Mutants with Fc fragments exhibiting increased affinity for FcRn and increased affinity for at least one Fc fragment receptor

Abstract

The present invention relates to variants of a parent polypeptide comprising an Fc fragment, which have increased affinity for the FcRn receptor and increased affinity for at least one Fc fragment receptor (FcR) selected from among FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (CD32a) compared to the affinity of the parent polypeptide, characterized in that the variants comprise (i) four mutations: 334N, 352S, 378V, and 397M; and (ii) at least one mutation selected from among 434Y, 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T, and 389K, and wherein the numbering is that of the EU index in Kabat or an equivalent. [Selection diagram] None

Description

[Technical Field]

[0001] The present invention relates to a polypeptide (also called a mutant) containing a mutated Fc region that has increased affinity for the FcRn receptor and increased affinity for at least one Fc fragment receptor (FcR) compared to the parent polypeptide. [Background technology]

[0002] Antibodies are composed of a tetramer consisting of heavy and light chains. Two light chains are identical to each other, and two heavy chains are identical, linked by disulfide crosslinks. There are five types of heavy chains (alpha, gamma, delta, epsilon, and mu) that determine the immunoglobulin class (IgA, IgG, IgD, IgE, IgM). The light chain group includes two subtypes: lambda and kappa.

[0003] IgG is a soluble antibody found in blood and other bodily fluids. IgG is a Y-shaped glycoprotein with an approximate molecular weight of 150 kDa, composed of two heavy chains and two light chains. Each chain is divided into a constant region and a variable region. The two carboxyl-terminal domains of the heavy chain form an Fc fragment, while the amino-terminal domains of both the heavy and light chains recognize antigens and are named the Fab fragment.

[0004] Fc fusion proteins are created by combining an antibody's Fc fragment with a protein domain that provides specificity for a given therapeutic target. Examples include any type of therapeutic protein or combinations of their fragments with an Fc fragment.

[0005] Fc polypeptides, particularly Fc fragments, therapeutic antibodies, and Fc fusion proteins are used today to treat a variety of diseases, including polyarthritis rheumatica, psoriasis, multiple sclerosis, and many forms of cancer. Therapeutic antibodies can be monoclonal or polyclonal antibodies. Monoclonal antibodies are obtained from a single antibody-producing cell lineage that exhibits identical specificity to a single antigen. Therapeutic Fc fusion proteins, such as etanercept (Enbrel d'Amgen, which is a TNF receptor bound to an Fc fragment) or alefacept (Amevive de Biogen Idec, which is LFA-3 bound to the Fc portion of human IgG1), are being used or developed as agents for diseases with inflammatory elements and / or autoimmune diseases.

[0006] Fc polypeptides, such as Fc fragments, Fc antibodies, and fusion proteins, have activity that depends on the binding of their Fc moiety to their receptors, namely FcRn and Fc fragment receptors (FcR), such as the FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (CD32a).

[0007] One of the desired effects in therapies that intervene in the interaction between Fc polypeptides and Fc fragment receptors (FcRs) is the inhibition of immune system activation by binding to Fc receptors present on the surface of effector cells. In particular, as part of the treatment of inflammatory and / or autoimmune diseases involving autoantibodies and / or cytokines, Fc fragment-based therapies can act by blocking Fc receptors and thus by competition with autoantibodies for access to these receptors. This leads to inhibition of direct activity normally mediated by autoantibodies (e.g., antibody-dependent cytotoxicity, complement-dependent cytotoxicity, or antibody-dependent phagocytosis) and a reduction in immune system activation, especially cytokine release. Furthermore, since FcRn receptors are involved in antibody recycling, blocking these receptors by Fc polypeptides allows for more rapid removal of autoantibodies, thus shortening their half-life. For these reasons, Fc fragment-based therapies are particularly well-suited for autoimmune and / or inflammatory diseases caused by uncontrolled stimulation of immune system cells, especially by autoantibodies and / or cytokines.

[0008] The basic therapy proposed for the treatment of these diseases is intravenous immunoglobulin (IgIV) therapy, which involves administering immunoglobulins (mostly IgG) derived from a pool of donated human plasma to patients intravenously. It is generally accepted that these IgIVs act by blocking Fc receptors in particular, thus entering into competition with autoantibodies for access to these receptors. More recently, Fc fragments have been developed to modify their binding properties to Fc receptors. However, their efficacy still needs to be demonstrated.

[0009] In particular, there is still a need to optimize these Fc fragments to increase their half-life and / or therapeutic efficacy.

[0010] The applicant has now developed special Fc fragments with improved activity, particularly due to improved binding affinity to FcRn. These Fc fragments can be used in therapy and are particularly suited to the treatment of inflammatory and / or autoimmune diseases, with the aim of bringing greater efficacy to products containing them.

[0011] More specifically, these fragments can offer more effective blocking of Fc receptors present on immune system cells, in which case these Fc receptors become less accessible or no longer accessible for binding to autoantibodies whose activity is inhibited at that point.

[0012] Furthermore, Fc fragments can more effectively block FcRn receptors, thus enabling more rapid elimination of autoantibodies.

[0013] Furthermore, some of these specific Fc fragments exhibit superior complement-dependent cytotoxicity (CDC) inhibition compared to IgIV, as demonstrated in the examples. Therefore, these Fc fragments are thought to be able to reduce the toxicity of pathogenic autoantibodies, such as those involved in inflammatory and / or autoimmune diseases. [Overview of the Initiative] [Problems that the invention aims to solve]

[0014] Therefore, the present invention provides a variant of the parent polypeptide having optimized properties with respect to functional activity mediated by the Fc region. [Means for solving the problem]

[0015] Therefore, the present invention relates to a variant of a parent polypeptide containing an Fc fragment, having increased affinity for the FcRn receptor and increased affinity for at least one Fc fragment receptor (FcR) selected from among the FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (CD32a), compared to the affinity of the parent polypeptide. (i) Four mutations 334N, 352S, 378V and 397M, (ii) At least one mutation selected from 434Y, 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K, Includes, This concerns variants characterized by having a numbering scheme that is the EU index or equivalent numbering scheme in Kabat.

[0016] According to one embodiment, the variant according to the present invention is Y296W, K290G, V240H, V240I, V240M, V240N, V240S, F241H, F241Y, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, F243L, F243S, E258G, E258I, E258R, E258M, E258Q, E258Y, V259C, V259I, V259L, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V262S, V263T, V264L, V264S, V264T, V266L, S267A, S267Q, S267V, K290D, K290E, K290H, K290L, K290N, K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E293Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y296H, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and at least one mutation (iii) within the Fc fragment selected from V305S, and further comprises The numbering is the EU index in Kabat or the equivalent numbering.

[0017] Such variants are referred to as "variants according to the present invention", "mutant variants according to the present invention" or "polypeptides according to the present invention".

[0018] Preferably, the variant according to the present invention simultaneously has an increased affinity for the FcRn receptor and an increased affinity for all FcγRI receptors (CD64), FcγRIIIa receptors (CD16a) and FcγRIIa receptors (CD32a).

[0019] Preferably, the variant according to the invention further has the ability to inhibit complement-dependent cytotoxicity (CDC) which is due to modification of the binding to complement, particularly the protein C1q. This inhibition is significantly improved compared to the inhibition conferred by IgIV.

[0020] Preferably, the variant according to the invention has five mutations N434Y, K334N, P352S, V397M and A378V, as numbered according to the EU index in Kabat or its equivalents, and is produced in HEK293 cells, and is different from the variant consisting particularly of the Fc fragment of IgG1. Thus, preferably, the variant according to the invention is produced in the cell HEK293, as numbered according to the EU index in Kabat or its equivalents, and is different from the Fc fragment of IgG1, N434Y / K334N / P352S / V397M / A378V.

[0021] Throughout the present application, the numbering of residues within the Fc region is the numbering of the heavy chain of immunoglobulins according to the EU index in Kabat et al. (Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, in Maryland, 1991) or its equivalents. The expression "EU index in Kabat or its equivalents" means the EU numbering of the residues of human antibodies IgG1, IgG2, IgG3 or IgG4. This is illustrated on the IMGT site (http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu IGHGnber.html).

[0022] "Polypeptide" or "protein" means a sequence containing at least 100 amino acids linked by covalent bonds.

[0023] "Amino acid" means one of the 20 natural amino acids or unnatural analogs.

[0024] The term "position" refers to the location within the sequence of a polypeptide. For Fc regions, the position is numbered according to the EU index or equivalent in Kabat.

[0025] The term "antibody" is used in a general sense. It corresponds to a tetramer containing at least one Fc region and two variable regions. Antibodies specifically include full-length immunoglobulins, monoclonal antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. The amino-terminus of each heavy chain contains a variable region of approximately 100-110 amino acids responsible for antigen recognition. Within each variable region, three rings assemble to form a binding site to the antigen. Each ring is called a complementarity-determining region (hereinafter referred to as "CDR"). The carboxyl-terminus of each heavy chain defines a constant region that primarily performs effector functions.

[0026] IgG has several subclasses, particularly IgG1, IgG2, IgG3, and IgG4. The subclasses of IgM are, in particular, IgM1 and IgM2. Thus, "isotype" means one of the subclasses of immunoglobulin defined by the chemical and antigenic properties of its constant region. The known isotypes of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgGA1, IgGA2, IgGM1, IgGM2, IgD, and IgE.

[0027] Full-length IgG is a tetramer, composed of two identical pairs of two immunoglobulin chains, each pair having one light chain and one heavy chain. Each light chain contains a VL domain and a CL domain, and each heavy chain contains a VH domain, Cγ1 (also called CH1), Cy2 (also called CH2), and Cy3 (also called CH3). Within the framework of human IgG1, "CH1" refers to positions 118-215 according to the Eu index or equivalent in Kabat, "CH2" refers to positions 231-340, and "CH3" refers to positions 341-447. The heavy chain of IgG also contains an N-terminal flexible hinge domain, which in the case of IgG1 refers to positions 216-230. The lower hinge domain refers to positions 226-230 according to the Eu index or equivalent in Kabat.

[0028] The term "variable region" refers to a region of immunoglobulin that contains one or more Ig domains substantially encoded by one of the genes VK, Vλ, and / or VH, which constitute the kappa, lambda, and heavy chains of the immunoglobulin, respectively. The variable region includes the complementarity-determining region (CDR) and the framework region (FR).

[0029] The term "Fc" or "Fc region" refers to the constant region of an antibody, excluding the first domain (CH1) of the immunoglobulin's constant region. Therefore, Fc refers to the last two domains (CH2 and CH3) of the constant region of IgG1, and the N-terminal flexible hinge of these domains. For human IgG1, the Fc region corresponds to residue C226 up to its carboxyl terminal, i.e., residues at positions 226–447, where the numbering is the EU index or equivalent numbering in Kabat. The Fc region used may further include the upper hinge region portion located between positions 216–226 according to the EU index or equivalent in Kabat. In this case, the Fc regions used correspond to residues at positions 216-447, 217-447, 218-447, 219-447, 220-447, 221-447, 222-447, 223-447, 224-447, or 225-447, with the numbering being the EU index or equivalent numbering in Kabat. Preferably, in this case, the Fc regions used correspond to residues at positions 216-447, with the numbering being according to the Eu index or equivalent in Kabat.

[0030] Preferably, the Fc region used is selected from the sequences of sequence numbers 1 to 10 and 14.

[0031] "Parent polypeptide" refers to a standard parent polypeptide. This parent polypeptide may be of natural or synthetic origin. In relation to the present invention, the parent polypeptide includes an Fc region called the "parent Fc region." This Fc region may be selected from the group consisting of wild-type Fc regions, their fragments, and their mutants. Preferably, the parent polypeptide includes a human Fc fragment, preferably a human IgG1 or human IgG2 Fc fragment. The parent polypeptide may include modifications of amino acids pre-existing within the Fc region in relation to the wild-type Fc region (e.g., Fc mutants).

[0032] Advantageously, the parent polypeptide may be an isolated Fc region (i.e., an as-is Fc fragment), a sequence derived from an isolated Fc region, an antibody, an antibody fragment containing an Fc region, or a fusion protein or Fc conjugate containing an Fc region, but this list is not limited.

[0033] "Sequence derived from isolated Fc regions" means a sequence containing at least two isolated Fc regions linked to each other, such as scFc (single-chain Fc) or Fc polymers. "Fusion protein containing an Fc region" means a polypeptide sequence fused to an Fc region, which is preferably selected from the variable region of any antibody, receptor and ligand binding sequences, adhesion molecules, ligands, enzymes, cytokines, and chemokines. "Fc conjugate" means a compound resulting from the chemical coupling of an Fc region and a conjugation partner. The conjugation partner may be a protein or a non-protein. Coupling reactions generally use functional groups on the Fc region and a conjugation partner. In the prior art, various binding groups are known to be suitable for the synthesis of conjugates. For example, homo- or hetero-bifunctional binding groups are well known (see Pierce Chemical Company catalog, 2005-2006, technical section on crosslinking agents, pp. 321-350). Suitable conjugation partners include therapeutic proteins, labels, cytotoxic agents such as chemotherapeutic agents, toxins, and their active fragments. Suitable toxins and their fragments include, in particular, diphtheria toxin, exotoxin A, lysine, abrin, saporin, geronin, calycheolin, auristatin E and F, and meltansine.

[0034] Advantageously, the parent polypeptide, and therefore the polypeptide according to the present invention, consists of an Fc region.

[0035] Advantageously, the parent polypeptide, and therefore the polypeptide according to the present invention, is an antibody.

[0036] "Mutation" refers to a change in at least one amino acid in the sequence of a polypeptide, particularly a change in at least one amino acid in the Fc region of the parent polypeptide. The mutated polypeptide thus obtained is a mutant polypeptide, which is the polypeptide according to the present invention. Such a polypeptide contains a mutated Fc region compared to the parent polypeptide. Preferably, the mutation is a substitution, insertion, or deletion of at least one amino acid.

[0037] "Substitution" means replacing an amino acid at a specific position within the parent polypeptide sequence with another amino acid. For example, substitution N434S means a mutant polypeptide, in this case, a mutant in which the asparagine at position 434 is replaced by serine.

[0038] "Amino acid insertion" or "insertion" refers to the addition of an amino acid at a specific position within the parent polypeptide sequence. For example, insertion G>235~236 represents the insertion of glycine between positions 235 and 236.

[0039] "Amino acid deletion" or "deletion" refers to the removal of an amino acid at a specific position within the parent polypeptide sequence. For example, E294del represents the removal of glutamic acid at position 294.

[0040] Preferably, the mutation label “434S” or “N434S” is used, meaning that the parent polypeptide contains asparagine at position 434, which is replaced by serine in the mutant. In the case of a combination of substitutions, the preferred format is: “259I / 315D / 434Y” or “V259I / N315D / N434Y”. This means that in the mutant there are three substitutions at positions 259, 315 and 434, and that the amino acid at position 259 of the parent polypeptide, i.e. valine, is replaced by isoleucine, the amino acid at position 315 of the parent polypeptide, i.e. asparagine, is replaced by aspartic acid, and the amino acid at position 434 of the parent polypeptide, i.e. asparagine, is replaced by tyrosine.

[0041] As used herein, “FcRn” or “neonatal Fc receptor” refers to the protein that binds to the Fc region of IgG and is at least partially encoded by the FcRn gene. As is well known in the art, functional FcRn proteins consist of two polypeptides, often referred to as the heavy chain and the light chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise indicated, FcRn or FcRn protein refers to the complex of beta-2-microglobulin and the α chain. In humans, the gene encoding FcRn is called FCGRT.

[0042] Preferably, the mutant according to the present invention has an affinity for the FcRn receptor that is increased by a ratio of at least 2 compared to the affinity of the parent polypeptide, preferably by a ratio greater than 5, preferably greater than 10, preferably greater than 15, preferably greater than 20, preferably greater than 25, and preferably greater than 30.

[0043] Preferably, the mutant according to the present invention has a half-life that is increased compared to the half-life of the parent polypeptide. Preferably, the mutant according to the present invention has a half-life that is increased by a ratio of at least 2, preferably greater than 5, preferably greater than 10, preferably greater than 15, preferably greater than 20, preferably greater than 25, and preferably greater than 30 compared to the half-life of the parent polypeptide.

[0044] One of the primary functions of FcRn is known as IgG recycling. This involves extracting IgG from the endothelial catabolic pathway of plasma proteins and restoring these IgGs to the circulation in an intact state. This recycling explains the long half-life of these IgGs under normal physiological conditions (3 weeks for IgG), while enabling the maintenance of high plasma concentrations. Transcytosis of IgG from one pole of the epithelium or endothelium to the other is a second primary function of FcRn, enabling their biodistribution in living organisms.

[0045] Preferably, the mutant according to the present invention has an affinity for the receptor (FcR) of at least one Fc fragment selected from FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (IC32a) that is increased by a ratio of at least 2 compared to the affinity of the parent polypeptide, preferably greater than 5, preferably greater than 10, preferably greater than 15, preferably greater than 20, preferably greater than 25, and preferably greater than 30.

[0046] The FcγRI receptor (CD64) is involved in phagocytosis and cell activation. The FcγRIIIIa receptor (CD16a) is similarly involved in Fc fragment-dependent activity, particularly ADCC and phagocytosis. This receptor has polymorphism V / F at position 158. The FcγRIIa receptor (CD32a) is involved in platelet activity and phagocytosis. This receptor has polymorphism H / R at position 131.

[0047] Preferably, the mutant according to the present invention simultaneously has increased affinity for the FcRn receptor and increased affinity for all of the FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (CD32a).

[0048] The affinity of polypeptides containing the Fc region to FcR can be evaluated by well-known prior art methods. For example, those skilled in the art can determine the affinity (Kd) using surface plasmon resonance (SPR). Alternatively, those skilled in the art can perform a suitable ELISA test. A suitable ELISA test allows for a comparison of the binding affinity of parental Fc and mutated Fc. The signals detected specifically for mutated Fc and parental Fc are compared. Binding affinity can be determined indiscriminately by evaluating the entire polypeptide or by evaluating the Fc region isolated from the entire polypeptide. Alternatively, those skilled in the art can perform a suitable competitive test. A suitable competitive test allows for the determination of the ability of mutated Fc to inhibit the binding of FcR-labeled ligands when incubated with cells expressing the receptor. Binding of the labeled ligand to FcR is evaluated, for example, by flow cytometry. The binding affinity of mutated Fc to FcR is then determined by evaluating the variability of the average fluorescence intensity emitted by the labeled ligand bound to FcR.

[0049] Preferably, the mutated Fc region of the polypeptide according to the present invention contains 3 to 20 mutations, preferably 4 to 20 mutations, compared to the parent polypeptide. "3 to 20 amino acid modifications" includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acid mutations. Preferably, this region contains 4 to 15 mutations, preferably 4 to 10 mutations, compared to the parent polypeptide.

[0050] More preferably, the mutated Fc region of the polypeptide according to the present invention comprises at least one combination of five mutations, the combination comprising the four mutations (i) described above and at least one mutation (ii) described above, where the numbering is the Eu index or equivalent numbering in Kabat.

[0051] More preferably, the mutated Fc region of the polypeptide according to the present invention comprises one combination of six mutations, the combination comprising four mutations (i) as described above, at least one mutation (ii) as described above, and at least one mutation (iii) as described above, where the numbering is the Eu index or equivalent numbering in Kabat.

[0052] Preferably, the mutated Fc region of the polypeptide according to the present invention is (i) Four mutations 334N, 352S, 378V and 397M, (ii) 434Y at least one mutation selected from 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K, Includes, If mutation (iii) exists, this mutation is selected from K290G and Y296W. The numbering here refers to the numbering of the EU index or equivalent in Kabat.

[0053] Preferably, the mutated Fc region of the polypeptide according to the present invention is (i) Four mutations 334N, 352S, 378V and 397M, (ii) 434Y at least one mutation selected from 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K, (iii) At least one mutation selected from K290G and Y296W, Includes, The numbering here refers to the numbering of the EU index or equivalent in Kabat.

[0054] Preferably, the mutated Fc region of the polypeptide according to the present invention includes a combination of mutations selected from the combinations N434Y / K334N / P352S / V397M / A378V and N434Y / K334N / P352S / V397M / A378V / Y296W.

[0055] Preferably, the polypeptide according to the present invention is produced in mammary epithelial cells of a transgenic non-human mammal.

[0056] Preferably, the polypeptide according to the present invention is produced in non-human transgenic animals, preferably in transgenic non-human mammals, and more preferably in their mammary epithelial cells.

[0057] "Transgenic non-human mammal" refers to a mammal selected from among cattle, pigs, goats, sheep, and rodents, preferably from among goats, mice, sows, female rabbits, female sheep, and female cows. Preferably, the non-human transgenic animal or transgenic non-human mammal is a transgenic goat.

[0058] Preferably, the mutant according to the present invention contains at least five mutations N434Y, K334N, P352S, V397M, and A378V within its Fc fragment, and is produced in mammary epithelial cells of transgenic non-human mammals or in non-human transgenic animals, preferably in transgenic non-human mammals such as goats. Such mutants simultaneously have increased affinity for the FcRn receptor and increased affinity for all of the FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (CD32a).

[0059] Therefore, preferably, the mutant according to the present invention is the Fc mutant N434Y / K334N / P352S / V397M / A378V produced in mammary epithelial cells of transgenic non-human mammals. Alternatively, preferably, the mutant according to the present invention is the Fc mutant N434Y / K334N / P352S / V397M / A378V produced in non-human transgenic animals, preferably in transgenic non-human mammals such as goats. Such mutants simultaneously have increased affinity for the FcRn receptor and increased affinity for all of the FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (CD32a). Preferably, the mutant according to the present invention contains the sequence of SEQ ID NO: 11 or the sequence of SEQ ID NO: 15.

[0060] Alternatively, preferably, the mutant according to the present invention is the Fc mutant N434Y / K334N / P352S / V397M / A378V / Y296W produced in mammary epithelial cells of transgenic non-human mammals. Alternatively, preferably, the mutant according to the present invention is the Fc mutant N434Y / K334N / P352S / V397M / A378V / Y296W produced in non-human transgenic animals, preferably in transgenic non-human mammals such as goats. Such mutants simultaneously possess increased affinity for the FcRn receptor and increased affinity for all of the FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (CD32a).

[0061] Preferably, the method for producing the mutant according to the present invention includes the expression of the mutant in mammary epithelial cells of a transgenic non-human mammal.

[0062] Therefore, the present invention similarly relates to a method for producing a variant of a parent polypeptide comprising an Fc fragment, wherein the variant has an increased affinity for the FcRn receptor and an increased affinity for the receptor (FcR) of at least one Fc fragment selected from among the FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (CD32a), compared to the affinity of the parent polypeptide, and the variant is (i) Four mutations 334N, 352S, 378V and 397M, (ii) At least one mutation selected from 434Y, 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K and Includes, Here, the numbering refers to the numbering of the EU index or equivalent in Kabat. Methods including the expression of the aforementioned mutants in mammary epithelial cells of transgenic non-human mammals are also covered by the present invention.

[0063] Preferably, the variants are Y296W, K290G, V240H, V240I, V240M, V240N, V240S, F241H, F241Y, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, F243L, F243S, E258G, E258I, E258R, E258M, E2 58Q, E258Y, V259C, V259I, V259L, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V262S, V 263T, V264L, V264S, V264T, V266L, S267A, S267Q, S267V, K290D, K290E, K290H, K290L, K290N, K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E293Q, E293S , E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y296H, S298A, S298R, Y300 The following further comprises (iii) at least one mutation within an Fc fragment selected from I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S: The numbering is for EU indexes or equivalents in Kabat.

[0064] In detail, this method involves the following steps: a) A step of preparing a DNA sequence comprising a sequence encoding a mutant, a sequence encoding a mammalian casein promoter or a mammalian whey promoter, and a sequence encoding a signal peptide that enables the secretion of the mutant. b) The step of introducing the DNA sequence obtained in a) into a non-human mammalian embryo to obtain a transgenic non-human mammal that expresses a mutant encoded by the DNA sequence obtained in a) in the mammary gland, and c)b) A step of recovering mutants from milk produced by transgenic non-human mammals obtained in step c)b).

[0065] Therefore, step a) includes preparing a DNA sequence comprising a sequence encoding the mutant, a sequence encoding the mammalian casein promoter or the mammalian whey promoter, and a sequence encoding the signal peptide that enables the secretion of the mutant. Such a step is illustrated in Figure 1.

[0066] The sequence encoding the mutant is the DNA sequence encoding the mutant according to the present invention.

[0067] For example, this mutant has sequence number 11 as its sequence. Along with the signal peptide, the corresponding sequence is sequence number 13.

[0068] In another example, this variant has sequence number 15 as its sequence. Along with the signal peptide, the corresponding sequence is sequence number 16.

[0069] Sequences encoding mammalian casein promoters or mammalian whey promoters enable mutant expression in milk. Those skilled in the art can select such promoters.

[0070] Within the scope of this application, the signal peptide is an amino acid sequence of preferably 2 to 30 amino acids located at the N-terminal end of an Fc polypeptide variant, which helps direct the Fc polypeptide variant into the milk of a mammalian host. Preferably, the sequence encoding the signal peptide is located between the sequence encoding the variant and the promoter. Without such a sequence, the variant would remain in the mammary tissue, making purification difficult and requiring the sacrifice of a mammalian host. The signal peptide can be cleaved upon secretion. The sequence encoding the signal peptide may be a sequence that naturally associates with the parent polypeptide according to the present invention. Alternatively, the sequence encoding the signal peptide may be a sequence of the milk protein from which the promoter originates; that is, when the gene for the milk protein is digested for the purpose of isolating the promoter, a DNA fragment containing both the promoter and the sequence encoding the signal peptide immediately downstream of this promoter is selected. Another option is to use a signal sequence derived from another secreted protein, which is neither a milk protein normally expressed from the promoter nor a polypeptide according to the present invention.

[0071] Preferably, the signal peptide has sequence number 12.

[0072] The DNA sequence used may include optimized codons.

[0073] The goal of codon optimization is to replace native codons with the codons that are most frequently encountered by the amino acid-carrying transfer RNA (tRNA) in the target cell type. The main advantage of recruiting frequently encountered tRNAs is to accelerate the translation rate of messenger RNA (mRNA) and thus increase its final titer (Carton JM et al., Protein Expr Purif, 2007). Sequence optimization also affects the prediction of mRNA secondary structure, which is thought to slow down reading by the ribosome complex. Sequence optimization also affects mRNA half-life and therefore the G / C percentage, which is directly related to its likelihood of being translated (Chechetkin, J. of theoretical biology 242, 2006, pp. 922-934).

[0074] Codon optimization can be performed by substituting natural codons using codon usage tables for mammals, and more specifically for Homo sapiens. Algorithms that enable this sequence optimization exist and are available on the internet from synthetic gene suppliers (DNA2.0, GeneArt, MWG, Genscript).

[0075] Preferably, step a) includes the following steps: (a1) A step of preparing a DNA sequence comprising a sequence encoding a mutant according to the present invention, which is directly fused at its N-terminal end to a sequence encoding a signal peptide that enables the secretion of the mutant according to the present invention. (a2) The step of introducing the DNA sequence obtained in (a1) into a vector containing a sequence encoding a mammalian casein promoter or a mammalian whey promoter, and (a3) Digesting the vector obtained in (a2) in order to obtain a DNA sequence comprising a DNA sequence comprising a sequence encoding a mammalian casein promoter or a mammalian whey promoter, and a sequence encoding a variant according to the present invention that is directly fused at the N-terminal end to a sequence encoding a signal peptide.

[0076] In other words, preferably, at the end of step a), a DNA sequence is obtained that includes, from the N-terminus to the C-terminus, a sequence encoding a mammalian casein promoter or a mammalian whey promoter, which is itself fused to a sequence encoding a variant according to the present invention, to a sequence encoding a signal peptide.

[0077] Next, the method according to the present invention includes step b) introducing the DNA sequence obtained in a) into a non-human mammalian embryo in order to obtain a transgenic non-human mammal that expresses a mutant encoded by the DNA sequence obtained in a) in the mammary gland.

[0078] Finally, the method according to the present invention includes step c) of recovering the mutant in milk produced by the transgenic non-human mammal obtained in b).

[0079] Steps b) and c) are known from the prior art, particularly from European Patent No. 0264166.

[0080] Preferably, such a method includes step d) purifying the recovered milk after step c). Purification step d) can be achieved by any known method of the prior art, in particular purification on protein A. Again, such a step is described in particular in European Patent No. 0264166.

[0081] The present invention similarly relates to a DNA sequence comprising a gene encoding a variant of a parent polypeptide containing an Fc fragment, wherein the variant has increased affinity for the FcRn receptor and increased affinity for at least one Fc fragment receptor (FcR) selected from FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (CD32a) compared to the affinity of the parent polypeptide, and the variant is (i) Four mutations 334N, 352S, 378V and 397M, (ii) 434Y at least one mutation selected from 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K, Includes, The numbering is the numbering of the EU index or equivalent in Kabat, The aforementioned gene is under the control of a mammalian whey or casein transcription promoter that does not naturally regulate the transcription of this gene. The sequence further comprises a sequence that encodes a signal peptide, which is placed between the sequence encoding the mutant and the promoter, enabling the secretion of the mutant. DNA sequences are also included as subjects of this invention.

[0082] In a particular embodiment, the variants are Y296W, K290G, V240H, V240I, V240M, V240N, V240S, F241H, F241Y, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, F243L, F243S, E258G, E258I, E258R, E258M, E258Q, E258Y, V259C, V259I, V259L, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V262S, V263T, V26 4L, V264S, V264T, V266L, S267A, S267Q, S267V, K290D, K290E, K290H, K290L, K290N, K290Q, K290R, K290S, K 290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E293Q, E293S, E293T, E294A, E294 G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y296H, S298A, S298R, Y300I, Y300V, Y300W, R301A, R3 It further comprises (iii) at least one mutation within an Fc fragment selected from 01M, R301P, R301S, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R, and V305S, where the numbering is the EU index or equivalent numbering in Kabat.

[0083] The present invention similarly relates to a DNA sequence comprising a gene encoding a variant of a parent polypeptide containing an Fc fragment, wherein the variant has increased affinity for the FcRn receptor and increased affinity for at least one Fc fragment receptor (FcR) selected from FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (CD32a) compared to the affinity of the parent polypeptide, and the variant is (i) Four mutations 334N, 352S, 378V and 397M, (ii) At least one mutation selected from 434Y, 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K, Includes, The numbering is the numbering of the EU index or equivalent in Kabat, Optionally, the sequence includes a sequence encoding a signal peptide that enables the secretion of the mutant. DNA sequences are also included as subjects of this invention.

[0084] In a particular embodiment, the variants are Y296W, K290G, V240H, V240I, V240M, V240N, V240S, F241H, F241Y, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, F243L, F243S, E258G, E258I, E258R, E258M, E258Q, E258Y, V259C, V259I, V259L, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V262S, V263T, V26 4L, V264S, V264T, V266L, S267A, S267Q, S267V, K290D, K290E, K290H, K290L, K290N, K290Q, K290R, K290S, K 290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E293Q, E293S, E293T, E294A, E294 G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y296H, S298A, S298R, Y300I, Y300V, Y300W, R301A, R3 It further comprises (iii) at least one mutation within an Fc fragment selected from 01M, R301P, R301S, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R, and V305S, where the numbering is the EU index or equivalent numbering in Kabat.

[0085] Alternatively, the polypeptides according to the present invention may be produced in mammalian cells in culture. Preferred cells include rat YB2 / 0, hamster CHO, more specifically CHO dhfr- and CHO Lec13, PER.C6® cells (Crucell), NS0, SP2 / 0, HeLa, BHK or COS cells, and HEK293 cells. Hamster CHO is preferably used.

[0086] Therefore, the present invention similarly relates to a method for producing a variant of a parent polypeptide comprising an Fc fragment, wherein the variant has an increased affinity for the FcRn receptor and an increased affinity for at least one Fc fragment receptor (FcR) selected from among the FcγRI receptor (CD64), FcγRIIIa receptor (CD16a), and FcγRIIa receptor (CD32a), compared to the affinity of the parent polypeptide. (i) Four mutations 334N, 352S, 378V and 397M, (ii) 434Y at least one mutation selected from 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K, Includes, The numbering is the numbering of the EU index or equivalent in Kabat, This includes the expression of the aforementioned mutant in mammalian cells during culture. The focus is on the method.

[0087] In a particular embodiment, the variants are Y296W, K290G, V240H, V240I, V240M, V240N, V240S, F241H, F241Y, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, F243L, F243S, E258G, E258I, E258R, E25 8M, E258Q, E258Y, V259C, V259I, V259L, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V26 2S, V263T, V264L, V264S, V264T, V266L, S267A, S267Q, S267V, K290D, K290E, K290H, K290L, K290N, K29 0Q, K290R, K290S, K290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E293Q, E2 93S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y296H, S298A, S298R, Y3 Further comprising (iii) at least one mutation within an Fc fragment selected from 00I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S, The numbering is for EU indexes or equivalents in Kabat.

[0088] In detail, this method involves the following steps: a) A step of preparing a DNA sequence that encodes the mutant, b) The step of introducing the DNA sequence obtained in a) into cultured mammalian cells (this introduction can be achieved transiently or stably (i.e., integration of the DNA sequence obtained in a) into the cell's genome)), and The step of expressing mutants from cells obtained in c)b), and d) Optionally, a step of recovering the mutants in the culture medium.

[0089] The present invention also covers a pharmaceutical composition comprising (i) a polypeptide according to the present invention and (ii) at least one pharmaceutically acceptable excipient.

[0090] The present invention also covers pharmaceutical compositions comprising (i) variants consisting of Fc fragments of IgG1 in particular having five mutations N434Y, K334N, P352S, V397M, and A378V, with the numbering being EU index or equivalent numbering in Kabat, and (ii) at least one pharmaceutically acceptable excipient. Preferably, the composition of the present invention comprises (i) variants consisting of Fc fragments of IgG1 in particular having six mutations N434Y, K334N, P352S, V397M, and A378V, Y296W, with the numbering being EU index or equivalent numbering in Kabat, and ii) at least one pharmaceutically acceptable excipient.

[0091] The present invention also applies to polypeptides or compositions as described above according to the present invention for use as pharmaceuticals.

[0092] The present invention also covers the use as a drug of mutants consisting of Fc fragments of IgG1 in particular, having five mutants N434Y, K334N, P352S, V397M, and A378V, with the numbering being the EU index or equivalent numbering in Kabat (i.e., mutants N434Y / K334N / P352S / V397M / A378V). In a particular embodiment, the present invention also covers the use as a drug of mutants consisting of Fc fragments of IgG1 in particular, having six mutants N434Y, Y296W, K334N, P352S, V397M, A378V, and Y296W, with the numbering being the EU index or equivalent numbering in Kabat (i.e., mutants N434Y / K334N / P352S / V397M / A378V / Y296W).

[0093] As described above, advantageously, the parent polypeptide, and therefore the polypeptide of the present invention, is an antibody. In this case, the antibody may be directed to an antigen selected from tumor antigens, viral antigens, bacterial antigens, fungal antigens, toxins, membrane cytokines or circulating cytokines, and membrane receptors.

[0094] When antibodies are directed against tumor antigens, their use is particularly appropriate in cancer treatment. "Cancer" refers to any physiological condition characterized by abnormal cell proliferation. Examples of cancer include, but are not exhaustive, epitheliomas, lymphomas, blastomas, sarcomas (including liposarcomas), neuroendocrine tumors, mesotheliomas, meningiomas, adenocarcinomas, melanomas, leukemias, and malignant lymphoid pathologies.

[0095] When antibodies are directed against viral antigens, their use is particularly appropriate in the treatment of viral infections. Viral infections include, but are not exhaustive, infections caused by HIV, retroviruses, coxsackieviruses, smallpox virus, influenza virus, yellow fever virus, West Nile virus, cytomegalovirus, rotavirus, or hepatitis B or C virus.

[0096] When antibodies are directed against toxins, their use is particularly appropriate in the treatment of bacterial infections, such as those caused by tetanus toxin, diphtheria toxin, and anthrax toxin, as well as infections caused by botulinum toxin, ricin toxin, and Shiga toxin, although this list is not exhaustive.

[0097] When antibodies are directed against cytokines, their use is particularly appropriate in the treatment of inflammatory and / or autoimmune diseases. Inflammatory and / or autoimmune diseases include, in particular, thrombotic thrombocytopenic purpura (TTP), graft or organ rejection, graft-versus-host disease, polyarthritis rheumatica, systemic lupus erythematosus, various types of sclerosis, first-onset Sjögren's syndrome (or Gougerot-Sjögren's syndrome), autoimmune polyneuropathy such as multiple sclerosis, type 1 diabetes mellitus, autoimmune hepatitis, ankylosing spondyloarthritis, Reiter's syndrome, gouty arthritis, abdominal diseases, Crohn's disease, and Hashimoto's chronic thyroiditis. Adenitis (hypothyroidism), Addison's disease, autoimmune hepatitis, Graves' disease (hyperthyroidism), ulcerative colitis, vasculitis such as systemic vasculitis linked to ANCA (anti-cytoplasmic antibodies against neutrophils), autoimmune cytopenia and other hematological complications in adults and children, such as acute or chronic autoimmune thrombocytopenia, autoimmune hemolytic anemia, neonatal hemolytic disease (MHN), cold agglutinin disease, autoimmune acquired hemophilia; Goodpasture syndrome, extramembrane nephropathy, autoimmune bullous skin Diseases, including refractory myasthenia gravis, mixed cryoglobulinemia, psoriasis, juvenile chronic arthritis, inflammatory myositis, dermatomyositis and antiphospholipid syndrome, autoimmune systemic diseases in children, connective tissue diseases, autoimmune pneumonia, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), autoimmune thyroiditis, diabetes mellitus, myasthenia gravis, autoimmune inflammatory eye disease, neuromyelitis optica (Devic's disease), scleroderma, pemphigus, insulin-resistant diabetes mellitus, polymyositis, Bomer anemia, glomerulonephritis, Wegener's disease, This includes Horton's disease, tuberous periarthritis and Churg-Strauss syndrome, Still's disease, atrophic polychondritis, Behçet's disease, monoclonal immunoglobulinemia, Wegner granulomatosis, lupus, hemorrhagic colitis, psoriatic arthritis, sarcoidosis, collagenous colitis, herpetiform dermatitis, familial Mediterranean fever, IgA-depositing glomerulonephritis, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmitis, Fiessinger-Leroy-Reiter syndrome, and uveal meningoencephalitis syndrome.

[0098] For example, other inflammatory diseases such as acute respiratory distress syndrome (ARDS), acute septic arthritis, adjuvant arthritis, allergic encephalomyelitis, allergic rhinitis, allergic vasculitis, allergies, asthma, atherosclerosis, chronic inflammation resulting from chronic bacterial or viral infections, chronic obstructive bronchopulmonary disease (COPD), coronary artery disease, encephalitis, inflammatory bowel disease, inflammatory osteolysis, acute and delayed hypersensitivity reaction-related inflammation, tumor-related inflammation, peripheral nerve lesions or demyelinating diseases, inflammation associated with traumatic tissue injury such as burns and ischemia, inflammation resulting from meningitis, multiple organ dysfunction syndrome (MODS), pulmonary fibrosis, sepsis and septic shock, Stevens-Johnson syndrome, undifferentiated arthritis, and undifferentiated spondyloarthropathy are also included. In certain embodiments of the present invention, autoimmune diseases are idiopathic thrombocytopenic purpura (ITP) and chronic inflammatory demyelinating polyneuropathy (CIDP).

[0099] Preferably, the autoimmune or inflammatory pathology is selected from immunopathological thrombocytopenic purpura (also known as idiopathic thrombocytopenic purpura, or ITP), neuromyelitis optica or Devic's disease (NMO), and multiple sclerosis. Multiple sclerosis has been studied in particular using the experimental autoimmune encephalomyelitis (EAE) model.

[0100] The sequences described in this application can be summarized as follows:

[0101] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0102] The present invention will be better understood by reading the following examples.

[0103] The legend for the figure is as follows: [Brief explanation of the drawing]

[0104] [Figure 1] Production of mutant A3A-184AY in goat and mouse milk using vector Bc451.

[0105] A) Beta-casein vector Bc451 was digested with XhoI. In vector Bc451, the NotI-NotI fragment is a prokaryotic fragment. The NotI(15370)-Xhol fragment is the 3' genome sequence containing the polyA signal. The BamHI-Xhol fragment is the promoter region of beta-casein.

[0106] B) A Sall fragment containing the coding region of the Fc mutant A3A-184AY (i.e., FC3179 A3A-184AY 884 bp) was inserted into the vector to generate the genetic structure BC3180 FC A3A-184AY(C).

[0107] D) Next, DNA fragments for microinjection were isolated from the prokaryotic vector. For this purpose, BC3180 was digested with Notl and Nru1. A 16.4kb free fragment containing the Fc gene (encoding mutant A3A-184AY) was then purified by gel elution under the control of a beta-casein promoter.

[0108] [Figure 2] Results of a study in a preventive model of arthritis induced by K / BxN mouse serum transfer.

[0109] Disease was induced in C57 / BI / 6J mice by transferring 10 μl of K / BxN mouse serum intravenously on day 0. On day 0, the test molecule was administered intraperitoneally once, 2 hours prior to the injection of K / BxN mouse serum.

[0110] The following clinical scores were obtained by adding the indices of the four limbs: 0 = normal, 1 = swelling of one joint, 2 = swelling of two or more joints, and 3 = severe swelling of all joints (arbitrary unit).

[0111] [Figure 3] Results of a treatment model for arthritis induced by K / BxN mouse serum transfer.

[0112] Disease was induced in C57 / BI / 6J mice by intravenous transfer of 10 μl of K / BxN mouse serum on day 0. On day 0, 72 hours after the injection of K / BxN mouse serum, the test molecule was administered intraperitoneally once (indicated by the dotted line).

[0113] The following clinical scores were obtained by adding the indices of the four limbs: 0 = normal, 1 = swelling of one joint, 2 = swelling of two or more joints, and 3 = severe swelling of all joints (arbitrary unit).

[0114] [Figure 4] Results of Fc and IgIV binding tests to blood cells

[0115] The Fc mutant or IgIV according to the present invention, labeled with Alexa, was incubated with target cells at 65 nM (10 μg / ml for Fc in PBS with 2% CSF) on ice for 20 minutes. After washing twice in PBS with 2% CSF, the cells were suspended in 500 μl of Isoflow before flow cytometry analysis.

[0116] The results are as follows: A) B cells labeled with anti-CD19 ("% positive B cells") B) Anti-CD56 labeled NK cells ("% positive NK cells") C) Monocytes labeled with anti-CD14 in the presence of IgIV ("% positive cells + IgIV") D) CD16+ monocytes labeled with anti-CD14 and anti-CD16 antibodies 3G8 in the presence of IgIV ("% positive cells + IgIV") E) Neutrophils labeled with anti-CD15 in the presence of IgIV ("% positive cells + IgIV") F) NK cells labeled with anti-CD56 in the presence of IgIV or WT Fc ("% cell-positive").

[0117] [Figure 5] ADCC test results for Jurkat CD64 cells and CDC activation

[0118] A) Inhibition of Jurkat CD64 cell activation Raji cells (5×10 6 50 μl of cells / ml, Rituxan (2 μg / ml, 50 μl), Jurkat cells expressing human CD64 (Jurkat-H-CD64) (5 × 10 6 The mixture (25 μl at cell / ml) was mixed with PMA (50 μl at 40 ng / ml) and then incubated at 1950 nM with the mutant according to the present invention (RFC A3A-184AY) or IgIV. After incubation overnight, the plates were centrifuged (125g for 1 minute), and IL2 in the supernatant was evaluated by ELISA. The results are expressed as a percentage in relation to IgIV using the following formula: (IL-2 IgIV / IL-2 of the sample) × 100

[0119] B) Inhibition of ADCC Effector cells (mononuclear cells) (8 x 10 7 (25 μl at cells / ml) and Rh (Rhesus) positive erythrocytes (final 4 × 10⁶ cells) 725 μl of cells / ml were incubated with anti-RhD antibodies at different concentrations (0-75 ng / ml) in an effector / target ratio of 2 / 1. After 16 hours of incubation, lysis was estimated by quantifying the hemoglobin released into the supernatant using a specific substrate (DAF). The results are expressed as specific lysis percentages corresponding to the antibody quantity. ADCC inhibition was induced by the Fc variant according to the present invention (RFC A3A-184AY) or IgIV added at 33 nM. The results are expressed as percentages, with 100% and 0% being values ​​obtained using IgIV at 650 nM and 0 nM, respectively, according to the following formula. [(ADCC with sample at 33nM - ADCC without IVIg) / (ADCC with IgIV at 33nM - ADCC without IVIg) × 100]

[0120] C) Inhibitory activity of CDC: Raji cells were incubated with rituximab at a final concentration of 50 ng / ml for 30 minutes. A 1 / 10 dilution was added, and serum solution from young rabbits, which had been previously incubated with the Fc mutant of the present invention (rFc A3A-184AY) or IgIV (vol / vol) at 37°C for 1 hour, was added. After incubation at 37°C for 1 hour, the plates were centrifuged (125 g for 1 minute), and CDC was estimated by measuring the intracellular LDH released into the culture medium. The results were expressed as inhibition percentages and compared with IgIV and negative control groups (Fc without function, i.e., rFc neg). 100% corresponded to complete inhibition of solubility, and 0% corresponded to the control value obtained without Fc or IgIV.

[0121] [Figure 6] Results of tests on binding to blood cells

[0122] IgIV, Fc-Rec (wild-type Fc), Fc MST-HN, or Fc variants according to the present invention (A3A-184AY_CHO, A3A-184EY_CHO) labeled with Alexa-Fluor® were incubated with target cells at 65 nM (10 μg / ml for Fc in PBS with 2% CSF (Colony Stimulating Factor)) for 20 minutes on ice. After washing twice in PBS with 2% CSF, the cells were suspended in 500 μl of Isoflow before flow cytometry analysis. The tests were performed on the following target cells. - Anti-CD56 labeled natural killer (NK) cells, - Monocytes labeled with anti-CD14, - Monocytes CD16+ labeled with anti-CD14 and anti-CD16 3G8 antibodies, - Neutrophils labeled with anti-CD15.

[0123] [Figure 7] Test results in a biological model of idiopathic thrombocytopenic purpura (ITP)

[0124] Disease was induced in mice expressing humanized FcRn by intravenous injection of the anti-platelet antibody 6A6-hlgG1 (0.3 μg per g of body weight) to deplete platelets, also known as mouse thrombocytes. Negative controls ("CTL PBS"), IgIV (1000 mg / kg), Fc-Rec fragment (wild-type Fc) (380 and 750 mg / kg), Fc fragment MST-HN (190 mg / kg), and the Fc mutant A3A-184AY_CHO of the present invention (190 mg / kg and 380 mg / kg) were administered intraperitoneally 2 hours before platelet depletion. Platelet counts were determined using the Advia Hematology (Bayer) system. The number of platelets before antibody injection was set to 100%. [Modes for carrying out the invention] [Examples]

[0125] Example 1: Preparation of a mutant (mutated Fc fragment) according to the present invention produced in the milk of a transgenic animal, and characterization of this mutant. I. Materials and Methods principle: The Fc fragment according to the present invention can be produced in the milk of transgenic animals by placing the coding sequence of the Fc fragment within a milk-specific expression vector. The vector can be introduced into the genome of transgenic goats or mice by microinjection. After screening and identifying animals carrying the trans gene, females are bred. After calving, the females can be milked to collect milk that may have been secreted after the expression of the milk-specific promoter.

[0126] [Table 2]

[0127] To obtain the sequence of SEQ ID NO: 13, a signal peptide (MRWSWIFLLLLSITSANA, SEQ ID NO: 12) is attached to the N-terminal end of the protein sequence. This allows the protein to be secreted from milk once it is expressed.

[0128] Nucleotide sequence optimization The nucleotide sequence was optimized for expression in the mammary glands of goats. To achieve this, the sequence was optimized for the Bos taurus (cattle) species using an algorithm from a synthetic gene supplier (such as GeneArt).

[0129] Expression vector To produce the mutant A3A-184AY in mouse and goat milk, an expression vector of goat beta-casein (Bc451) was used. (See Figure 1) The beta-casein vector Bc451 was digested with Xhol (Figure 1A). A Sall fragment containing the coding region of the Fc mutant A3A-184AY was inserted to generate the genetic structure BC3180 FC A3A-184AY (Figures 1B and 1C).

[0130] Next, DNA fragments for microinjection were isolated from the prokaryotic vector.

[0131] BC3180 was digested with NotI and NruI (Figure 1D). Next, a 16.4 kb free fragment containing the Fc gene was purified by gel elution under the control of a beta-casein promoter. This DNA was then used in the microinjection step.

[0132] Production in mice DNA fragments were inserted into pre-implantation mouse embryos by microinjection. Next, the embryos were transplanted into female mice in a pseudo-pregnancy state. The subsequent generations were screened for the presence of trans genes by PCR analysis.

[0133] Expression in goats DNA fragments prepared for microinjection can also be used for the production of the Fc mutant A3A-184AY in goat's milk.

[0134] Example 2: Preparation of a mutant (mutated Fc fragment) according to the present invention produced in HEK cells, and characterization of this mutant. I. Materials and methods for production Using two primer sets adapted to incorporate one or more targeted mutations at codons encoding desired amino acids, each target mutation within the Fc fragment of sequence SEQ ID NO: 14 was inserted by overlap PCR. Advantageously, if the mutations to be inserted are close together on the Fc sequence, these mutations are added via the same oligonucleotide. The fragments thus obtained by PCR were associated, and the resulting fragments were amplified by PCR using a standard protocol. The PCR products were purified on a 1% (w / v) agarose gel, digested with appropriate restriction enzymes, and cloned.

[0135] Recombinant Fc fragments were produced by transient transfection (lipofection) in HEK293 cells (293-F cells, freestyle InvitroGen) in an F17 medium supplemented with L-glutamine using the vector pCEP4. After culturing for 8 days, the supernatant was clarified by centrifugation and filtered through a 0.2 μm filter. Subsequently, the Fc fragments were purified on a Protein A Hi-Trap, eluted with 25 mM citrate buffer at pH=3.0, neutralized in PBS, dialyzed, and then sterilized by filtration (0.2 μm).

[0136] II. Coupling tests on Octet® (BLI "Bio-Layer Interferometry" technology, Octet RED96 instrument, Fortebio, Pall) Protocol: Binding to human FcRn (hFcRn): Biotinylated hFcRn receptors were immobilized on a streptavidin biosensor after being diluted at 0.7 μg / ml in running buffer (0.1 M phosphate buffer, 150 mM NaCl, 0.05% Tween20, pH 6). The mutants according to the present invention, WT and IgIV, were tested in running buffer at 200, 100, 50, 25, 12.5, 6.25, 3.125 and 0 nM (200 nM = 10 μg / ml for Fc).

[0137] - Test design: Baseline 1 x 120 seconds, running buffer, Loading 300 seconds: The receptor is placed onto the biosensor. Baseline 2 x 60 seconds, during running buffer, Meeting time 60 seconds: Add the sample (Fc or IVIg) to the biosensor into which hFcRn has been added. Dissociation 30 seconds, running buffer, Regeneration time: 120 seconds, in regeneration buffer (0.1M phosphate buffer, 150mM NaCl, 0.05% Tween20, pH 7.8).

[0138] - Interpretation of results: Using the association and dissociation curves (first 10 seconds), the association rate constant (kon) and dissociation rate constant (koff) are calculated using association model 1 / 1. Then, KD(nM) is calculated (kon / koff).

[0139] Binding to hCD16aV and hCD32aH receptors: The hCD16aV receptor (R&D system) or hCD32aH receptor (PX therapeutics) His Tag was immobilized on an anti-Penta-HIS biosensor (HIS 1K) after being diluted to 1 μ / ml in kinetic buffer (Pall). The Fc mutant, WT, and IgIV according to the present invention were tested in kinetic buffer at concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15, and 0 nM.

[0140] - Loading before each sample, - Test design: All steps are performed in a kinetic buffer (Pall). Baseline 1 x 60 seconds Loading time: 400 seconds Baseline 2 x 60 seconds Meeting 60 seconds Dissociation 30 seconds Regeneration: Regeneration buffer (glycine 10mM pH 1.5 / neutralized: PBS) for 5 seconds.

[0141] - Interpretation of results: Using the association and dissociation curves (first 5 seconds), the association rate constant (kon) and dissociation rate constant (koff) are calculated using association model 1 / 1. Then, KD(nM) is calculated (kon / koff).

[0142] result: The results are shown in Table 1 below:

[0143] [Table 3]

[0144] The results reveal that the Fc mutant A3A_184AY(HEK) according to the present invention simultaneously exhibits increased affinity for the hFcRn receptor, as well as increased affinity for the FcγRIIIa receptor (CD16a) and FcγRIIa receptor (CD32a), compared not only to the unmutated parental Fc (Fc-WT) but also to IgIV.

[0145] III. Tests on arthritis models induced by K / BxN mouse serum transfer Protocol: A K / BxN model was generated by crossing transgenic mice with T cell receptor KRNs with a NOD mouse strain. K / BxN F1 mice spontaneously develop the disease at 3-5 weeks of age and share many clinical features with human rheumatoid arthritis.

[0146] Disease was induced in C57 / BI / 6J mice by intravenous transfer of 10 μl of K / BxN mouse serum on day 0. On day 0, the test molecule was administered intraperitoneally once, either 2 hours prior to or 72 hours after the injection of K / BxN mouse serum.

[0147] Mice were monitored daily to pursue and identify signs of arthritis, with the index of all four limbs added up to assess morbidity and severity: 0 = normal, 1 = swelling of one joint, 2 = swelling of two or more joints, and 3 = severe swelling of all joints.

[0148] result Mice treated with K / BxN serum developed intraarticular arthritis. The disease was characterized by increased ankle size and induced an increase in clinical score. These mice showed a significant increase in clinical score and ankle thickness compared to control mice treated with physiological serum.

[0149] 1. Prevention Model Treatment with 750 mg / kg of wild-type Fc fragment (Fc WT), administered two hours prior to injection of K / BxN mouse serum, significantly reduced clinical scores compared to the group treated with K / BxN mouse serum.

[0150] Treatment with the Fc variant A3A-184AY(HEK) according to the present invention significantly reduced clinical scores in a manner similar to that of Fc WT, but in this case, the dose was 1 / 15th (50 mg / kg) (Figure 2).

[0151] 2- Treatment Models 72 hours after K / BxN mouse serum injection, IgIV administered at 2 g / kg did not significantly reduce clinical scores compared to the group treated with K / BxN mouse serum.

[0152] However, treatment with Fc fragment WT at 750 mg / kg (equivalent to a molecular dose of 2 g / kg IVIg) significantly reduced clinical scores compared to the group treated with K / BxN mouse serum. Furthermore, treatment with the Fc mutant A3A-184AY (HEK) according to the present invention significantly reduced clinical scores in a similar manner to that of the Fc-WT fragment, but at one-quarter the dose (190 mg / kg) (Figure 3).

[0153] IV. In vitro studies on cells Protocol: Evaluation of the binding of Fc fragments and IgIV to blood cells Fc mutants or IgIV cells labeled with Alexa according to the present invention were incubated with target cells at 65 nM (10 μg / ml for Fc in 2% CSF PBS) on ice for 20 minutes. After washing twice in 2% CSF PBS, the cells were suspended in 500 μl of Isoflow before flow cytometry analysis. B cells, NK cells, monocytes, and neutrophils were specifically labeled with anti-CD19, anti-CD56, anti-CD14, and anti-CD15 antibodies, respectively. The FcγRIII receptor (CD16) was identified using an anti-CD16 3G8 antibody.

[0154] Inhibition of ADCC: To mimic the erythrolysis observed in idiopathic thrombocytopenic purpura (ITP), which involves autoantibodies from patients with ITP, we performed effector cell-mediated erythrolysis in the presence of anti-RhesusD (RhD) monoclonal antibodies and evaluated the ability of different quantities of polyvalent immunoglobulin (IVIg) or mutated or non-mutated recombinant Fc fragments to inhibit this lysis, for example, by competition with anti-RhD for the fixation of Fc receptors on the surface of effector cells.

[0155] The cytotoxicity of anti-RhD antibodies was studied using ADCC technology. Simply put, effector cells (mononuclear cells) (8 × 10⁻¹⁰) were treated with anti-RhD antibodies at different concentrations (0-75 ng / ml). 7 (25 μl at cells / ml) and Rh-positive red blood cells (final 4 × 10) 7 25 μl of cells / ml were incubated at an effector / target ratio of 2 / 1. After 16 hours of incubation, lysis was estimated by quantifying the hemoglobin released into the supernatant using a specific filtration factor (DAF).

[0156] The results were expressed as specific lysis percentages corresponding to the antibody quantity. Inhibition of ADCC induced by the Fc variant (RFC A3A-184AY) or IgIV according to the present invention, added at 33 nM, was evaluated.

[0157] The results are expressed as percentages, with 100% and 0% being values ​​obtained using IgIV at 650 nM and 0 nM, respectively, according to the following formula. [(ADCC with sample at 33nM - ADCC without IVIg) / (ADCC with IgIV at 33nM - ADCC without IVIg) × 100]

[0158] Inhibition of CD64 activation in Jurkat cells: This test estimates the ability (total IgG) of the Fc mutant or IgIV according to the present invention to inhibit IL2 secretion by human CD64-expressing Jurkat cells (Jurkat-H-CD64) induced by the Raji cell line using rituxan.

[0159] Simply put, Raji cells (5 x 10 6 (50 μl of cells / ml), Rituxan (50 μl of 2 μg / ml), Jurkat cells H-CD64 (5 × 10 6 The mixture (25 μl at cell / ml) was mixed with phorbol ester (PMA, 50 μl at 40 ng / ml) and then incubated with the Fc mutant or IgIV according to the present invention at 1950 nM.

[0160] After overnight incubation, the plates were centrifuged (125g for 1 minute), and the IL2 content in the supernatant was evaluated by ELISA.

[0161] The results were expressed as a percentage in relation to IgIV using the following formula: (IL-2IgIV / IL-2 in sample) × 100

[0162] Inhibitory activity of CDC: This test estimates the ability of the Fc variant or IgIV according to the present invention to inhibit rituximab-mediated CDC activity in Raji cell lines in the presence of rabbit serum as a complement source. Briefly, Raji cells were incubated with rituximab at a final concentration of 50 ng / ml for 30 minutes. A solution of rabbit serum, pre-incubated with the variant or IgIV according to the present invention (vol / vol) and diluted 1 / 10, was added and incubated at 37°C for 1 hour. After incubation at 37°C for 1 hour, the plates were centrifuged (125 g for 1 minute), and CDC was estimated by measuring the intracellular LDH released into the culture medium.

[0163] The results were expressed as percent inhibition and were compared to IgIV and the negative control group (Fc without Fc function), where 100% corresponded to complete inhibition of lysis activity and 0% corresponded to the control values obtained without IgIV or Fc.

[0164] Results: The results are shown in Figures 4 and 5.

[0165] As shown in Figure 5, the Fc mutant according to the present invention (A3A-184AY (HEK)) exhibits better inhibition of ADCC and CDC activities of Jurkat cells expressing CD64 compared to IgIV. These results indicate that mutants according to the present invention, such as A3A-184AY, may be effective in the treatment of pathologies involving patient autoantibodies, particularly by blocking Fc receptors on patient effector cells (see Figure 4).

[0166] Example 3: Preparation of the mutant according to the present invention (mutated Fc fragment) produced in CHO cells A recombinant Fc fragment can be obtained from SEQ ID NO: 14 in the same manner as described in Example 2. This mutated Fc fragment can be produced by transfection in CHO-S cells using a lipofection reagent such as Freestyle Max Reagent (Thermofisher) with a vector optimized for expression in the CHO-S cell line. CHO-S cells are cultured in a medium of CDFortiCHO + 8 mM glutamine under conditions of being stirred at 135 rpm in a controlled atmosphere (CO2 8%) at 37°C. One day before the transfection day, seed the cells at a density of 6×10 5 cells / ml.

[0167] On the day of transfection, linearized DNA (50 μg) and 50 μl of transfection reagent (TA) are pre-incubated separately in Opti-Pro SFM medium, then mixed and incubated for 20 minutes to allow formation of the DNA / AT complex. Next, the whole is added at a volume of 30 ml to 1×106 Add to cell preparation at a concentration of cells / ml. After 48 hours of incubation, add transfection reagents (neomycin 1g / L and methotrexate 200nM) to the cells. Determine cell density and viability every 3-4 days, and set the cell density to 6 × 10⁶. 5 The culture volume is adapted to maintain a cell / ml higher than 90%. If the viability exceeds 90%, the resulting stable pool is stored by freezing at low temperatures, and production is carried out in "Fedbatch" mode under agitated conditions for 10 days while adding 4 g / L or 6 g / L of glucose during production. At the end of production, the cells and supernatant are separated by centrifugation. The cells are removed, supernatant a is harvested, concentrated, and filtered through 0.22 μm.

[0168] Subsequently, the Fc fragments are purified by affinity chromatography on protein A resin (HiTrap Protein A, GE Healthcare). After capturing the fragments on the resin equilibrated in PBS buffer, the Fc fragments are eluted in 25 mM citrate buffer at pH=3.0, then the pH is rapidly neutralized with Tris 1M, followed by dialyzing in PBS buffer and sterilization by filtration (0.2 μm).

[0169] Example 4: Binding tests of mutants produced in CHO cells and transgenic goat milk to FcRn, CD16aH, CD16aV, CD64, and CD32a receptors. The binding test to the Fc receptor will be performed using the following molecules. - Mutants of the present invention, A3A-184AY_CHO(K334N / P352S / A378V / V397M / N434Y), A3A-184EY_CHO(Y296W / K334N / P352S / A378V / V397M / N434Y) produced in CHO cells according to the method described in Example 3, A3A-184AY_TGg produced in transgenic goats according to the method described in Example 1, - Fc fragment MST-HN, containing the mutant M252Y / S254T / T256E / H433K / N434F, described in the literature (Ulrichts et al., JCI, 2018) as having optimized binding only to the FcRn receptor, was produced in HEK-293 cells (293-F cells, freestyle Invitro Gen). - Wild Fc fragments "Fc-WT" or "Fc-Rec" obtained by digesting IgG1 produced in the milk of transgenic goats with papain. -IgIV.

[0170] Binding to human FcRn (hFcRn): We will study the binding to FcRn using competitive testing with rituxan labeled with the A488 marker (Rituxan-A488) and Jurkat cells expressing the FcRn receptor (Jurkat-FcRn).

[0171] 2 x 10 per well 5 Jurkat-FcRn cells were seeded in a 96-well plate (V-shaped base) at the specified cell concentration. The cells were then incubated at 4°C for 20 minutes with the test molecule, diluted in buffer at the final concentrations of 167 μg / ml, 83 μg / ml, 42 μg / ml, 21 μg / ml, 10 μg / ml, 5 μg / ml, 3 μg / ml, 1 μg / ml, and 0 μg / ml, along with 25 μg / mL of Rituxan-A488.

[0172] Next, wash the cells with 100 μL of pH 6 PBS and centrifuge at 1700 rpm for 3 minutes at 4°C. Then, remove the supernatant and add 300 μL of cold PBS at pH 6.

[0173] The binding of Rituxan-A488 to FcRn expressed by Jurkat-FcRn cells is evaluated by flow cytometry. The observed mean fluorescence intensity (MFI) is expressed as a percentage, where 100% is the value obtained with Rituxan-A488 alone, and 0% is the value in the absence of Rituxan-A488. The molecular concentration required to induce 50% inhibition of Rituxan-A488 binding to FcRn in Jurkat-FcRn cells is calculated using the software "Prism Software".

[0174] The results are shown in Table 2 below.

[0175] [Table 4]

[0176] The results reveal that the Fc mutants A3A-184AY_CHO, A3A-184EY_CHO, and A3A-184AY-TGg exhibit increased inhibition of Rituxan-A488 binding (100 times compared to IVIg). The mutants of the present invention exhibit a binding affinity for FcRn equivalent to the binding affinity observed with the Fc fragment MST-HN described in the literature (Ulrichts et al., JCI, 2018) as a binding affinity optimized only for FcRn.

[0177] Binding to hCD64 and hCD16aH, hCD16aV, hCD32aH, and hCD32aR receptors: Binding to human CD64 (hCD64) We will study the binding of Rituxan-A488 and the CD64 receptor to human CD64 using Jurkat cells expressing these receptors (Jurkat-CD64) through competitive testing.

[0178] 2 x 10 per well 5Jurkat-CD64 cells were seeded in a 96-well plate (V-shaped base) at the specified cell concentration. The cells were then incubated at 4°C for 20 minutes with the test molecule, diluted in buffer at the final concentrations of 167 μg / ml; 83 μg / ml; 42 μg / ml; 21 μg / ml; 10 μg / ml; 5 μg / ml; 3 μg / ml; 1 μg / ml; and 0 μg / ml, along with 25 μg / mL of Rituxan-A488.

[0179] Next, wash the cells with 100 μL of pH 6 PBS and centrifuge at 1700 rpm for 3 minutes at 4°C. Then, remove the supernatant and add 300 μL of cold PBS at pH 6.

[0180] The binding of Rituxan-A488 to CD64 expressed by Jurkat-CD64 cells is evaluated by flow cytometry. The observed mean fluorescence intensity (MFI) is expressed as a percentage, where 100% is the value obtained with Rituxan-A488 alone, and 0% is the value in the absence of Rituxan-A488. The molecular concentration required to induce 50% inhibition of Rituxan-A488 binding to CD64 in Jurkat-CD64 cells is calculated using the software "Prism Software".

[0181] Binding to CD32aH and CD32aR We will study the binding of HEK cells transfected with CD32aH and CD32aR receptors (HEK-CD32) and Rituxan-A488 to the human CD32 receptor through competitive testing.

[0182] 2 x 10 per well 5HEK-CD32 cells were seeded in a 96-well plate (V-shaped base) at the specified cell concentration. The cells were then incubated at 4°C for 20 minutes with the test molecule, diluted in buffer at the final concentrations of 333 μg / ml; 167 μg / ml; 83 μg / ml; 42 μg / ml; 21 μg / ml; 10 μg / ml; 5 μg / ml; 3 μg / ml; 1 μg / ml; and 0 μg / ml, along with 30 μg / mL of Rituxan-A488.

[0183] Next, wash the cells with 100 μL of pH 6 PBS and centrifuge at 1700 rpm for 3 minutes at 4°C. Then, remove the supernatant and add 300 μL of cold PBS at pH 6.

[0184] The binding of Rituxan-A488 to CD32aH and CD32aR expressed by HEK-CD32 cells is evaluated by flow cytometry. The observed mean fluorescence intensity (MFI) is expressed as a percentage, where 100% is the value obtained with Rituxan-A488 alone, and 0% is the value in the absence of Rituxan-A488. The molecular concentration required to induce 50% inhibition of Rituxan-A488 binding to CD32aH and CD32aR in HEK-CD32 cells is calculated using the software "Prism Software".

[0185] Binding to hCD16aH We will study the binding of a mouse anti-CD16 3G8 antibody labeled with phycoelthrin (3G8-PE) to human CD16aH using competitive testing with Jurkat cells transfected with the human CD16aH receptor (Jurkat-CD16aH).

[0186] 2 x 10 per well 5Jurkat-CD16aH cells were seeded in a 96-well plate (V-shaped base) at the specified cell concentration. The cells were then incubated at 4°C for 20 minutes with the test molecule, diluted in buffer at final concentrations of 83 μg / ml; 42 μg / ml; 21 μg / ml; 10 μg / ml; 5 μg / ml; 3 μg / ml; 1 μg / ml; and 0 μg / ml, along with 0.5 μg / mL of mAb 3G8-PE.

[0187] Next, wash the cells with 100 μL of pH 6 PBS and centrifuge at 1700 rpm for 3 minutes at 4°C. Then, remove the supernatant and add 300 μL of cold PBS at pH 6.

[0188] The binding of mAb 3G8-PE to CD16aH expressed by Jurkat-CD16aH cells is evaluated by flow cytometry. The observed mean fluorescence intensity (MFI) is expressed as a percentage, where 100% is the value obtained with mAb 3G8-PE alone, and 0% is the value in the absence of mAb 3G8-PE. The molecular concentration required to induce 50% inhibition of mAb 3G8-PE binding to CD16aH in Jurkat-CD16aH cells is calculated using the software "Prism Software".

[0189] The results are shown in Table 3 below.

[0190] [Table 5]

[0191] The results show that the Fc mutants A3A-184AY_CHO, A3A-184EY_CHO, and A3A-184AY_TGg exhibit increased affinity for the FcγRIIIa receptor (CD16a), FcγRI receptor (CD64), and FcγRIIa receptor (CD32a) compared not only with non-mutated Fc (Fc-WT) but also with IgIV.

[0192] The mutant of the present invention exhibits significantly increased affinity for the FcγRIIIa receptor (CD16a), FcγRI receptor (CD64), and FcγRIIa receptor (CD32a) compared to MST-HN.

[0193] Binding to human CD16aV: The hCD16aV receptor (R&D system) His Tag was immobilized on an anti-Penta-HIS biosensor (HIS 1K) after being diluted to 1 μ / ml in kinetic buffer (Pall). The molecules were tested in kinetic buffer at concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15, and 0 nM.

[0194] - Loading before each sample, - Test design: All steps are performed in a kinetic buffer (Pall). Baseline 1 x 60 seconds Loading time: 400 seconds Baseline 2 x 60 seconds Meeting 60 seconds Dissociation 30 seconds Regeneration: 5 seconds in regeneration buffer (glycine 10mM pH 1.5 / neutralized: PBS)

[0195] - Interpretation of results: Using the association and dissociation curves (first 5 seconds), the association rate constant (kon) and dissociation rate constant (koff) are calculated using association model 1 / 1. Then, KD(nM) is calculated (kon / koff).

[0196] The results are shown in Table 4 below.

[0197] [Table 6]

[0198] The results reveal that the Fc mutants A3A-184AY_CHO, A3A-184EY_CHO, and A3A-184AY_TGg show increased binding to the human FcγRIIIa-V receptor (CD16a-V) not only to unmutated Fc (Fc-WT) but also to IgIV and the Fc fragment MST-HN containing the mutations M252Y / S254T / T256E / H433K / N434F.

[0199] Example 5: Testing of ADCC inhibition and Jurkat cell activation of mutants produced in CHO cells and transgenic goat milk. ADCC inhibition and Jurkat cell activation tests will be performed using the following molecules: - Mutants of the present invention, A3A-184AY_CHO(K334N / P352S / A378V / V397M / N434Y), A3A-184EY_CHO(Y296W / K334N / P352S / A378V / V397M / N434Y), produced in CHO cells according to the method described in Example 3, Fc fragment MST-HN, containing the mutant M252Y / S254T / T256E / H433K / N434F described in the literature (Ulrichts et al., JCI, 2018) as having optimized binding only to the FcRn receptor, was produced in HEK-293 cells (293-F cells, freestyle Invitro Gen). - Wild Fc fragments "Fc-Rec" or "Fc-WT" obtained by digesting IgG1 produced in the milk of transgenic goats with papain. -IgIV.

[0200] ADCC inhibition test: To mimic the erythrolysis observed in idiopathic thrombocytopenic purpura (ITP), which involves autoantibodies from patients with ITP, we performed effector cell-mediated erythrolysis in the presence of anti-RhesusD (RhD) monoclonal antibodies and evaluated the ability of different quantities of polyvalent immunoglobulin (IVIg) or mutated or non-mutated recombinant Fc fragments to inhibit this lysis, for example, by competition with anti-RhD for the fixation of Fc receptors on the surface of effector cells.

[0201] The cytotoxicity of anti-RhD antibodies was studied using ADCC technology. Simply put, effector cells (mononuclear cells) (8 × 10⁻¹⁰) were treated with anti-RhD antibodies at different concentrations (0-75 ng / ml). 7 (25 μl at cells / ml) and Rh-positive red blood cells (final 4 × 10) 7 25 μl of cells / ml were incubated at an effector / target ratio of 2 / 1. After 16 hours of incubation, lysis was estimated by quantifying the hemoglobin released into the supernatant using a specific filtration factor (DAF).

[0202] The results were expressed as specific lysis percentages corresponding to the antibody quantity. ADCC inhibition was induced by the test molecules (IgIV, MST-HN, Fc-WT A3A-184AY_CHO, A3A-184EY_CHO) at concentrations of 500 μg / ml, 50 μg / ml, 5 μg / ml, and 0.5 μg / ml for MST-HN, Fc-WT A3A-184AY_CHO, and A3A-184EY_CHO, and at concentrations of 1500 μg / ml, 150 μg / ml, 15 μg / ml, and 1.5 μg / ml for IgIV. The molecular concentrations required to induce 25% or 50% inhibition were calculated using the software "Prism Software".

[0203] The results are shown in Table 5 below.

[0204] [Table 7]

[0205] The results reveal that the Fc mutants A3A-184AY_CHO and A3A-184EY_CHO exhibit increased inhibition of erythrolysis by anti-RhesusD antibodies, even when compared to non-mutated Fc (Fc-WT) and IgIV.

[0206] Furthermore, inhibition of A3A-184AY_CHO or A3A-184EY_CHO is significantly increased compared to Fc fragment MST-HN containing the mutations M252Y / S254T / T256E / H433K / N434F.

[0207] Inhibition of CD64 activation in Jurkat cells: This test estimates the ability (total IgG) of the Fc mutant or IgIV according to the present invention to inhibit IL2 secretion by human CD64-expressing Jurkat cells (Jurkat-H-CD64) induced by the Raji cell line using rituxan.

[0208] Simply put, Raji cells (5 x 10 6 (50 μl of cells / ml), Rituxan (50 μl of 2 μg / ml), Jurkat cells H-CD64 (5 × 10 6 The mixture (25 μl at cell / ml) was mixed with phorbol ester (PMA, 50 μl at 40 ng / ml) and then incubated with the Fc mutant or IgIV according to the present invention at 1950 nM.

[0209] After overnight incubation, the plates were centrifuged (125g for 1 minute), and the IL2 content in the supernatant was evaluated by ELISA.

[0210] Inhibition of IL2 secretion was induced by IgIV, Fc-WT, MST-HN, or the Fc variant according to the present invention (A3A-184AY_CHO or A3A-184EY_CHO) at concentrations of 50 and 100 μg / ml, and by IgV at concentrations of 150 and 300 μg / ml.

[0211] The molecular concentrations required to induce 25% or 50% inhibition were calculated using the software "prism Software".

[0212] The results are shown in Table 6 below.

[0213] [Table 8]

[0214] The results reveal that the Fc mutants A3A-184AY_CHO and A3A-184EY_CHO exhibit increased inhibition of IL2 secretion, not only compared to non-mutated Fc (Fc-WT) but also to IgIV.

[0215] Furthermore, inhibition of RFC A3A-184AY_CHO or A3A-184EY_CHO is significantly increased compared to Fc fragment MST-HN containing mutations M252Y / S254T / T256E / H433K / N434F.

[0216] Example 6: Binding test of Fc variant to blood cells Binding tests to blood cells are performed using the following molecules: - Variants of the present invention, A3A-184AY_CHO (K334N / P352S / A378V / V397M / N434Y) produced in CHO cells according to the method described in Example 3, A3A-184EY_CHO (Y296W / K334N / P352S / A378V / V397M / N434Y), A3A-184AY_TGg produced in transgenic goats according to the method described in Example 1, - The Fc fragment MST-HN containing the mutations M252Y / S254T / T256E / H433K / N434F described in the literature (Ulrichts et al., JCI, 2018) as having optimized binding only to the FcRn receptor, produced in HEK-293 cells (293-F cells, freestyle Invitro Gen), - Wild-type Fc fragment "Fc-Rec" or "Fc-WT" obtained by digesting IgG1 produced in the milk of transgenic goats with papain, - IgIV.

[0217] The test molecule labeled with the marker Alexa Fluor® (a highly fluorescent protein marker) was incubated with the target cells at 65 nM (10 μg / ml for Fc in PBS with 2% CSF2) for 20 minutes on ice.

[0218] After washing twice in PBS with 2% CSF2, the cells were resuspended in 500 μl of Isoflow before analysis by flow cytometry. The tests were performed on the following cells. - Natural killer (NK) cells labeled with anti-CD56 ("% positive NK cells"), - Monocytes labeled with anti-CD14 ("% positive cells"), - Monocyte CD16+ labeled with anti-CD14 and anti-CD16 3G8 antibody ("% positive cells"), - Neutrophils labeled with anti-CD15 ("% positive cells").

[0219] The FcγRIII receptor (CD16) was revealed using the anti-CD16 3G8 antibody.

[0220] The results revealed that the Fc variants A3A-184AY_CHO, A3A-184EY_CHO, and A3A-184AY_TGg exhibited increased binding not only to the unmutated Fc (Fc-Rec) but also compared to IgIV, regardless of the production mode. Moreover, the binding of A3A-184AY or A3A-184EY was significantly increased for NK cells, monocytes CD16+, and neutrophils compared to the fragment MST-HN (see Figure 6).

[0221] Example 7: Tests on a Biomodel of Idiopathic Thrombocytopenic Purpura (ITP) Disease was induced in mice expressing humanized FcRn (heterozygous mFcRn- / -hFcRnTg 276 with a genetic background of B6, The Jackson Laboratory) by injecting anti-platelet antibody 6A6-hlgG1 (0.3 μg per gram of body weight) intravenously to deplete mouse platelets. Four hours after disease induction, blood analysis (platelet count) was performed 24 hours before the injection of 6A6-hlgG1. IgIV (1000 mg / kg), Fc-Rec (380 and 750 mg / kg), Fc MST-HN (190 mg / kg), and Fc A3A-184AY_CHO (190 mg / kg and 380 mg / kg) were administered intraperitoneally 2 hours before platelet depletion.

[0222] Platelet numerical expressions were determined using the Advia Hematology (Bayer) system. The platelet count before antibody injection was set as 100%.

[0223] The anti-platelet antibody 6A6-hlgG1 (0.3 μg / g) enables depletion of 90% of platelets.

[0224] Administration of the candidate drug 2 hours before platelet depletion enables the following recovery (Figure 7). · 100% platelets in the case of A3A 184AY_CHO at a dose of 380 mg / kg • In the case of A3A-184AY_CHO at a dose of 190 mg / kg, 106% of platelets were produced. • In the case of IgIV at a dose of 1000 mg / kg, 90% of platelets • In the case of Fc-WT at a dose of 750g / kg, 64% of platelets • In the case of Fc-WT with a dose of 380 mg / kg, 75% of platelets • 61% of platelets in the MST-HN variant at a dose of 190 mg / kg.

Claims

1. In a variant of a parent polypeptide containing a human IgG1 Fc fragment, the parent polypeptide is selected from sequences of SEQ ID NOs: 1, 5, 6, 10, and 14, and the variant has increased affinity for the FcRn receptor and increased affinity for at least one Fc fragment receptor (FcR) selected from FcγRI receptor (CD64), FcγRIIIIa receptor (CD16a), and FcγRIIIa receptor (CD32a), compared to the affinity of the parent polypeptide. (i) Four mutations 334N, 352S, 378V and 397M, (ii) At least mutation 434Y and (iii) At least mutation Y296W and Includes, The numbering is based on the EU index numbering system. A variant characterized by the following:

2. A variant according to claim 1, having affinity for the FcRn receptor increased by a ratio of at least 2, preferably greater than 5, preferably greater than 10, preferably greater than 15, preferably greater than 20, preferably greater than 25, and preferably greater than 30, compared to the affinity of the parent polypeptide.

3. A variant according to claim 1 or 2, having affinity for the receptor (FcR) of at least one Fc fragment selected from FcγRI receptor (CD64), FcγRIIIIa receptor (CD16a), and FcγRIIIIa receptor (CD32a), increased by a ratio of at least 2 compared to the affinity of the parent polypeptide, preferably greater than 5, preferably greater than 10, preferably greater than 15, preferably greater than 20, preferably greater than 25, and preferably greater than 30.

4. A variant according to any one of claims 1 to 3, characterized in that it is produced in mammary epithelial cells of a transgenic non-human mammal.

5. The mutant according to any one of claims 1 to 4, characterized in that it is produced in a non-human transgenic animal, preferably in a transgenic non-human mammal.

6. The variant according to claim 5, characterized in that the non-human transgenic animal is a transgenic goat.

7. A mutant according to any one of claims 1 to 6, characterized in that it is an isolated Fc fragment.

8. A fusion protein comprising the mutant described in any one of claims 1 to 6.

9. A variant according to any one of claims 1 to 7, or a fusion protein according to claim 8, for use as a pharmaceutical agent.

10. Preferably, a variant according to any one of claims 1 to 7, or a fusion protein according to claim 8, for use in the treatment of an autoimmune or inflammatory pathology selected from immunogenic thrombocytopenic purpura, neuromyelitis optica, or Devic's disease and multiple sclerosis.

11. A pharmaceutical composition comprising a variant according to any one of claims 1 to 7, or a fusion protein according to claim 8, and at least one pharmaceutically acceptable excipient.

12. In a method for producing a variant of a parent polypeptide containing a human IgG1 Fc fragment, the parent polypeptide is selected from sequences of SEQ ID NOs: 1, 5, 6, 10, and 14, and the variant has increased affinity for the FcRn receptor and increased affinity for at least one Fc fragment receptor (FcR) selected from FcγRI receptor (CD64), FcγRIIIIa receptor (CD16a), and FcγRIIIa receptor (CD32a) compared to the affinity of the parent polypeptide. (i) Four mutations 334N, 352S, 378V and 397M, (ii) At least mutation 434Y, (iii) At least mutation Y296W and Includes, The numbering is based on the EU index numbering system. It is characterized by, This includes the expression of the mutant in transgenic non-human mammalian mammary epithelial cells, or the expression of the mutant in mammalian cells in culture. method.

13. a) A step of preparing DNA comprising a sequence encoding a mutant, a sequence encoding a mammalian casein promoter or a mammalian whey promoter, and a sequence encoding a signal peptide that enables the secretion of the mutant, b) The step of introducing the DNA obtained in a) into the embryo of a non-human mammal to obtain a transgenic non-human mammal that expresses a mutant encoded by the DNA obtained in a) in the mammary gland, and c) A step of collecting mutants in milk produced by transgenic non-human mammals obtained in b) and A method for producing a polypeptide variant containing the Fc fragment according to claim 12, including the above.

14. A method for producing a polypeptide variant containing an Fc fragment according to claim 12 or 13, wherein the transgenic non-human mammal is preferably selected from among cattle, pigs, goats, sheep, and rodents, and more preferably from among goats, mice, sows, female rabbits, female sheep, and female cows.

15. a) A step of preparing DNA that encodes the mutant, b) A step of introducing the DNA obtained in a) into cultured mammalian cells transiently or stably, c) The step of expressing mutants from cells obtained in b), and d) A step of recovering the mutant in the culture medium and A method for producing a polypeptide variant containing the Fc fragment according to claim 12, including the above.

16. In DNA containing a gene encoding a variant of a parent polypeptide containing a human IgG1 Fc fragment, the parent polypeptide is selected from sequences SEQ ID NOs: 1, 5, 6, 10, and 14, and the variant has increased affinity for the FcRn receptor and increased affinity for at least one Fc fragment receptor (FcR) selected from FcγRI receptor (CD64), FcγRIIIIa receptor (CD16a), and FcγRIIIa receptor (CD32a) compared to the affinity of the parent polypeptide. (i) Four mutations 334N, 352S, 378V and 397M, (ii) At least mutation 434Y and (iii) At least mutation Y296W and Includes, The numbering is based on the EU index numbering system. It is characterized by, Optionally, DNA containing a sequence encoding a signal peptide that enables the secretion of the aforementioned mutant.

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