Fc variants and their preparation

Abstract

The present invention relates to FcRn antagonist or Fc variant proteins and preparations thereof. The Fc variants have altered binding affinity to FcRn. In one embodiment, an Fc variant according to the invention comprises the following amino acid substitutions: L234A, L235A, T307N, V308P, L309Y, P329G, H433R, and N434W. In another embodiment, an Fc variant according to the invention comprises the following amino acid substitutions: T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, and T256E. In a further embodiment, the Fc variants of the invention comprise the following amino acid substitutions: T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E, L234A, L235A, P329G. The Fc variants prepared according to the invention can be used to generate FcRn antagonist compositions or to generate Fc variant-containing drugs or molecules with altered effector functions.

Description

[Technical Field]

[0001] The present invention relates to Fc variant proteins and their preparation. [Background technology]

[0002] The neonatal Fc receptor (FcRn) is structurally similar to the major histocompatibility complex (MHC) class I heterodimeric molecule, consisting of a type I transmembrane heavy chain noncovalently associated with a soluble light chain, β2-microglobulin (β2m). Monoclonal antibody-based antagonists have been developed to inhibit endogenous IgG-FcRn interactions. One approach is the use of monoclonal antibodies directed against FcRn that act via a 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 accelerating the clearance of endogenous IgG. A mean reduction of endogenous IgG ranging from 25 to 80% was observed in a dose-dependent manner with M281. Furthermore, a single dose of M281 at 30 mg / kg or 60 mg / kg maintained serum IgG at or below 50% of baseline for 18 and 27 days, respectively. 1 In healthy subjects, serum IgG concentrations were reduced by 50% with a single IV dose of 7 mg / kg UCB7665. The observed reduction in serum IgG concentrations with UCB7665 persisted for several weeks, with the maximum reduction achieved by days 7–10, followed by a gradual return to baseline by day 57. 2 . [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2021234655(PCT / IB2021 / 054423) [Patent Document 2] U.S. Patent No. 7,083,970 [Patent Document 3] U.S. Patent No. 7,524,647 [Patent Document 4] WO2007 / 017903 [Patent Document 5] WO2012 / 046255 [Patent Document 6] WO2022101839; PCT / IB2021 / 060495 Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention provides improved FcRn-binding agents that address various unmet needs, such as minimal toxicity, down-regulation of FcRn activity, increasing the circulating half-life of various drugs, targeting drugs and / or vaccines to specific cell types, etc. The FcRn-binding agents of the present invention can be used to treat a wide variety of diseases. WO2021234655 discloses various FcRn-binding molecules. The present invention provides therapeutically effective FcRn-binding molecules that can be used in the treatment of autoimmune diseases. [Means for solving the problem]

[0005] The present invention provides novel Fc variants with altered binding affinity to FcRn, preferably higher binding affinity to FcRn. In one embodiment, an Fc variant according to the invention comprises amino acid substitutions L234A, L235A, T307N, V308P, L309Y, P329G, H433R, and N434W. In another embodiment, an Fc variant according to the invention comprises amino acid substitutions T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, and T256E. In a further embodiment, an Fc variant according to the invention comprises amino acid substitutions T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E, L234A, L235A, and P329G. The amino acid substitutions referred to in the present invention herein are according to the EU numbering system (EU positions). The Fc variants can be used to develop drug compositions with FcRn antagonist function or increased circulating half-life, or for targeting specific cells and / or tissues. The present invention also provides methods for making the novel Fc variants of the present invention. The Fc variants according to the present invention are further used in the preparation of drugs for treating diseases in which FcRn activity is deleterious, or for increasing the circulating half-life of drugs, or for targeting drugs to certain cells or tissues. [Brief explanation of the drawings]

[0006] [Figure 1] FIG. 1 depicts a map of the vectors used for the production of the Fc monomers and Fc dimers of the present invention. [Figure 2] FIG. 2 depicts a map of the vector used for the production of monoclonal antibodies containing the Fc monomers of the present invention. [Figure 3] FIG. 3 depicts the relative percentage IgG concentrations with respect to pre-dose levels across the various treatment groups—A3H11A4.0, A3H11A4.1, B14H11A4.0, and B14H11A4.1. [Figure 4]FIG. 4 depicts the relative percentage IgG concentrations with respect to pre-dose levels across the various treatment groups of antibody molecules - A3H11A4.0, A3H11A4.2, A3H11A4.3. [Figure 5] FIG. 5 depicts the relative percentage IgG concentrations with respect to pre-dose levels across the various treatment groups of candidate-H11A4.1, H11A4.0, H11A4.2, H11A4.3 Fc. [Figure 6] FIG. 6 depicts the relative percentage IgG concentrations across study time points versus pre-dose levels for the H11A4.1 ​​Fc candidate. [Figure 7] FIG. 7 depicts the relative percentage IgG concentrations with respect to pre-dose levels across study time points for the A3H11A4.1 ​​antibody candidate. [Figure 8] FIG. 8 depicts the relative percentage IgG concentrations with respect to pre-dose levels across study time points for the B14H11A4.1 ​​antibody candidate. DETAILED DESCRIPTION OF THE INVENTION

[0007] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. However, the meaning and scope of terms shall be clear in the event of any unidentified ambiguity, and the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, the nomenclature used in connection with and in the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization techniques described herein is that well known and commonly used in the art.

[0008] As used herein, the term "Fc domain" refers to that portion of a single immunoglobulin heavy chain beginning with 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 at least a portion of the hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, and a CH3 domain.

[0009] As used herein, the term "amino acid modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence.

[0010] As used herein, the term "amino acid substitution" or "substitution" refers to the replacement of an amino acid with another amino acid at a particular position in a parent polypeptide sequence. For example, the substitution T307N refers to a variant polypeptide, in this case an Fc variant, in which the threonine at position 307 is replaced with an asparagine, where "T" and "N" represent the one-letter codes for threonine and asparagine, respectively.

[0011] According to the present invention, the term "antigen-binding molecule" 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, peptibodies, fusion proteins, monomers, or dimers comprising the Fc variants of the present invention.

[0012] The term "FcRn antagonist" refers to any agent containing an Fc region that specifically binds to FcRn through the Fc region and inhibits the binding of immunoglobulins to FcRn. The terms "FcRn antagonist," "Fc variant protein," "Fc protein variant," "Fc variant," "Fc variant-containing molecule," "Fc variant-containing protein," and the like can be used interchangeably in the present invention.

[0013] As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") containing the Fc portion. An "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain contains a heavy chain variable region (referred to herein as V H The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as V L The light chain constant region consists of one domain, C L It is composed of: V H and V L The regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L Each antibody 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, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to factors including host tissues or 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.

[0014] 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 the gene.

[0015] The term "ka" is the association rate of an interaction between two molecules, and the term "kd" is the dissociation rate of an interaction between two molecules. D" is the affinity rate constant, which is obtained from the ratio of kd to ka. Affinity rate constants can be measured using surface plasma resonance techniques, which are well known in the art.

[0016] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer 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.

[0017] 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.

[0018] The term "recombinant antibody," as used herein, includes all antibodies that are prepared, expressed, generated, or isolated by recombinant means. However, in certain embodiments, such recombinant antibodies may be obtained by in vitro mutagenesis, and thus the V of the recombinant antibodies described herein may be used. H and V L The amino acid sequence of the region is a sequence that may not naturally occur within the human antibody germline repertoire in vivo.

[0019] The term "Fc" fragment reflects its ability to readily crystallize. The crystal structure of the human IgG Fc region has been determined. 3 In human IgG molecules, the Fc region can be separated from the rest of the molecule by digestion with papain, which cleaves from the N-terminus to Cys226. The Fc region is central to the effector functions of antibodies.

[0020] As used herein, the term "Fc protein" refers to the portion of a single immunoglobulin heavy chain beginning at the hinge immediately upstream of the papain cleavage site and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes at least a portion of the hinge (e.g., upper, middle, and / or lower hinge region), a CH2 domain, and a CH3 domain. An Fc protein comprises two Fc chains linked together via two or more disulfide bridges. Fc proteins include, but are not limited to, "Fc protein variants known in the art."

[0021] 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 referred to herein include any approved or clinically or preclinically active antibodies, fusion proteins, and peptibodies.

[0022] As used herein, the term "Fc variant-containing molecule" refers to any molecule comprising an Fc variant of the invention. This molecule 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.

[0023] As used herein, the term "Fc variant protein," or "Fc protein variant," or "Fc variant" refers to an Fc protein that differs from the wild-type Fc protein by virtue of 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 the amino acid sequence encoding the Fc variant. Preferably, the Fc variant comprises a monomer, dimer, or multimer. Preferably, the Fc variant has at least one amino acid modification compared to the wild-type Fc protein, e.g., about one to about 11 amino acid modifications, preferably about one to about eight amino acid modifications compared to the wild-type Fc protein. The term "Fc variant proteins known in the art" or "Fc protein variants known in the art" or "Fc variants known in the art" as used herein in the present invention refers to "Fc variant proteins" or "Fc protein variants" or "Fc variants" disclosed in the public domain before the priority date of this patent specification, preferably one or more "Fc variant proteins" or "Fc protein variants" or "Fc variants" disclosed in PCT / IB2021 / 054423 (WO2021234655).

[0024] As used herein, the term "EU position" refers to an amino acid position in the EU numbering convention for the Fc region as described in the reference. 4 .

[0025] As used herein, the term "CH1 domain" refers to the first (amino-terminal) constant region domain of an immunoglobulin heavy chain, extending from about EU position 118 to 215. The CH1 domain is H domain and adjacent amino terminal to the hinge region of an immunoglobulin heavy chain molecule, and does not form part of the Fc region of an immunoglobulin heavy chain.

[0026] 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, middle, and lower hinge domains. 5 The Fc variants of the present invention may comprise all or part of the hinge region.

[0027] As used herein, the term "CH2 domain" refers to the portion of a heavy chain immunoglobulin molecule that extends from about EU positions 231 to 340.

[0028] As used herein, the term "CH3 domain" refers to the portion of a heavy chain immunoglobulin molecule extending from the N-terminus of the CH2 domain, for example, approximately 110 residues from about positions 341-447 (EU numbering system).

[0029] As used herein, the term "about" refers to variations in numerical quantities that may occur through, for example, typical measuring and handling procedures used to make a compound, composition, concentrate, or formulation; through inadvertent errors in these procedures; differences in manufacture, source, or purity of starting materials or components used in carrying out the method, and other similar considerations.

[0030] 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 encoded, at least in part, 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 comprises two polypeptides, often 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. Unless otherwise specified herein, FcRn or FcRn protein refers to a complex of the FcRn heavy chain and beta-2-microglobulin.

[0031] As used herein, the term "wild-type Fc" refers to an unmodified Fc polypeptide that is subsequently 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 the unmodified Fc polypeptide.

[0032] As used herein, the term "position" refers to a site in a protein sequence. Positions may be numbered sequentially or according to established formats, such as the EU numbering system or EU numbering or EU index or Kabat numbering. For example, position 105 refers to the 105th amino acid residue from the N-terminus of the heavy or light chain in human antibody IgG1.

[0033] The terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to a living organism suffering from or susceptible to a condition that can be prevented or treated by administering the compositions of the present invention, including both humans and non-human animals. Examples of subjects include, but are not limited to, humans, chimpanzees and other apes and monkey species; livestock such as cows, sheep, pigs, goats, and horses; domestic animals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; and birds, including domestic, wild, and game birds such as chickens, turkeys, and other pheasants, ducks, geese, and the like. This term does not denote a specific age. Thus, adult, juvenile, and newborn individuals are subjects.

[0034] As used herein, the term "residue" refers to a position in a protein and its associated amino acid identity. For example, threonine 307 (also called Thr307, also called T307) is a residue in the human antibody IgG1.

[0035] As used herein, the term "immunoconjugate" or "conjugate" refers to a compound or derivative thereof linked to a cell-binding agent (i.e., an antibody or Fc variant described herein) and defined by the general formula ALD, where A = cell-binding agent or antibody or Fc variant of the invention, L = linker, and D = drug (toxin or pharmaceutical). Immunoconjugates can also be defined by the general formula DLA in the reverse order:

[0036] The term "linker" refers to any chemical moiety capable of stably and covalently linking a compound, typically a drug such as a maytansinoid or auristatin, to a cell-binding agent (i.e., an antibody or Fc variant described herein). The linker can be susceptible to or substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, provided that 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, photolabile groups, peptidase-labile groups, and esterase-labile groups. Linkers also include charged linkers, as described herein and known in the art, and hydrophilic forms thereof.

[0037] The term "antagonist" refers to an agent that inhibits or reduces the biological activity of an antigen, such as FcRn, to which it binds. 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%.

[0038] As used herein, the term "treatment" or "therapy" refers to any treatment of a disease in a mammal, particularly a human. This term includes (a) preventing a disease from occurring in a subject prone to or at risk of having the disease but who has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., halting the development of the disease; and (c) palliating the disease, i.e., causing regression of the disease.

[0039] 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.

[0040] The term "non-naturally encoded amino acid" refers to an amino acid that is not one of the common or natural amino acids or pyrrolysine, pyrroline-carboxy-lysine, or selenocysteine. Other terms that may be used synonymously with "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 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 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.

[0041] [Table 1]

[0042] Other abbreviations used in this patent application %:percentage ℃: 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% effective concentration of any drug EC 75 : 75% effective concentration of any drug ELISA: Enzyme-linked immunosorbent assay Fc: crystallizable fragment FcGRT: Fc fragment of IgG receptor and transporter FcRn: neonatal Fc receptor G: Guanine h: time H2SO4: sulfuric acid HRP: horseradish peroxidase IC: immune complex IgG: immunoglobulin G IPTG: Isopropyl β-D-1-thiogalactopyranoside ITP: Acute 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:minutes mL: milliliter mM: millimolar mAb: 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: seconds SCIg: Subcutaneous immunoglobulin SEQ / seq: Sequence SPR: surface plasmon resonance T: Thymine YT media: Yeast extract tryptone medium

[0043] Embodiments of the present invention The present disclosure provides novel FcRn antagonists that comprise the Fc variants disclosed in the description herein.

[0044] In one embodiment the Fc variant protein or Fc variant containing protein or Fc variant containing molecule of the invention comprises L234A, L235A, T307N, V308P, L309Y, P329G, H433R, N434W amino acid substitutions.

[0045] In one embodiment the Fc variant protein or Fc variant containing protein or Fc variant containing molecule of the invention comprises T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E amino acid substitutions.

[0046] In one preferred embodiment, an Fc variant of the invention comprises T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E, L234A, L235A, P329G amino acid substitutions.

[0047] In one embodiment, an Fc variant of the invention comprises a single chain of an Fc domain or two chains of an Fc domain, preferably two identical chains of an Fc domain or multiple chains of an Fc domain.

[0048] In one embodiment, the Fc variant protein or Fc variant-containing protein or Fc variant-containing molecule of the invention binds with high affinity to human FcRn.

[0049] In one embodiment, the Fc variant protein or Fc variant-containing protein or Fc variant-containing molecule of the invention reduces serum IgG levels in a subject undergoing treatment.

[0050] In one embodiment, the Fc variant protein or Fc variant-containing protein or Fc variant-containing molecule of the invention binds with higher affinity to human FcRn compared to wild-type Fc protein.

[0051] In one embodiment, the Fc variants of the invention have a 10 -8 M or less, preferably 10 -9 M or less, more preferably 10 -10 M or less K D K D The value is a measure of the binding affinity of the drug candidate to FcRn. In one embodiment, the Fc variants of the invention have a binding affinity of 10 to FcRn. -11 M~10 -8 K in the M range D It has.

[0052] In one embodiment, the amino acid sequence of the Fc variant is l, IgG2, IgG3, IgG4 or IgG2 / G4 isotype, preferably IgG1 isotype.

[0053] In one embodiment, the Fc variants of the invention do not significantly interfere with the binding properties of FcRn to albumin.

[0054] In one embodiment, the Fc variants of the invention cross-react with mouse, monkey and human FcRn.

[0055] In a preferred embodiment, the Fc variants of the invention have a higher binding affinity for human FcRn compared to the wild-type Fc protein or one or more protein variants known in the art.

[0056] In a preferred embodiment, the Fc variants of the present invention have a higher binding affinity to mouse FcRn compared to the wild-type Fc protein or one or more protein variants known in the art.

[0057] In a preferred embodiment, the Fc variants of the invention have a higher binding affinity for monkey FcRn compared to the wild-type Fc protein or one or more protein variants known in the art.

[0058] In one embodiment, the Fc variants or Fc variant-containing proteins or Fc variant-containing molecules of the present invention have a longer half-life in a subject compared to one or more protein variants known in the art. In certain embodiments, polyethylene glycol or human serum albumin may be linked to the Fc variants of the present invention to further increase the half-life of the Fc variants. In another embodiment, the Fc variants of the present invention may contain mutations that increase their half-life in a subject. In another embodiment, the Fc variants of the present invention can be expressed in a monomeric, dimeric, or multimeric form to increase half-life by increasing molecular size and affinity through higher binding activity of the Fc variants. As used herein, the term "multimeric form" refers to a form of a protein containing more than one unit of a protein, preferably an Fc protein of the present invention. For example, the protein form can be a monomer, dimer, trimer, tetramer, pentamer, hexamer, etc. For example, a monomeric Fc protein has two binding sites for FcRn. Similarly, an Fc dimer according to the present invention has four binding sites for FcRn.

[0059] In another embodiment, the Fc variants of the invention are capable of binding to mouse and monkey FcRn, allowing for facile drug discovery by providing a suitable animal model for pharmacology and toxicology studies.

[0060] In one embodiment, the invention provides a composition comprising an Fc variant protein or Fc variant-containing protein or molecule and an acceptable carrier.

[0061] In another embodiment, the Fc variant proteins or Fc variant containing proteins of the present invention can be used for the treatment of diseases such as infectious diseases, various cancers, autoimmune disorders and the like.

[0062] In certain embodiments, the Fc variant or Fc variant-containing protein or Fc variant-containing molecule of the invention 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.

[0063] In another embodiment, the Fc variants of the invention can be used to generate full-length antibodies, in which the antibody has any of the Fc variants of the invention in place of a conventional Fc region. The antibodies can be modified versions of already approved or newly discovered antibodies, where the modified version of an existing antibody has an Fc variant of the invention. Such antibodies can be selected and / or designed so that they do not bind to any target in the treated subject via their paratope.

[0064] In one embodiment, the Fc variant or Fc variant-containing protein or Fc variant-containing molecule of the invention comprises the amino acid sequence shown in SEQ ID NO:3 and SEQ ID NO:5.

[0065] In one embodiment, the Fc variant or Fc variant-containing protein or Fc variant-containing molecule of the invention comprises the amino acid sequence shown in SEQ ID NO:7 and SEQ ID NO:9.

[0066] In one embodiment, the Fc variant or Fc variant-containing protein or Fc variant-containing molecule of the invention comprises the amino acid sequence shown in SEQ ID NO:22 and SEQ ID NO:24.

[0067] In one embodiment, the Fc variant or Fc variant-containing protein or Fc variant-containing molecule of the invention comprises the amino acid sequence shown in SEQ ID NO:26 and SEQ ID NO:28.

[0068] In one embodiment, the Fc variant or Fc variant-containing protein or Fc variant-containing molecule of the invention comprises the amino acid sequence shown in SEQ ID NO:30 and SEQ ID NO:32.

[0069] In one embodiment, the Fc variant or Fc variant-containing protein or Fc variant-containing molecule of the invention comprises the amino acid sequence shown in SEQ ID NO:34 and SEQ ID NO:36.

[0070] In one embodiment, the Fc variants of the invention can be used to create drugs with longer half-lives in which the Fc variants of the invention are fused, linked or conjugated to a drug that initially has a short half-life in the circulation.

[0071] In another embodiment, the Fc variants of the invention can be used to increase the half-life of an ADC by incorporating novel variants into the antibody of the ADC, thereby decreasing its off-target toxicity by increasing its recycling through the FcRn receptor.

[0072] 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.

[0073] In one embodiment, the Fc variants of the present invention can be used to replace albumin in an albumin fusion drug to improve the pharmacokinetics of the drug.

[0074] 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 about pH 6, increasing circulating half-life by reducing metabolism.

[0075] In one embodiment, Fc variant-containing antibody molecules can be used as scavengers to remove pathogenic proteins or toxins such as TNF alpha, VEGF, etc. from the circulation.

[0076] In one embodiment, Fc variant-containing antibody molecules or proteins can be used as trappers to capture desired proteins, peptides, carbohydrates or drugs from the environment and bring them inside FcRn-expressing target cells.

[0077] In one embodiment, the Fc variants of the present invention can be used to develop monomeric Fc fusion proteins fused to any therapeutic peptide to retain their FcRn binding activity and improve their tissue penetration.

[0078] In one embodiment, the Fc variants of the invention, when present in a functional antibody molecule, can aid in the transcytosis of a target antigen bound to said antibody, thereby increasing its tissue penetration, such as a peptide, protein, carbohydrate, lipid or other molecule that needs to be delivered into a tissue to mediate its effect.

[0079] In one embodiment, the Fc variants of the invention block the IgG binding site on FcRn and compete with endogenous IgG for FcRn, resulting in greater clearance of endogenous IgG.

[0080] In one embodiment, the improved Fc variants of the invention cause less morbidity when compared to one or more Fc variants disclosed in WO2021234655 (PCT / IB2021 / 054423). In a preferred embodiment, the improved Fc variants of the invention cause no morbidity when given at the same dose.

[0081] In one embodiment, the Fc variants of the invention bind to human, mouse, and monkey FcRn in vitro. The advantageous feature of cross-reactivity with monkey and mouse FcRn means that the Fc variants of the invention can be tested in non-human primates and mice, and the data obtained may be highly useful in predicting their pharmacokinetics, pharmacodynamics, and toxicity profile in humans.

[0082] In one embodiment, the Fc variants of the present invention fused to an appropriate immunogen / antigen can improve the delivery of said antigen to FcRn-expressing APCs, such as dendritic cells, resulting in an improved immune response.

[0083] Detailed Description of the Invention The present invention relates to improved Fc variants or Fc variant-containing proteins comprising the L234A, L235A, T307N, V308P, L309Y, P329G, H433R, and N434W amino acid substitutions. In one embodiment, the Fc variant proteins or Fc variant-containing proteins or molecules of the invention comprise the T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, and T256E amino acid substitutions. In a further embodiment, the Fc variant proteins or Fc variant-containing proteins or molecules of the invention further comprise the T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E, L234A, L235A, and P329G amino acid substitutions. The amino acid substitutions in the Fc variants of the invention are according to the EU numbering system. The amino acid substitutions of the invention are in the Fc region. The Fc domain comprises at least a portion of the hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, and a CH3 domain.

[0084] In one embodiment, the improved Fc variants of the present invention cause less morbidity when compared to one or more Fc variants disclosed in WO2021234655 (PCT / IB2021 / 054423). At some doses, intravenous administration of the Fc variants of WO2021234655 (PCT / IB2021 / 054423) caused severe morbidity in animals. The inventors of the present invention surprisingly found that introducing additional mutations (amino acid substitutions) into the Fc region referred to in the invention herein helps to solve this problem. The improved Fc variants of the present invention cause significantly less morbidity when compared to one or more Fc variants disclosed in WO2021234655 (PCT / IB2021 / 054423) when administered at the same dose. Preferably, the improved Fc variants of the present invention do not cause morbidity. In some embodiments, the improved Fc variants of the invention have a higher reduction in endogenous IgG when compared to one or more Fc variants disclosed in WO2021234655 (PCT / IB2021 / 054423). Accordingly, the improved Fc variants of the invention cause less, and preferably no, morbidity when compared to one or more Fc variants disclosed in WO2021234655 (PCT / IB2021 / 054423). Furthermore, the Fc variants of the invention reduce serum IgG and thus retain their efficacy. In one embodiment, the Fc variants of the invention reduce serum IgG.

[0085] In another embodiment, the improved Fc variants of the present invention have significant binding to the FcRn receptors of humans, mice, and other non-human primates, such as monkeys. The advantageous characteristics of cross-reactivity with monkey and mouse FcRn and the resulting data may be useful in predicting the pharmacokinetics, pharmacodynamics, and toxicity profile of drugs in humans.

[0086] In one embodiment, the Fc variant proteins or Fc variant-containing proteins or molecules of the invention bind with high affinity to human FcRn. The Fc variants of the invention have a lower K for FcRn at about pH 6.0 than the wild-type Fc. D The Fc variants of the present invention have a 10 binding value (i.e., higher binding) to FcRn at about pH 6.0. -8 M or less, preferably 10 -9 M or less, more preferably 10 -10 M or less K D In one embodiment, the Fc variants of the invention have a 10 -11 M~10 -8 K in the M range D K D The value is a measure of the binding affinity of a binder (in this case, an Fc variant) for its target antigen (in this case, FcRn).

[0087] Amino acid sequences of Fc variants The amino acid and nucleotide sequences of the Fc variants of the present invention are set forth in Table 2 herein.

[0088] [Table 2A]

[0089] [Table 2B]

[0090] [Table 2C]

[0091] [Table 2D]

[0092] [Table 2E]

[0093] Table 2F

[0094]

Table 2G

[0095] Table 2H

[0096] Table 2I

[0097]

Table 2J

[0098]

Table 2K

[0099]

Table 2L

[0100]

Table 2M

[0101]

Table 2N

[0102] Table 2O

[0103] [Table 2P]

[0104] [Table 2Q]

[0105] [Table 2R]

[0106] [Table 2S]

[0107] The Fc variants of the present invention are IgG l The Fc variant constructs may be in the IgG1, IgG2, IgG3, IgG4 or IgG2 / IgG4 isotype, preferably the IgG1 isotype. Fc variant constructs of the IgG4 isotype may further 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 .

[0108] In one aspect, the Fc variants of the invention may comprise one or more unnatural amino acids at one or more positions. The introduction of such unnatural amino acids into peptides is well known to those of skill in the art. Methods for generating and introducing non-naturally occurring amino acids into proteins are known, for example, from U.S. Patent Nos. 7,083,970 and 7,524,647. In one aspect, the Fc variants according to the invention have increased FcRn binding and increased half-life compared to one or more protein variants known in the art.

[0109] Nucleic acid molecules encoding Fc variants, vectors and host cells In one embodiment, the present invention provides nucleic acid molecules encoding Fc variants or Fc variant-containing proteins, suitable expression vectors containing such nucleic acids, and suitable host cells containing such nucleic acid molecules encoding the Fc variants from the expression vectors. Suitable vectors for producing the Fc variants or Fc variant-containing proteins of the present invention by recombinant methods are known to those skilled in the art. Examples of such known vectors are described in patent documents WO2007 / 017903 and WO2012 / 046255, which are incorporated herein by reference. Host cells according to the present invention may be prokaryotic cells such as E. coli or eukaryotic cells such as CHO cells.

[0110] Pharmaceutical Composition Pharmaceutical compositions containing the Fc variants or Fc variant-containing proteins or Fc variant-containing molecules of the invention, formulated together with a suitable pharmaceutically acceptable carrier, can be developed according to suitable methods known in the art. Such compositions may also contain one or a combination of (e.g., two or more different) Fc variants or Fc variant-containing proteins, or immunoconjugates or bispecific molecules of the invention. For example, a pharmaceutical composition of the invention may comprise a combination of an Fc variant or Fc variant-containing protein and an antibody (or immunoconjugate or bispecific) that binds to different epitopes on one or more target antigens.

[0111] therapeutic use Fc variants or Fc variant-containing proteins or molecules can be used for the treatment of diseases, including therapeutic methods requiring binding of drugs to FcRn.

[0112] In one embodiment of the present invention, the Fc variants or Fc variant-containing proteins of the invention can be used to inhibit FcRn in vivo in subjects, including humans, suffering from diseases such as, but not limited to, autoimmune diseases and inflammation, whose binding reduces endogenous IgG levels.

[0113] In one embodiment of the present invention, the Fc variants or Fc variant-containing proteins of the invention can be used to increase the circulating half-life of therapeutic drugs, thereby making them available in the body for a longer period of time, thereby reducing the frequency of dosing, increasing compliance and improving therapeutic efficacy in, for example, cancer, infectious diseases, autoimmune diseases, etc.

[0114] In one embodiment of the invention, the Fc variants or Fc variant-containing proteins of the invention can be used to target antigens to dendritic cells or other APCs to create better prophylactic or therapeutic vaccines, for example in infectious diseases or cancer.

[0115] In certain embodiments, the Fc variant or Fc variant containing protein or Fc variant containing molecule is used to treat Autoimmune Hemolytic Anemia, Pernicious Anemia, Idiopathic Thrombocytopenic Purpura, Goodpasture's Syndrome, Bullous Pemphigoid, Pemphigus Vulgaris, Hashimoto's Thyroiditis, ANCA-Associated Vasculitis, Insulin-Dependent Diabetes Mellitus (IDDM), Graves' Disease, Myasthenia Gravis, CIDP Diabetes, Antianemics (Beta Thalassemia), Hematological Agents, Ophthalmic Agents, Cold Agglutinin Disease, Antiphospholipid Syndrome (APS), Lupus Nephritis, Bone Diseases, Neurological Genetic Disorders, Complement 3 Glomerulopathy, Age-Related Macular Degeneration, Immunoglobulin A Nephropathy (IgAN) Disease, Diabetic Retinopathy, Macular Diseases, Non-Small Cell Lung Cancer Treatment, Ocular Genetic Disorders, Disorders, Hemophilia B, (Coagulation Factor IX Deficiency), Drugs for Cardiovascular Disease, 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, Genitourinary Cancer, Macular Disease, 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, Painkillers, Atypical Hemolytic Dysfunction Syndrome, Hematologic Genetic Disorders, Multisystem Genetic Disorders and rheumatoid arthritis, osteoarthritis, scleroderma, treatment of autoimmune diseases, treatment of gout, urticaria, ankylosing spondylitis, asthma treatment, dermatological medications, idiopathic inflammatory myopathies, immunosuppressants, inflammatory bowel disease, interstitial lung disease, multiple myeloma treatment, multiple sclerosis, nephritis, psoriatic arthritis, systemic lupus erythematosus, medications for acute alcoholic hepatitis, Alzheimer's dementia, anti-allergy / anti-asthmatic medications, anti-arthritic medications, antipsoriatic medications, breast cancer treatment, cancer associated disorders, dermatological medications, heart failure treatment, lymphoma treatment, nephritis, neurological medications (various), respiratory disorders, treatment of inborn errors of metabolism.

[0116] Preferred embodiments of the present invention include the following. 1. An FcRn antagonist comprising an Fc variant, said Fc variant comprising L234A, L235A, T307N, V308P, L309Y, P329G, H433R, N434W amino acid substitutions according to EU numbering. 2. The FcRn antagonist claimed in claim 1 is a monoclonal antibody, a monomer, a dimer, or a multimer. 3. The FcRn antagonist claimed in claim 2 is a monomer having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 11. 4. The FcRn antagonist claimed in 2 is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO:3 and SEQ ID NO:5. 5. The FcRn antagonist claimed in 2 is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO:7 and SEQ ID NO:9. 6. The FcRn antagonist claimed in 2 is a dimer having the amino acid sequence shown in SEQ ID NO:13. 7. The FcRn antagonist claimed in claim 1 further comprises M252Y, S254T, T256E amino acid substitutions according to EU numbering. 8. The FcRn antagonist claimed in claim 7 is a monoclonal antibody, a monomer, a dimer, or a multimer. 9. The FcRn antagonist claimed in claim 8 is a monomer having the amino acid sequence shown in SEQ ID NO:38. 10. The FcRn antagonist claimed in 8 is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO:30 and SEQ ID NO:32. 11. The FcRn antagonist claimed in 8 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 T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E amino acid substitutions according to EU numbering. 13. The FcRn antagonist claimed in claim 12 is a monoclonal antibody, a monomer, a dimer, or a multimer. 14. The FcRn antagonist claimed in claim 13 is a monomer having the amino acid sequence shown in SEQ ID NO:20. 15. The FcRn antagonist claimed in claim 13 is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO:22 and SEQ ID NO:24. 16. The FcRn antagonist claimed in claim 13 is a monoclonal antibody comprising the amino acid sequences shown in SEQ ID NO:26 and SEQ ID NO:28. 17. 10 for FcRn -11 M~10 -8 K in the range of M D 17. An FcRn antagonist as claimed in claims 1 to 16, comprising: 18. An FcRn antagonist as claimed in claims 1 to 17, which cross-reacts with mouse, monkey and human FcRn. 19. A composition comprising an FcRn antagonist as claimed in any one of claims 1-18 and an acceptable carrier. [Example]

[0117] 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 may be practiced. The following examples 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 appropriate modifications well known in the art and well within the capabilities of one of ordinary skill in the art. Such modifications will be known to those of skill in the art.

[0118] Example 1 Construction of full-length antibodies using Fc variants, pDGV / A3H11A4.1 ​​(mAb) and pDGV / B14H11A4.1 ​​(mAb) The Fc variant of the present invention, H11A4.1, was prepared as a complete antibody molecule by fusing the H11A4.1 ​​fragment to the heavy chain variable region of an anti-Cov2 Spike monoclonal antibody (not cross-reactive to human / mouse / NHP protein or antigen) combined with the light chain of an anti-Cov2 Spike monoclonal antibody (not cross-reactive to human / mouse / NHP protein or antigen) (patent application no. WO2022101839; PCT / IB2021 / 060495).

[0119] Briefly, the anti-Cov2 Spike monoclonal antibody A3 (SEQ ID NOs: 15 and 16) and B14 (SEQ ID NOs: 17 and 18) genes were chemically synthesized and contained L234A, L235A, and P329G mutations. Additionally, H11A4 mutations, i.e., T307N, V308P, L309Y, H433R, and N434W, were engineered into A3 and B14 using mutagenic oligonucleotides in sequential overlapping polymerase chain reactions. PCR was also used to incorporate an ApaI restriction site at the 5' end and an EcoR1 restriction site at the 3' end to facilitate in-frame cloning of the heavy chain variable region into the pDGV / A3 (pDual cov2 A3) and pDGV / B14 (pDual cov2 B14) vectors.

[0120] The amplified approximately 1.0 kb heavy chain fragment (constant region) was cloned into pDGV / A3 and pDGV / B14 plasmids digested with ApaI and EcoRI to replace the Fc region with the H11A4.1 ​​mutant Fc. After ligation, the reaction mixture was transformed into E. coli Top10F' cells, and transformants were scored for carbenicillin resistance. The sequences of the pDGV / A3 H11A4.1 ​​(mAb) (light chain SEQ ID NOs: 2 and 3; heavy chain SEQ ID NOs: 4 and 5) and pDGV / B14 H11A4.1 ​​(mAb) (light chain SEQ ID NOs: 6 and 7; heavy chain SEQ ID NOs: 8 and 9) plasmid vectors were confirmed by restriction digestion and Sanger analysis. The vectors were linearized with PvuI for transfection into CHO-GS cells (Lonza).

[0121] Example 2 Construction of monomeric pXC 17.4 / H11A4.1 ​​(monomeric) Fc variant The H11A4.1 ​​(monomeric) 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). PCR also added a HindIII restriction site at the 5' end and an EcoR1 restriction site at the 3' end to facilitate cloning into the pXC17.4 vector (Lonza).

[0122] The approximately 0.75 kb HindIII- and EcoRI-digested PCR amplicon was cloned into HindIII- and EcoRI-digested pXC17.4 vector (Lonza). After ligation, the reaction mixture was transformed into E. coli Top10F', and transformants were scored for carbenicillin resistance. Clones were analyzed by restriction digestion with HindIII and EcoRI. The sequence of the pXC17.4 / H11A4.1 ​​(monomer) clone was confirmed by restriction digestion and Sanger analysis. The vector was linearized with PvuI for transfection into CHO-GS cells (Lonza).

[0123] Example 3 Construction of multimeric (dimeric) pXC 17.4 / W11A4.1 ​​(dimeric) Fc variant For the W11A4.1 ​​variant (dimer; SEQ ID NO: 13), an Fc variant dimer was formed by linking two chains of an Fc variant monomer (H11A4.1) via a glycine-serine linker (SEQ ID NO: 14: GGGSGGGSGGGSGGGSSGGGSS). Furthermore, C226S and C229S mutations were incorporated into one of the two chains of the Fc variant to prevent self-dimerization. A codon-optimized gene for the Fc variant dimer with L234A, L235A, and P329G mutations was chemically synthesized at GeneArt GmbH, Germany. The W11A4 (dimer) gene was isolated from the pMA Geneart construct by restriction digestion with HindIII and EcoRI. Approximately 1.5 kb of HindIII- and EcoRI-digested Fc variant W11A4.1 ​​(dimer) was ligated into HindIII- and EcoRI-digested pXC-17.4 vector (Lonza). The ligation mixture was transformed into E. coli Top10F', and transformants were scored for carbenicillin resistance. Clones were analyzed by restriction digestion with HindIII and EcoRI. The sequence of the pXC17.4 / W11A4.1 ​​(monomer) clone (SEQ ID NOs: 12 and 13) was confirmed by restriction digestion and Sanger analysis. The vector was linearized with PvuI for transfection into CHO-GS cells (Lonza).

[0124] Example 4 Construction of monomeric pXC 17.4 / H11A4.2 Fc variant Amino acid substitutions were made at residues M252Y, S254T, and T256E in H11A4.0 to generate the H11A4.2 variant (SEQ ID NOs: 19 and 20). Briefly, sequential overlap PCR was performed using pXC17.4 / H11A4.0 as a template to incorporate the desired substitutions at amino acids 252, 254, and 256 (EU numbering). The approximately 0.75 kb PCR amplicon was digested with HindIII and EcoRI and cloned into the pXC-17.4 plasmid (Lonza). After ligation, the reaction mixture was transformed into E. coli Top10F', and transformants were scored for 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 PvuI for transfection into CHO-GS cells (Lonza).

[0125] Example 5 Construction of full-length antibodies using Fc variants, pDGV / A3H11A4.2 (mAb) and pDGV / B14 H11A4.2 (mAb) The Fc variant of the present invention, H11A4.2, was incorporated into a whole antibody format by replacing the constant regions of A3H11A4.0 and B14H11A4.0 (patent application no. WO2022101839; PCT / IB2021 / 060495) with the H11A4.2 fragment.

[0126] Briefly, for construct preparation, an ApaI restriction site at the 5' end and an EcoR1 restriction site at the 3' end were engineered by PCR into the H11A4.2 nucleotide sequence adjacent to the coding sequence representing amino acids 20 to 247 (SEQ ID NO: 20) to facilitate in-frame cloning of the heavy chain variable region into the pDGV / A3 and pDGV / B14 vectors. The amplified approximately 1.0 kb H11A4.2 fragment was digested with ApaI and EcoRI and cloned into the pDGV / A3 and pDGV / B14 plasmids. Transformants in E. coli Top10F' were scored for carbenicillin resistance. The sequences of the pDGV / A3 H11A4.2 (light chain SEQ ID NOs: 21 and 22; heavy chain SEQ ID NOs: 23 and 24) and pDGV / B14 H11A4.2 (light chain SEQ ID NOs: 25 and 26; heavy chain SEQ ID NOs: 27 and 28) plasmid vectors were confirmed by restriction digestion and Sanger analysis. The vectors were linearized with PvuI for transfection into CHO-GS cells (Lonza).

[0127] Example 6 Construction of full-length antibodies using Fc variants, pDGV / A3H11A4.3 and pDGV / B14H11A4.3 Amino acid substitutions were made at residues M252Y, S254T, and T256E of H11A4.1 ​​to generate the H11A4.3 variant. The pDGV / A3 and pDGV / B14 constructs encoding H11A4.3 in mAb format were generated by sequential overlapping polymerase chain reactions to incorporate the desired substitutions at amino acid positions 252, 254, and 256 (EU numbering). MluI and EcoR1 restriction sites were subsequently added to the 5' and 3' ends to facilitate insertion of PCR-amplified fragments into the pDGV / A3 and pDGV / B14 vectors.

[0128] PCR amplicons containing the desired substitutions were digested with MluI and EcoR1 restriction enzymes and subsequently cloned into pDGV / A3 and pDGV / B14 plasmids, respectively, which had been digested with MluI and EcoR1. After ligation, the reaction mixture was transformed into E. coli Top10F' cells, and transformants were scored for carbenicillin resistance. The sequences of the pDGV / A3 H11A4.3 (light chain SEQ ID NOs: 29 and 30; heavy chain SEQ ID NOs: 31 and 32) and pDGV / B14 H11A4.3 (light chain SEQ ID NOs: 33 and 34; heavy chain SEQ ID NOs: 35 and 36) plasmid vectors were confirmed by restriction digestion and Sanger sequencing. The vectors were linearized with PvuI for transfection into CHO-GS cells (Lonza).

[0129] Example 7 Construction of monomeric pXC 17.4 / H11A4.3 variant A nucleotide sequence encoding a secretory signal peptide was inserted upstream of the nucleotide sequence encoding H11A4.3 by PCR. This was followed by another PCR to add a HindIII restriction site at the 5' end and an EcoRI restriction site at the 3' end to facilitate cloning into the pXC17.4 vector (Lonza). The resulting approximately 0.75 kb amplicon (SEQ ID NO: 37) encoding H11A4.3 (SEQ ID NO: 38) was digested with HindIII and EcoRI and ligated into HindIII- and EcoRI-digested pXC17.4 vector (Lonza). After ligation, the reaction mixture was transformed into E. coli Top10F', and transformants were scored for carbenicillin resistance. Clones were verified by restriction digestion with HindIII and EcoRI. The sequence of the pXC17.4 / H11A4.3 construct was confirmed by restriction digestion and Sanger analysis. The vector was linearized with PvuI for transfection into CHO-GS cells (Lonza).

[0130] Example 8 Production of Fc variant proteins All vector constructs described in Examples 1-7 were used for transfection. All plasmids prepared in Examples 1-7 above were linearized with PvuI restriction enzyme and used for transfection. Chinese hamster ovary (CHO) cells were used as hosts for recombinant protein expression. CHO cells were seeded at a density of 0.5 million / mL approximately 24 hours before transfection to have cells in exponential phase. Transfection was performed using the Neon transfection system (Invitrogen) via electroporation according to the manufacturer's instructions. After transfection, cells were plated into 24-well cell culture plates containing 1 mL of prewarmed ProCHO5 serum-free medium (Lonz, Switzerland) containing 25 μM MSX and incubated at 37°C in the presence of 5% CO2 in a humidified incubator. The cell numbers of all transfected pools (i.e., heterogeneous mixtures of separately expressing cells) were monitored regularly, and medium changes were performed periodically. After the cells recovered from transfection, they were further expanded sequentially into 6-well culture plates, T-flasks, and cultitubes (TPPs).

[0131] Fed-batch cultures were performed on transfected pools of all Fc variant candidates in culti-tubes (TPP) for recombinant protein production. Cells were grown at 0.3 × 10 in ActiPro production medium (Hyclone™, Cytiv). 6 The cultures were seeded at a density of 1000 cells / mL. The culture tubes were incubated in a humidified Kuhner shaker at 37°C with a 5% CO2 level and a shaking speed of 230 RPM. All pools followed a fixed daily feeding regimen during culture using a chemically defined feed from Hyclone, Cytiv. After 72 hours of culture, feeding was initiated and continued until the batch was harvested.

[0132] At harvest, 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.

[0133] Example 9 Determination of kinetic rate constants for binding of Fc variants to recombinant human neonatal Fc receptor (rhFcRn) at pH 6.0 The kinetic constants for binding of 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). A CM5 sensor chip was activated by immobilizing an anti-histidine antibody on its surface using standard amine coupling chemistry, followed by blocking with 1 M ethanolamine, pH 8.0, for 7 minutes. Recombinant human FcRn receptor (rhFcRn) at a concentration of 0.1 μg / mL was captured on the anti-histidine antibody-immobilized chip surface by injection at a flow rate of 10 μL / min for 60 seconds. Five dilutions of Fc variant samples (ranging from 55 nM to 0.68 nM) in dPBS, pH 6.0, containing 0.05% (v / v) polysorbate 20 were injected over the captured FcRn protein on the chip at 25 °C, a flow rate of 30 μL / min, with an association time of 60 s and a dissociation time of 60 s. Following each sample run, the chip surface was regenerated with 10 mM glycine, pH 1.5, for 30 s. The rate constant, k d (dissociation constant) and k a The association constants were calculated using a one-to-one binding model in Biacore Insight Evaluation software (v 3.0.12.15655). The KD values ​​(affinity constants) were calculated using the k d k a The ratio to K D =k d / k a The affinity constants for binding of candidate Fc variants to rhFcRn were measured at pH 6.0 and are shown in Table 3.

[0134] [Table 3]

[0135] Example 10 Determination of kinetic rate constants for binding of Fc variants to recombinant human neonatal Fc receptor (rhFcRn) at pH 7.4 To examine the effect of pH on sample binding to and dissociation from FcRn, samples were analyzed for FcRn binding at pH 7.4. In this experiment, the affinity constants for binding of Fc variant candidates to 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 described in Example 9. Kinetic measurements were performed using a running buffer with pH 7.4. To measure affinity constants, five dilutions of Fc variant samples (ranging from 550 nM to 6.79 nM) were prepared and injected at a flow rate of 30 μL / min with an association time of 60 seconds and a dissociation time of 60 seconds. All reactions were performed at 25°C. The affinity constants for binding of candidate Fc variants to rhFcRn were measured at pH 7.4 and are shown in Table 4.

[0136] [Table 4]

[0137] As can be seen from Tables 3 and 4, the Fc variants of the present invention can bind to FcRn with higher affinity at pH 6.0 compared to pH 7.4. Thus, the Fc variants of the present invention retain the pH-dependent FcRn interaction (higher affinity at pH 6.0 than at near-neutral pH) characteristic of the wild-type Fc.

[0138] Example 11 Determination of kinetic rate constants for binding of Fc variants to recombinant neonatal Fc receptor (rFcRn) from different species In this experiment, the binding of candidate Fc variants of the present invention to FcRn of different species was determined by surface plasmon resonance-based measurements using a Biacore 8K+ instrument (Cytiva). To investigate the kinetic rate constants, experiments were performed using recombinant mouse, monkey, and human FcRn for binding with the candidate Fc variants. FcRn (monkey and human species) was captured on a CM5 chip as mentioned in Example 9. Mouse FcRn was immobilized on the CM5 sensor chip surface 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 to perform kinetic measurements. The association rate constant (k a ) and dissociation rate constant (k d To measure Fc muteins, five dilutions (ranging from 55 nM to 0.027 nM) of Fc muteins were prepared in the running buffer and injected at a flow rate of 30 μL / min with an association time of 60 seconds. The dissociation time was extended to 1800 seconds. All reactions were performed at 25°C. After each sample run, the chip surface was regenerated using glycine pH 1.5 / 3M MgCl2. Data were in the form of sensorgrams and analyzed using Biacore Insight Evaluation software (v 3.0.12.15655).

[0139] The kinetic constants for binding of Fc variants to FcRn receptors of different species are shown in Table 5.

[0140] [Table 5]

[0141] As can be seen from Table 5, the Fc variants of the invention can bind not only to human FcRn but also to mouse and monkey FcRn in vitro. Due to the advantageous feature of cross-reactivity with monkey and mouse FcRn, it is expected that the Fc variants of the invention can be tested in non-human primates and mice, and the obtained data may be very useful for predicting their pharmacokinetics, pharmacodynamics, and toxicity profile in humans.

[0142] Example 12 Effect of Fc variants on total serum IgG levels in wild-type C57BL / 6 mice In part 1 of this study, two Fc variants of the present invention in full-length antibody format, namely A3H11A4.1 ​​(SEQ ID NOs: 3 and 5, i.e., light chain and heavy chain amino acid sequences, respectively) and B14H11A4.1 ​​(SEQ ID NOs: 7 and 9, i.e., light chain and heavy chain amino acid sequences, respectively), were compared with two full-length antibodies, namely A3H11A4.0 (SEQ ID NOs: 15 and 16, i.e., heavy chain and light chain amino acid sequences, respectively) and B14H11A4.0 (SEQ ID NOs: 17 and 18, i.e., heavy chain and light chain amino acid sequences, respectively).

[0143] Antibodies A3H11A4.1 ​​and B14H11A4.1 ​​of the invention were compared to A3H11A4.0 and B14H11A4.0, respectively, to determine the effect of the Fc variants of the invention on circulating total serum IgG levels. Wild-type C57BL / 6 mice were randomized based on their body weight. On day -2 (i.e., 48 hours prior to dosing), blood was collected. On day 0 (0 hours), the doses of antibody noted in Table 6 were administered intraperitoneally to each group of study animals (6 mice per group).

[0144] [Table 6]

[0145] Blood samples were collected at the following time points: days 1, 2, 5, and 8. Endogenous serum IgG levels were determined using ELISA from samples collected at the noted time points. All four treatment groups [A3H11A4.1, B14H11A4.1, A3H11A4.0, and B14H11A4.0] showed a decrease in mouse IgG concentrations compared to normal subjects (placebo group). When compared between treatment groups, A3H11A4.1 ​​and B14H11A4.1 ​​showed a greater decrease in IgG than A3H11A4.0 and B14H11A4.0 over the study period. A graphical representation of the results using relative IgG concentrations (%) with respect to pre-dose levels across all treatment groups is shown in Figure 3.

[0146] In part 2 of this study, two Fc variants of the invention in full-length antibody format, A3H11A4.2 (SEQ ID NOs: 22 and 24, i.e., light and heavy chain amino acid sequences, respectively) and B14H11A4.3 (SEQ ID NOs: 30 and 32, i.e., light and heavy chain amino acid sequences, respectively), were compared with two full-length antibodies, A3H11A4.0 (SEQ ID NOs: 15 and 16, i.e., heavy and light chain amino acid sequences, respectively). The full-length amino acid and nucleotide sequences of the antibodies and potential Fc formats are listed in Table 2.

[0147] Antibodies A3H11A4.2 and A3H11A4.3 of the invention were compared to A3H11A4.0 to determine the effect of Fc variants of the invention on circulating total serum IgG levels. Wild-type C57BL / 6 mice were randomized based on their body weight. On day -2 (i.e., 48 hours prior to dosing), blood was collected. On day 0 (0 hours), the candidate doses noted in Table 7 were administered intraperitoneally to each group of study animals (6 mice per group).

[0148] [Table 7]

[0149] Blood samples were collected at the following time points: days 1, 2, 5, and 8. Endogenous serum IgG levels were determined using ELISA from samples collected at the noted time points. All three treatment groups [A3H11A4.0, A3H11A4.2, and A3H11A4.3] showed a decrease in mouse IgG concentrations compared to normal subjects (placebo group). Comparing among the antibodies tested in this study, A3H11A4.2 and A3H11A4.3 showed a greater decrease in IgG over the study period than A3H11A4.0. A graphical representation of the results using relative IgG concentrations (%) with respect to pre-dose levels across the different antibody candidates is shown in Figure 4.

[0150] In part 3 of this study, three Fc variants of the invention, H11A4.1, H11A4.2, and H11A4.3, in an Fc format were evaluated to determine their effect on circulating total serum IgG levels. The full-length amino acid and nucleotide sequences of the candidate Fc formats are listed in Table 2.

[0151] Wild-type C57BL / 6 mice were randomized based on their body weight. On day -2 (i.e., 48 hours before dosing), blood was collected. On day 0 (0 hours), the doses of the candidates mentioned in Table 8 were administered intraperitoneally to each group of study animals (6 mice per group).

[0152] [Table 8]

[0153] Blood samples were collected at the following time points: days 1, 2, 5, and 8. Endogenous serum IgG levels were determined using ELISA from samples collected at the mentioned time points. All three treatment groups [H11A4.1, H11A4.2, and H11A4.3] showed a decrease in mouse IgG concentrations compared to normal subjects (placebo group), with the highest IgG depletion observed on day 2. A graphical representation of the results using relative IgG concentrations (%) with respect to pre-dose levels across the different Fc candidates is shown in Figure 5.

[0154] Example 13 Effect of Fc variants on total serum IgG levels in rhesus monkeys The effect of the Fc variants of the present invention in Fc format, i.e., H11A4.1 ​​(SEQ ID NO: 1 and SEQ ID NO: 11), on circulating total serum IgG levels was determined in a single-dose study in rhesus monkeys. H11A4.1 ​​was administered to animals (N=2) at a dose of 10 mg / kg via the intravenous infusion route. Blood samples were collected at the following time points: pre-dose, 6 hours, 24 hours (day 1), days 2, 3, 4, 5, 7, 9, 11, 14, 17, 21, 28, 35, 42, 49, and 56. Endogenous serum IgG levels were determined using an ELISA-based method from samples collected at the indicated time points. A relative decrease in endogenous IgG compared to pre-dose values ​​was observed in both animals, with the highest IgG depletion observed on day 5. A graphical representation of the results using relative IgG concentrations (%) with respect to pre-dose levels is shown in Figure 6.

[0155] Example 14 Effect of Fc variants on total serum IgG levels in rhesus monkeys The effect of an Fc variant of the present invention in a full-length antibody format, i.e., A3H11A4.1 ​​(SEQ ID NOs: 3 and 5, i.e., light chain and heavy chain amino acid sequences, respectively), on circulating total serum IgG levels was determined in a single-dose study in rhesus monkeys. A3H11A4.1 ​​was administered to animals (N=2) at a dose of 30 mg / kg via the intravenous infusion route. Blood samples were collected at the following time points: pre-dose, 6 hours, 24 hours (day 1), days 2, 3, 4, 5, 7, 9, 11, 14, 17, 21, 28, 35, 42, 49, and 56. Endogenous serum IgG levels were determined using an ELISA-based method from samples collected at the indicated time points. A relative decrease in endogenous IgG compared to pre-dose values ​​was observed in both animals, with the highest IgG depletion observed on day 9. A graphical representation of the results using relative IgG concentrations (%) with respect to pre-dose levels is shown in Figure 7.

[0156] Example 15 Effect of Fc variants on total serum IgG levels in rhesus monkeys The effect of an Fc variant of the present invention in a full-length antibody format, i.e., B14H11A4.1 ​​(SEQ ID NOS: 7 and 9, i.e., light chain and heavy chain amino acid sequences, respectively), on circulating total serum IgG levels was determined in a single-dose study in rhesus monkeys. B14H11A4.1 ​​was administered to animals (N=2) at a dose of 30 mg / kg via the intravenous infusion route. Blood samples were collected at the following time points: pre-dose, 6 hours, 24 hours (day 1), days 2, 3, 4, 5, 7, 9, 11, 14, 17, 21, 28, 35, 42, 49, and 56. Endogenous serum IgG levels were determined using an ELISA-based method from samples collected at the noted time points. A relative decrease in endogenous IgG compared to pre-dose values ​​was observed in both animals, with the highest IgG depletion observed on day 4. A graphical representation of the results using relative IgG concentrations (%) with respect to pre-dose levels is shown in Figure 8.

[0157] Incorporation by Reference The entire disclosure of each patent document and scientific article referred to herein is incorporated by reference for all purposes.

[0158] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments, therefore, are to be considered in all respects illustrative rather than 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.

[0159] References incorporated into this patent application (References) TIFF2026501180000027.tif134160

Claims

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

2. The FcRn antagonist of claim 1, which is a monoclonal antibody, a monomer, a dimer, or a 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 of claim 2, which is a dimer having the amino acid sequence shown in SEQ ID NO:

13.

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

8. The FcRn antagonist according to claim 7, which is a monoclonal antibody, a monomer, a dimer, or a 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 said Fc variant comprises T307N, V308P, L309Y, H433R, N434W, M252Y, S254T, T256E amino acid substitutions according to EU numbering.

13. The FcRn antagonist of claim 12, which is a monoclonal antibody, a monomer, a dimer, or a 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. 10 for FcRn -11 M to 10 -8 K in the range of M D The FcRn antagonist according to any one of claims 1 to 16, having the following structure:

18. The FcRn antagonist according to any one of claims 1 to 17, which cross-reacts with mouse, monkey and human FcRn.

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

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