FC variants and preparation thereof

Abstract

The present invention relates to FcRn antagonists or Fc variant proteins and their preparation. The Fc variants have an altered binding affinity for FcRn. In one aspect, the Fc variants according to the invention comprise amino acid substitutions of L234A, L235A, T307N, V308P, L309Y, P329G, H433R, N434W. In another aspect, the Fc variants according to the invention comprise amino acid substitutions of T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E. In yet another aspect, the Fc variants of the present invention comprise amino acid substitutions of T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E, L234A, L235A, P329G. The Fc variant prepared according to the invention can be used for preparing an FcRn antagonist composition or can be used for preparing a drug or molecule containing the Fc variant with a changed effector function.

Description

[0001] Field of the Invention

[0002] The present invention relates to Fc variant proteins and their preparation. Background of the Invention

[0004] The neonatal Fc receptor (FcRn) is structurally homologous to the major histocompatibility complex (MHC) class I heterodimeric molecule consisting of a type I transmembrane heavy chain that non-covalently binds to the soluble light chain β2-microglobulin (β2m).

[0005] Monoclonal antibody-based antagonists have been developed to inhibit the endogenous IgG-FcRn interaction. One approach is to use 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 to inhibit the binding of IgG to FcRn, thereby accelerating the clearance of endogenous IgG. An average reduction of endogenous IgG ranging from 25 - 80% was observed with M281 in a dose-dependent manner. In addition, single doses of 30 mg / kg or 60 mg / kg of M281 maintained serum IgG at 50% or below of baseline for 18 days and 27 days, respectively. 1 In healthy subjects, at a single IV dose of 7 mg / kg of UCB7665, the IgG concentration in serum decreased by up to 50%. The decrease in serum IgG concentration observed with UCB7665 persisted for several weeks, reaching a maximum reduction between days 7 and 10 and then gradually returning to baseline by day 57. 2 .

[0006] The present invention provides improved FcRn binders that address various unmet needs, such as minimal toxicity, downregulating FcRn activity, increasing the circulating half-life of various drugs, targeting drugs and / or vaccines to specific cell types, etc. The FcRn binders of the present invention can be used to treat a variety of diseases. WO2021234655 discloses various FcRn binding molecules. The present invention provides therapeutically effective FcRn binding molecules that can be used to treat autoimmune diseases. Summary of the Invention

[0007] The present invention provides novel Fc variants having an altered binding affinity for FcRn, preferably a higher binding affinity for FcRn. In one aspect, the Fc variant according to the present invention comprises the L234A, L235A, T307N, V308P, L309Y, P329G, H433R, N434W amino acid substitutions. In another aspect, the Fc variant according to the present invention comprises the T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E amino acid substitutions. In yet another aspect, the Fc variant of the present invention comprises the T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E, L234A, L235A, P329G amino acid substitutions. The amino acid substitutions mentioned herein in the present invention are according to the EU numbering system (EU positions). The Fc variant can be used for developing pharmaceutical compositions having an FcRn antagonist function or an increased circulatory half-life, or for targeting specific cells and / or tissues. The present invention also provides a method for preparing the novel Fc variant of the present invention. The Fc variant according to the present invention is further used for preparing a medicament for treating a disease in which the activity of FcRn is adverse, or for increasing the circulatory half-life of a medicament or for targeting the medicament to certain cells or tissues. Brief Description of the Drawings

[0009] Figure 1 : It depicts a map of the vector for generating the Fc monomer and Fc dimer of the present invention.

[0010] Figure 2 : It depicts a map of the vector for generating monoclonal antibodies containing the Fc monomer of the present invention.

[0011] Figure 3 : It depicts the percentage of relative IgG concentration relative to the pre-dose level in the following various treatment groups: A3H11A4.0, A3H11A4.1, B14H11A4.0 and B14H11A4.1.

[0012] Figure 4 : It depicts the percentage of relative IgG concentration relative to the pre-dose level in the antibody molecules of the following various treatment groups: A3H11A4.0, A3H11A4.2, A3H11A4.3.

[0013] Figure 5 : It depicts the percentage of relative IgG concentration relative to the pre-dose level in the candidates of the following various treatment groups: H11A4.1, H11A4.0, H11A4.2, H11A4.3 Fc.

[0014] Figure 6: It describes the percentage of relative IgG concentration of the H11A4.1 Fc candidate at the study time points relative to the pre - dosing level.

[0015] Figure 7 : It describes the percentage of relative IgG concentration of the A3H11A4.1 antibody candidate at the study time points relative to the pre - dosing level.

[0016] Figure 8 : It describes the percentage of relative IgG concentration of the B14H11A4.1 antibody candidate at the study time points relative to the pre - dosing level.

[0017] Definition

[0018] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. The meanings and scopes of the terms should be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are well - known and commonly used in the art.

[0019] As used herein, the term "Fc domain" refers to the portion of a single immunoglobulin heavy chain that begins just upstream of the papain cleavage site and terminates at the C - terminus of the antibody. Thus, the complete Fc domain contains at least a portion of the hinge (e.g., upper, middle, and / or lower hinge regions) domain, CH2 domain, and CH3 domain.

[0020] The term "amino acid modification" as used herein is an amino acid substitution, insertion, and / or deletion in a polypeptide sequence.

[0021] The term "amino acid substitution" or "substitution" as used herein is the replacement of an amino acid at a specific position in a parental polypeptide sequence with another amino acid. For example, substitution T307N refers to a variant polypeptide (in this case, an Fc variant) in which the threonine at position 307 is replaced by asparagine, where "T" and "N" represent the single - letter codes for threonine and asparagine, respectively.

[0022] The term "antigen - binding molecule" according to the present invention refers to a protein that is capable of binding to a target antigen and contains an "FcRn - binding domain". The FcRn - binding domain according to the present invention is present in an Fc protein, preferably present 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 or monomers or dimers containing an Fc variant of the present invention.

[0023] The term "FcRn antagonist" refers to any reagent that contains an Fc region and specifically binds to FcRn through the Fc region and inhibits the binding of immunoglobulins to FcRn. The terms "FcRn antagonist", "Fc variant protein" or "Fc protein variant" or "Fc variant", "molecule containing an Fc variant", "protein containing an Fc variant", etc. are used interchangeably in the present invention.

[0024] As used herein, the term "antibody" includes intact antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") that contains an Fc portion. An "antibody" refers to a glycoprotein that contains at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region (Fc). The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region consists of one domain C L . V H and V L regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each V H and V L consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (such as effector cells, such as NK cells, T cells, macrophages, and dendritic cells, etc.) and the first component (C1q) of the classical complement system.

[0025] The term "operably linked" is intended to mean that a gene is linked into a vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of such gene.

[0026] The term "k a " is the association rate of the interaction between two molecules, while the term "k d " is the dissociation rate of the interaction between two molecules. The term "K D " is the affinity rate constant obtained from the ratio of k d to k a . It can be measured by using surface plasmon resonance methods well known in the art.

[0027] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0028] The term "bispecific antibody" refers to a homogeneous group of antibodies that participate in the highly specific recognition and binding of two different antigenic determinants or epitopes.

[0029] As used herein, the term "recombinant antibody" includes all antibodies prepared, expressed, produced, or isolated by recombinant means. However, in certain embodiments, such recombinant antibodies can be obtained by in vitro mutagenesis, and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies described herein are sequences that may not naturally occur in the human antibody germline repertoire in vivo.

[0030] The term "Fc" fragment, the name of which reflects its ability to crystallize easily. The crystal structure of the human IgG Fc region has been determined 3 . In the human IgG molecule, the Fc region can be separated from the rest of the molecule by papain digestion that cleaves the N-terminus of Cys226. The Fc region is central to the effector functions of the antibody.

[0031] As used herein, the term "Fc protein" refers to the portion of a single immunoglobulin heavy chain that begins just upstream of the papain cleavage site and terminates at the C-terminus of the antibody. Thus, the complete Fc domain contains at least a portion of the hinge (e.g., upper, middle, and / or lower hinge regions) domain, CH2 domain, and CH3 domain. The Fc protein contains two Fc chains attached to each other via two or more disulfide bonds. The Fc protein includes, but is not limited to, "Fc protein variants" known in the art.

[0032] As used herein, the term "protein containing an Fc variant" refers to any molecule that contains an Fc variant of the present invention. Preferably, the protein containing an Fc variant includes antibodies, fusion proteins, and peptibodies. The antibodies, fusion proteins, and peptibodies mentioned herein include any approved or under clinical trial or under preclinical trial antibodies, fusion proteins, and peptibodies.

[0033] As used herein, the term "molecule containing an Fc variant" refers to any molecule that contains an Fc variant of the present invention. It can be a fusion product in which the Fc variant of the present invention is linked or conjugated to a drug, where the drug can be a small peptide or a receptor or a toxin or any chemical molecule.

[0034] As used herein, the term "Fc variant protein" or "Fc protein variant" or "Fc variant" refers to an Fc protein that is different from the wild-type Fc due to at least one amino acid modification in the wild-type Fc. The Fc variant may refer to the Fc variant itself, a composition comprising the Fc variant, or an amino acid sequence encoding the Fc variant. Preferably, the Fc variant includes a monomer, dimer, or multimer. Preferably, the Fc variant has at least one amino acid modification compared to the wild-type Fc protein, for example, about 1 to about 11 amino acid modifications, and preferably about 1 to about 8 amino acid modifications compared to the wild-type Fc protein. As used herein, the term "Fc variant protein known in the art" or "Fc protein variant known in the art" or "Fc variant known in the art" refers to the "Fc variant protein" or "Fc protein variant" or "Fc variant" disclosed in the public domain prior to the priority date of the present patent specification, preferably one or more "Fc variant proteins" or "Fc protein variants" or "Fc variants" disclosed in PCT / IB2021 / 054423 (WO2021234655).

[0035] As used herein, the term "EU position" refers to the amino acid position in the EU numbering convention of the Fc region as described in the reference 4 herein.

[0036] As used herein, the term "CH1 domain" refers to the first (amino-terminal) constant region domain of the immunoglobulin heavy chain extending from about EU position 118 - 215. The CH1 domain is adjacent to the amino terminus of the V H domain of the immunoglobulin heavy chain molecule and the hinge region and does not form part of the Fc region of the immunoglobulin heavy chain.

[0037] As used herein, the term "hinge region" refers to the portion that connects the CH1 domain of the heavy chain molecule to the CH2 domain. The hinge region contains about 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three different domains: the upper hinge region, the middle hinge region, and the lower hinge region 5 . The Fc variant of the present invention may include all or part of the hinge region.

[0038] As used herein, the term "CH2 domain" refers to the portion of the heavy chain immunoglobulin molecule extending from about EU position 231 - 340.

[0039] As used herein, the term "CH3 domain" refers to the portion of the heavy chain immunoglobulin molecule that extends about 110 residues from the N-terminus of the CH2 domain, for example, from about position 341 - 447 (EU numbering system).

[0040] As used herein, the term "about" refers to variations in numerical quantities which can occur, for example, through typical measurement and handling procedures used in the preparation of compounds, compositions, concentrates or formulations; through inadvertent error in these procedures; through differences in the manufacture, source or purity of starting materials or ingredients used to carry out the methods, and other similar considerations.

[0041] As used herein, the term "FcRn" or "neonatal Fc receptor" is a protein that binds to the Fc region of an IgG antibody and is encoded at least in part by the FcRn gene. FcRn can be from any organism, including but not limited to human, mouse, rat, rabbit, and monkey. As is known in the art, a functional FcRn protein comprises two polypeptide chains commonly referred to as a heavy chain and a light chain. The light chain is β-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein refers to a complex of the FcRn heavy chain and β-2-microglobulin.

[0042] As used herein, the term "wild-type Fc" is an unmodified Fc polypeptide that is subsequently modified to generate variants. Wild-type Fc can be a naturally occurring polypeptide or a recombinant form of a naturally occurring polypeptide. Wild-type Fc can refer to the unmodified Fc polypeptide itself, a composition comprising the unmodified Fc polypeptide, or an amino acid sequence encoding the unmodified Fc polypeptide.

[0043] As used herein, the term "position" is a position in a protein sequence. Positions can be numbered sequentially or according to an established format, such as the EU numbering system or EU number or EU index or Kabat numbering. For example, position 105 is the position of the 105th amino acid residue from the N-terminus of the heavy or light chain of a human antibody IgG1.

[0044] The terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to a living organism that has or is susceptible to a condition that can be prevented or treated by administration of a composition of the present invention, and includes both human and non-human animals. Examples of subjects include but are not limited to humans, chimpanzees, and other ape and monkey species; farm animals 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; avians, including poultry, game birds, and fowl such as chickens, turkeys, and other galliformes, ducks, geese, etc. The term does not denote a particular age. Thus, adult, juvenile, and neonatal individuals are of interest.

[0045] As used herein, the term "residue" is a position in a protein and its associated amino acid identity. For example, threonine 307 (also referred to as Thr307, also referred to as T307) is a residue in a human antibody IgG1.

[0046] As used herein, the term "immunoconjugate" or "conjugate" refers to a compound or derivative thereof that is linked to a cell-binding agent (i.e., an antibody or Fc variant as described herein) and is defined by the general formula: A-L-D, where A = the cell-binding agent or antibody or Fc variant of the present invention, L = a linker, and D = a drug (a toxin or a medicament). The immunoconjugate may also be defined by the general formula in the reverse order: D-L-A.

[0047] The term "linker" is any chemical moiety capable of stably covalently linking a compound (usually a drug such as a maytansinoid or an auristatin) to a cell-binding agent (i.e., an antibody or Fc variant as described herein). The linker may be sensitive or substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide cleavage under conditions where the compound and / or antibody remain active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photo-labile groups, peptidase-labile groups, and esterase-labile groups. Linkers also include charged linkers and their hydrophilic forms as described herein and known in the art.

[0048] The term "antagonist" is an antagonist that inhibits or reduces the biological activity of an antigen it binds to, such as FcRn. In certain embodiments, the 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%.

[0049] As used herein, the term "treatment" or "therapeutic agent" refers to any treatment of a disease in a mammal, particularly a human. It includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease or at risk of developing the disease but has not been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its progression; and (c) alleviating the disease, i.e., causing the regression of the disease.

[0050] The term "wild type" or "WT" herein refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0051] The term "non-naturally encoded amino acid" refers to an amino acid that is not one of the common amino acids or natural 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 their various hyphenated and non-hyphenated forms. 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 a naturally encoded amino acid (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 a 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.

[0052] Table 1: Amino Acid Abbreviations Used in This Application

[0053] Other abbreviations used in this patent application: %: Percentage ℃: Degrees Celsius μg: Microgram μL: Microliter A: Adenine ANCA: Anti-neutrophil cytoplasmic autoantibody APC: Antigen-presenting cell ADC: Antibody-drug conjugate C: Cytosine CFU: Colony-forming unit CHO: Chinese hamster ovary DNA: Deoxyribonucleic acid EC 50 : 50% of the maximum effective concentration of any drug EC 75 : 75% of the maximum effective concentration of any drug ELISA: Enzyme-linked immunosorbent assay F C : Fragment crystallizable FcGRT: Fc fragment of IgG receptor and transporter protein FcRn: Neonatal Fc receptor G: Guanine h: Hour H2SO4: Sulfuric acid HRP: Horseradish peroxidase IC: Immune complex IgG: Immunoglobulin G IPTG: Isopropyl β-D-1-thiogalactopyranoside ITP: Immune thrombocytopenia IVIg: Intravenous immunoglobulin k a / k assoc : Association constant k d / k dissoc : Dissociation constant K D : Equilibrium dissociation constant M: Mole mg: Milligram MgCl2: Magnesium chloride min: Minute mL: Milliliter mM: Millimole mAb: Monoclonal antibody 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 DETAILED DESCRIPTION OF THE INVENTION The disclosure of the present invention provides novel FcRn antagonists comprising Fc variants disclosed herein.

[0054] In one embodiment, the Fc variant protein, protein containing an Fc variant, or molecule containing an Fc variant of the present invention comprises the amino acid substitutions L234A, L235A, T307N, V308P, L309Y, P329G, H433R, N434W.

[0055] In one embodiment, the Fc variant protein, protein containing an Fc variant, or molecule containing an Fc variant of the present invention comprises the amino acid substitutions T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E.

[0056] In a preferred embodiment, the Fc variant of the present invention comprises the amino acid substitutions T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E, L234A, L235A, P329G.

[0057] In one embodiment, the Fc variant of the present invention comprises a single chain of the Fc domain or a double chain of the Fc domain, preferably two identical chains of the Fc domain or multiple chains of the Fc domain.

[0058] In one embodiment, the Fc variant protein, protein containing an Fc variant, or molecule containing an Fc variant of the present invention binds to human FcRn with high affinity.

[0059] In one embodiment, the Fc variant protein, protein containing an Fc variant, or molecule containing an Fc variant of the present invention reduces the serum IgG level in a subject being treated.

[0060] In one embodiment, the Fc variant protein, protein containing an Fc variant, or molecule containing an Fc variant of the present invention binds to human FcRn with higher affinity relative to the wild-type Fc protein.

[0061] In one embodiment, the Fc variant of the present invention has a K D of 10 -8 M or lower, preferably 10 -19 M or lower, more preferably 10 -10 M or lower. The K D value is a measure of the binding affinity of a drug candidate for FcRn. In one embodiment, the Fc variant of the present invention has a K D of 10 -11 M to 10 -8 M.

[0062] In one embodiment, the amino acid sequence of the Fc variant is IgG l, IgG2, IgG3, IgG4 or IgG2 / G4 isotype, preferably IgG1 isotype.

[0063] In one embodiment, the Fc variant of the present invention does not significantly interfere with the binding characteristics of FcRn to albumin.

[0064] In a preferred embodiment, the Fc variant of the present invention cross-reacts with FcRn of mice, monkeys and humans.

[0065] In a preferred embodiment, the Fc variant of the present invention has a higher binding affinity for human FcRn compared to the wild-type Fc protein or one or more protein variants known in the art.

[0066] In a preferred embodiment, the Fc variant of the present invention has a higher binding affinity for mouse FcRn compared to the wild-type Fc protein or one or more protein variants known in the art.

[0067] In a preferred embodiment, the Fc variant of the present invention has a higher binding affinity for monkey FcRn compared to the wild-type Fc protein or one or more protein variants known in the art.

[0068] In one embodiment, compared to one or more protein variants known in the art, the Fc variant of the present invention, or a protein or molecule containing the Fc variant, has a longer half-life in a subject. In certain embodiments, polyethylene glycol or human serum albumin can be linked to the Fc variant of the present invention to further increase the half-life of the Fc variant. In alternative embodiments, the Fc variant of the present invention can contain mutations to increase its half-life in a subject. In another alternative embodiment, the Fc variant of the present invention can be expressed in monomeric, dimeric or multimeric forms to increase the 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 protein form that includes more than one protein unit, preferably the Fc protein of the present invention. For example, it can be monomer, dimer, trimer, tetramer, pentamer, hexamer, etc. For example, a monomeric Fc protein has two binding sites for FcRn. In a similar manner, an Fc dimer according to the present invention has four binding sites for FcRn.

[0069] In another embodiment, the Fc variant of the present invention is capable of binding to mouse and monkey FcRn, thereby facilitating drug development by providing animal models for relevant pharmacological and toxicological studies.

[0070] In one embodiment, the present invention provides a composition comprising an Fc variant protein or a protein or molecule containing the Fc variant and an acceptable carrier.

[0071] In another embodiment, the Fc variant protein of the present invention or a protein containing an Fc variant can be used to treat diseases such as infections, various cancers, autoimmune disorders, and the like.

[0072] In certain embodiments, the Fc variant of the present invention, or a protein containing an Fc variant, or a molecule containing an Fc variant comprises an amino acid sequence selected from: SEQ ID No. 1, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 20, and SEQ ID No. 38.

[0073] In another embodiment, the Fc variant of the present invention can be used to prepare a full-length antibody, wherein the antibody has any Fc variant of the present invention in place of the conventional Fc region. The antibody can be a modified form of an already approved or newly discovered antibody, wherein the modified form of the existing antibody has the Fc variant of the present invention. Such an antibody can be selected and / or designed in such a way that it does not bind to any target in the subject being treated via its paratope.

[0074] In one embodiment, the Fc variant of the present invention, or a protein containing an Fc variant, or a molecule containing an Fc variant comprises the amino acid sequences shown in SEQ ID No. 3 and SEQ ID No. 5.

[0075] In one embodiment, the Fc variant of the present invention, or a protein containing an Fc variant, or a molecule containing an Fc variant comprises the amino acid sequences shown in SEQ ID No. 7 and SEQ ID No. 9.

[0076] In one embodiment, the Fc variant of the present invention, or a protein containing an Fc variant, or a molecule containing an Fc variant comprises the amino acid sequences shown in SEQ ID No. 22 and SEQ ID No. 24.

[0077] In one embodiment, the Fc variant of the present invention, or a protein containing an Fc variant, or a molecule containing an Fc variant comprises the amino acid sequences shown in SEQ ID No. 26 and SEQ ID No. 28.

[0078] In one embodiment, the Fc variant of the present invention, or a protein containing an Fc variant, or a molecule containing an Fc variant comprises the amino acid sequences shown in SEQ ID No. 30 and SEQ ID No. 32.

[0079] In one embodiment, the Fc variant of the present invention, or a protein containing an Fc variant, or a molecule containing an Fc variant comprises the amino acid sequences shown in SEQ ID No. 34 and SEQ ID No. 36.

[0080] In one embodiment, the Fc variants of the present invention can be used to prepare drugs with a longer half-life, wherein the Fc variants of the present invention are fused or linked or conjugated with a drug having a short original half-life in circulation.

[0081] In another embodiment, the Fc variants of the present invention can be used to increase the half-life of an ADC by incorporating the novel variant into the antibody of the ADC, thereby reducing its off-target toxicity by increasing its recycling through the FcRn receptor.

[0082] In one embodiment, the Fc variants of the present invention can be fused with any drug to improve its half-life and in vivo stability.

[0083] In one embodiment, the Fc variants of the present invention can be used to replace albumin in albumin fusion drugs to improve the pharmacokinetics of the drugs.

[0084] In one embodiment, the mutations in the Fc variants of the present invention can be used to increase the affinity of a full antibody for FcRn at about pH 6, in order to increase its circulating half-life by reducing catabolism.

[0085] In one embodiment, an antibody molecule containing an Fc variant can be used as a scavenger to remove pathogenic proteins or toxins, such as TNFα, VEGF, etc., from circulation.

[0086] In one embodiment, an antibody molecule or protein containing an Fc variant can be used as a trapper to trap a desired protein, peptide, carbohydrate, or drug from the environment and bring it into target cells expressing FcRn.

[0087] In one embodiment, the Fc variants of the present invention can be used to develop monomeric Fc fusion proteins fused with any therapeutic peptide to improve its penetrability in tissues and retain its FcRn binding activity.

[0088] In one embodiment, when present in a functional antibody molecule, the Fc variants of the present invention can contribute to the transcytosis of a target antigen bound by the antibody, thereby increasing their tissue penetrability. Such target antigens are peptides, proteins, carbohydrates, lipids, or such molecules that need to be delivered to tissues to mediate their effects.

[0089] In one embodiment, the Fc variants of the present invention block the IgG binding site on FcRn and compete with endogenous IgG for FcRn, resulting in a higher clearance rate of endogenous IgG.

[0090] In one embodiment, compared to one or more Fc variants disclosed in WO2021234655 (PCT / IB2021 / 054423), the improved Fc variants of the present invention produce a lower incidence. In a preferred embodiment, the improved Fc variants of the present invention do not cause morbidity when administered at the same dose.

[0091] In one embodiment, the Fc variants of the present invention bind to human FcRn, mouse FcRn, and monkey FcRn in vitro. The advantageous feature of cross-reactivity with monkey and mouse FcRn indicates that the Fc variants of the present invention can be tested in non-human primates and mice, and the resulting data can be very useful in predicting their pharmacokinetic, pharmacodynamic, and toxicity profiles in humans.

[0092] In one embodiment, the Fc variants of the present invention fused to a suitable immunogen / antigen can improve the delivery of the antigen to APCs expressing FcRn, such as dendritic cells, etc., resulting in an improved immune response. DETAILED DESCRIPTION OF THE INVENTION

[0094] The present invention relates to improved Fc variants comprising the amino acid substitutions of L234A, L235A, T307N, V308P, L309Y, P329G, H433R, N434W or proteins containing the Fc variants. In one embodiment, the Fc variant proteins or proteins containing the Fc variants or molecules containing the Fc variants of the present invention comprise the amino acid substitutions of T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E. In additional embodiments, the Fc variant proteins or proteins containing the Fc variants or molecules containing the Fc variants of the present invention further comprise the amino acid substitutions of T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E, L234A, L235A, P329G. The amino acid substitutions in the Fc variants of the present invention are made according to the EU numbering system. The amino acid substitutions are in the Fc region. The Fc domain comprises at least a part of the hinge (e.g., upper, middle, and / or lower hinge regions) domain, CH2 domain, and CH3 domain.

[0095] In one embodiment, compared to one or more Fc variants disclosed in WO2021234655 (PCT / IB2021 / 054423), the improved Fc variants of the present invention result in a lower incidence rate. At some doses, intravenous administration of the Fc variants in WO2021234655 (PCT / IB2021 / 054423) causes severe incidence in animals. The inventors of the present invention surprisingly found that introducing additional mutations (amino acid substitutions) in the Fc region as mentioned herein in the present invention helps to solve this problem. When administered at the same dose, compared to one or more Fc variants disclosed in WO2021234655 (PCT / IB2021 / 054423), the improved Fc variants of the present invention cause significantly lower incidence rates. Preferably, the improved Fc variants of the present invention do not cause incidence. In some embodiments, compared to one or more Fc variants disclosed in WO2021234655 (PCT / IB2021 / 054423), the improved Fc variants of the present invention have a higher reduction of endogenous IgG. Therefore, compared to one or more Fc variants disclosed in WO2021234655 (PCT / IB2021 / 054423), the improved Fc variants of the present invention have a lower incidence rate, preferably no incidence. In addition, the Fc variants of the present invention reduce serum IgG and thus retain their efficacy. In one embodiment, the Fc variants of the present invention reduce serum IgG.

[0096] In another embodiment, the improved Fc variants of the present invention have significant binding to the FcRn receptor of humans, mice, and other non-human primates such as monkeys. The advantageous properties of cross-reactivity with monkey and mouse FcRn and the resulting data can be used to predict the pharmacokinetic, pharmacodynamic, and toxicological characteristics of drugs in humans.

[0097] In one embodiment, the Fc variant protein of the present invention, or a protein containing the Fc variant, or a molecule containing the Fc variant binds to human FcRn with high affinity. At about pH 6.0, the Fc variant of the present invention has a lower K D value for FcRn than wild-type Fc (i.e., higher binding). At about pH 6.0, the Fc variant of the present invention has a K D value of 10 -8 M or lower, preferably 10 -9 M or lower, more preferably 10 -10 M or lower for FcRn. In one embodiment, the K D of the Fc variant of the present invention for FcRn is from 10 D M to 10 -11 M. The K -8 D ​The value is a measure of the binding affinity of the binder (in this case an Fc variant) for its target antigen (in this case FcRn).

[0098] Amino acid sequence of the Fc variant

[0099] The amino acid sequence and nucleotide sequence of the Fc variant of the present invention are mentioned in Table 2 herein.

[0100] Table 2: List of amino acid and nucleotide sequences

[0101] The Fc variants of the present invention can be present in IgG1, IgG2, IgG3, IgG4 or IgG2 / G4 isotypes, preferably in the IgG1 isotype. The Fc variant construct of the IgG4 isotype may also contain a single amino acid substitution (i.e., S228P) in the hinge region of the Fc variant to reduce disruption of the disulfide bond between the two Fc chains 6 .

[0102] In one aspect, the Fc variants of the present invention can contain one or more non-natural amino acids at one or more positions. The introduction of such non-natural amino acids into peptides is well known to those skilled in the art. Methods for preparing non-naturally occurring amino acids and introducing them into proteins are known from, for example, U.S. Patent Nos. 7,083,970 and 7,524,647. In one aspect, the Fc variants according to the present invention have higher FcRn binding and longer half-lives relative to one or more protein variants known in the art.

[0103] Nucleic acid molecules, vectors, and host cells encoding Fc variants

[0104] In one embodiment, the present invention provides nucleic acid molecules encoding Fc variants or proteins containing Fc variants, suitable expression vectors containing such nucleic acids, and suitable host cells containing such nucleic acid molecules encoding Fc variants from the expression vectors. Suitable vectors for producing the Fc variants or proteins containing Fc variants of the present invention by recombinant methods are known to those skilled in the art. Examples of such known vectors are described in the patent documents WO 2007 / 017903, WO 2012 / 046255 (which are incorporated herein by reference). The host cells according to the present invention can be prokaryotic cells such as Escherichia coli (E-coli) or eukaryotic cells such as CHO cells.

[0105] Pharmaceutical compositions

[0106] Pharmaceutical compositions formulated with suitable pharmaceutically acceptable carriers can be developed by methods well known in the art, which contain the Fc variants of the present invention or proteins containing Fc variants or molecules containing Fc variants. Such compositions can also contain one or a combination of the (e.g., two or more different) Fc variants of the present invention or proteins containing Fc variants or immunoconjugates or bispecific molecules. For example, the pharmaceutical compositions of the present invention can contain a combination of an Fc variant or a protein containing an Fc variant and an antibody (or immunoconjugate or bispecific) that binds to different epitopes on one or more target antigens.

[0107] Therapeutic uses

[0108] Fc variants or proteins containing Fc variants or molecules containing Fc variants can be used to treat diseases involving therapeutic methods that require drug binding to FcRn.

[0109] In one embodiment of the present invention, the Fc variants or proteins containing Fc variants of the present invention can be used to inhibit FcRn in a subject (including a human) suffering from a disease (such as but not limited to autoimmune diseases and inflammation), wherein the binding results in a decrease in endogenous IgG levels.

[0110] In one embodiment of the present invention, the Fc variant of the present invention or a protein containing the Fc variant can be used to increase the circulating half-life of a therapeutic drug (which is a part that enables the drug to be available in the body for a long period of time), resulting in a reduced dosing frequency, increased compliance, and better therapeutic effects in diseases such as cancer, infectious diseases, autoimmune diseases, etc.

[0111] In one embodiment of the present invention, the Fc variant of the present invention or a protein containing the Fc variant can be used to target an antigen to dendritic cells or other APCs to prepare a better prophylactic or therapeutic vaccine in diseases such as infectious diseases or cancer, etc.

[0112] In certain embodiments, the Fc variant, or a protein containing the Fc variant, or a molecule containing the Fc variant is used to treat diseases selected from: autoimmune hemolytic anemia, pernicious anemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome, bullous pemphigoid, pemphigus vulgaris, Hashimoto's thyroiditis, ANCA vasculitis, insulin-dependent diabetes mellitus (IDDM), Graves' disease, myasthenia gravis, CIDP diabetes, anti-anemia drugs (β-thalassemia), hematopoietic agents, ophthalmic drugs, cold agglutinin disease, antiphospholipid syndrome (APS), lupus nephritis, bone diseases, neurogenetic disorders, complement 3 glomerulopathy, age-related macular degeneration, immunoglobulin A nephropathy (IgAN) disease, diabetic retinopathy, macular diseases, non-small cell lung cancer therapy, ocular genetic disorders, hemophilia B, agents for (factor IX deficiency), cardiovascular diseases, type 2 diabetes, immunosuppressants, rheumatoid arthritis, treatment of transplant rejection, brain cancer, breast cancer, colorectal cancer, diabetic retinopathy, digestive / gastrointestinal cancer, endocrine cancer, female reproductive system cancer, gastric cancer, urogenital cancer, macular diseases, melanoma, multiple myeloma, treatment of myelodysplastic syndromes, treatment of myeloid leukemia, treatment of non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer therapy, renal cancer therapy, retinopathy, aplastic anemia, painkillers, atypical hemolytic uremic syndrome, blood genetic disorders, multi-system genetic disorders, osteoarthritis, scleroderma, treatment of autoimmune diseases, treatment of gout, urticaria, ankylosing spondylitis, asthma therapy, dermatological drugs, idiopathic inflammatory myopathy, immunosuppressants, inflammatory bowel disease, interstitial lung disease, multiple myeloma therapy, multiple sclerosis, nephritis, psoriatic arthritis, systemic lupus erythematosus, agents for acute alcoholic hepatitis, Alzheimer's dementia, anti-allergy drugs / anti-asthma drugs, anti-arthritis drugs, anti-psoriatic drugs, breast cancer therapy, cancer-related disorders, dermatological drugs, heart failure therapy, lymphoma therapy, nephritis, neurological drugs (miscellaneous), respiratory disorders, treatment of inborn errors of metabolism.

[0113] Preferred embodiments of the present invention include: 1. An FcRn antagonist comprising an Fc variant, wherein the Fc variant comprises amino acid substitutions L234A, L235A, T307N, V308P, L309Y, P329G, H433R, and N434W according to EU numbering.

[0114] 2. The FcRn antagonist as claimed in claim 1, which is a monoclonal antibody, monomer, dimer or multimer.

[0115] 3. The FcRn antagonist as claimed in claim 2, which is a monomer having the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID No. 11.

[0116] 4. The FcRn antagonist as claimed in claim 2, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 3 and SEQ ID No. 5.

[0117] 5. The FcRn antagonist as claimed in claim 2, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 7 and SEQ ID No. 9.

[0118] 6. The FcRn antagonist as claimed in claim 2, which is a dimer having the amino acid sequence shown in SEQ ID NO. 13.

[0119] 7. The FcRn antagonist as claimed in claim 1, which further comprises amino acid substitutions M252Y, S254T, and T256E according to EU numbering.

[0120] 8. The FcRn antagonist as claimed in claim 7, which is a monoclonal antibody, monomer, dimer or multimer.

[0121] 9. The FcRn antagonist as claimed in claim 8, which is a monomer having the amino acid sequence shown in SEQ ID NO. 38.

[0122] 10. The FcRn antagonist as claimed in claim 8, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 30 and SEQ ID No. 32.

[0123] 11. The FcRn antagonist as claimed in claim 8, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 34 and SEQ ID No. 36.

[0124] 12. An FcRn antagonist comprising an Fc variant, wherein the Fc variant comprises amino acid substitutions of T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, and T256E according to EU numbering.

[0125] 13. The FcRn antagonist according to claim 12, which is a monoclonal antibody, monomer, dimer, or multimer.

[0126] 14. The FcRn antagonist according to claim 13, which is a monomer having the amino acid sequence shown in SEQ ID NO. 20.

[0127] 15. The FcRn antagonist according to claim 13, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 22 and SEQ ID No. 24.

[0128] 16. The FcRn antagonist according to claim 13, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 26 and SEQ ID No. 28.

[0129] 17. The FcRn antagonist according to claims 1-16 has a K for FcRn in the range of 10 -11 M to 10 -8 M. D .

[0130] 18. The FcRn antagonist according to claims 1-17 cross-reacts with FcRn of mice, monkeys, and humans.

[0131] 19. A composition comprising an FcRn antagonist according to any one of claims 1-18 and an acceptable carrier.

[0132] Examples

[0133] The following examples are presented to provide those of ordinary skill in the art with a disclosure and description of how to practice the methods and Fc variants claimed herein. They are for illustrative purposes only 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 appropriate modifications, which are well known in the art and fully within the capabilities of those skilled in the art. Such modifications are known to those skilled in the art.

[0134] Example 1: Construction of full-length antibodies pDGV / A3H11A4.1 (mAb) and pDGV / B14H11A4.1 (mAb) with Fc variants.

[0135] The Fc variant H11A4.1 of the present invention was prepared as a full antibody molecule by fusing the H11A4.1 fragment with the heavy chain variable region of an anti-Cov2 spike monoclonal antibody (not cross-reactive with human / mouse / NHP proteins or antigens) and the light chain of an anti-Cov2 spike monoclonal antibody (not cross-reactive with human / mouse / NHP proteins or antigens). (Patent Publication No. WO2022101839; PCT / IB2021 / 060495).

[0136] Briefly, the genes of anti-Cov2 spike monoclonal antibodies A3 (Sequence Id Nos. 15 and 16) and B14 (Sequence Id Nos. 17 and 18) were chemically synthesized, which had L234A, L235A, and P329G mutations. In addition, the H11A4 mutations, namely, T307N, V308P, L309Y, H433R, and N434W, were incorporated into the Fc regions of A3 and B14 using mutagenesis-induced oligonucleotides in a consecutive overlapping polymerase chain reaction. The Apa I restriction site at the 5' end and the Eco R1 restriction site at the 3' end were also incorporated to facilitate in-frame cloning with the heavy chain variable regions of the pDGV / A3 (pDual cov2 A3) and pDGV / B14 (pDual cov2 B14) vectors.

[0137] The amplified ~1.0 kb heavy chain fragment (constant region) was cloned into the pDGV / A3 and pDGV / B14 plasmids digested with Apa I and Eco RI to replace the Fc region with the Fc of H11A4.1 mutant. After ligation, the reaction mixture was transformed into Escherichia coli Top10F' cells, and the transformants were scored based on carbenicillin resistance. The sequences of the pDGV / A3 H11A4.1 (mAb) (for the light chain, Sequence Id Nos. 2 and 3; for the heavy chain, Sequence Id Nos. 4 and 5) and pDGV / B14 H11A4.1 (mAb) (for the light chain, Sequence Id Nos. 6 and 7; for the heavy chain, Sequence Id Nos. 8 and 9) plasmid vectors were confirmed by restriction digestion and Sanger sequencing. The vectors were linearized with Pvu I for transfection in CHO-GS cells (Lonza).

[0138] Example 2: Construction of monomeric pXC 17.4 / H11A4.1 (monomer) Fc variant.

[0139] The H11A4.1 (monomer) variant (SEQ ID Nos. 10 and 11) contains the CH2-CH3 domains of IgG1 Fc with the following mutations: L234A, L235A, T307N, V308P, L309Y, P329G, H433R, and N434W. PCR was performed to incorporate a signal peptide upstream of the CH2 domain of the pDGV / A3 H11A4.1 heavy chain constant region (Example 1). The PCR also added Hin the dIII restriction site at the 5' end and EcoR the I restriction site at the 3' end to facilitate cloning into the pXC17.4 vector (Lonza).

[0140] The approximately 0.75 kb HindIII and EcoRI digested PCR amplicon was cloned into the HindIII and EcoRI digested pXC-17.4 vector (Lonza). After ligation, the reaction mixture was transformed into Escherichia coli Top10F' and the transformants were scored based on carbenicillin resistance. The clones were analyzed by HindIII and EcoRI restriction digestion. The sequence of the pXC17.4 / H11A4.1 (monomer) clone was confirmed by restriction digestion and Sanger sequencing. The vector was linearized with PvuI for transfection into CHO-GS cells (Lonza).

[0141] Example 3: Construction of the multimer (dimer) pXC 17.4 / W11A4.1 (dimer) Fc variant.

[0142] In the W11A4.1 variant (dimer; SEQ ID NO.13), an Fc variant dimer was formed by connecting two chains of the Fc variant monomer (H11A4.1) via a glycine-serine linker (SEQ ID NO. 14: GGGSGGGSGGGSGGGSSGGGSS). Additionally, to prevent self-dimerization, the C226S and C229S mutations were incorporated into one of the two chains of the Fc variant. The codon-optimized gene of the Fc variant dimer with L234A, L235A, and P329G mutations was chemically synthesized by GeneArt in Germany. The W11A4 (dimer) gene was isolated from the pMA Geneart construct by digestion with Hind III and EcoR I. The approximately 1.5 kb Hind III and EcoR I digested Fc variant W11A4.1 (dimer) was ligated with Hind III and EcoRLigation of the digested pXC-17.4 vector (Lonza). The ligation mixture was transformed into Escherichia coli Top10F’, and the transformants were scored based on carbenicillin resistance. Clones were analyzed by restriction digestion with HindIII and EcoRI. The sequence of the pXC17.4 / W11A4.1 (dimer) clone (SEQ ID 12 and 13) was confirmed by restriction digestion and Sanger sequencing. The vector was linearized with Pvu I for transfection in CHO-GS cells (Lonza).

[0143] Example 4: Construction of monomeric pXC 17.4 / H11A4.2 Fc variant.

[0144] Amino acid substitutions were made at the M252Y, S254T, and T256E residues of H11A4.0 to generate the H11A4.2 variant (SEQ ID No. 19 and 20). Briefly, sequential overlap PCR was performed using pXC17.4 / H11A4.0 as a template to incorporate the desired substitutions at amino acid positions 252, 254, and 256 (EU numbering). The approximately 0.75 kb PCR amplicon was digested with Hin dIII and EcoR I and cloned into the pXC-17.4 plasmid (Lonza). After ligation, the reaction mixture was transformed into Escherichia coli Top10F’, and the transformants were scored based on carbenicillin resistance. Clones were analyzed by restriction digestion with HindIII and EcoRI. The sequence of the pXC17.4 / H11A4.2 plasmid was confirmed by restriction digestion and Sanger sequencing. The vector was linearized with Pvu I for transfection in CHO-GS cells (Lonza).

[0145] Example 5: Construction of full-length antibodies pDGV / A3H11A4.2 (mAb) and pDGV / B14 H11A4.2 (mAb) with Fc variants.

[0146] The Fc variant H11A4.2 of the present invention was incorporated into the full antibody form, replacing the constant regions of A3H11A4.0 and B14H11A4.0 with the H11A4.2 fragment (Patent Publication No. WO2022101839; PCT / IB2021 / 060495).

[0147] Briefly, for construct preparation, the Apa I restriction site at the 5’ end and the EcoThe RI restriction sites were incorporated into the H11A4.2 nucleotide sequences flanking the coding region of amino acids 20 - 247 (SEQ ID No: 20) to facilitate in-frame cloning with the heavy chain variable regions of the pDGV / A3 and pDGV / B14 vectors. The amplified ~1.0 kb H11A4.2 fragment was digested with Apa I and Eco RI and cloned into the pDGV / A3 and pDGV / B14 plasmids. Transformants in E. coli Top10F’ were scored based on carbenicillin resistance. The sequences of the pDGV / A3H11A4.2 (for light chain, SEQ ID no. 21 and 22; for heavy chain, SEQ ID No. 23 and 24) and pDGV / B14H11A4.2 (for light chain, SEQ ID no. 25 and 26; for heavy chain, SEQ ID no. 27 and 28) plasmid vectors were confirmed by restriction digestion and Sanger sequencing. The vectors were linearized with Pvu I for transfection in CHO - GS cells (Lonza).

[0148] Example 6: Construction of full - length antibodies pDGV / A3H11A4.3 and pDGV / B14H11A4.3 with Fc variants

[0149] Amino acid substitutions were made at the M252Y, S254T, and T256E residues of H11A4.1 to generate the H11A4.3 variant. The pDGV / A3 and pDGV / B14 constructs encoding H11A4.3 in the form of mAb were generated by sequential overlapping polymerase chain reaction to incorporate the desired substitutions at amino acid positions 252, 254, and 256 (EU numbering). Subsequently, Mlu I and Eco R1 restriction sites were also added at the 5’ and 3’ ends to facilitate the insertion of the PCR - amplified fragments into the pDGV / A3 and pDGV / B14 vectors.

[0150] The PCR amplicons containing the desired substitutions were digested with Mlu I and Eco RI restriction enzymes and then cloned separately into the vectors digested with Mlu I and Ecoin the RI digested pDGV / A3 and pDGV / B14 plasmids. After ligation, the reaction mixture was transformed into E. coli Top10F’ cells and the transformants were scored based on carbenicillin resistance. The sequences of the pDGV / A3 H11A4.3 (for the light chain, SEQ ID no. 29 and 30; for the heavy chain, SEQ ID no: 31 and 32) and pDGV / B14 H11A4.3 (for the light chain, SEQ ID no. 33 and 34; for the heavy chain, SEQ ID No: 35 and 36) plasmid vectors were confirmed by restriction digestion and Sanger sequencing. The vector was linearized with Pvu I for transfection into CHO-GS cells (Lonza).

[0151] Example 7: Construction of monomeric pXC 17.4 / H11A4.3 variants.

[0152] The nucleotide sequence encoding the secretion signal peptide was incorporated upstream of the nucleotide sequence encoding H11A4.3 by PCR. This was followed by another PCR to add Hin the dIII restriction site at the 5’ end and EcoR the I restriction site at the 3’ end to facilitate cloning into the pXC17.4 vector (Lonza). The resulting ~0.75 kb amplicon (SEQ ID. 37) encoding H11A4.3 (SEQ ID 38) was digested with Hin dIII and EcoR I and ligated to Hind III and EcoR I digested pXC-17.4 vector (Lonza). After ligation, the reaction mixture was transformed into E. coli Top10F’ and the transformants were scored based on carbenicillin resistance. The clone was confirmed by digestion with Hind III and EcoR I restriction digestion. The sequence of the pXC17.4 / H11A4.3 construct was confirmed by restriction digestion and Sanger sequencing. The vector was linearized with Pvu I for transfection into CHO-GS cells (Lonza).

[0153] Example 8: Production of Fc variant proteins

[0154] All vector constructs as described in Examples 1 to 7 were used for transfection. All plasmids generated in Examples 1 to 7 above were used with PvuIThe restriction enzyme was linearized and used for transfection. Chinese hamster ovary (CHO) cells were used as the host for expressing recombinant proteins. CHO cells were seeded at a density of 0.5 million / mL approximately 24 hours before transfection to make the cells in the exponential phase. Transfection was carried out by electroporation technology using the Neon Transfection System (Invitrogen) according to the manufacturer's instructions. After transfection, the cells were plated in a 24-well cell culture plate containing 1 mL of pre-warmed ProCHO5 serum-free medium (Lonza, Switzerland) containing 25 µM MSX; and incubated in a humidified incubator at 37°C in the presence of 5% CO2. The cell numbers of all transfection pools (i.e., heterogeneous mixtures of different expressing cells) were monitored regularly and medium replacement was carried out regularly. Once the cells recovered from transfection, the cells were further successively amplified into 6-well culture plates, T-flasks, and culture tubes (TPP).

[0155] Fed-batch cultures of transfection pools of all Fc variant candidates were carried out in culture tubes (TPP) to produce recombinant proteins. The cells were seeded at a density of 0.3×10 6 cells / mL in ActiPro production medium from Hyclone Tm , Cytiva. The culture tubes were incubated in a humidified Kuhner shaker at a temperature of 37°C, a CO2 level of 5%, and a shaking speed of 230 RPM. A fixed daily feeding protocol was followed during the culture of all pools using chemically defined feeds from Hyclone, Cytiva. After 72 hours of culture, feeding was started and continued until the harvest batch.

[0156] After harvest, the candidate proteins were isolated from the culture supernatant by protein A affinity chromatography. These proteins were further tested in various in vitro assays to analyze their various properties.

[0157] Example 9: Determination of the kinetic rate constants for the binding of Fc variants to recombinant human neonatal Fc receptor (rhFcRn) at pH 6.0

[0158] The kinetic constants of the binding of the Fc variant candidates (expressed in Example 8) to recombinant human neonatal Fc receptor (rhFcRn) were determined by surface plasmon resonance-based measurements using a Biacore 8K+ instrument (Cytiva). The CM5 sensor chip was activated by immobilizing an anti-histidine antibody on the surface of the CM5 sensor chip using standard amine coupling chemistry, followed by blocking with a 1 M, pH 8.0 ethanolamine solution for 7 minutes. Recombinant human FcRn receptor (rhFcRn) at a concentration of 0.1 μg / mL was captured onto the anti-histidine antibody-immobilized chip surface by injecting for 60 s at a flow rate of 10 μL / min. Five dilutions (ranging from 55 nM - 0.68 nM) of the Fc variant sample in dPBS at pH 6.0 containing 0.05% (v / v) polysorbate 20 were injected onto the FcRn protein captured on the chip at a flow rate of 30 μL / min, with a binding time of 60 s and a dissociation time of 60 s. After each sample run, the chip surface was regenerated with a 10 mM, pH 1.5 glycine solution for 30 s. Rate constant values k d (dissociation constant) and k a (association constant) were calculated using a 1:1 binding model in Biacore Insight Evaluation software (v 3.0.12.15655). The K D value (affinity constant) was calculated as the ratio of k d to k a , i.e., K D = k d / k a . The affinity constants of the binding of the Fc variant candidates to rhFcRn were measured at pH 6.0 and are shown in Table 3.

[0159] Table 3: Kinetic rate constants of FcRn binders at pH 6.0

[0160] Example 10: Determination of the kinetic rate constants of the binding of Fc variants to recombinant human neonatal Fc receptor (rhFcRn) at pH 7.4

[0161] The FcRn binding of the samples was analyzed at pH 7.4 to examine the effect of pH on their binding and dissociation with FcRn. In this experiment, the affinity constants of the Fc variant candidates for recombinant human neonatal Fc receptor (rhFcRn) were determined by surface plasmon resonance-based measurements using a Biacore 8K+ instrument (Cytiva). rhFcRn was captured on an anti-histidine antibody immobilized on a CM5 sensor chip as mentioned in Example 9. Kinetic measurements were carried out using a running buffer at pH 7.4. To measure the affinity constants, five dilutions of the Fc variant (ranging from 550 nM - 6.79 nM) were prepared and injected at a flow rate of 30 μL / min, with a binding time of 60 s and a dissociation time of 60 s. All reactions were conducted at 25 °C. The affinity constants of the Fc variant candidates for binding to rhFcRn were measured at pH 7.4 and are shown in Table 4.

[0162] Table 4: Affinity constants of FcRn binders at pH 7.4

[0163] As can be seen from Tables 3 and 4, compared with pH 7.4, the Fc variants of the present invention can bind to FcRn with higher affinity at pH 6.0. Therefore, 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), like wild-type Fc.

[0164] Example 11: Determination of kinetic rate constants for the binding of Fc variants to recombinant neonatal Fc receptors (rFcRn) of different species

[0165] In this experiment, the binding of the Fc variant candidates of the present invention to FcRn of different species was determined by surface plasmon resonance-based measurements using a Biacore 8K+ instrument (Cytiva). To examine the kinetic rate constants, experiments were conducted using recombinant FcRn from mice, monkeys, and humans to bind to the Fc variant candidates. FcRn (from monkey and human species) was captured on a CM5 chip as mentioned in Example 9. Mouse FcRn was immobilized on the surface of a CM5 sensor chip using standard amine coupling chemistry according to the manufacturer's protocol. 10 mM phosphate buffered saline (PBS) (10 mM phosphate buffer, pH 6.0, 150 mM NaCl, 0.005% Tween 20) was used as the running buffer for kinetic measurements. To measure the association rate constant (k a ) and the dissociation rate constant (k d) Five dilutions of the Fc mutant protein (ranging from 55 nM - 0.027 nM) were prepared in the above running buffer and injected at a flow rate of 30 μL / min, with a binding time of 60 s. The dissociation time was maintained up to 1800 s. All reactions were carried out at 25 °C. After each sample run, the chip surface was regenerated using glycine / 3M MgCl2 at pH 1.5. Sensorgram-form data was analyzed using Biacore Insight Evaluation software (v 3.0.12.15655).

[0166] The kinetic constants of the binding of the Fc variants to FcRn receptors of different species are shown in Table 5.

[0167] Table 5: Kinetic constants of Fc variants binding to FcRn of different species

[0168] As can be seen from Table 5, the Fc variants of the present invention can not only bind to human FcRn in vitro, but also bind to mouse FcRn and monkey FcRn in vitro. The favorable feature of cross-reactivity with monkey and mouse FcRn implies that the Fc variants of the present invention can be tested in non-human primates and mice, and the resulting data may be very useful in predicting their pharmacokinetic, pharmacodynamic and toxicity characteristics in humans.

[0169] Example 12: Effect of Fc variants on serum total IgG levels in wild-type C57BL / 6 mice

[0170] In Part 1 of this study, two Fc variants of the present invention in the form of full-length antibodies, namely A3H11A4.1 (SEQ ID Nos. 3 and 5, i.e., the light chain amino acid sequence and the heavy chain amino acid sequence respectively) and B14H11A4.1 (SEQ ID Nos. 7 and 9, i.e., the light chain amino acid sequence and the heavy chain amino acid sequence respectively), were compared with two full-length antibodies, namely A3H11A4.0 (SEQ ID Nos. 15 and 16, i.e., the heavy chain amino acid sequence and the light chain amino acid sequence respectively) and B14H11A4.0 (SEQ ID Nos. 17 and 18, i.e., the heavy chain amino acid sequence and the light chain amino acid sequence respectively).

[0171] The antibodies A3H11A4.1 and B14H11A4.1 of the present invention were compared with A3H11A4.0 and B14H11A4.0 respectively to determine the effect of the Fc variants of the present invention on serum total IgG levels in circulation. Wild-type C57BL / 6 mice were randomized based on their body weight. On day -2 (i.e., 48 hours before dosing), blood collection was performed. On day 0 (0 hours), the antibodies at the doses mentioned in Table 6 were administered to the study animals in each group (6 mice per group) via the intraperitoneal route.

[0172] Table 6: Administration of Antibodies via Intraperitoneal Route

[0173] Blood samples were collected at the following time points: Day 1, Day 2, Day 5, and Day 8. Endogenous serum IgG levels were measured in the samples collected at the mentioned time points using ELISA. Compared to the normal control (placebo group), all four treatment groups [A3H11A4.1, B14H11A4.1, A3H11A4.0, and B14H11A4.0] showed a decrease in mouse IgG concentration. When comparing between the treatment groups, A3H11A4.1 and B14H11A4.1 showed a higher IgG decrease than A3H11A4.0 and B14H11A4.0 throughout the study period. A graphical representation of the results of the relative IgG concentration (%) in all treatment groups relative to the pre-dose level is shown in Figure 3 in.

[0174] In Part 2 of this study, two Fc variants of the present invention in the form of full-length antibodies, namely A3H11A4.2 (SEQ ID 22 and 24, i.e., the light chain amino acid sequence and the heavy chain amino acid sequence, respectively) and A3H11A4.3 (SEQ ID 30 and 32, i.e., the light chain amino acid sequence and the heavy chain amino acid sequence, respectively), were compared with the full-length antibody, namely A3H11A4.0 (SEQ ID 15 and 16, i.e., the heavy chain amino acid sequence and the light chain amino acid sequence, respectively). The full-length amino acid and nucleotide sequences of the antibodies and Fc form candidates are described in Table 2.

[0175] The antibodies A3H11A4.2 and A3H11A4.3 of the present invention were compared with A3H11A4.0 to determine the effect of the Fc variants of the present invention on the total serum IgG level in circulation. Wild-type C57BL / 6 mice were randomized based on their body weight. On Day -2 (i.e., 48 hours before dosing), blood collection was performed. On Day 0 (0 hours), the candidates at the doses mentioned in Table 7 were administered via the intraperitoneal route to the study animals in each group (6 mice per group).

[0176] Table 7: Administration of Antibodies via Intraperitoneal Route

[0177] Blood samples were collected at the following time points: Day 1, Day 2, Day 5, and Day 8. Endogenous serum IgG levels were measured in the samples collected at the time points mentioned using ELISA. Compared to the normal control (placebo group), all three treatment groups [A3H11A4.0, A3H11A4.2, and A3H11A4.3] showed a decrease in mouse IgG concentration. When compared between the antibodies tested in the study, treatment groups A3H11A4.2 and A3H11A4.3 showed a higher IgG reduction than A3H11A4.0. A graphical representation of the results of the relative IgG concentration (%) in different antibody candidates relative to the pre-dose level is shown in Figure 4 in.

[0178] In Part 3 of this study, three Fc variants of the present invention in Fc form (i.e., H11A4.1, H11A4.2, and H11A4.3) were evaluated to examine their effects on the total serum IgG level in circulation. The full-length amino acid and nucleotide sequences of the Fc form candidates are described in Table 2.

[0179] Wild-type C57BL / 6 mice were randomized based on their body weight. On Day -2 (i.e., 48 hours before dosing), blood collection was performed. On Day 0 (0 hours), the candidates at the doses mentioned in Table 8 were administered to the study animals in each group (6 mice per group) via the intraperitoneal route.

[0180] Table 8: Administration of Antibodies via the Intraperitoneal Route

[0181] Blood samples were collected at the following time points: Day 1, Day 2, Day 5, and Day 8. Endogenous serum IgG levels were measured in the samples collected at the time points mentioned using ELISA. Compared to the normal control (placebo group), all three treatment groups [H11A4.1, H11A4.2, and H11A4.3] showed a decrease in mouse IgG concentration, and the highest IgG depletion was observed on Day 2. A graphical representation of the results of the relative IgG concentration (%) in different Fc candidates relative to the pre-dose level is shown in Figure 5 in.

[0182] Example 13: Effects of Fc Variants on Total Serum IgG Levels in Rhesus Macaques

[0183] The effect of the Fc variants of the present invention in Fc form, namely H11A4.1 (SEQ ID NO: 1 and SEQ ID NO: 11), on the total serum IgG level in circulation was determined in rhesus monkeys by a single-dose study. H11A4.1 was administered to the animals (N = 2) at a dose of 10 mg / kg via the intravenous infusion route. Blood samples were collected at the following time points: before dosing, 6 hours, 24 hours (day 1), day 2, day 3, day 4, day 5, day 7, day 9, day 11, day 14, day 17, day 21, day 28, day 35, day 42, day 49, and day 56. The endogenous serum IgG levels were determined in the samples collected at the mentioned time points using an ELISA-based method. A relative decrease in endogenous IgG compared to the pre-dose values was observed in both animals, with the highest IgG depletion observed on day 5.

[0184] Graphical representation of the results of the relative IgG concentration (%) relative to the pre-dose level is shown in Figure 6 the

[0185] Example 14: Effect of Fc variants on total serum IgG levels in rhesus monkeys

[0186] The effect of the Fc variants of the present invention in full-length antibody form, namely A3H11A4.1 (SEQ ID NO: 3 and 5, i.e., the light chain amino acid sequence and the heavy chain amino acid sequence, respectively), on the total serum IgG level in circulation was determined in rhesus monkeys by a single-dose study. A3H11A4.1 was administered to the animals (N = 2) at a dose of 30 mg / kg via the intravenous infusion route. Blood samples were collected at the following time points: before dosing, 6 hours, 24 hours (day 1), day 2, day 3, day 4, day 5, day 7, day 9, day 11, day 14, day 17, day 21, day 28, day 35, day 42, day 49, and day 56. The endogenous serum IgG levels were determined in the samples collected at the mentioned time points using an ELISA-based method. A relative decrease in endogenous IgG compared to the pre-dose values was observed in both animals, with the highest IgG depletion observed on day 9.

[0187] Graphical representation of the results of the relative IgG concentration (%) relative to the pre-dose level is shown in Figure 7 the

[0188] Example 15: Effect of Fc variants on total serum IgG levels in rhesus monkeys

[0189] The effect of the Fc variant of the invention in full-length antibody form, i.e., B14H11A4.1 (SEQ ID NOs: 7 and 9, which are the light chain amino acid sequence and the heavy chain amino acid sequence, respectively), on the total serum IgG level in circulation was determined in a single-dose study in rhesus monkeys. B14H11A4.1 was administered to animals (N = 2) via the intravenous infusion route at a dose of 30 mg / kg. Blood samples were collected at the following time points: before dosing, 6 hours, 24 hours (Day 1), Day 2, Day 3, Day 4, Day 5, Day 7, Day 9, Day 11, Day 14, Day 17, Day 21, Day 28, Day 35, Day 42, Day 49, and Day 56. The endogenous serum IgG level was determined for the samples collected at the time points mentioned using an ELISA-based method. A relative decrease in endogenous IgG compared to the pre-dose value was observed in both animals, with the highest IgG depletion observed on Day 4.

[0190] A graphical representation of the results of the relative IgG concentration (%) relative to the pre-dose level is shown in Figure 8 .

[0191] Incorporated by reference

[0192] The entire disclosure of each patent document and scientific article mentioned herein is incorporated by reference for all purposes.

[0193] Equivalents

[0194] Without departing from the spirit or essential characteristics of the invention, the invention may be embodied in other specific forms. Accordingly, the above-described embodiments are to be considered in all respects as illustrative and not restrictive of the invention described herein. Thus, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalents of the claims are intended to be embraced therein.

[0195] References incorporated into this patent application: 1. Leona E. Lingl, Jan L. Hillson, M281, an Anti-FcRn Antibody: Pharmacodynamics, Pharmacokinetics, and Safety Across the Full Range of IgG Reduction in a First-in-Human Study, Clinical Pharmacology & Therapeutics, Volume 105 Number 4, April 2019 2. Peter Kiessling et al., The FcRn inhibitor rozanolixizumab reduces human serum IgG concentration: A randomized phase 1 study, Sci. Transl. Med. 9, eaan1208, November 2017 3. Deisenhofer, Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-.ANG. resolution, Biochemistry 20: 2361-2370, 1981 4. Edelman, G.M. et al., The covalent structure of an entire gamma G immunoglobulin 5 molecule. Proc. Natl. Acad. USA, 63, 78-85, 1969 5. Roux et al., Comparisons of the Ability of Human IgG3 Hinge Mutants, IgM, IgE, and IgA2, to Form Small Immune Complexes: A Role for Flexibility and Geometry, J. Immunol. 161: 4083, 1998 6. Angal S, King DJ, Bodmer MW, Turner A, Lawson AD, Roberts G, Pedley B, Adair JR. A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody. Mol Immunol. 30(1): 105-8, 1993.

Claims

1. An FcRn antagonist comprising an Fc variant, wherein the Fc variant comprises amino acid substitutions of L234A, L235A, T307N, V308P, L309Y, P329G, H433R and N434W according to EU numbering.

2. The FcRn antagonist according to claim 1, which is a monoclonal antibody, monomer, dimer or multimer.

3. The FcRn antagonist according to claim 2, which is a monomer having the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO.

11.

4. The FcRn antagonist according to claim 2, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 3 and SEQ ID No.

5.

5. The FcRn antagonist according to claim 2, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 7 and SEQ ID No.

9.

6. The FcRn antagonist according to claim 2, which is a dimer having the amino acid sequence shown in SEQ ID NO.

13.

7. The FcRn antagonist according to claim 1, which further comprises amino acid substitutions of M252Y, S254T and T256E according to EU numbering.

8. The FcRn antagonist according to claim 7, which is a monoclonal antibody, monomer, dimer or multimer.

9. The FcRn antagonist according to claim 8, which is a monomer having the amino acid sequence shown in SEQ ID NO.

38.

10. The FcRn antagonist according to claim 8, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 30 and SEQ ID No.

32.

11. The FcRn antagonist according to claim 8, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 34 and SEQ ID No.

36.

12. An FcRn antagonist comprising an Fc variant, wherein the Fc variant comprises amino acid substitutions of T307N, V308P, L309Y, H433R, N434W, M252Y, S254T and T256E according to EU numbering.

13. The FcRn antagonist according to claim 12, which is a monoclonal antibody, monomer, dimer or multimer.

14. The FcRn antagonist according to claim 13, which is a monomer having the amino acid sequence shown in SEQ ID NO.

20.

15. The FcRn antagonist according to claim 13, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 22 and SEQ ID No.

24.

16. The FcRn antagonist according to claim 13, which is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID No. 26 and SEQ ID No.

28.

17. The FcRn antagonist as claimed in claims 1-16, which has a K value for FcRn in the range of 10 -11 M to 10 -8 M D .

18. An FcRn antagonist as claimed in any one of claims 1-17, which cross-reacts with FcRn of mice, monkeys and humans.

19. A composition comprising an FcRn antagonist as claimed in any one of claims 1-18 and an acceptable carrier.

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