.1% Triton X, and 1 U / 100 mL benzonase and incubated at 37°C for 1 hour. The resuspended pellet was centrifuged at 10,800 g for 15 minutes, and the supernatant was collected as a cell lysate containing soluble His-tagged Fc variants. This cell lysate fraction was used in an immunoassay to quantify soluble Fc in the periplasmic fraction, followed by FcRn binding.

[0128] Quantification of soluble Fc in the periplasmic fraction Clones were individually cultured to produce the soluble Fc variants described above. Total Fc in cell lysates or periplasmic fractions was quantified by immobilizing soluble Fc on maxisorp plates seeded with mouse anti-human IgG antibody (Sigma, catalog no. I6760). Mouse anti-human IgG antibody was coated overnight at 2.5 μg / mL (100 μL / well) in coating buffer (0.1 M NaHCO3, pH 9.6). After washing the plates twice, 100 μL of cell lysate (1 / 20 v / v in 1x PBS) was added and incubated for 1 hour at 25°C. A serially diluted wild-type Fc IgG1 of known concentration was used as a standard to calculate the amount of Fc protein in the cell lysate. The plates were washed three times with PBST (0.1% Tween 20 in 1x PBS). 100 μL (1:10000) of HRP-conjugated goat anti-human IgG Fc, F(ab)'2 fragment (Invitrogen, Cat. No. A24476) was added to each well of the plate and incubated for 1 hour. The plate was then washed five times with PBST, followed by the addition of the substrate o-phenylenediamine dihydrochloride (OPD) (100 μL / well) for 15 minutes at 37°C. The reaction was stopped with 1N H2SO4 (100 μL / well), and the optical density (OD) was measured at 450 nm using a TECAN INFINITE® M1000pro. The Fc concentration in the cell lysate was then calculated using a reference standard.

[0129] Qualitative analysis of Fc variants FcRn antigen was coated onto polystyrene plates (100 ng / 100 μL / well) in coating buffer (0.1 M NaHCO3, pH 9.6) overnight at 4°C. After washing the plates twice, 100 ng of Fc variant diluted in 100 μL PBS pH 6.0 was added to each well and incubated at 25°C for 1 hour. The plates were washed four times with PBST (1x PBS pH 6.0 with 0.1% Tween 20), followed by the addition of 100 μL (1:10,000) of HRP-conjugated goat anti-human IgG Fc, F(ab)'2 fragment (Invitrogen, catalog no. A24476) and incubation at 25°C for 1 hour. The plates were then washed five times with PBST, followed by the addition of the substrate o-phenylenediamine dihydrochloride (OPD) (100 μL / well) for 15 minutes at 37°C. The reaction was stopped with 1N H2SO4 (100 μl / well), and the optical density (OD) was measured at 450 nm using a TECAN INFINITE® M1000pro. Individual clones with relatively high OD signals compared to wild-type Fc were selected for further characterization.

[0130] Example 5 Determination of kinetic rate constants for Fc variant binding to recombinant human neonatal Fc receptor (rhFcRn) The kinetic constants for binding of Fc variants to recombinant human neonatal Fc receptor (rhFcRn) were determined by surface plasmon resonance-based measurements using a ProteOn™ XPR36 (Bio-Rad). rhFcRn receptor (Sino Biologics) was immobilized on a GLC chip according to the manufacturer's instructions. Kinetic measurements were performed using 10 mM phosphate buffer saline (PBS) (10 mM phosphate buffer, pH 6.0, 150 mM NaCl, 0.005% Tween 20) as the running buffer. The binding rate constant (K a ) and dissociation rate constant (K dTo measure rhFcRn binding, five dilutions of Fc variants were prepared in the running buffer described above and injected at a flow rate of 100 μL / min with an association time of 180 s and a dissociation time of 600 s. Reactions were performed in PBS (pH 6.0, 0.005% surfactant P20) at 25°C. After each sample run, the chip surface was regenerated with two pulses of PBS (0.005% surfactant P20) pH 7.4, followed by one pulse of glycine buffer pH 1.5. Data in the form of sensograms were analyzed using the data fitting program of the ProteOn™ system. The kinetic constants of rhFcRn binding to different Fc variants are shown in Table 7.

[0131] [Table 7]

[0132] Example 6 Determination of kinetic rate constants for Fc variant binding to recombinant human neonatal Fc receptor (rhFcRn) under different pH conditions In this experiment, the affinity constants for binding of Fc variants to recombinant human neonatal Fc receptor (rhFcRn) were determined under two different conditions by surface plasmon resonance-based measurements using a ProteOn™ XPR36 (Bio-Rad). Kinetic measurements were performed using 10 mM phosphate-buffered saline (PBS) (containing 10 mM phosphate buffer, 150 mM NaCl, and 0.005% Tween 20) as the running buffer. To measure affinity constants, five dilutions of the Fc variants were prepared and injected at a flow rate of 100 μL / min with an association time of 180 s and a dissociation time of 600 s. All reactions were performed at 25°C. To confirm the effect of pH on FcRn binding and dissociation of molecules from FcRn, samples were analyzed for FcRn binding under different pH conditions. The affinity constants for rhFcRn of the Fc variant with the sequence shown in SEQ ID NO: 22 (H11+A4) were measured. The buffer for the binding phase of the interaction was the sample dilution buffer, and the buffer for the dissociation phase of the interaction was the running buffer. In condition 1, the buffer for the binding phase was PBST pH 6.0, and the buffer for the dissociation phase was PBST pH 7.4. In condition 2, the buffer for the binding phase was PBST pH 7.4, and the buffer for the dissociation phase was PBST pH 6.0. The Fc variants used to perform this experiment were produced in a CHO cell line. The affinity constants for rhFcRn binding to the Fc variants under two different pH conditions are shown in Table 8.

[0133] [Table 8]

[0134] As can be seen from Table 8, the Fc variants of the present invention are able to block FcRn with higher affinity at pH 6.0 compared to pH 7.4. Thus, the Fc variants of the present invention retain the pH-dependence characteristic of FcRn interaction (higher affinity at pH 6.0 than at near-neutral pH). Thus, the Fc variants prepared according to the present invention are able to reduce circulating total serum IgG levels.

[0135] Example 7 Determination of kinetic rate constants for Fc variant binding to recombinant neonatal Fc receptor (rFcRn) from different species In this experiment, the binding of the Fc variants of the present invention to FcRn of different species was determined by surface plasmon resonance-based measurements using a ProteOn™ XPR36 (Bio-Rad). To confirm the kinetic rate constants, experiments were performed using recombinant mouse, monkey, and human FcRn containing the Fc variant having the sequence shown in SEQ ID NO: 22 (H11+A4). The Fc variants used to perform this experiment were produced using CHO cells as an expression system. FcRn (of different species) was immobilized on a GLC sensor chip surface using standard amine coupling chemistry essentially according to the manufacturer's instructions. Kinetic measurements were performed using 10 mM phosphate buffer saline (PBS) (10 mM phosphate buffer, pH 6.0, 150 mM NaCl, 0.005% Tween 20) as the running buffer. The binding rate constant (k assoc ) and dissociation rate constant (k dissoc To measure Fc muteins, five dilutions of the Fc muteins were prepared in the running buffer described above and injected at a flow rate of 100 μL / min with an association time of 180 seconds and a dissociation time of 600 seconds. All reactions were performed at 25°C. After each sample run, the chip surface was regenerated with PBST, pH 7.4. Data in the form of sensorgrams were analyzed using the ProteOn™ system's data fitting program.

[0136] The kinetic constants for FcRn binding (of different species) to Fc variants are shown in Table 9.

[0137] [Table 9]

[0138] As is clear from Table 9, the Fc variants of the invention are able to block not only human FcRn, but also mouse and monkey FcRn in vitro. The advantageous feature of cross-reactivity with monkey FcRn means that the Fc variants of the invention can be tested in non-human primates, and the data obtained may be very useful for predicting the pharmacokinetics, pharmacodynamics, and toxicity of the Fc variants of the invention in humans.

[0139] Example 8 Determination of kinetic rate constants for Fc variant binding to Fc gamma receptors In this experiment, the kinetic constants for binding of Fc variants to recombinant Fc gamma receptors were determined by surface plasmon resonance measurements using a ProteOn™ XPR36 (Bio-Rad). For this experiment, FcγRIIIa(Phe) and FcγRIIIa(Val) Fcγ receptors were analyzed for binding to an Fc variant of the present invention having the sequence shown in SEQ ID NO: 22 (H11+A4). All recombinant human Fc receptors were directly immobilized onto a GLC sensor chip surface using standard amine coupling chemistry essentially according to the manufacturer's instructions. Kinetic measurements of all Fc receptors were performed using 10 mM phosphate buffer saline (PBS) (10 mM phosphate buffer, pH 7.4, containing 150 mM NaCl, 0.005% Tween 20) as the running buffer. The binding rate constants (k assoc ) and dissociation rate constant (k dissocTo measure the Fc-specific binding activity (Fc-specific binding activity), five dilutions of Fc variants were prepared in the running buffer described above and injected at a flow rate of 50 μL / min. The association and dissociation times for each interaction are listed in the table below. All reactions were performed at 25°C. Data in the form of sensorgrams were analyzed using a data fitting program available on the ProteOn™ system.

[0140] [Table 10]

[0141] The Fc variants used to perform this experiment were produced in a CHO cell line. A control molecule representing a human IgG1 control (lacking the substituents of the invention) was maintained to determine the affinity level of the Fc variants of the invention for Fc gamma receptors.

[0142] [Table 11]

[0143] As can be seen from Table 11, the Fc variants of the invention are able to block FcγRIIIa (including allotypes V158 and F158) with higher affinity compared to molecules representing IgG1 controls. This demonstrated activity of the Fc variants of the invention to block Fc gamma receptors indicates that the Fc variants of the invention are able to inhibit immune complex-mediated FcγR activation.

[0144] Example 9 Construction of monomeric and multimeric (dimeric) DGV-H11A4 Fc variants with SEQ ID NO: 22 The two chains of the Fc variant monomer were linked with a glycine-serine linker (SEQ ID NO: A-GGGSGGGSGGGSGGGSSGGGSS), and the cysteine ​​residues at EU positions 226 and 229 of the second chain were changed to serine to prevent self-dimerization. Chemically synthesized genes for an Fc variant monomer having the amino acid sequence of SEQ ID NO: 22 (H11+A4; also referred to as H11A4) and an Fc dimer having the amino acid sequence of SEQ ID NO: 22 (H11+A4; also referred to as H11A4) were obtained in pMK vectors obtained from Geneart GmbH, Germany. The Fc variant monomer and dimer genes were isolated from the pMK Geneart constructs by restriction digestion with HindIII and EcoRI. The approximately 0.7 kb Fc variant monomer H11A4 gene and the approximately 1.5 kb Fc variant dimer gene were individually ligated into pXC-17.4 vector (Lonza) and pXC-18.4 vector (Lonza) digested with HindIII and EcoRI. The ligation products were transformed into E. coli Top10F', and transformants were scored for ampicillin resistance. Clones were analyzed by restriction digestion with HindIII and EcoRI. Positive clones from pXC-17.4 H11A4 monomer and dimer and pXC-18.4 H11A4 monomer and dimer, respectively, were digested with SalI and NotI. The digestion products were separated on a 1% agarose gel. The final DGV H11A4 monomer carrying two expression assemblies was prepared by ligating a 6.9 kb fragment from pXCH11A4 monomer with a 2.7 kb fragment from pXC-18.4 H11A4 monomer. Similarly, the final DGV H11A4 dimer carrying two expression assemblies was prepared by ligating a 7.7 kb fragment from pXCH11A4 dimer with a 3.5 kb fragment from pXC-18.4 H11A4 dimer. The ligation product was transformed into E. coli Top 10F', and transformants were scored for ampicillin resistance. The final DGV-H11A4 monomer and dimer vectors were confirmed by restriction digestion characterization and Sanger sequencing.The vector was linearized with PvuI for transfection into CHO-GS cells (Lonza). A map of the vector used to prepare the Fc monomer and Fc dimer is shown here as Figure 2.

[0145] Example 10 Construction of full-length antibody DGV-H11A4 with Fc variant having SEQ ID NO: 22 The Fc variant H11A4 was prepared as a full-length monoclonal antibody molecule by combining the Fc region with an unrelated (human / mouse / NHP-non-cross-reactive) heavy chain variable domain and an unrelated (human / mouse / NHP-non-cross-reactive) light chain. The Fc variant H11A4 region was amplified and inserted into the CH1 domain, with an ApaI restriction site at the 5' end and an EcoRI restriction site at the 3' end. This approximately 1.0 kb insert was digested with ApaI and EcoRI to facilitate in-frame cloning with the heavy chain variable region. The approximately 0.5 kb gene, the heavy chain variable region digested with MluI and ApaI and the ApaI-EcoRI-digested H11A4 PCR fragment, were ligated into the MluI- and EcoRI-digested DGV vector carrying the light chain gene. The ligation product was transformed into E. coli Top10F' cells, and transformants were scored for ampicillin resistance. The final DGV-H11A4 mAb vector was characterized by restriction digestion and confirmed by Sanger sequencing. The vector was linearized with PvuI for transfection into CHO-GS cells (Lonza). A map of the vector used to produce full-length antibody containing the Fc monomer is shown here as Figure 3.

[0146] Example 11 Generation of cell line-expressing Fc constructs This example describes the generation of stable transfected cell lines expressing Fc variants. All vector constructs described in Examples 9 and 10 were used for transfection. Plasmids were linearized with PvuI restriction enzyme before transfection. Chinese hamster ovary (CHO), a suitable host for recombinant protein expression, was used to generate the cell lines. CHO cells were seeded at a density of 500,000 cells / mL 24 hours before transfection to ensure that the cells were in logarithmic growth phase. Transfection was performed by electroporation using the Neon Transfection system (Invitrogen) according to the manufacturer's instructions. After transfection, the cells were seeded into 24-well cell culture plates containing 1 mL of prewarmed ProCHO5 serum-free medium (Lonza, Switzerland) containing selective pressure and incubated in a humidified incubator at 37°C in the presence of 5% CO2. The cell numbers of all transfected pools were monitored periodically, and the medium was replaced periodically. Once the cells recovered from transfection, they were further expanded into 6-well culture plates, T-flasks, and culti-tubes (TPP).

[0147] Fed-batch cultures were performed on the transfected pools of all Fc variant candidates in cultitubes for recombinant protein production (TPP). Cells were grown in ActiPro production medium (Hyclone, GE) at 0.3 × 10 6 Cultures were seeded at a density of 1000 cells / mL. The culture tubes were incubated in a humidified Kuhner shaker at 37°C, 5% CO2, and 230 RPM. A chemically defined diet from Hyclone (GE) was used, and all pools followed a consistent daily feeding schedule during culture. Feeding began after 72 hours of culture and continued until the batch was harvested.

[0148] After harvesting, the culture supernatant was collected for protein purification by Protein A affinity chromatography. These purified protein candidates were further tested in various in vitro assays, as described in the above examples. It has been confirmed that the Fc variant in multimeric form (SEQ ID NO: 22) and in full-length antibody form has the same technical effect as the Fc monomer. Therefore, the Fc variant of the present invention can achieve the same technical effect in all constructed forms (monomer, multimer, and full-length antibody).

[0149] Example 12 Effect of Fc variants on total serum IgG levels in wild-type C57BL / 6 mice In this study, the Fc dimer prepared according to Example 9 was compared with commercially available IVIg to determine the effect of the Fc variant of the present invention on circulating total blood IgG levels. Wild-type C57BL / 6 mice were randomized based on body weight. Blood was collected at time zero (pre-dose). Within one hour, 50 mg / kg of Fc dimer and 1 g / kg of IVIg were administered intravenously to each group of test animals (6 mice per group). Blood samples were collected at 5 hours, 24 hours (day 1), 48 hours (day 2), 72 hours (day 3), 144 hours (day 6), 216 hours (day 9), 288 hours (day 12), and 312 hours (day 13). Endogenous IgG levels were determined using ELISA from samples collected at each of the indicated time points. The graphed results are shown in Figure 4. Both IVIg and the Fc dimer demonstrated a reduction in endogenous mouse IgG levels compared to controls, with the Fc dimer demonstrating greater IgG clearance by day 6 compared to IVIg. The lowest % IgG reduction was observed with the Fc dimer, approximately 47% compared to pre-administration IgG, compared to approximately 66% with IVIg. Albumin levels were also determined at the indicated time points. Overall, no differences were observed across time points and groups (Figure 5). The Fc dimer prepared according to the present invention did not affect serum albumin levels. It is clear that the Fc variant prepared according to the present invention enables FcRn to maintain serum albumin levels without interfering with the albumin binding site on FcRn. Similar experiments were performed using full-length antibodies containing the Fc variants of the present invention, as well as the monomeric form of the Fc variants of the present invention having SEQ ID NO: 22, according to the same method as described in Example 12 herein.

[0150] Example 13 Effect of Fc variants on Fc gamma receptor-dependent ADCC activity SKBR3 cells were stained with calcein AM dye and EC 50 Concentration and EC 75The cells were seeded with trastuzumab at concentrations of 10.39 ng / mL and 31.17 ng / mL, respectively, and incubated at 37°C and 5% CO2 for 30 minutes. To determine whether the Fc dimers prepared according to Example 9 could block ADCC, serial dilutions of the Fc dimers, starting at 100 μg / mL, and human PBMCs at a target cell to effector cell ratio of 1:30 were added to wells containing SKBR3 cells and incubated at 37°C and 5% CO2 for 4 hours. After incubation, the plate was centrifuged at 2000 rpm for 5 minutes at 25°C, and 100 μL of supernatant was collected and transferred to a 96-well black clear-bottom plate. The plate was read on a plate reader at excitation 494 nm and emission 515 nm, with a cutoff set at 515 nm. The results are shown in Figure 6. As is clear from Figure 6, it is confirmed that the Fc dimer of the present invention can reduce the ADCC activity mediated by trastuzumab, and its strong binding to Fc gamma receptors can block the binding of trastuzumab to NK cells present in PBMCs.

[0151] Example 14 Effects of Fc variants in an animal model of chronic idiopathic thrombocytopenic purpura (ITP) In this study, the therapeutic efficacy of H11+A4 (SEQ ID NO: 22) was tested in a mouse model of acute immune thrombocytopenia. Specifically, C57BL / 6 mice were randomized based on body weight and subsequently treated intravenously with two different doses of H11+A4 (i.e., 0.5 mg / 20 gm mouse and 1 mg / 20 gm mouse) or phosphate buffered saline (7 animals / group). One hour later, the mice were treated intravenously with the anti-platelet antibody MWReg30 (5 μg / 20 gm mouse) or phosphate buffered saline. Twenty-four hours later, the mice were again treated intraperitoneally with MWReg30 (5 μg / 20 gm mouse) or phosphate buffered saline. 72 hours after the first injection of MWReg30, blood samples were collected to measure platelet counts. Platelet counts were determined from the collected samples using a hematology analyzer and plotted. The results are shown in Figure 7. As can be seen from Figure 7, pretreatment with H11+A4 reduced MWReg30 (anti-platelet antibody)-induced thrombocytopenia in a dose-dependent manner at both doses, indicating that the molecules have the potential to effectively reverse the thrombocytopenic state.

[0152] References incorporated into this patent application: (References) TIFF2025166155000028.tif177168

[0153] Incorporation by Reference The entire disclosure of each patent document and scientific article referred to herein is incorporated by reference for all purposes.

[0154] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. An Fc variant that binds to human FcRn with higher affinity than human Fc protein and comprises a combination of substituted amino acids at specific EU positions of the wild-type Fc protein, comprising the amino acid sequence of SEQ ID NO:

22.

2. The Fc variant of claim 1, which has a higher binding affinity to human FcRn at pH 6.0 compared to its binding affinity at neutral pH.

3. 10 for human FcRn -8 K below M D The Fc variant of claim 1, having:

4. The Fc variant of claim 1, which cross-reacts with FcRn from species other than human.

5. IgG 1 , IgG 2 , IgG 3 , IgG 4 or IgG 2 / G 4 The Fc variant of claim 1 present in an Fc protein of any isotype.

6. The Fc variant of claim 1, which is expressed in a multimeric form and has an extended half-life by increasing molecular size and affinity via the Fc variant's higher avidity for human FcRn.

7. The Fc variant of claim 6, which is in a multimeric form selected from a dimer, trimer, tetramer, pentamer, and hexamer.

8. The Fc variant of claim 1, which exists in a full-length antibody form.

9. Human IgG to Fc gamma receptors 1 2. The Fc variant of claim 1, having an altered affinity for Fc gamma receptors compared to the affinity of the Fc region.

10. Human IgG against FcγRIIIa containing allotype V158 or F158 1 10. The Fc variant of claim 9, having increased affinity for FcγRIIIa (CD16a) compared to the affinity of the Fc region.

11. The following features: a) has an extended half-life in the subject; b) have reduced or no ADCC activity compared to the human protein; c) inhibiting FcγR-mediated phagocytosis, and d) inhibiting FcγR-mediated cytokine release The Fc variant of claim 10, comprising at least one of:

12. The Fc variant of claim 1 , fused to a drug or therapeutic peptide or polyethylene glycol or immunogen or neutralizing antibody.

13. The Fc variant of claim 1, comprising a defucosylated N-linked glycan at EU position 297.

14. 2. The Fc variant of claim 1, wherein SEQ ID NO: 22 comprises the substitutions T307N, V308P, L309Y, H433R and N434W.

15. 15. The Fc variant of claim 14, wherein SEQ ID NO: 22 has an amino acid sequence selected from SEQ ID NO: 22a, SEQ ID NO: 22b, SEQ ID NO: 22c, SEQ ID NO: 22d, SEQ ID NO: 22e, SEQ ID NO: 22f, and SEQ ID NO: 22g.

16. A composition comprising an Fc variant according to any one of claims 1 to 15 and an acceptable carrier.

17. 16. An Fc variant according to any one of claims 1 to 15 for use in the preparation of a drug for treating a disease in which FcRn activity is deleterious or for extending the circulating half-life of a drug.

18. 18. The Fc variant of claim 17, wherein the disease is selected from infectious diseases, cancer, and autoimmune disorders.

Images