MUTANTS FC WITH ENHANCED FUNCTIONAL ACTIVITY

Abstract

Fc MUTANTS WITH ENHANCED FUNCTIONAL ACTIVITY The present invention relates to a variant of a parent polypeptide comprising an Fc fragment, said variant having an increased affinity for at least one of the Fc fragment receptors (FcR) selected from FcγRIIIa (CD16a), FcγRIIa (CD32a), and FcγRIIb (CD32b) compared to that of the parent polypeptide, characterized in that it comprises at least one mutation selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W,S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc, the numbering being that of the EU index or equivalent in Kabat.

Description

Title of the invention: MUTANTS Fc A IMPROVED FUNCTIONAL ACTIVITY

[0001] The present invention relates to a polypeptide comprising a mutated Fc region and having an increased affinity for at least one of the Fc fragment receptors (FcR) selected from the FcyRIIIa (CD16a), FcyRIIa (CD32a), and FcyRIIb (CD32b) receptors.

[0002] Therapeutic antibodies and Fc fusion proteins are used today to treat various diseases, such as rheumatoid arthritis, psoriasis, multiple sclerosis, and many forms of cancer. Therapeutic antibodies can be monoclonal or polyclonal. Monoclonal antibodies are obtained from a single antibody-producing cell line that exhibits identical specificity for a single antigen.

[0003] The therapeutic effect of antibodies targeting membrane antigens relies in particular on the recruitment of effector cells expressing receptors for the crystallizable fragment of the antibodies (the "Fc receptors"). Fc receptors are proteins present on the surface of certain cells that contribute to the functions of the immune system, in particular NK ("natural killer") cells, macrophages, neutrophils, and mast cells. Several types exist, which are classified according to the type of antibody they recognize: Fc gamma receptors (FcyR) bind to IgG, Fc alpha receptors (FcaR) bind to IgA, and Fc epsilon receptors (FeR) bind to IgE.

[0004] The binding of the Fc receptor to the Fc region of an antibody triggers different mechanisms depending on the type of cell on which the receptor is expressed. The functional activity of antibodies is notably mediated by the binding of the Fc fragment to Fc receptors. Given the importance of the mechanisms involved in the binding of Fc receptors to antibodies, it would be particularly advantageous to have variants with modified, preferably increased, affinity for Fc receptors and, in particular, improved Fc-mediated functional activity (e.g., antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, or antibody-dependent cellular phagocytosis).

[0005] The Applicant has now developed specific Fc fragments exhibiting enhanced affinity for Fc receptors, in particular for at least one Fc receptor selected from among the FcyRIIIa (CD16a), FcyRIIa (CD32a), FcyRIIb (CD32b), FcyRI (CD64), and complement Clq receptors. Advantageously, such Fc fragments possess a single mutation allowing optimal control of the impact of said A mutation affecting the binding of the Fc fragment to at least one of the Fc receptors and / or its effector activities. Alternatively, such fragments may include at least one additional mutation, conferring advantageous properties such as increased binding to FcRn and / or an increased half-life. These fragments can be used in therapy to improve the efficacy of the product containing them. LEGEND FOR FIGURES

[0006] Figure 1 shows alignments of native human IgGl sequences referring to positions 216 to 447 (according to the EU index) with the corresponding sequences of human IgG2 (SEQ ID NO: 7), human IgG3 (SEQ ID NO: 8), and human IgG4 (SEQ ID NO: 9). The IgGl sequences refer to allotype Glml,17 (SEQ ID NO: 6) and allotype Glm3 (SEQ ID NO: 10). The lower CH2-CH3 hinge domain of IgGl begins at cysteine ​​226 (see arrow). The CH2 domain is highlighted in gray, and the CH3 domain is italicized.

[0007] Figure 2 shows the G0, G0F, G1 and GIF forms of the glycan structures that may be present on the Fc fragments of the invention. DESCRIPTION OF THE INVENTION

[0008] The present invention relates to a variant of a parent polypeptide comprising an Fc fragment, said variant having a modified, preferably increased, affinity for at least one of the Fc fragment receptors (FcR) selected from FcyRIIIa (CD16a), FcyRIIa (CD32a), and FcyRIIb (CD32b) receptors compared to that of the parent polypeptide, characterized in that it comprises at least one mutation selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R,Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc; the numbering being that of the EU index or equivalent in Kabat.

[0009] Such a variant of a parent polypeptide is referred to in the present application as a "variant according to the invention".

[0010] Throughout this application, the residue numbering in the Fc region is that of the immunoglobulin heavy chain according to the EU index or equivalent in Kabat et al. (Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991). The expression "EU index or equivalent in Kabat" refers to the EU numbering of residues of the human antibody IgG1, IgG2, IgG3, or IgG4. This is illustrated on the IMGT website (http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html).

[0011] FcRs allow immune cells to take advantage of the specificity of the antibodies that bind to them to direct their cellular functions on antigens and pathogens specific to that antibody.

[0012] FcyRs represent the most diverse group of FcRs and are the main mediators of antibody functions in the body. There are three families of human FcyRs: FcyRI (CD64); FcγRII (CD32); and FcγRIII(CD16).

[0013] Three of them (FcyRI, FcyRIIa, FcyRIIIa) are activating receptors, which differ in their binding affinities and in their cellular expressions.

[0014] The FcyRIIIa receptor (CD16a) is involved in antibody-dependent cell-mediated cytotoxicity (ADCC); it exhibits a V / F polymorphism at position 158. The FcyRIIa receptor (CD32a), on the other hand, is involved in platelet activation and phagocytosis; it exhibits an H / R polymorphism at position 131. The FcyRI receptor (CD64) is also involved in antibody-dependent cell-mediated cytotoxicity (ADCC) as well as in phagocytosis mechanisms.

[0015] Finally, the FcyRIIb receptor (CD32b) is involved in the inhibition of cellular activity. Among the Fc receptors defined within the scope of the invention, complement Clq is involved in CDC or Complement Dependent Cytotoxicity activity.

[0016] By "polypeptide" or "protein" is meant a sequence comprising at least 100 covalently attached amino acids.

[0017] By "amino acid" is meant one of the 20 natural amino acids or non-natural analogues.

[0018] The term "position" means a position in the sequence of a polypeptide. For the Fc region, positions are numbered according to the EU index or equivalent in Kabat.

[0019] The term "antibody" is used in its common sense. It refers to a tetramer comprising at least one Fc region and two variable regions. Antibodies These include full-length immunoglobulins, monoclonal antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. The amino-terminal portion of each heavy chain comprises a variable region of approximately 100 to 110 amino acids responsible for antigen recognition. Within each variable region, three loops are assembled to form an antigen-binding site. Each of these loops is called a complementarity-determining region (CDR). The carboxy-terminal portion of each heavy chain defines a constant region primarily responsible for effector function.

[0020] IgG has several subclasses, including IgG1, IgG2, IgG3, and IgG4. The IgM subclasses include IgM1 and IgM2. Thus, "isotype" refers to one of the immunoglobulin subclasses defined by the chemical and antigenic characteristics of their constant regions. The known isotypes of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.

[0021] Full-length IgG is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having a light chain and a heavy chain. Each light chain comprises the VL and CL domains, and each heavy chain comprises the VH, Cyl (also called CH1), Cy2 (also called CH2), and Cy3 (also called CH3) domains. In the context of human IgG1, "CH1" refers to positions 118 to 215, "CH2" refers to positions 231 to 340, and "CH3" refers to positions 341 to 447 according to the EU index or equivalent in Kabat. The heavy chain of IgG also includes a flexible N-terminal hinge domain, which refers to positions 216 to 230 in the case of IgG1. The lower hinge domain refers to positions 226 to 230 according to the EU index or equivalent in Kabat.

[0022] By "variable region" is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any one of the VK, VX and / or VH genes that make up the kappa, lambda, and heavy chains of immunoglobulin, respectively. Variable regions include complementarity-determining regions (CDRs) and framework regions (FRs).

[0023] The term "Fc" or "Fc region" designates the constant region of an antibody, excluding the first constant region domain of immunoglobulin (CH1). Thus, Fc refers to the last two constant region domains (CH2 and CH3) of IgGl, and to the flexible N-terminal hinge of these domains. For a human IgGl, the Fc region corresponds to residue C226 up to its carboxy-terminal end, i.e., residues from position 226 to 447, where the numbering is according to the EU index or equivalent in Kabat. The Fc region used may further include a portion of the upper hinge region, located between positions 216 and 226 according to the EU index or equivalent in Kabat; in this case, the Fc region used corresponds to the residuals from position 216 to 447, 217 to 447, 218 to 447, 219 to 447, 220 to 447, 221 to 447, 222 to 447, 223 to 447, 224 to 447, or 225 to 447, where the numbering follows the EU index or its equivalent in Kabat. Preferably, in this case, the Fc region used corresponds to the residuals from position 216 to 447, where the numbering follows the EU index or its equivalent in Kabat. Preferably, the Fc region used is chosen from SEQ ID NO: 1 to 10. Preferably, the Fc fragment used is chosen from SEQ ID NO: 1, 2, 3, 4, and 5. Preferably, the Fc fragment of the parent antibody has the sequence SEQ ID NO: 1. The sequences represented in SEQ ID NO: 1, 2, 3, 4, and 5 are free of an N-terminal hinge region. The sequences represented in SEQ ID NO: 6, 7, 8, 9, and 10 correspond respectively to the sequences represented in SEQ ID NO: 1, 2, 3, 4, and 5 with their N-terminal hinge regions.Also, in a particular embodiment, the Fc fragment of the parent antibody is chosen from the SEQ ID NO sequences: 6, 7, 8, 9 and 10. Preferably, the Fc fragment of the parent antibody has a sequence corresponding to positions 1-232, 2-232, 3-232, 4-232, 5-232, 6-232, 7-232, 8-232, 9-232, 10-232 or 11-232 of the SEQ ID NO sequence: 6.

[0024] By "parent polypeptide," we mean a reference polypeptide. Said parent polypeptide may be of natural or synthetic origin. In the context of the present invention, the parent polypeptide comprises an Fc region, referred to as the "parent Fc region." This Fc region may be selected from the group of wild-type Fc regions, their fragments, and their mutants. Preferably, the parent polypeptide comprises a human Fc region, preferably an Fc region from a human IgG1. The parent polypeptide may include pre-existing amino acid modifications in the Fc region (e.g., an Fc mutant) compared to wild-type Fc regions. Advantageously, the parent polypeptide is an isolated Fc region (i.e., an Fc fragment as such), a sequence derived from an isolated Fc region, an antibody, a fusion protein comprising an Fc region, or an Fc conjugate, this list not being exhaustive.By "sequence derived from an isolated Fc region," we mean a sequence comprising at least two isolated Fc regions linked together, such as a single-chain Fc (scFc) or an Fc multimer. By "fusion protein comprising an Fc region," we mean a polypeptide sequence fused to an Fc region, said polypeptide sequence preferably being chosen from the variable regions of any antibody, the binding sequences of a receptor to its ligand, adhesion molecules, ligands, enzymes, cytokines, and chemokines. By "Fc conjugate," we mean a compound that is the result of the chemical coupling of an Fc region with a conjugation partner. The conjugation partner may be protein or non-protein. The coupling reaction typically uses functional groups on the Fc region and the conjugation partner. Various linker groups are known. in the prior art as being suitable for the synthesis of a conjugate; for example, homo- or heterobifunctional linker groups are well known (see Pierce Chemical Company catalog, 2005-2006, technical section on crosslinking agents, pages 321-350). Suitable conjugation partners include therapeutic proteins, tags, cytotoxic agents such as chemotherapeutic agents, toxins, and their active fragments.

[0025] Advantageously, the parent polypeptide - and therefore the variant according to the invention - consists of an Fc region.

[0026] Advantageously, the parent polypeptide - and therefore the variant according to the invention - is an antibody.

[0027] Finally, preferably, the parent polypeptide - and therefore the variant according to the invention - is a polypeptide produced in the milk of transgenic animals.

[0028] By "mutation" is meant a change in at least one amino acid in the sequence of a polypeptide, in particular a change in at least one amino acid in the Fc region of the parent polypeptide. The mutated polypeptide thus obtained is a variant polypeptide; it is a variant according to the invention. Such a polypeptide comprises a mutated Fc region, relative to the parent polypeptide. Preferably, the mutation is a substitution, insertion, or deletion of at least one amino acid. By "substitution" is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence by another amino acid. For example, the N434S substitution refers to a variant polypeptide, in this case a variant in which asparagine at position 434 is replaced by serine. By "amino acid insertion" or "insertion" is meant the addition of an amino acid at a particular position in a parent polypeptide sequence.For example, the insertion G>235-236 denotes a glycine insertion between positions 235 and 236. An "amino acid deletion" or "deletion" refers to the removal of an amino acid at a specific position in a parent polypeptide sequence. For example, E294del denotes the deletion of glutamic acid at position 294. Preferably, the following mutation label is used: "434S" or "N434S", indicating that the parent polypeptide contains asparagine at position 434, which is replaced by serine in the variant. In the case of a combination of substitutions, the preferred format is "2591 / 315D / 434Y" or "V259I / N315D / N434Y".This means that there are three substitutions in the variant, at positions 259, 315 and 434, and that the amino acid at position 259 of the parent polypeptide, namely valine, is replaced by isoleucine, that the amino acid at position 315 of the parent polypeptide, namely asparagine, is replaced by aspartic acid and that the amino acid at position 434 of the parent polypeptide, namely asparagine, is replaced by tyrosine.

[0029] The variant according to the invention has a functional activity mediated by the modified Fc region, preferably increased compared to that of the parent polypeptide.

[0030] The term "Fc region-mediated functional activity" refers in particular to effector functions. Fc region-mediated functional activity thus includes, in particular, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADCP), endocytic activity, cytokine secretion, or a combination of at least two of these activities. Preferably, the Fc region-mediated functional activity considered in the invention is selected from ADCC, ADCP, CDC, and combinations thereof. This functional activity can be evaluated by methods well known in the prior art.The functional activity mediated by the Fc region of the variant according to the invention is increased relative to that of the parent polypeptide, typically 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.

[0031] Preferably, said mutated Fc region has a modified, preferably increased, affinity for at least one of the FcRs. Preferably, the affinity is increased, relative to that of the parent Fc, 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. In other words, the affinity of the mutated Fc region for an FcR is greater than that of the parent polypeptide.

[0032] The affinity of a polypeptide comprising an Fc region for an FcR can be evaluated by methods well known in the prior art. 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 assay. A suitable ELISA assay allows for the comparison of the binding strengths of the parent Fc and the mutated Fc. The signals detected specific to the mutated Fc and the parent Fc are compared. The binding affinity can be determined by evaluating either the entire polypeptides or isolated Fc regions thereof.

[0033] Preferably, the mutated Fc region of the variant according to the invention comprises from 1 to 20 mutations compared to the parent polypeptide, preferably from 2 to 20 mutations. "From 1 to 20 amino acid modifications" encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acid mutations. Preferably, it comprises from 1 to 15 mutations, preferably from 2 to 15 mutations, preferably from 1 to 10 mutations compared to the parent polypeptide, preferably from 2 to 10 mutations.

[0034] Preferably, the variant according to the invention is characterized in that the mutation is chosen from an insertion, a substitution, preferably point-based, and a deletion.

[0035] Preferably, the variant according to the invention comprises at least one mutation i) selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc; the numbering being that of the EU index or equivalent in Kabat.

[0036] In a particular embodiment, the invention relates to a variant of a parent polypeptide comprising an Fc fragment, said variant having a modified, preferably increased, affinity for at least one of the Fc fragment receptors (FcR) selected from FcyRIIIa (CD16a), FcyRIIa (CD32a), and FcyRIIb (CD32b) receptors compared to that of the parent polypeptide, characterized in that it comprises a single mutation i) selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc; the numbering being that of the EU index or equivalent in Kabat.

[0037] Thus, in a particular embodiment, the variant according to the invention comprises a single mutation i) selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc; the numbering being that of the EU index or equivalent in Kabat.

[0038] In another particular embodiment, said Fc variant comprises at least two mutations i), said mutations being selected from i) 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc;the numbering being that of the EU index or equivalent in Kabat and with the condition that the mutations are not identical. ;

[0039] In a more particular embodiment, said Fc variant comprises at least three mutations i), said mutations i) being selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc;the numbering being that of the EU index or equivalent in Kabat and with the condition that the mutations are not identical. ;

[0040] In a more particular embodiment, said variant Fc comprises at least four mutations i), said mutations i) being chosen from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said Fc fragment; the numbering being that of the EU index or equivalent in Kabat and with the condition that the mutations are not identical.

[0041] In one embodiment, the variant according to the invention has an increased affinity for the FcyRIIIa receptor (CD 16a). In this particular embodiment, said variant comprises at least one mutation i) selected from S298A, S298R, F243S, F243L, L242A, L242F, L242G, L242I, L242K, L242S, L242V, V240I, V240M, V240N, V240S, E258I, T260A, K290D, K290E, K290G, K290H, K290Q, K290S, K290Y, Y296H, Y296W of said Fc fragment;

[0042] the numbering being that of the EU index or equivalent in Kabat.

[0043] In another embodiment, the variant according to the invention has an increased affinity for the FcyRIIa (CD32a) receptor. In this particular embodiment, said variant comprises at least one mutation i) selected from F241H, F241Y, F243L, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, V240H, V240I, V240M, V240S, E258G, E258I, E258R, E258M, E258Q, E258Y, S267A, S267Q, S267V, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V259C, V259I, V259L, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, E293A, E293D, E293G, E293M, E293Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, K290D, K290E, K290G, K290H, K290L, K290N, K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, Q295I, Q295M, R292I, R292L, R301A, R301P, R301S, S304T, V302A, V302F, V302L, V302M, V302R, V302S, V303Y, V305A, V305F, V305L, V305R, V305S, Y300I, Y300V or Y300W; the numbering being that of the EU index or equivalent in Kabat.

[0044] In another embodiment, the variant according to the invention has an increased affinity for the FcyRIIb (CD32b) receptor. In this particular embodiment, said variant comprises at least one mutation i) selected from E258R, E258Y, V262A, S267A, S267Q, S267V, V264S, V266L, V266M, K290R, R301A, R301M, S304T, V302A, V302L, V302R, V303S, V305A, V305F, V305I, V305R, Y300V of said fragment Fc; the numbering being that of the EU index or equivalent in Kabat.

[0045] Preferably, the variant according to the invention is characterized in that the Fc fragment of the parent polypeptide already comprises at least:

[0046] (ii) a mutation selected from 378V, 378T, 434Y and 434S; and

[0047] (iii) at least one mutation selected from 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 378V, 378T, 389T, 389K, 434Y and 434S,

[0048] the numbering being that of the EU index or equivalent in Kabat and with the condition that mutations (ii) and (iii) do not take place on the same amino acids.

[0049] Thus, according to a particular aspect, the invention relates to a variant of a parent polypeptide comprising an Fc fragment, said variant having a modified, preferably increased, affinity for at least one of the Fc fragment receptors (FcR) selected from FcyRIIIa (CD16a), FcyRIIa (CD32a), and FcyRIIb (CD32b) receptors compared to that of the parent polypeptide, characterized in that it comprises at least one mutation i) selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc; and further comprising at least: ,

[0050] (ii) a mutation selected from 378V, 378T, 434Y and 434S; and

[0051] (iii) at least one mutation selected from 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 378V, 378T, 389T, 389K, 434Y and 434S, with the condition that mutations (ii) and (iii) do not occur on the same amino acids,

[0052] the numbering being that of the EU index or equivalent in Kabat,

[0053] In a particular embodiment, said variant comprises at least one combination of mutations selected from the group consisting of 226G / 315D / 434Y, 230S / 315D / 434Y, 230T / 315D / 434Y, 230T / 264E / 434S, 230T / 389T / 434S, 241L / 264E / 378V, 241L / 264E / 434S, 250A / 389K / 434Y, 2591 / 315D / 434Y, 264E / 378T / 396L, 264E / 378V / 416K, 264E / 378V / 434S, 264E / 396L / 434S, 294del / 307P / 434Y, 307P / 378V / 434Y, 315D / 330V / 434Y, 315D / 382V / 434Y and 378V / 383N / 434Y,

[0054] it being understood that the enumeration of the positions of the amino acids of the fragment Fc is that of the EU index or equivalent in Kabat.

[0055] In a particular embodiment, said variant further comprises at least one mutation selected from the group consisting of 226G, 227L, 230S, 230T, 230L, 231T, 241L, 243L, 250A, 256N, 2591, 264E, 265G, 267R, 290E, 294del, 303A, 305A, 307P, 307A, 3081, 315D, 322R, 325S, 327V, 330V, 342R, 347R, 352S, 361D, 362R, 362E, 370R, 378V, 378T, 382V, 383N, 386R, 386K, 387T, 389T, 389K, 392R, 395A, 396L, 397M, 403T, 404L, 415N, 416K, 421T, 426T, 428L, 433R, 434Y, 434S and 439R,

[0056] it being understood that the enumeration of the positions of the amino acids of the fragment Fc is that of the EU index or equivalent in Kabat.

[0057] In a particular embodiment, said variant comprises at least one combination of mutations selected from the group consisting of 307A / 315D / 330V / 382V / 389T / 434Y, 256N / 378V / 383N / 434Y, 315D / 330V / 361D / 378V / 434Y, 2591 / 315D / 434Y, 230S / 315D / 428L / 434Y, 241L / 264E / 307P / 378V / 433R, 250A / 389K / 434Y, 305A / 315D / 330V / 395A / 434Y, 264E / 386R / 396L / 434S / 439R, 315D / 330V / 362R / 434Y, 294del / 307P / 434Y, 305A / 315D / 330V / 389K / 434Y, 315D / 327V / 330V / 397M / 434Y,

[0058] 230T / 241L / 264E / 265G / 378V / 421T, 264E / 396L / 415N / 434S, 227L / 264E / 378V / 434S, 264E / 378T / 396L, 230T / 315D / 362R / 426T / 434Y, 226G / 315D / 330V / 434Y,

[0059] 230L / 241L / 243L / 264E / 307P / 378V, 250A / 315D / 325S / 330V / 434Y, 290E / 315D / 342R / 382V / 434Y, 241L / 315D / 330V / 392R / 434Y, 241L / 264E / 307P / 378V / 434S, 230T / 264E / 403T / 434S, 264E / 378V / 416K, 230T / 315D / 362E / 434Y, 226G / 315D / 434Y, 226G / 315D / 362R / 434Y, 226G / 264E / 347R / 370R / 378V / 434S, 3081 / 315D / 330V / 382V / 434Y, 230T / 264E / 378V / 434S, 231T / 241L / 264E / 378T / 397M / 434S, 230L / 264E / 378V / 434S, 230T / 315D / 330V / 386K / 434Y, 226G / 315D / 330V / 389T / 434Y, 267R / 307P / 378V / 421T / 434Y, 230S / 315D / 387T / 434Y, 230S / 264E / 352S / 378V / 434S and 230T / 303A / 322R / 389T / 404L / 434S,

[0060] it being understood that the numbering of the positions of the amino acids of the fragment Fc is that of the EU index or equivalent in Kabat.

[0061] In a particular embodiment, the variant according to the invention comprises at least one mutation (i), preferably a single mutation (i), selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said Fc fragment; and a combination of mutations ii) and iii) selected from 315D / 330V / 361D / 378V / 434Y, 230S / 315D / 428L / 434Y, 307A / 315D / 330V / 382V / 389T / 434Y, 2591 / 315D / 434Y, 256N / 378V / 383N / 434Y.

[0062] Preferably, the variant according to the invention is characterized in that the Fc fragment of the parent polypeptide already comprises at least:

[0063] (iv) a mutation selected from 307N, 326E, 326T, 334N, 334R, 352L, 378V, 378T, 394P, 396L, 397M, 421T; and

[0064] (v) at least one mutation selected from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 26IR, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 2861, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 3081, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 3231, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 3631, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 3931, 394P, 396L, 3971, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N,

[0065] the numbering being that of the EU index or equivalent in Kabat and

[0066] with the condition that mutations (iv) and (v) do not take place on the same amino acids.

[0067] Thus, according to a particular aspect, the invention relates to a variant of a parent polypeptide comprising an Fc fragment, said variant having a modified, preferably increased, affinity for at least one of the Fc fragment receptors (FcR) selected from FcyRIIIa (CD16a), FcyRIIa (CD32a), and FcyRIIb (CD32b) receptors compared to that of the parent polypeptide, characterized in that it comprises at least one mutation i) selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc; and further comprising at least:

[0068] (iv) a mutation selected from 307N, 326E, 326T, 334N, 334R, 352L, 378V, 378T, 394P, 396L, 397M, 421T; and

[0069] (v) at least one mutation selected from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 26IR, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 2861, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 3081, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 3231, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 3631, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 3931, 394P, 396L, 3971, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N,

[0070] the numbering being that of the EU index or equivalent in Kabat and

[0071] with the condition that the mutations (i), (iv) and (v) do not take place on the same amino acids.

[0072] Preferably, mutation (iv) is selected from 378V, 396L and 397M. Preferably, the polypeptide further comprises a mutation selected from 248E, 326T, 333G and 423Y.

[0073] Preferably, the mutation (v) according to the invention is chosen from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 26IR, 262A, 263A, 266M, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 2861, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 3081, 308G, 309P, 312G, 316D, 319H, 320T, 320R, 320M, 322E, 3231, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 3631, 366A, 373D, 375R, 377T, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 3931, 394P, 396L, 3971, 398P, 405V, 405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N.

[0074] In one embodiment, mutation (iv) is chosen from 378V, 396L and 397M and mutation (v) is chosen from 248E, 316D, 326E, 333G, 378T, 396L and 421T.

[0075] In another embodiment, mutation (iv) is 378V and mutation (v) is chosen from 298N and 336T.

[0076] In another embodiment, mutation (iv) is chosen from 378V, 396L and 397M; and mutation (v) is chosen from 231V, 2861, 286Y, 290E, 315D, 334N, 352S, 361H, 366A, 378T, 397M, 412M, 421T and 423Y.

[0077] In another embodiment, mutation (iv) is 378V; and mutation (v) is chosen from 248E, 308A, 334R, 447N.

[0078] In another embodiment, mutation (iv) is chosen from 378V, 326E, 397M, 334N and 396L; and mutation (v) is chosen from 316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P, 307P, 378V, 378T, 2861, 286Y and 298N.

[0079] In another embodiment, mutation (iv) is chosen from 378V, 326E, 397M, 307N, 394P, 326T, 396L and 334N; and mutation (v) is chosen from 316D, 334R, 334N, 3231, 231V, 246R, 336T, 378T, 286Y, 2861, 352S, 383R, 359A, 421T, 361H, 315D, 366A, 290E, 307P and 439R. Preferably, the mutation (v) is chosen from: 316D, 334R, 334N, 3231, 231V, 246R, 336T, 378T, 286Y, 2861, 352S, 383R, 359A, 421T, 361H, 366A, 290E, 307P and 439R.

[0080] In another embodiment, mutation (iv) is chosen from 326E, 326T, 378V, 397M, 352L, 394P, 396L and 421T; and mutation (v) is chosen from 316D, 334R, 248E, 334N, 418P, 231V, 320E, 402D, 359A, 383R, 421T and 361H.

[0081] In another embodiment, mutation (iv) is chosen from 378V, 378T, 396L, 421T, 334R and 326E; and mutation (v) is chosen from 361H, 290E, 316D, 248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T, 396L, 2861, 315D and 397M.

[0082] In another embodiment, mutation (iv) is chosen from 378V, 326E, 397M, 334N and 396L; and mutation (v) is chosen from 316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P, 307P, 378V, 378T, 2861, 286Y and 298N.

[0083] In a different authentication mode, the mutation (iv) is chosen by 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P, and the mutation (v) consists of au moins 2 mutations choisies parmi 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 26IR, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 2861, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 3081, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 3231, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S 349H, 350A, 352S, 359A, 361H, 362R, 3631, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 3931, 394P, 396L, 3971, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N.

[0084] Preferably, the at least 2 mutations (v) are chosen from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 26IR, 262A, 263A, 266M, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 2861, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 3081, 308G, 309P, 312G, 316D, 319H, 320T, 320R, 320M, 322E, 3231, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 3631, 366A, 373D, 375R, 377T, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 3931, 394P, 396L, 3971, 398P, 405V, 405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P, and 447N.

[0085] Preferably, the mutated Fc region of the polypeptide according to the invention comprises a combination of mutations selected from the following combinations:

[0086] K320E / T394P / G402D;

[0087] K290E / K320E / T350A / P396L;

[0088] T359A / S383R / V397M.

[0089] According to another aspect of the invention, a composition is used comprising a plurality of variants of a parent polypeptide comprising an Fc fragment, all of which have substantially the same sequence, said variants comprising Fc fragments which, taken together, exhibit a particular glycosylation profile.

[0090] According to a particular aspect, the Fc fragments of the variants within a composition used in the context of the invention have on their glycosylation site (Asn 297, the numbering being that of the EU index or equivalent in Kabat) N-glycans, characterized in that said N-glycans of the Fc fragments have a fucosylation rate of less than 65%, preferably less than 60%, preferably less than 55%, preferably less than 50%, preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%.

[0091] According to yet another aspect, the Fc fragments of the variants within a composition used in the context of the invention have on their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans of the Fc fragments have a glycan structure of the biantenne type, with short chains, low sialylation, exhibiting non-intercalated terminal N-acetylglucosamines.

[0092] According to a more particular aspect, the Fc fragments of the variants within a composition used in the context of the invention possess on their glycosylation site (Asn 297) N-glycans, characterized in that said N- glycans of the Fc fragments have a content greater than 60% for the G0+G1+G0F+G1F forms, the G0F+G1F forms being less than 50%.

[0093] According to another more particular aspect, the Fc fragments of the variants within a composition used in the context of the invention have on their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans of the Fc fragments have a content greater than 60% for the forms G0+G1+G0F+G1F, the fucose content being less than 65%.

[0094] According to another even more particular aspect, the Fc fragments of the variants within a composition used in the context of the invention have on their glycosylation site (Asn 297) N-glycans, characterized in that said N-glycans of the Fc fragments have a content of less than 40% for the G1F+G0F forms.

[0095] In a more particular aspect, the Fc fragments of the variants within a composition used in the context of the invention possess N-glycans at their glycosylation site (Asn 297), said N-glycans of the Fc fragments having a fucosylation level of 0%. The invention thus provides a composition comprising variants of a parent polypeptide including an Fc fragment, the Fc fragments of said variants possessing N-glycans at the Asn297 glycosylation site, characterized in that said N-glycans of the Fc fragments are devoid of fucose.

[0096] Also, according to a particular aspect, the Fc fragments of the variants within a composition used in the context of the invention have N-glycans at the Asn297 glycosylation site, characterized in that said N-glycans of the Fc fragments exhibit a fucosylation level of between 20% and 55%. In particular, the invention provides a composition comprising variants of a parent polypeptide including an Fc fragment, the Fc fragments of said variants having N-glycans at the Asn297 glycosylation site, characterized in that said N-glycans of the Fc fragments exhibit a fucosylation level of between 20% and 50%, between 25% and 55%, between 25% and 50%, between 20% and 45%, or between 25% and 45%.

[0097] According to a more particular aspect, a useful composition according to the invention comprises variants of a parent polypeptide comprising an Fc fragment, the Fc fragments of said variants having N-glycans on the Asn297 glycosylation site, characterized in that said N-glycans of the Fc fragments have a content greater than 60%, preferably greater than 80%, for the G0+G1+G0F +G1F forms, the G0F+G1F forms being less than 50%, preferably less than 40%, or 30%.

[0098] According to another more particular aspect, the N-glycans of the Fc fragments within the composition have a content greater than 60% for the G0+G1+G0F +G1F forms, the fucose content being less than 65%.

[0099] According to yet another more particular aspect, the N-glycans of the Fc fragments within the composition have a content of less than 50% for the GIF +G0F forms, preferably less than 40%, or 30%.

[0100] The shapes G0, G0F, G1 and GIF are selected from the shapes shown in Figure 2.

[0101] Advantageously, the N-glycans of the Fc fragments within the variant composition have an average sialic acid content of less than 25%, 20%, 15%, or 10%, preferably 5%, 4%, 3%, or 2%.

[0102] A composition that can be used within the framework of the invention comprises variants of a parent polypeptide comprising an Fc fragment, the Fc fragments of said variants having on their glycosylation site (Asn 297) N-glycans, said N-glycans of the Fc fragments having a biantene-type glycan structure, with short chains, low sialylation, and low fucosylation, the N-glycans having, for example, a content greater than 60% for the G0 + G1 + G0F + GIF forms, and a fucosylation less than 60%, preferably less than 55%, the N-glycans having, for example, a content less than 50% for the G0F + GIF forms and a fucosylation less than 55%.

[0103] In a particular embodiment, the Fc fragments according to the invention have glycan structures as described in patent application WO01 / 77181.

[0104] According to an advantageous embodiment, the Fc fragments used in the invention comprise at least one amino acid mutation compared to a parent Fc fragment, and have N-glycans on their glycosylation site (Asn 297), said N-glycans of the Fc fragments having a fucosylation rate of less than 65%, preferably less than 60%, preferably less than 55%, preferably less than 50%, preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%.Preferably, the Fc fragments carry at least one mutation i) selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc; . the numbering being that of the EU index or equivalent in Kabat, and furthermore possess on their glycosylation site (Asn 297) N-glycans, exhibiting a fucosylation rate of less than 55%, preferably less than 50%, preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%.

[0105] Preferably, the fragments Fc of the composition according to the invention carry a mutation i) selected from V240H, F241H, F241Y, L242H, L242P, L242T, E258G, E258R, E258M, E258Q, E258Y, V259C, V259I, V259L, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290L, K290N, K290R, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E293Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S, of said fragment Fc;the numbering being that of the EU index or equivalent in Kabat, and furthermore possess on their glycosylation site (Asn 297) N-glycans, exhibiting a fucosylation rate of less than 55%, preferably less than 50%, preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%. ;

[0106] Advantageously, Fc fragments having modified glycosylation at the glycosylation site at position 297, in particular weak fucosylation, exhibit increased binding of said fragment to Fcgamma receptors (FcyR), in particular the FcyRIIIa receptor (CD 16a).

[0107] Preferably, said Fc fragments have an affinity for CD16a of at least 2x106 M', at least 2xl07 M1, 2xl08 M1 or 2x109 M', as determined by Scatchard analysis or BIAcore technology (Label-free surface plasmon resonance based technology).

[0108] Preferably, the variant according to the invention is characterized in that it comprises from 1 to 20 mutations of said Fc fragment, preferably from 1 to 10 mutations.

[0109] Preferably, the variant according to the invention is characterized in that the parent polypeptide comprises a parent Fc fragment which is a human Fc fragment, preferably an Fc fragment of a human IgG1 or a human IgG2.

[0110] Preferably, the variant according to the invention is characterized in that it is selected from an isolated Fc fragment, a sequence derived from an isolated Fc fragment, an antibody and a fusion protein comprising an Fc fragment.

[0111] Preferably, the variant according to the invention is characterized in that it is an antibody.

[0112] The present invention also relates to a composition of polypeptides according to the invention.

[0113] The present invention also relates to a pharmaceutical composition comprising (1) a variant according to the invention or a composition as described in the preceding paragraph, and (2) at least one pharmaceutically acceptable excipient.

[0114] The present invention also relates to the variant according to the invention or the composition as described above, for its use as a medicinal product.

[0115] As previously stated, advantageously, the parent polypeptide – and therefore the variant according to the invention – is an antibody. In this case, the antibody can be directed against an antigen selected from among a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, a toxin, a membrane or circulating cytokine, and a membrane receptor.

[0116] When the antibody is directed against a tumor antigen, its use is particularly suitable in the treatment of cancers. "Cancer" is defined as any physiological condition characterized by abnormal cell proliferation. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas (including liposarcomas), neuroendocrine tumors, mesotheliomas, meningiomas, adenocarcinomas, melanomas, leukemias, and malignant lymphoid disorders.

[0117] When the antibody is directed against a viral antigen, its use is particularly suitable in the treatment of viral infections. Viral infections include, but are not limited to, infections due to HIV, a retrovirus, a Coxsackie virus, smallpox virus, influenza virus, yellow fever virus, West Nile virus, cytomegalovirus, rotavirus, or hepatitis B or C virus.

[0118] When the antibody is directed against a toxin, its use is particularly suitable in the treatment of bacterial infections, for example infections by tetanus toxin, diphtheria toxin, anthrax toxins Bacillus anthracis, or in the treatment of infections by botulinum toxins, ricin toxins, Shiga toxins, this list not being exhaustive.

[0119] When the antibody is directed against a cytokine, its use is particularly suitable in the treatment of inflammatory and / or autoimmune diseases. Inflammatory and / or autoimmune diseases include, but are not limited to, thrombotic thrombocytopenic purpura (ITP), organ or transplant rejection, graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, various types of sclerosis, primary Sjögren's syndrome (or Gougerot-Sjögren syndrome), autoimmune polyneuropathies such as multiple sclerosis, type 1 diabetes, autoimmune hepatitis, ankylosing spondylitis, Reiter's syndrome, gout, celiac disease, and other diseases. Crohn's disease, Hashimoto's chronic thyroiditis (hypothyroidism), Addison's disease, autoimmune hepatitis, Graves' disease (hyperthyroidism), ulcerative colitis, vasculitis such as ANCA-associated (anti-neutrophil cytoplasmic antibody) systemic vasculitis, autoimmune cytopenias and other hematological complications in adults and children, such as acute or chronic autoimmune thrombocytopenia, autoimmune hemolytic anemias, hemolytic disease of the newborn (HDN), cold agglutinin disease, autoimmune acquired hemophilia; Goodpasture syndrome, extramembranous nephropathies, autoimmune bullous skin diseases, refractory myasthenia gravis, mixed cryoglobulinemias, psoriasis, juvenile chronic arthritis, inflammatory myositis, dermatomyositis, and systemic autoimmune diseases of children including antiphospholipid syndrome and connective tissue disease,autoimmune pulmonary inflammation, Guillain-Barré syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), autoimmune thyroiditis, diabetes mellitus, myasthenia gravis, autoimmune inflammatory disease of the eye, neuromyelitis optica (Dévie's disease), scleroderma, pemphigus, insulin-resistant diabetes, polymyositis, pernicious anemia, glomerulonephritis, Wegener's granulomatosis, Horton's disease, periarthritis nodosa and Churg-Strauss syndrome, Still's disease, relapsing polychondritis, Behçet's disease, monoclonal gammopathy, Wegener's granulomatosis, lupus, ulcerative colitis, psoriatic arthritis, sarcoidosis, collagenous colitis, dermatitis herpetiformis, familial Mediterranean fever, IgA glomerulonephritis, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmia,Fiessinger-Leroy-Reiter syndrome and uveomeningoencephalic syndrome.

[0120] Other inflammatory diseases are also included, such as acute respiratory distress syndrome (ARDS), acute septic arthritis, adjuvant arthritis, allergic encephalomyelitis, allergic rhinitis, allergic vasculitis, allergy, asthma, atherosclerosis, chronic inflammation due to chronic bacterial or viral infections, chronic obstructive pulmonary disease (COPD), coronary artery disease, encephalitis, inflammatory bowel disease, inflammatory osteolysis, inflammation associated with acute and delayed hypersensitivity reactions, inflammation associated with tumors, peripheral nerve injury or demyelinating diseases, inflammation associated with tissue trauma such as burns and ischemia, inflammation due to meningitis, multiple organ dysfunction syndrome (MODS), pulmonary fibrosis,septicemia and septic shock, the Stevens-Johnson syndrome, undifferentiated arthritis, and undifferentiated spondyloarthropathies.

[0121] In a particular embodiment of the invention, the autoimmune disease is idiopathic thrombotic purpura (ITP) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).

[0122] The present invention also relates to a method for producing a variant of a parent polypeptide comprising an Fc fragment, said variant having an increased affinity for at least one of the receptors of the Fc fragment (FcR) selected from the receptors FcyRIIIa (CD16a), FcyRIIa (CD32a), and FcyRIIb (CD32b) compared to that of the parent polypeptide, which comprises a mutation step of at least one amino acid, the mutation being selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc; the numbering being that of the EU index or equivalent in Kabat.

[0123] The present invention relates in particular to a method for producing a variant of a polypeptide comprising an Fc fragment, said variant having an increased affinity for at least one of the receptors of the Fc fragment (FcR) selected from the receptors FcyRIIIa (CD16a), FcyRIIa (CD32a), and FcyRIIb (CD32b), relative to that of the parent polypeptide, 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, preferably greater than 30, which comprises a mutation step of at least one amino acid, the mutation being selected from 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, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V,K290D, K290E, K290G, 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, Y296W, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S of said fragment Fc; the numbering being that of the EU index or equivalent in Kabat.

[0124] The sequences described in this application can be summarized as follows: SEQ ID NO: Protein 1 Human IgG1 Fc region G 1ml, 17 (residues 226-447 according to the EU index or equivalent in Kabat) without upper N-terminal hinge region 2 Human IgG2 Fc region without upper N-terminal hinge region 3 Human IgG3 Fc region without upper N-terminal hinge region 4 Human IgG4 Fc region without upper N-terminal hinge region 5 Human IgG1 Fc region Glm3 without upper N-terminal hinge region 6 Human IgG1 Fc region G 1ml, 17 with upper N-terminal hinge region (residues 216-447 according to the EU index or equivalent in Kabat) 7 Human IgG2 Fc region with upper N-terminal hinge region 8 Human IgG3 Fc region with 9 Human IgG4 Fc region with N-terminal upper hinge region 10 Human IgGl Glm3 Fc region with N-terminal upper hinge region

[0125] EXAMPLES The following examples are given to illustrate various embodiments of the invention.

[0126] Example 1: Production of Fc variants according to the invention by site-directed mutagenesis 1. Construction of Fc variants:

[0127] Each mutation of interest in the Fc fragment was independently inserted into an expression vector containing the anti-CD20 heavy chain by PCR of Overlap was achieved using two sets of primers adapted to integrate a deletion or a degenerate codon (NNN or NNK) at the targeted position (240 to 243, 258 to 267, 290 to 296, 298 to 305). The fragments thus obtained by PCR were combined, and the resulting fragment was amplified by PCR using standard protocols. The PCR product was purified on 1% (w / v) agarose gels, digested with appropriate restriction enzymes, and cloned into the eukaryotic expression vector pMGM05-CD20 (pCEP4 InvitroGen), which contains cloning sites for the Fc fragment (BamHI and Notl) and the variable chain VH of the anti-CD20 antibody. This construct results in the mutation of two amino acids in the Fc (aa224 and 225, HT changed to GS) and the addition of the EFAAA sequence to the C-terminus of the Fc, but allows for very rapid testing of a very large number of clones. Initially, it was verified that these mutations did not alter the binding of IgG-WT to the various receptors.

[0128] The DNA of the isolated clones was sequenced after PCR on colonies. After bioinformatic analyses, the clones with novel mutations were frozen at -80°C in XL1-Blue bacteria and the sequences included in our database.

[0129] 2. Production of variant IgG in HEK293 cells:

[0130] The anti-CD20 light chain was inserted into a pCEP4 vector identical to the vector used for the heavy chain, designated pMGM01-CDC20 (pCEP4 InvitroGen). HEK293-F Freestyle™ cells (Invitrogen), cultured in 24-well plates, were co-transfected with the pMGM01-CD20 and pMGM05-CD20 vectors (Fc-WT and variants) in equimolar amounts (250 ng / ml) with a transfection reagent (1 µl / ml) using standard protocols (Invitrogen). The cells were cultured in suspension in serum-free medium for 7–9 days post-transfection, and the IgG-containing supernatants (1 ml) were harvested after centrifugation of the cells at 100 g for 10 min. The IgG secreted in the supernatants were quantified using an ELISA test (FastELISA, R&D biotech).

[0131] Table 1: List of generated mutants Nom du mutant mutations ZAC 1-36 V240H ZAC1-136 V240I ZAC1-123 V240M ZAC1-78 V240N ZAC 1-226 V240S ZAC1-1OO F241H ZAC 1-220 F241Y ZAC1-134 L242A ZAC1-121 L242F ZAC1-110 L242G ZAC1-150 L242H ZAC 1-09 L242I ZAC 1-66 L242K ZAC1-17 L242P ZAC 1-224 L242S ZAC 1-229 L242T ZAC1-177 L242V ZAC 1-08 F243L ZAC1-115 F243S ZAC2-158 E258G ZAC2-167 E258I ZAC2-210 E258R ZAC2-30 E258M ZAC2-111 E258Q ZAC2-86 E258Y ZAC2-150 V259C ZAC2-74 V259I ZAC2-180 V259L ZAC2-36 T260H ZAC2-114 T260I ZAC2-250 T260M ZAC2-162 T260N ZAC2-124 T260R ZAC2-110 T260S ZAC2-258 T260W ZAC2-85 T260A ZAC2-226 V262A ZAC2-153 V262S ZAC2-39 V263T ZAC2-107 V264L ZAC2-42 V264S ZAC2-156 V264T ZAC2-148 V266L ZAC2-122 V266M ZAC2-225 S267A ZAC2-64 S267Q ZAC2-121 S267V ZAC3-182 K290D ZAC3-174 K290E ZAC3-83 K290G ZAC3-70 K290H ZAC3-62 K290L ZAC3-246 K290N ZAC3-54 K290Q ZAC3-41 K290R ZAC3-203 K290S ZAC3-172 K290Y ZAC3-39 P291G ZAC3-08 P291Q ZAC3-185 P291R ZAC3-13 R292I ZAC3-71 R292L ZAC3-196 E293A ZAC3-178 E293D ZAC3-61 E293G ZAC3-126 E293M ZAC3-120 E293Q ZAC3-10 E293S ZAC3-15 E293T ZAC3-118 E294A ZAC3-53 E294G ZAC3-82 E294P ZAC3-80 E294Q ZAC3-02 E294R ZAC3-105 E294T ZAC3-66 E294V ZAC3-48 Q295I ZAC3-254 Q295M ZAC3-110 Y296H ZAC3-42 Y296W ZAC4-192 S298A ZAC4-130 S298R ZAC4-233 Y300I ZAC4-14 Y300V ZAC4-71 Y300W ZAC4-187 R301A ZAC4-218 R301M ZAC4-255 R301P ZAC4-03 R301S ZAC4-268 V302A ZAC4-131 V302F ZAC4-237 V302L ZAC4-53 V302M ZAC4-236 V302R ZAC4-29 V302S ZAC4-208 V303S ZAC4-144 V303Y ZAC4-219 S304T ZAC4-33 V305A ZAC4-229 V305F ZAC4-262 V305I ZAC4-139 V305L ZAC4-36 V305R ZAC4-179 V305S

[0132] Example 2: Binding tests on Fc receptors: 1. Recombinant Fc receptors used:

[0133] CD16a is an activating receptor that exhibits a V / F polymorphism at position 158, at the Fc binding site. CD16aV has a higher affinity. CD16aV is commercially available (R&D System).

[0134] CD32a is an activating receptor that exhibits an H / R polymorphism at position 131, at the Fc binding site. CD32aH has a higher affinity. CD32aH was produced by PX'Therapeutics. CD32aR and CD32b are commercially available (R&D System).

[0135] 2. ELIS A tests of IgG variants produced in cell supernatants HEK293-F:

[0136] IgG variants were tested for their binding to several human FcRs and FcRn by ELISA. Maxisorp immunoplates were coated with 0.1 pg CD32aH / well, or 0.2 pg CD16aV / well in PBS, or 0.25 pg FcRn in P6 (100 mM sodium phosphate, 50 mM sodium chloride, pH 6.0). NiNTA (HisGrab Pierce) plates were coated with 0.05 pg CD32aR / well, or 0.2 pg CD32b / well in PBS. After coating overnight at 4°C, the plates were washed twice with PBS (or P6) / 0.05% Tween-20 and saturated with PBS / 4% BSA (or P6). 4% skimmed milk) for 2 hours at 37°C. In parallel, the supernatants were diluted in PBS (or P6 for the FcRn assay) to a final concentration of 0.5 pg IgG / ml and mixed with F(ab')2 of anti-human goat HRP IgG at the same concentration for 2 hours at room temperature. The IgG aggregated with the F(ab')2 was then incubated with gentle shaking for 1 hour at 30°C on undiluted saturated ELISA plates for CDlôaV, CD32aR, and CD32b (i.e., IgG at 0.5 pg / ml), diluted in PBS to 0.25 pg / ml for CD32aH, and diluted in P6 to 0.035 pg / ml for FcRn. The plates are then developed with TMB (Pierce) and the absorbance is read at 450 nm.

[0137] Using this ELISA test, the constructed variants were tested in comparison with the wild-type Fc (Fc-WT) and their variant / Fc-WT ratio was calculated, as shown in Table 2 below. The ELISA tests performed on these variants show a ratio greater than 2 for at least one of the FcyRs tested.

[0138] Table 2: ELISA tests for binding to the receptors CD1αV, CD32αH, CD32αR, and CD32β. The results are expressed as a ratio of Fc varying according to the invention / Fc-WT Nom du va riant Mutations Résultats tests ELISA CDlôaV CD32aH CD32aR CD32b ZAC 1-36 V240H 1,91 2,00 0,82 0,78 ZAC1-136 V240I 3,91 3,59 1,71 1,45 ZAC1-123 V240M 4,10 2,16 1,08 1,03 ZAC1-78 V240N 2,44 1,42 0,64 0,89 ZAC 1-226 V240S 3,82 2,51 0,82 0,81 ZAC1-100 F241H 0,77 3,47 1,44 1,44 ZAC 1-220 F241Y 1,60 5,50 1,84 1,10 ZAC1-134 L242A 3,75 3,06 1,75 1,41 ZAC1-121 L242F 5,89 5,31 1,47 1,38 ZAC1-110 L242G 4,40 2,71 1,43 1,35 ZAC1-150 L242H 1,19 2,04 1,14 1,36 ZAC 1-09 L242I 5,11 4,99 1,77 1,58 ZAC 1-66 L242K 7,27 2,87 3,04 1,32 ZAC1-17 L242P 1,47 2,50 1,19 1,14 ZAC 1-224 L242S 3,34 2,00 0,88 0,91 ZAC 1-229 L242T 1,49 2,11 1,65 1,40 ZAC1-177 L242V 2,49 6,68 1,96 1,70 ZAC 1-08 F243L 5,15 2,23 1,60 1,46 ZAC1-115 F243S 2,25 1,48 1,50 1,38 ZAC2-158 E258G 1,16 6,81 1,45 1,29 ZAC2-167 E258I 2,03 6,78 1,83 1,29 ZAC2-30 E258M 1,28 4,52 1,56 1,52 ZAC2-111 E258Q 1,88 8,75 1,55 0,85 ZAC2-210 E258R 1,66 8,17 3,70 2,60 ZAC2-86 E258Y 1,53 5,86 2,30 2,70 ZAC2-150 V259C 1,23 2,91 2,03 1,44 ZAC2-74 V259I 1,06 2,20 1,47 1,10 ZAC2-180 V259L 1,19 3,08 2,14 1,84 , ZAC2-85 T260A 4.68 3.89 1.65 1.55 ZAC2-36 T260H 1.16 2.43 1.19 0.91 ZAC2-114 T260I 1.91 7.06 1.46 1.00 ZAC2-250 T260M 1.06 3.80 1.29 1.71 ZAC2-162 T260N 0.94 2.99 1.57 1.27 ZAC2-124 T260R 1.09 3.45 2.60 1.42 ZAC2-110 T260S 1.44 3.71 1.74 0.91 ZAC2-258 T260W 1.49 3.54 1.40 1.24 ZAC2-226 V262A 0.91 5.84 3.78 2.42 ZAC2-153 V262S 1.01 3.64 1.14 1.03 ZAC2-39 V263T 1.13 4.80 1.27 1.01 ZAC2-107 V264L 0.82 2.67 2.13 1.34 ZAC2-42 V264S 0.67 1.40 2.30 2.07 ZAC2-156 V264T 0.91 6.24 1.86 1.42 ZAC2-148 V266L 1.12 2.10 4.67 3.68 ZAC2-122 V266M 0.47 0.34 2.44 2.32 ZAC2-225 S267A 1.26 4.26 5.82 4.75 ZAC2-64 S267Q 0.63 0.43 2.49 3.00 ZAC2-121 S267V 0.59 0.29 2.36 2.02 ZAC3-172 K290Y 4.79 6.72 2.16 1.00 ZAC3-203 K290S 2.28 4.70 1.76 1.21 ZAC3-41 K290R 1.12 1.58 2.15 2.39 ZAC3-54 K290Q 2.47 3.85 1.52 1.50 ZAC3-246 K290N 1.36 3.22 1.71 NA ZAC3-62 K290L 1.51 2.65 1.35 0.63 ZAC3-70 K290H 3.49 6.48 2.64 1.66 ZAC3-83 K290G 4.20 5.78 1.86 1.83 ZAC3-174 K290E 2.83 4.89 1.66 1.83 ZAC3-182 K290D 2.04 3.38 2.23 NA ZAC3-185 P291R 0.64 2.57 1.93 NA , ZAC3-08 P291Q 1.61 2.32 0.99 0.96 ZAC3-39 P291G 1.32 2.39 1.28 1.65 ZAC3-71 R292L 1.67 2.22 0.71 0.41 ZAC3-13 R292I 0.81 2.19 0.48 0.53 ZAC3-15 E293T 0.41 1.40 2.07 1.81 ZAC3-10 E293S 1.02 2.95 1.18 1.51 ZAC3-120 E293Q 1.78 2.17 1.49 NA ZAC3-126 E293M 1.32 2.42 1.81 NA ZAC3-61 E293G 0.48 1.37 2.44 0.89 ZAC3-178 E293D 0.79 2.68 1.80 NA ZAC3-196 E293A 0.89 2.99 1.91 NA ZAC3-66 E294V 1.00 1.91 3.03 0.93 ZAC3-105 E294T 0.80 2.34 1.13 NA ZAC3-02 E294R 0.71 0.90 2.09 1.58 ZAC3-80 E294Q 0.88 1.26 2.78 1.01 ZAC3-82 E294P 0.87 1.32 2.43 0.70 ZAC3-53 E294G 0.57 3.30 2.34 0.52 ZAC3-118 E294A 1.86 5.10 1.57 1.25 ZAC3-254 Q295M 1.74 2.81 1.10 NA ZAC3-48 Q295I 0.92 5.36 1.29 0.58 ZAC3-42 Y296W 3.83 1.49 1.20 1.60 ZAC3-110 Y296H 2.17 0.86 0.98 NA ZAC4-192 S298A 5.53 0.24 0.48 0.51 ZAC4-130 S298R 4.37 0.66 1.09 0.66 ZAC4-233 Y300I 0.69 2.25 1.01 1.04 ZAC4-14 Y300V 0.76 2.37 0.78 2.05 ZAC4-71 Y300W 0.87 2.34 1.14 1.06 ZAC4-187 R301A 0.81 1.25 2.06 2.11 ZAC4-218 R301M 0.95 1.22 1.78 2,07 ZAC4-255 R301P 0.63 4.64 0.06 0.24, ZAC4-03 R301S 1.29 2.42 1.16 1.06 ZAC4-268 V302A 1.00 2.38 1.86 3.09 ZAC4-131 V302F 0.76 2.82 1.06 0.71 ZAC4-237 V302L 0.46 0.31 3.15 4.69 ZAC4-53 V302M 0.63 1.50 2.25 1.56 ZAC4-236 V302R 0.45 0.38 5.08 10.56 ZAC4-29 V302S 1.10 2.51 1.87 1.71 ZAC4-208 V303S 0.96 1.39 1.64 2.27 ZAC4-144 V303Y 1.00 3.50 2.10 1.03 ZAC4-219 S304T 0.98 1.82 2.14 2.34 ZAC4-33 V305A 0.55 1.00 2.20 2.04 ZAC4-229 V305F 1.34 1.11 2.15 2.40 ZAC4-262 V305I 1.22 1.26 1.87 2.22 ZAC4-139 V305L 1.19 2.72 1.87 0.82 ZAC4-36 V305R 1.33 3.50 1.71 2.42 ZAC4-179 V305S 1.29 1.65 2.03 1.66

[0139] Example 3: Production of additional variant IgG in HEK293 cells

[0140] Combinations of mutants comprising at least one mutation i) according to the invention were produced from an Fc fragment comprising the starting mutations N315D / A330V / N361D / A378V / N434Y (mutant T5A-74).

[0141] Table 3: Additional variants generated within the framework of the invention Starting Variant Name Mutation i) according to the added invention List of combined mutations T5A-74I T5A-74 T260A T260A / N315D / A330V / N361D / A378V / N434Y T5A-74J T5A-74 E258I E258EN315D / A330V / N361D / A378V / N434Y T5A-74K T5A-74 K290Y K290Y / N315D / A330V / N361D / A378V / N434Y T5A-74L T5A-74 E294A E294A / N315D / A330V / N361D / A378V / N434Y T5A-74M T5A-74 Y296W Y296W / N315D / A330V / N361D / A3 78V / N434Y

Claims

Demands

1. A variant of a parent polypeptide comprising a human Fc fragment, said variant having increased affinity for at least one of the Fc fragment receptors (FcR) selected from FcyRIIIa (CD 16a), FcyRIIa (CD32a), and FcyRIIb (CD32b) compared to that of the parent polypeptide, characterized in that it comprises at least the K290G or Y296W mutation of said Fc fragment; numbering being that of the EU index or equivalent in Kabat, the human Fc fragment of the parent polypeptide already comprising at least: (ii) a mutation selected from 378V, 378T, 434Y, and 434S;and (iii) at least one mutation selected from 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 378V, 378T, 389T, 389K, 434Y and 434S, numbering being that of the EU index or equivalent in Kabat and with the condition that mutations (ii) and (iii) do not occur on the same amino acids, or the human Fc fragment of the parent polypeptide already comprising at least: (iv) one mutation selected from 307N, 326E, 326T, 334N, 334R, 352L, 378V, 378T, 394P, 396L, 397M, and 421T;and (v) at least one mutation selected from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 26IR, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 2861, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 3081, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 3231, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 3631, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 3931, 394P, 396L, 3971, 397M, 398P, 405V, 405L, 410R, 412M, 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434K, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N, the numbering being that of the EU index or equivalent in Kabat and with the condition that mutations (iv) and (v) do not take place on the same amino acids.;

2. Variant according to claim 1, having an increased affinity for at least one of the receptors of the Fc fragment (FcR) selected from the receptors FcyRIIIa (CD 16a), FcyRIIa (CD32a), and FcyRIIb (CD32b), relative to that of the parent polypeptide, of 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, preferably greater than 30.

3. 3. Variant according to any one of claims 1 to 2, characterized in that it comprises from 1 to 20 mutations of said human Fc fragment, preferably from 1 to 10 mutations.

4. Variant according to any one of claims 1 to 3, characterized in that the parent polypeptide comprises a parent human Fc fragment which is an Fc fragment of a human IgG1 or a human IgG2.

5. Variant according to any one of claims 1 to 4, characterized in that it is selected from an isolated Fc fragment, a sequence derived from an isolated Fc fragment, an antibody and a fusion protein comprising an Fc fragment.

6. 6. Variant of a parent polypeptide according to any one of claims 1 to 5, characterized in that it is an antibody.

7. Variant according to claim 1 to 6, directed against an antigen selected from a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, a toxin, a membrane or circulating cytokine, a membrane receptor.

8. Variant according to any one of claims 1 to 7, for its use as a medicinal product.

9. Pharmaceutical composition comprising (1) a variant according to any one of claims 1 to 7, and (2) at least one pharmaceutically acceptable excipient.

10. A method for producing a variant of a polypeptide comprising a human Fc fragment according to claim 1, said variant having an increased affinity for at least one of the Fc fragment receptors (FcR) selected from the FcyRIIIa (CD 16a), FcyRIIa (CD32a), and FcyRIIb (CD32b) receptors compared to that of the parent polypeptide, characterized in that it comprises a mutation step of at least one amino acid, the mutation being K290G or Y296W of said human Fc fragment; the numbering being that of the EU index or equivalent in Kabat.

11. A method for producing a variant of a polypeptide comprising a human Fc fragment according to claim 10, said variant having an increased affinity for at least one of the Fc fragment receptors (FcR) selected from FcyRIIIa (CD 16a), FcyRIIa (CD32a), and FcyRIIb (CD32b) receptors, relative to that of the parent polypeptide, of 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, preferably greater than 30.