MUTANTS FC WITH MODIFIED FUNCTIONAL ACTIVITY

Abstract

The present invention relates to a polypeptide comprising a mutated Fc region and exhibiting functional activity mediated by the modified Fc region compared to that of a parent polypeptide, characterized in that said Fc region comprises at least one combination of 2 mutations, said combination being selected from: (i) a mutation selected from 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and (ii) at least one mutation chosen from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 379A, 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, 333G, 339T, 349S, 325S, 334N, 334R, 336T, 340E, 343S, 345G, 349H, 350A 352S, 359A, 361H, 362R, 3631, 366A, 373D, 375R, 377T, 378V, 378T, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 3931, 394P, 396L, 3971, 397M,398P, 405V, 405L, 410R, 412M, 414R, 421T, 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 mutation (i) does not occur on the same amino acid as mutation (ii). The present invention also relates to the use of such a polypeptide, to compositions comprising it, and to methods of preparing such a polypeptide.

Description

1. MUTANTS Fc WITH MODIFIED FUNCTIONAL ACTIVITY The present invention relates to a polypeptide comprising a mutated Fc region and exhibiting functional activity mediated by the modified Fc region compared to a parent polypeptide. An antibody consists of a tetramer of heavy and light chains. The two light chains are identical to each other, and the two heavy chains are identical and linked by disulfide bonds. There are five types of heavy chains (alpha, gamma, delta, epsilon, mu), which determine the immunoglobulin classes (IgA, IgG, IgD, IgE, IgM). The light chain group comprises two subtypes, lambda and kappa. IgG is a soluble antibody that can be found in blood and other body 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 carboxy-terminal domains of the heavy chains form the Fc fragment, while the amino-terminal domains of the heavy and light chains recognize the antigen and are called the Fab fragment. Fc fusion proteins are created by combining an Fc fragment of an antibody with a protein domain that provides specificity for a given therapeutic target. Examples include combinations of the Fc fragment with any type of therapeutic protein or its fragments. 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 shows identical specificity for a single antigen. Therapeutic Fc fusion proteins are used or being developed as drugs against autoimmune diseases, such as etanercept (Amgen's Enbrel, which is a TNF receptor linked to an Fc fragment) or Alefacept (Biogen Idec's Amevive, which is LFA-3 linked to the Fc portion of human IgG1). For antibodies and Fc fusion proteins to exert their effects, it is necessary that the Fc fragment exhibit the best possible activity (e.g., antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, or antibody-dependent cellular phagocytosis).i.e., that it be optimized. Such optimization would make it possible to obtain a protein with improved activity and / or efficacy and / or reduced side effects. 10 The Applicant has now developed specific Fc fragments exhibiting enhanced activity. These fragments can be used in therapy to improve the efficacy of the product containing them. 15 The present invention thus provides a variant of a parent polypeptide with optimized properties relating to functional activity mediated by the Fc region. The present invention relates to a polypeptide comprising a mutated Fc region and exhibiting functional activity mediated by the modified Fc region compared to that of a parent polypeptide, said Fc region comprising at least one mutation 20 selected from: G316D, K326E, N315D, N361H, P396L, T350A, V284L, V323I, P352S, A378V, Y436H, V266M, N421T, G385R, K326T, H435R, K447N, N434K, K334N, V397M, E283G,A378T, F423L, A431V, F423S, N325S, P343S, 1(290E, S375R, F405V, K322E, K340E, N389S, F243I, T307P, N389T, S442F, K248E, Y349H, N286I, T359A, 25 S383R, K334R, T394P, V259A, T393A, P352L, Q418P, V302A, L398P, F423P, S442P, V363I, S383N, S254F, K320E, G402D, I253F, V284A, A431T, N315H Y319H, C226Y, F405L, T393I, N434S, R255W, A287T, N286Y, A231V, K274R, V308G, K414R, M428T, E345G, F243L, P247T, Q362R, S440N, Y278H, D312G, V262A, V305A, K246R, V3081, E380G, N276S, K439Q, S267G, F423Y, A231T, K320R, L410R, K320M, V412M, T307N, T366A, P230S, Y349S, A339T, K246E K274E, A231P, I336T, S298N, L234P, S267N, V263A, E333G, V308A, K439R, K392R, S440G, V397I, I336V, Y373D, K288E, L309P, P227S, V379A, K288R, K320T, V282A, I377T, N421S et C261R,the numbering being that of the EU index or equivalent in Kabat. 3035879 3 Such a polypeptide is referred to as the "polypeptide according to the invention" in the present application. The present invention relates more specifically to a polypeptide comprising a mutated Fc region and having functional activity mediated by the modified Fc region compared to that of a parent polypeptide, said Fc region comprising at least a combination of 2 mutations, said combination being selected from: (i) a mutation selected from 307N, 326E, 326T, 334N, 334R, 378V, 378T, 394P, 396L, 397M and 421T and; 352L, 10 (ii) at least one mutation chosen from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 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, 15 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, numbered according to the EU index or equivalent in Kabat, and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii). 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 30 website (http: / / www.imgcorg / IMGTScientificChart / Numbering / Hu IGHGnber.html). By "polypeptide" or "protein" is meant a sequence comprising at least 100 covalently linked amino acids. By "amino acid" is meant one of the 20 naturally occurring amino acids or non-natural analogs. The term "position" means a position within the sequence of a polypeptide. For the Fc region, positions are numbered according to the EU index or equivalent in 5 Kabat. The term "antibody" is used in the common sense. It refers to a tetramer that includes at least one Fc region and two variable regions. Antibodies include, but are not limited to, full-length immunoglobulins, monoclonal antibodies, multispecific antibodies, and chimeric antibodies.Humanized antibodies and fully human antibodies are distinguished. 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 (hereafter referred to as a "CDR"). The carboxy-terminal portion of each heavy chain defines a constant region primarily responsible for effector function. IgG has several subclasses, including IgG1, IgG2, IgG3, and IgG4. IgM subclasses include IgM1 and IgM2. Thus, "isotype" refers to one of the immunoglobulin subclasses defined by the chemical and antigenic characteristics of its constant regions. The known isotypes of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2,IgD and IgE. Full-length IgG are tetramers and consist of two identical pairs of 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, Cy1 (also called CH1), Cy2 (also called CH2), and Cy3 (also called CH3) domains. In 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. By "variable region",The term Fc refers to the region of an immunoglobulin that includes one or more Ig domains substantially encoded by any 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). The term "Fc" or "Fc region" designates the constant region of an antibody excluding the first immunoglobulin constant region domain (CH1). Thus, Fc refers to the last two (CH2 and CH3) constant region domains of IgG1, and the N-terminal flexible hinge of these domains. For human IgG1, the Fc region corresponds to residue C226 up to its carboxy-terminal end, i.e., residues from position 226 to 447, where the numbering follows the EU index or equivalent in Kabat. The Fc region used may also include a portion of the upper hinge region,located 10 between positions 216 to 226 according to the EU index or equivalent in Kabat; in this case, the Fc region used corresponds to residues from positions 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 is according to the EU index or equivalent in Kabat. Preferably, in this case, the Fc region used corresponds to residues from positions 216 to 447, where the numbering is according to the EU index or equivalent in Kabat. Preferably, the Fc region used is chosen from the SEQ ID NO: 1 to 10 sequences. By "parent polypeptide" is meant a reference polypeptide. The 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 (for example, 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" is meant 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" is meant a polypeptide sequence fused to an Fc region, said polypeptide sequence preferably being selected 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. Suitable toxins and their fragments include, in particular, diphtheria toxin, exotoxin A, ricin, abrin, saporin, gelonin, calicheamicin, auristatins E and F, and mertansin. Advantageously, the parent polypeptide—and thus the polypeptide according to the invention—consists of an Fc region. Advantageously, the parent polypeptide—and thus the polypeptide according to the invention—is an antibody. Finally, preferably, the parent polypeptide—and thus the polypeptide according to the invention—is a polypeptide produced in the milk of transgenic animals. 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; 25 is a polypeptide according to the invention. Such a polypeptide comprises a mutated Fc region,compared 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 one amino acid at a particular position in a parent polypeptide sequence with 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 G>235-236 insertion is meant the insertion of glycine between positions 235 and 236. By "amino acid deletion" or "deletion" is meant the removal of an amino acid at a particular position in a parent polypeptide sequence. For example, E294de1 denotes the deletion of glutamic acid at position 294. Preferably,The following mutation label is used: "434S" or "N434S", and means 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 "V2591 / 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. The polypeptide according to the invention exhibits functional activity mediated by the modified Fc region compared to that of the parent polypeptide. By "functional activity mediated by the Fc region,"This includes, in particular, effector functions. Functional activity mediated by the Fc region 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 functional activity mediated by the Fc region considered in the invention is selected from ADCC, CDC, and their combination. This functional activity can be evaluated by methods well known in the prior art.such as those described in the examples (see in particular points 4.4 and 4.5 of the examples). The Fc region-mediated functional activity of the polypeptide according to the invention is increased or decreased relative to that of the parent polypeptide. According to a first embodiment, the polypeptide according to the invention exhibits increased Fc region-mediated functional activity compared to that of the parent polypeptide. Preferably, the polypeptide according to the invention exhibits increased Fc region-mediated functional activity, 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, and preferably greater than 30. According to a second embodiment, the polypeptide according to the invention exhibits decreased Fc region-mediated functional activity compared to that of the parent polypeptide. Preferably,The polypeptide according to the invention exhibits decreased Fc region-mediated functional activity, compared 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 10 to 25, and preferably greater than 30. Preferably, the mutated Fc region of the polypeptide according to the invention has a modified affinity for at least one of the Fc region receptors (FcR) selected from complement Clq, FcgRIIIa (CD16a), FcgRIIa (CD32a), and FcgRIIb (CD32b). Complement Clq is indeed involved in CDC activity. The FcgRIIIa (CD16a) receptor, for its part, is involved in ADCC; It exhibits a V / F polymorphism at position 158. The FcgRIIa receptor (CD32a), on the other hand, is involved in platelet activation and phagocytosis; it exhibits an H / R polymorphism at position 131. Finally,The FcgRIIb receptor (CD32b) is involved in the inhibition of cellular activity. Preferably, the mutated Fc region has an increased affinity for at least one of the Fc receptors. 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 Fc receptor is greater than that of the parent polypeptide. Alternatively, the mutated Fc region has a decreased affinity for at least one of the Fc receptors. Preferably, the affinity is decreased, 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, 30, 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 lower than that of the parent polypeptide. 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, a person skilled in the art can determine the affinity (Kd) using surface plasmon resonance (SPR). Alternatively, a person 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 and mutated Fc regions. The signals detected specific to the mutated and parent Fc regions are compared. The binding affinity can be determined by evaluating either the whole polypeptides or isolated Fc regions thereof. Preferably, the mutated Fc region of the polypeptide according to the invention comprises 1 to 20 mutations compared to the parent polypeptide, and preferably 2 to 20 mutations. By "101 to 20 amino acid modifications", we include 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 relative to the parent polypeptide, preferably from 2 to 10 mutations. 15 Preferably, the mutated Fc region of the polypeptide according to the invention has an improved affinity for complement Clq, and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 378V, 378T, 396L, 421T, 334R and 326E; and ii) at least one mutation selected from 361H, 290E, 316D, 248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T, 396L, 2861, 315D and 397M, the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii). Preferably, the mutated Fc region of the polypeptide according to the invention has an improved affinity for the FcgRIIIa receptor (CD16a),and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 378V, 326E, 397M, 334N and 396L; and ii) at least one mutation selected from 316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P, 307P, 378V, 378T, 2861, 286Y and 30298N, the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii). 3035879 10 Preferably, the mutated Fc region of the polypeptide according to the invention has an increased affinity for the FcgRIIa receptor (CD32a), and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 378V, 326E, 397M, 307N, 394P, 326T, 396L and 334N; and ii) at least one mutation chosen from: 316D, 334R, 334N, 3231, 231V, 246R, 336T, 378T, 286Y, 2861, 352S, 383R, 359A, 421T, 361H, 315D, 366A, 290E, 307P and 439R,the numbering being that of the EU index or equivalent in Kabat and with the condition 10 that the mutation (i) does not take place on the same amino acid as the mutation (ii). Preferably, the mutated Fc region of the polypeptide according to the invention has an increased affinity for the FcgRIIb receptor (CD32b), and comprises at least a combination of 2 mutations, said combination comprising: 15 i) a mutation selected from 326E, 326T, 378V, 397M, 352L, 394P, 396L and 421T; and ii) at least one mutation selected from 316D, 334R, 248E, 334N, 418P, 231V, 320E, 402D, 359A, 383R, 421T, and 361H, the numbering being that of the EU index or equivalent in Kabat, and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii). Preferably, the mutated Fc region of the polypeptide according to the invention has increased CDC activity and comprises at least one combination of two mutations, said combination comprising: i) a mutation selected from 378V, 378T, 396L, 421T,334R and 326E; and ii) at least one mutation selected from 361H, 290E, 316D, 248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T, 396L, 2861, 315D and 397M, the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii). 30 Preferably, the mutated Fc region of the polypeptide according to the invention has increased ADCC activity, and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 378V, 326E, 397M, 334N and 396L; and 3035879 n ii) at least one mutation chosen from 316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P, 307P, 378V, 378T, 2861, 286Y and 298N, numbering being that of the EU index or equivalent in Kabat and with condition 5 that mutation (i) does not occur on the same amino acid as mutation (ii). Preferably,the mutated Fc region of the polypeptide according to the invention comprises at least one combination of 3 mutations, said combination comprising: 10 (i) a mutation selected from 326E, 326T, 352L, 378V, 378T, 397M, 421T, 334N, 334R, 307N and 394P; and 396L, (ii) at least 2 mutations chosen from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 2861, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 15 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, 20 414R, 421T, 421S, 423L, 423Y, 423S, 423P, 428T, 431V, 431T, 434S, 435R, 436H, 439R, 440G, 440N, 442F, 442P and 447N, 434K,the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii). 25 More preferably, the mutated Fc region of the polypeptide according to the invention comprises at least a combination of 4 mutations, said combination comprising at least one mutation (i) as described above, and at least 3 mutations (ii) as described above, the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii). 30 More preferably, the mutated Fc region of the polypeptide according to the invention comprises at least a combination of 5 mutations, said combination comprising at least one mutation (i) as described above, and at least 4 mutations (ii) such as those described above,The numbering being that of the EU index or equivalent in Kabat, and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii). 5 The present invention also relates to a polypeptide composition according to the invention, said purified polypeptides having N-glycans at their Asn297 glycosylation site with a fucosylation level of less than 65%, preferably less than 50%, and preferably even less than 40%. Preferably, said purified polypeptides have a 10-glycan structure of the biantenn type at their Asn297 glycosylation site, with short chains, low sialylation, and non-intercalated terminal mannoses and / or terminal N-acetylglucosamines. More preferably, the purified polypeptides have a content greater than 60% for the G0+G1+G0F+G1F forms, with the G0F+G1F forms having a content less than 50%. 15 More preferably,The purified polypeptides have a content greater than 60% for the G0+G1+G0F+G1F forms, with a fucose content less than 65%. More preferably, the purified polypeptides have a content less than 40% for the G1F+GOF forms. The present invention also relates to a pharmaceutical composition comprising (i) a polypeptide according to the invention or a composition as described in the preceding paragraph, and (ii) at least one pharmaceutically acceptable excipient. The present invention also relates to the polypeptide according to the invention or the composition as described above, for use as a medicinal product. As indicated above, advantageously, the parent polypeptide—and therefore the polypeptide 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. 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. When the antibody is directed against a viral antigen, its use is particularly suitable in the treatment of viral infections. Viral infections include infections caused by HIV, retroviruses, Coxsackie virus, smallpox virus, influenza virus, yellow fever virus, West Nile virus, and cytomegalovirus.to rotavirus or hepatitis B or C virus, this list not being exhaustive. 10 When the antibody is directed against a toxin, its use is particularly suitable in the treatment of bacterial infections, for example, infections caused by tetanus toxin, diphtheria toxin, anthrax toxins (Bacillus anthracis), or in the treatment of infections caused by botulinum toxin, ricin toxin, Shiga toxins, this list not being exhaustive. 15 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, in particular, thrombotic thrombocytopenic purpura (ITP), organ or transplant rejection, graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, and the 20 different 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, Crohn's disease, Hashimoto's 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), and disease of cold agglutinins, acquired autoimmune hemophilia; Goodpasture syndrome, extramembranous nephropathies,autoimmune bullous skin diseases, refractory myasthenia gravis, mixed cryoglobulinemia, psoriasis, juvenile chronic arthritis, inflammatory myositis, dermatomyositis, and systemic autoimmune diseases of children including antiphospholipid syndrome, connective tissue disease, autoimmune pulmonary inflammation, Guillain-Barré syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), autoimmune thyroiditis, mellitis, myasthenia gravis, autoimmune inflammatory disease of the eye, neuromyelitis optica (Devic's disease), scleroderma, pemphigus, insulin-resistant diabetes, polymyositis, pernicious anemia, glomerulonephritis, Wegener's granulomatosis, and other diseases of Horton's disease, polyarthritis 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. 15 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; sepsis and septic shock; Stevens-Johnson syndrome; undifferentiated arthritis; and undifferentiated spondyloarthropathies. In a particular embodiment of the invention, the autoimmune disease is idiopathic thrombotic purpura (ITP) and the other is chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). The present invention also relates to a method for producing a polypeptide comprising an Fc region and having functional activity mediated by the modified Fc region compared to that of a parent polypeptide.said method comprising a step of introducing at least one mutation into said Fc region selected from: G316D, K326E, N315D, N361H, P396L, T350A, V284L, V323I, P352S, A378V, Y436H, V266M, N421T, G385R, K326T, H435R, K447N, N434K, K334N, V397M, E283G, A378T, F423L, A431V, F423S, N325S, P343S, F290E, S375R, F405V, K322E, K340E, N389S, F243I, T307P, N389T, S442F, K248E, Y349H, N286I, T359A, S383R, K334R, T394P, V259A, T393A, P352L, Q418P, V302A, L398P, F423P, S442P, V363I, S383N, S254F, K320E, G402D, I253F, V284A, A431T, N315H, 10 Y319H, C226Y, F405L, T393I, N434S, R255W, A287T, N286Y, A231V, K274R, V308G, K414R, M428T, E345G, F243L, P247T, Q362R, S440N, Y278H, D312G, V262A, V305A, K246R, V3081, E380G, N276S, K439Q, S267G, F423Y, A231T, K320R, L410R, K320M, V412M, T307N, T366A, P230S, Y349S, A339T, K246E, K274E, A231P, I336T, S298N, L234P, S267N, V263A, E333G, V308A, K439R, 15 K392R, S440G, V397I, I336V, Y373D, K288E, L309P, P227S, V379A, K288R, K320T, V282A, I377T,N421S and C261R, the numbering being that of the EU index or equivalent in Kabat. Preferably, the present invention relates to a method for producing a 20 polypeptide comprising an Fc region and having functional activity mediated by the modified Fc region compared to that of a parent polypeptide, said method comprising a step of introducing at least one combination of 2 mutations, said combination being chosen from: 25 (i) a mutation chosen from 326E, 326T, 352L, 378V, 378T, 397M, 421T, 334N, 334R, 307N and 394P; and 396L, (ii) at least one mutation selected from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 2861, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 30 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, 3035879 16 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 mutation (i) does not take place on the same amino acid as mutation (ii). The present invention also relates to a method for increasing the binding of a polypeptide comprising an Fc region to at least one of the Fc region receptors (FcR) selected from the Clq receptor, FcgRIIIa (CD16a), and FcgRIIa (CD32a), and FcgRIIb (CD32b), said method comprising a step of introducing at least one mutation in said Fc region selected from: G316D, K326E, N315D, N361H, P396L, T350A, V284L, V323I, P352S, A378V, Y436H, V266M, N421T, G385R, K326T, H435R, K447N, N434K,K334N, V397M, E283G, A378T, F423L, A431V, F423S, N325S, P343S, 1(290E, S375R, F405V, 15 K322E, K340E, N389S, F243I, T307P, N389T, S442F, K248E, Y349H, N286I, T359A, S383R, K334R, T394P, V259A, T393A, P352L, Q418P, V302A, L398P, F423P, S442P, V363I, S383N, S254F, K320E, G402D, I253F, V284A, A431T, N315H, Y319H, C226Y, F405L, T393I, N434S, R255W, A287T, N286Y, A231V, K274R, V308G, K414R, M428T, E345G, F243L, P247T, Q362R, S440N, Y278H, D312G, 20 V262A, V305A, K246R, V3081, E380G, N276S, K439Q, S267G, F423Y, A231T, K320R, L410R, K320M, V412M, T307N, T366A, P230S, Y349S, A339T, K246E, K274E, A231P, I336T, S298N, L234P, S267N, V263A, E333G, V308A, K439R, K392R, S440G, V397I, I336V, Y373D, K288E, L309P, P227S, V379A, K288R, K320T, V282A, I377T, N421S and C261R, 25, the numbering being that of the EU index or equivalent in Kabat. Preferably,the present invention relates to a method for increasing the binding of a polypeptide comprising an Fc region to at least one of the Fc region receptors (FcR) selected from the Clq receptor, FcgRIIIa (CD16a), and FcgRIIa (CD32a), 30 and FcgRIIb (CD32b), said method comprising a step of introducing at least one combination of 2 mutations, said combination being selected from: (i) a mutation selected from 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and 3035879 17 (ii) at least one mutation selected from 226Y, 227S, 230S, 231V, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 234P, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 2861, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 5 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 mutation (i) does not occur on the same amino acid as mutation (ii). 15 The sequences described in this application can be summarized as follows: SEQ ID NO: Protein 1 Human IgG1 Fc region Glm1,17 (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 G1m3 without upper N-terminal hinge region 6 Human IgG1 Fc region Glm1,17 with upper N-terminal hinge region (residues 216-447 according to 3035879 18 EU index or equivalent in Kabat) 7 Human IgG2 Fc region with upper N-terminal hinge region 8 Human IgG3 Fc region with upper N-terminal hinge region 9 Human IgG4 Fc region with upper N-terminal hinge region 10 Human IgG1 G1m3 Fc region with upper N-terminal hinge region 11 and 12 MG 619 and MG 621 primers The present invention will be better understood by reading the following examples. 5 EXAMPLES EXAMPLE 1: Identification of polypeptides with a mutated Fc region according to the invention and characterization of said polypeptides 10 I. Materials and methods 1. Construction of libraries of the human Fc region The human Fc gene encoding amino acids 226-447 (EU index or equivalent in Kabat), i.e. a polypeptide comprising an Fc region, derived from a heavy chain 15 of human IgG1 and having the allotype Glm1.17 (SEQ ID NO: 1), (Poul MA et al,Eur J. Immunol 25 (7): 2005-2009, 1995) was cloned into the phagemid vector pMG58 (pMG58 Fc226) as a BamHI / EcoRI fragment using standard PCR protocols. Several completely randomized libraries were generated using the MUTAGENTm method (W002 / 038756), which uses low-fidelity human DNA polymerases (mutases) to introduce random mutations homogeneously across the entire target sequence. Three distinct mutases (pol(3, polri, and polt)303587919) were used under different conditions to create complementary mutation profiles. These human polymerases were produced and purified as previously described (Mondon et al. J. Biotechnol 2: 76–82 (2007), Emond et al. Protein Eng Des Sel 21:267–274, (2008)). 5 1.1. Mutagenesis with the MUTAGENTm Process The MUTAGENTm process was described in application W002 / 038756. Briefly,The human Fc gene (Fc gene) was replicated using mutases with the 5' primer MG 619: 5'-AGTACTGACTCTACCTAGGATCCTGCCCACCGTGC-3' 10 (SEQ ID NO: 11) and the 3' primer MG 621: 5'-ACTGCTCGATGTCCGTACTATGCGGCCGCGAATTC-3' (SEQ ID NO: 12). A mixture containing 0.61 tg of the pMG58 Fc226 plasmid as a template (wild-type Fc region or selected variants), the MG 619 and MG 621 primers (250 nM each), and the appropriate replication buffer (see details below) was processed for 5 to 15 min at 95°C and immediately cooled to 4°C to denature the DNA strands. For pol(3), the replication buffer was 50 mM Tris HCl pH 8.8, 10 mM MgCl2, 10 mM KCl, 1 mM DTT, and 1% (v / v) glycerol. The replication buffer for pol(ri) (or pol(ri) and pol(t)) was 25 mM Tris HCl pH 7.2, 5 mM MgCl2, 10 mM KCl, 1 mM DTT, and 2.5% (v / v) glycerol. After the denaturation step, mutagenic replications were performed by adding 501.. mM of dATP / dCTP.1001..1M of dTTP / dGTP and 1..tg of pol (3 or 100 1..1M of dNTP and 1 1..tg of pol ri (or pol ri and poli, 1 1..tg of each mutase). The replication reaction was carried out at 37°C for two hours. The replication products were then concentrated and desalted on Microcon (Millipore) columns. 25 1.2. Selective Amplification and Cloning of Mutated Fragments The previously obtained replication products were amplified by selective PCR with tail primers. The primers (MG 619 MG 621) were designed with a tail that is non-complementary to the template, allowing for the specific amplification of DNA fragments synthesized by the mutases. A fraction of the 30 replication products was added to a mixture containing PCR buffer (20mM Tris-HCl1 pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 1 µmol of 5' and 3' primers, 200 pM dNTPs, and 1.25 U of DNA Taq polymerase Platinum (Invitrogen). The PCR cycles were as follows: first cycle: 2 min. at 94°C, 10 sec. at 64°C,30 sec. at 75°C, 1 min. at 94°C, then 30 selective cycles: 20 sec. at 94°C and 30 sec. at 75°C. 3035879 20 The amplified replication products were purified on 1% (w / v) agarose gels, digested with BamHI and EcoRI, and cloned into the pMG58 vector. The ligation mixtures were transformed in electrocompetent E. coli XL1-Blue cells and then plated on 2YT solid medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl, 15 g / L agar) supplemented with 1001..tg / mL ampicillin and 1% (w / v) glucose. After growth, the number of colonies was determined to estimate the library size, and at least 48 clones per library were subjected to PCR and high-throughput DNA sequencing. The cells were resuspended in 2YT medium with 15% 10% glycerol, frozen, and stored at -80°C. 1.3. Construction of the Mut3 Library A first library was obtained using pol p on the wild-type Fc gene and contained 3,2 x 10⁶ clones (called Mut1.1). The DNA from this first library was used to generate the second and third libraries, using pol p (3.8 x 10⁶ clones, Mut1.2) and pol ri and i (3.0 x 10⁶ clones, Mut1.3), respectively. This two-step cumulative replication strategy increased the mutation rate. The fourth library was created using pol ri alone on the wild-type Fc gene (1.0 x 10⁶ clones, Mut1.4). Finally, these four libraries were proportionally mixed to obtain the final library, called Mutl, representing 1.1 x 10⁷ different clones. Two different libraries were then constructed using a DNA pool of 42 single and double mutants isolated on the FcRn receptor. A first bank was obtained using pol p (1.9x10⁷ clones, Mut2.1) and a second bank with pol ri 25 (1x10⁶ clones, Mut2.2). Finally, these two banks were proportionally mixed to obtain the final bank called Mut2.that is, 2 x 10⁷ different clones. A new library was obtained using pol p on the wild-type Fc gene, with a diversity of 4.7 x 10⁷ clones. 30 Finally, the Mutl, Mut2, and new libraries were proportionally mixed to obtain the final library called Mut3, containing 7.8 x 10⁷ different clones. Ultimately, the Mut3 library contains 3 x 10⁷ in-frame mutated clones with an average of 2.2 amino acids mutated per Fc. 3035879 21 1.4. Construction of the Mut4 library An Fc variant library was constructed from 36 Clq and / or CD16aV-enhanced binding clones selected from the Mut3 library (1.6 amino acids mutated per Fc on average). The Mut4 library was obtained using an equimolar mixture of the pol p and pol ri polymerases (in the pol p replication buffer). The Mut4 library has a diversity of 1.3 x 10⁷ clones, with 77% of clones in phase and an average of 2.6 amino acids mutated per Fc. This library was cloned into the pMG93 vector.A selection vector developed for Fc 10 allows the selection of coding sequences (ORFs) on ampicillin medium through fusion with the α-lactamase gene. This construct was transformed in the non-suppressive HB2151 bacteria, eliminating TAG codons, which are recognized as stop codons in this strain. Clones from this library were plated onto agar plates containing low concentrations of ampicillin at a low density to perform ORF selection. Viable clones were then collected and frozen. Plating onto 500 Petri dishes (120x120) covered the entire Mut4 library. The selected library was designated Mut4sel and contains 92% in-phase clones, representing 1x10⁷ in-phase mutated clones with an average of 2.5 amino acid mutations per Fc. The Mut4sel library was subsequently subcloned into the phagemid pMG58 in XL1-Blue bacteria,to enable selection by phage display on targets. 1.5. Construction of the Mut5 Library. An Fc variant library was constructed from 42 clones with enhanced binding for Clq and / or one of the FcgRs selected from the Mut4sel library (2.4 amino acids mutated by Fc on average). The Mut5 library was obtained using polymerase pol p alone to achieve a low mutation rate. Ultimately, the resulting Mut5 library contains 1.2 x 10⁷ clones with 95% of clones in phase, with an average of 3.1 amino acids mutated by Fc. The high quality of this library did not require ORF selection as performed for the Mut4 library. 2. Phage display expression of Fc libraries and selection of improved variants. During the selection steps, the Mut3, Mut4sel and Mut5 libraries were expressed on the surface of bacteriophage M13 using standard procedures (Smith GP,3035879 22 Science 228: 1315 (1985)). E. cou i XL1-Blue bacteria, containing the library to be expressed cloned in the pMG58 vector, were cultured in 60 ml of 2YT medium supplemented with 100 1..tg / ml of ampicillin, 15 1..tg / m1 of tetracycline and 1% (w / v) of glucose at 30°C. The cells were then infected with the helper phage M13 (M13K07, 5 Biolabs, bacteria / phage ratio = 1 / 3) at 37°C for 20 min, and phage-Fc production was continued overnight at 26°C, in 23 Orpm 2YT / ampicillin / glucose with 0.5 mM IPTG and 50 1..tg kanamycin / ml. The following day, the phages were precipitated with PEG6000 using standard protocols, resuspended in 1 ml of PBS buffer at pH 7.4, and titrated by infecting XL1-Blue cells. 2.1 Recombinant proteins used: Complement Clq is commercially available (Calbiochem). CD16a is an activating receptor that exhibits a V / F polymorphism at position 158.15 on the Fc binding site. CD16aV has a higher affinity. CD16aV is commercially available (R&D system). CD16aF was produced by PX'Therapeutics. CD32a is an activating receptor that exhibits an H / R polymorphism at position 131, 20 on the Fc binding site. CD32aH has a higher affinity. CD32aR is commercially available (R&D system). CD32aH was produced by PX'Therapeutics. CD32b is an inhibitory receptor that has a lower affinity for IgG1 than CD32aR. It is commercially available (R&D system). 25 2.2. Solid-phase selections: For solid-phase selections, phages-Fc diluted in PBS / 5% skimmed milk / 0.1% Tween 20 were incubated in 8 wells of Maxisorp plates (1-4x10¹¹ phages / well in 1000 final) previously coated with 50 ng / well of CD16aV, biotinylated CD16aV 30, biotinylated CD16aF, biotinylated Clq,Biotinylated CD32aR or biotinylated CD32b were blocked with 5% skimmed milk in PBS. After a 2-hour incubation at 37°C, the wells were washed 8 times with PBS / 0.1% Tween 20 and 2 times with PBS. For the CD32aR and CD32b selections, counter-selection rounds were also performed: before the binding step to the target immobilized on a Maxisorp plate, the 23 Fc-phages were similarly pre-incubated in 8 wells with the competitor receptor. Only the Fc-phages not bound to the first target were then transferred to the wells containing the second target as described previously. The selected phages were then eluted by infection with XL1-Blue bacteria in phase 5 exponential growth (2 x 1500 / well, 20 min at 37°C without shaking). The infected bacteria were then plated on 2YT / ampicillin / glucose solid medium. The following day, the cells were resuspended in 2YT medium with 15% glycerol.frozen and stored at -80°C until the next selection round. 10 2.3. Liquid-phase selections: For liquid-phase selections, 4 x 10¹¹ phages were first incubated with biotinylated CD16aV (250 nM), biotinylated CD16aF (1000 nM), or biotinylated Clq (250 nM) for 1 hour at room temperature with gentle stirring. Streptavidin-coated magnetic beads (Dynal), previously blocked with 5% skimmed milk PBS, were then added to the phages for 30 minutes at room temperature. The phage-bead complexes were washed 10 times with PBS / 0.1% Tween 20 using a magnet. The phage-bead complexes were then used to infect 5 ml of exponentially growing XL1-Blue bacteria, which were plated on 2YT / ampicillin / glucose solid medium. The following day, the cells were resuspended in 2YT medium with 15% glycerol.frozen and stored at -80°C until the next selection round. 2.4. Selections from the libraries: Mut3 Library: 25 For liquid-phase selections, 6 rounds were performed on 3 biotinylated targets: CD16aV, CD16aF, and Clq (clones labeled DL6A, DL6B, and QL6A). For solid-phase selection rounds, 6 rounds were performed on 3 targets: CD16aV, biotinylated CD16aV, and biotinylated Clq (clones labeled DS6A, DS6B, and QS6A). 30 Mut4sel Bank: Selection rounds were performed in solid phase only: 3 rounds of selection on biotinylated CD16aV, 3 rounds on biotinylated Clq, 3 rounds on biotinylated CD32aR (+1-depletion on CD32b for the 3rd round) and 3 rounds on biotinylated CD32b (+ / - depletion on CD32aR for the 3rd round), (clones labeled A3A, G3A, J3A / B and K3A / B). 3035879 24 Mut5 Bank: Selection rounds were performed as before: 3 rounds of selection on biotinylated CD16aF, 3 rounds on biotinylated Clq, 3 rounds on biotinylated CD32aR,3 out of 5 turns on CD32b-biotinylated, 2 turns on CD32aR-biotinylated and 2 turns with depletion on CD32b-biotinylated, and 2 turns on CD32b-biotinylated and 2 turns of depletion on CD32aR-biotinylated (clones labeled N3A, O3A, P3A, Q3A, P4B, and Q4B). Selection is then carried out as previously described. 10 2.5. Pool cloning in the pMGM05 vector: The clones selected after the selection rounds were then directly transferred in a mixture (approximately 104 clones per condition) into the eukaryotic vector pMGM05-CD20 (pCEP4 InvitroGen), which contains the same cloning sites as the phagemid pMG58 for the Fc fragment (BamHI and NotI) 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 different receptors. Subsequently, the positive controls were cloned into this system to validate it: - IgGl-S239D, 1332E, from the anti-CD19 antibody XmAb5574 from Xencor (Cl): positive control for CD16a; - IgGl-G236A, from Xencor (C4): positive control for CD32aH / R; - IgGl-K326W, E333S, from Abgenix / Genentech (C3): positive control for C lq; and - IgGl-S267E, L328F, from the anti-CD19 antibody XmAb5574 from Xencor (C5): positive control for CD32b. The DNA of approximately one hundred clones isolated per selection round was sequenced by colony PCR. After bioinformatic analysis, the clones containing 30 novel mutations were frozen at -80°C in XL1-Blue bacteria, and the sequences were included in our database. Thus, 158 clones were isolated from the Mut3 library.371 clones from the Mut4sel library and 171 clones from the Mut5 library. 2.6. Production of variants in HEK293 cells: 3035879 25 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 5 variants) in equimolar amounts (250 ng / ml) with Freestyle™ MAX reagent (10 / mL) using standard protocols (Invitrogen). The cells were cultured in suspension in serum-free medium for 7 days post-transfection, and the supernatants (1 ml) containing IgG were harvested after centrifugation of the cells at 100 g for 10 min. The IgG secreted into the supernatants was quantified using an ELISA assay on recombinant L protein (Pierce).with a purified anti-CD20 antibody produced in 293-F cells used as a standard. Supernatants and standard antibodies, serially diluted in PBS / 0.05% Tween-20, were tested on Maxisorp immunoplates (Nunc) pre-coated with 0.25 µg L protein / well and blocked with 5% skimmed milk in PBS. After incubation for 1 hour at 37°C, the wells were washed three times with PBS / 0.05% Tween-20. Bound IgG variants were detected with an anti-human goat HRP IgG F(ab')2 fragment (sigma). The IgG variants produced were quantified (1–41 µg / ml) using the standard curve. 2.7. ELISA assays of IgG variants produced in 293-F cell supernatants: IgG variants were tested for their binding to human complement Clq and several human FcRs by ELISA. Maxisorp immunoplates were coated with 0.5 tg complement Clq / well, 0.05 1.0 tg CD32aH / well, 0,2 1..tg CD16aF / well or 0.1 1..tg CD16aV / well in PBS. Immobilizing nickel chelating plates 25 (Nunc) were coated with 0.1 1..tg CD32aR / well or 0.41 1..tg CD32b / well in 0.01 M KC1. After coating overnight at 4°C, the plates were washed twice with PBS / 0.05% Tween-20 and saturated with PBS / 4% BSA for 2 hours at 37°C. In parallel, the supernatants were diluted in PBS to a final concentration of 0.5 µg IgG / m² and mixed with goat anti-human HRP IgG F(ab')2 at the same concentration for 2 hours at room temperature. The IgG aggregated with F(ab')2 was then incubated with gentle shaking for 1 hour at 30°C on undiluted saturated ELISA plates for Cl₂, CD₁₆aF, CD₃₂aR, and CD₃₂b (i.e., IgG at 0.5 µg / m²), diluted in PBS at 0.251..tg / m1 for CD16aV and CD32aH. The plates were then visualized with TMB (Pierce) and Pabsorbance readings at 450 nm. 3035879 26 Selections from the Mut3 library: Using this ELISA assay, the variants selected by phage display were tested for their binding to complement Clq and to the various receptors. They were tested in comparison with the wild-type Fc (Fc-WT) and positive controls. 36 clones positive for CD16aV and / or Clq were thus selected from the Mut3 library and used to construct the Mut4 library. Selections from the Mut4sel library: 10 ELISA tests performed on the 371 isolated clones identified 116 clones with a ratio greater than 2 for at least one FcR and 17 clones with a ratio greater than 3 for Clq only, corresponding to 133 clones of interest (Table 1). Table: 133 isolated clones of interest Name of the mutant Mutations ELISA test results Clq CD16a CD32aR CD32 Vb K3B-01 N315D, N361H, P396L 3.46 2.01 7.65 7.30 A3A-105 G316D,K326E 5.18 2.48 4.50 7.13 G3A-59 N315D, T350A, P396L 4.05 2.22 7.95 6.19 J3B-81 V284L, V323I, P352S, A378V, Y436H 4.32 2.52 13.17 6.12 J3A-123 V266M, P352S, A378V 0.77 0.35 7.76 5.93 J3B-118 P396L, N421T 3.88 2.58 9.00 4.99 A3A-46 T350A, P396L 2.19 2.40 6.17 4.79 J3A-129 T350A, G385R, P396L 1.33 2.84 6.56 4.33 QL4A-55 N315D, P396L 2.06 2.00 3.41 4.27 A3A-27 K326T, H435R 5.81 2.21 3.41 4.10 A3A-123 N315D, P396L, K447N 4.30 2.70 4.53 4.08 J3A-109 N315D, P396L, N434K 1.75 2.46 6.01 4.07 J3B-124 P396L 2.25 2.52 7.78 3.86 A3A-79 E283G, A378T, V397M 1.62 1.35 3.07 3.83 G3A-69 K326T, V397M, F423L, A431V 3.30 2.51 5.38 3.43 A3A-178 P396L, F423S 4.03 1.73 3.55 3.37 DS3A-39 V284L, A378V 2.76 3.18 3.48 3.30 J3A-89 V284L, N3255, A378V 0.80 0.51 4.50 3.29 DS3A-09 A378V 1.30 3.03 2.70 3.21 J3A-120 N315D, P343S, P396L 1.66 2.32 4.71 3.16 K3A-35 1(290E, 5375R, F405V 1.89 1.64 3.93 3.12 3035879 27 DL3A-58 S375R 1.85 2.25 3.95 3.11 A3A-50 V284L, K322E, A378V 0.67 1.50 5.08 3.10 DL4A-146 P352S, A378V 156 2.31 2.35 3.10 A3A-91 K340E, A378V, N389S, N421T 2.39 1.88 2.67 3.03 DS3A-93 F2431, P352S, A378V 1.29 1.71 4.19 2.97 A3A-184 K334N, P352S, V397M 1.44 2.90 3.06 2.91 A3A-112 T307P, N389T, V397M, S442F 3.17 1.74 2.39 2.91 A3A-132 K248E, N315D, P396L 5.03 2.12 3.70 2.79 DS3B-92 V284L, Y349H, A378V 2.52 1.98 2.57 2.79 QL3A-61 N2861, A378V 1.33 2.07 2.20 2.77 J3B-115 T359A, S383R, V397M 1.72 1.69 5.17 2.77 A3A-173 K248E, K334R, A378V OVER 1.63 2.96 2.76 (>30) A3A-164 A378V, K447N 1.64 2.41 2.82 2.71 G3A-118 N2861, P352S, A378V 1.21 2.45 4.83 2.68 QS5A-66 T394P 2.64 1.81 2.05 2.67 DL4A-55 A378V, N421T 1.20 1.87 2.34 2.66 G3A-176 V259A, V284L, A378V, N421T 1.05 1.68 2.10 2.61 G3A-09 A378T, V397M, K447N 1.58 2.23 3.12 2.45 DL3A-131 A378T, V397M 1.20 1.72 2.30 2.40 J3A-10 T307P, P396L 5.18 2.74 5.71 2.39 J3B-49 N315D, T393A, P396L 1.42 1.77 5.17 2.30 K3A-41 V302A, P352L, L398P 1.11 0.77 2.17 2.30 DL4A-54 P352L, Q418P 1.35 1.49 1.58 2.30 A3A-06 K248E, A378T, V397M 4.79 1.59 1.78 229 G3A-154 A378T, V397M, F423P, S442P 2.51 2.11 4.14 2.28 A3A-12 V3631, V397M 3.02 1.56 2.99 2.27 J3A-14 K326T 1.83 1.43 4.25 2.26 G3A-05 V3231, S383N 2.21 1.33 3.37 2.25 J3B-138 K334R, T394P 4.41 1.09 5.34 2.24 QL2A-11 V302A 0.97 0.68 1.52 2.22 K3B-32 S254F, A378T, V397M 5.80 1.42 3.72 2.22 G3A-88 K320E, T394P, G402D 1.32 1.88 4.94 2.21 K3B-30 1253F, K326T, F423L, A431V 7.34 1.58 5.99 2.20 A3A-176 V284A, A378V, A431T 3.32 1.82 2.01 2.20 K3A-59 N315H, Y319H, V3231 2.88 0.59 3.33 2.17 J3B-120 A378V, L398P 1.29 1.67 2.94 2.17 QL4B-10 S383R, V397M 1.40 1.35 1.95 2.08 J3B-89 1(290E, V3081, A327T, S383N 0.50 0.14 0.30 2.05 G3A-83 C226Y, A378V, N421T 0.90 2.26 2.00 2.04 G3A-165 V3231, T3931 1.33 1.52 4.15 2.04 J3B-135 K248E, S383R, V397M, N434S 6.36 1.51 3.99 2.04 A3A-137 1(290E, V3231, F405L 1.91 1.65 3.02 2.03 K3B-94 R255W, A287T, P352S, A378V 4.66 1.52 5.08 2.02 A3A-30 V284L, T350A 2.09 1.47 2.47 2.01 3035879 28 K3A-07 N315D, A378V 1.30 1.76 2.26 2.01 G3A-164 T394P, N434S 1.72 1.19 2.29 1.99 J3A-16 N286Y, P352S, A378V 1.46 1.83 3.54 1.98 G3A-43 A231V, A378V 1.13 2.44 3.57 1.96 A3A-09 V308G, V3231 3.65 1.23 2.26 1.95 G3A-106 S254F, V284L, A378V 1.57 1.95 3.96 1.94 J3A-08 K274R, A378V, N421T 1.17 1.53 3.47 1.94 QL3A-20 V397M 2.49 1.96 2.60 1.93 G3A-108 T394P, K414R 1.50 1.54 3.67 1.91 G3A-163 V3231, M428T 1.06 1.05 2.41 1.89 A3A-125 T394P, K447N 1.94 1.83 2.48 1.84 J3B-109 E345G, V397M 1.18 1.36 2.37 1.83 DS3B-33 K326T, F423L, A431V 5.29 1.31 1.73 1.80 QL4A-28 F243L, P247T, Q362R, G402D, S440N 0.74 1.65 2.88 1.80 J3B-101 Y278H, N315D, P396L 1.18 1.64 2.35 1.78 A3A-140 D312G, A378T, V397M 1.47 1.39 2.23 1.76 G3A-25 V262A, V305A, A378V 1.76 1.56 2.01 1.70 G3A-45 K246R, A378V 1.15 2.46 2.76 1.68 QS6A-78 V3231 1.41 1.31 2.03 1.68 K3B-91 V3081, K326T, F423L, A431V 4.84 1.13 2.71 1.65 G3A-103 K248E, A378V 2.54 2.00 1.96 1.64 G3A-07 N276S, T394P, K439Q 2.27 1.13 2.17 1.59 A3A-17 1(290E, E380G 1.64 2.02 1.91 1.59 J3B-16 S267G, A378T, V397M 0.85 0.57 4.95 1.58 J3B-23 N2861, A378V,F423Y 1.13 1.81 3.33 1.57 J3B-68 K320E, T350A 0.82 1.57 3.30 1.55 A3A-37 A231T, 1(290E, S383N, F423L 1.41 1.36 2.02 1.53 J3A-49 K320R, A378T, V397M 0.60 0.45 2.15 1.52 A3A-31 K334R, L410R 6.23 1.36 1.57 1.50 A3A-41 V3231, P352L, L398P 2.00 1.46 2.53 1.47 K3A-36 K248E, K320M 2.81 1.69 2.34 1.42 J3A-06 A378T, V397M, V412M 1.60 1.65 2.74 1.41 G3A-139 T307N, V3231 1.30 1.10 2.57 1.41 G3A-98 S375R, N434S 1.14 1.56 3.02 1.40 K3B-80 V284L, T366A, A378V 2.58 1.25 2.27 1.39 J3B-74 P230S, N389S, T394P 0.94 0.65 1.81 1.39 K3B-41 Y349S, V397M 1.82 1.27 2.11 1.38 G3A-13 K248E, A339T, T350A, S440N 2.10 1.28 2.89 1.37 J3B-44 K246E, K274E, V397M 1.59 1.05 2.38 1.37 K3B-43 A231P, K290E, S383N 1.66 1.43 2.15 1.36 J3B-107 T307P, A378T, N389T 1.50 1.32 2.05 1.36 K3B-33 K334R, K392R, S440G 4.87 0.77 0.97 1.33 K3A-11 K246E, K290E, T307P, N389T 1.73 1.65 2.00 1.33 K3B-49 D376G, S383R, V397M 2.54 1.34 2.57 1.33 3035879 29 G3A-95 1336T, A378V 1.05 2.25 1.82 1.31 K3B-90 V308A, K334R, A378V, K447N 1.32 1.30 3,72 1.27 A3A-146 1(290E, R355Q, S383N 1.49 1.38 2.15 1.26 A3A-11 S254F, K447N 4.78 1.18 1.02 1.22 G3A-31 K248E, K334Q 3.60 1.52 1.06 1.21 QL2A-16 K248E, N421T 5.30 0.79 0.77 1.19 J3A-43 K248E, P352L, Q418P 2.96 1.23 2.13 1.19 J3A-28 E333G, A378T, V397M 1.89 1.83 1.73 1.18 A3A-07 L365P, T366S, A378T 1.20 1.94 1.17 1.18 A3A-96 K248E, L365P, A378T 4.09 1.43 0.70 1.12 K3B-87 V3231, F405L 1.56 0.93 2.36 1.10 G3A-148 K248E, S375R 3.79 1.25 1.64 1.08 K3B-34 K334N, T394P, S408N, K414N 0.62 1.18 2.25 1.02 K3B-89 L309M, A378V 1.98 1.42 2.07 1.02 G3A-49 T307A, E380G 3.66 0.91 0.82 1.02 G3A-159 E382G, L432P, Q438DEL 3.00 0.91 1.13 0.98 G3A-28 T307P, H435R 3.49 1.06 0.95 0.96 K3B-78 H310R, P352L, Q418P 1.20 0.84 2.04 0.96 G3A-22 P352S, E382G, L432P, Y436N 3.11 0.95 0.62 0.94 G3A-56 K248E, N421T, K447N 3.25 0.71 1.03 0.89 G3A-145 E430G, K447N 4.34 1.01 0.81 0.88 A3A-21 S267N, N384D, N389S, P396L 4.32 1.00 0.56 0.88 G3A-33 K248E, S267N, I336M, P352L, P396L 5.67 0.79 0.39 0.76 G3A-51 V2401, K246E,P353L 3.90 0.95 0.51 0.64 Among these clones, 47 improved clones were used to construct the Mut5 library. From these results, 18 new variants were constructed by site-directed mutagenesis in order to accumulate mutations of interest (Table 2). Table 2: 18 new variants constructed and compared to reference variants NA = Not Determined Mutation Name ELISA Test Results Mutant Chi CD16aF CD16aV CD32aH CD32aR (I)32b A3A-105 G316D, K326E 5.18 1.31 2.48 1.50 4.50 7.13 A3A-105A G316D, K326E, T394P 15.33 1.09 1.45 0.66 5.60 1.21 A3A-105B G316D, K326E, P396L 14.30 2.25 3.52 3.92 12.64 4.16 A3A-105C G316D, K326E, V397M 9.61 2.90 1.86 1.81 11.00 2.22 A3A-105D G316D, K326E, A378V 13.32 2.46 3.68 1.39 8.20 5.96 3035879 30 A3A-184 K334N, P352S, V397M 1.44 1.71 2.90 1.34 3.06 2.91 A3A-184A K334N, P352S, A378V, V397M 1.75 3.40 4.74 3.25 5.03 2.32 DS3A-09 A378V 1.30 3.15 3.03 2.90 2.70 3.21 DS3A-09A A378V, T394P 1.24 2.29 2.83 2.26 2.00 1.26 DS3A-09B A378V, P396L NA 1,83 2.43 NA NA NA DS3A-09C A378V, V397M NA 1.48 2.25 NA NA NA G3A-43 A231V, A378V 1.13 1.79 2.44 1.42 3.57 1.96 G3A-43A A231V, A378V, T394P NA 1.13 1.65 NA NA NA G3A-43B A231V, A378V, P396L NA 1.85 2.42 NA NA NA G3A-43C A231V, A378V, V397M NA 1.81 2.68 NA NA NA G3A-45 K246R, A378V 1.15 1.92 2.46 1.38 2.76 1.68 G3A-45A K246R, A378V, T394P NA 1.34 2.34 NA NA NA G3A-45B K246R, A378V, P396L NA 1.37 1.75 NA NA NA G3A-45C K246R, A378V, V397M NA 1.51 2.21 NA NA NA G3A-95 1336T, A378V 1.05 1.29 2.25 1.17 1.82 1.31 G3A-95A 1336T, A378V, T394P NA 1.50 1.90 NA NA NA G3A-95B 1336T, A378V, P396L NA 2.00 2.54 NA NA NA G3A-95C 1336T, A378V, V397M NA 1.55 2.01 NA NA NA J3B-118 P396L, N421T 3.88 1.26 2.58 NA 9.00 4.99 J3B-118A A378V, P396L, N421T NA 1.76 3.37 NA NA NA QL3A-20 V397M 2.49 0.62 1.96 1.09 2.60 1.93 QS5A-66 T394P 2.64 NA 1.81 NA 2.05 2.67 Final, including 151 different test variants,26 variants of interest were selected to be produced on a larger scale and studied more precisely (Table 3). Table 3: 26 newly selected variants NA = Not Determined Name of Mutation ELISA Test Results Mutant Clq CD16 CD16a V CD32a H CD32n CD32 aF A3A-105 G316D, K326E 5.18 1.31 2.48 1.50 4.50 7.13 A3A-105D G316D, K326E, A378V 13.32 2.46 3.68 1.39 8.20 5.96 3035879 31 A3A-14 S298N, A378V 0.53 0.96 0.85 0.22 0.35 0.88 A3A-173 K248E, K334R, A378V NA NA 1.63 NA 2.96 2.76 A3A-184 K334N, P352S, V397M 1.44 1.71 2.90 1.34 3.06 2.91 A3A-184A K334N, P352S, A378V, V397M 1.75 3.40 4.74 3.25 5.03 2.32 A3A-31 K334R, L410R 6.23 NA 1.36 NA 1.57 1.50 DL4A-54 P352L, Q418P 1.35 1.18 1.49 1.09 1.58 2.30 G3A-103 K248E, A378V 2.54 1.17 2.00 0.83 1.96 1.64 G3A-139 T307N, V3231 1.30 0.81 1.10 0.65 2.57 1.41 G3A-43 A231V, A378V 1.13 1.79 2.44 1.42 3.57 1.96 G3A-45 K246R, A378V 1.15 1.92 2.46 1.38 2.76 1.68 G3A-88 K320E, T394P, G402D 1.32 NA 1.88 NA 4.94 2.21 G3A-95 1336T,A378V 1.05 1.29 2.25 1.17 1.82 1.31 J3A-06 A378T, V397M, V412M 1.60 1.24 1.65 1.58 2.74 1.41 J3A-14 K326T 1.83 0.52 1.43 0.99 4.25 2.26 J3A-16 N286Y, P352S, A378V 1.46 1.35 1.83 1.77 3.54 1.98 J3A-28 E333G, A378T, V397M 1.89 1.41 1.83 1.39 1.73 1.18 J3B-115 T359A, S383R, V397M 1.72 1.32 1.69 1.42 5.17 2.77 J3B-118 P396L, N421T 3.88 1.26 2.58 NA 9.00 4.99 J3B-118A A378V, P396L, N421T NA 1.76 3.37 NA NA NA J3B-16 S267G, A378T, V397M 0.85 0.59 0.57 0.42 4.95 1.58 J3B-23 N2861, A378V, F423Y 1.13 1.91 1.81 1.57 3.33 1.57 K3B-01 N315D, N361H, P396L 3.46 0.98 2.01 2.10 7.65 7.30 K3B-90 V308A, K334R, A378V, K447N 1.32 1.06 1.30 1.40 3.72 1.27 QL2A-16 K248E, N421T 5.30 0.69 0.79 0.33 0.77 1,19 Selections on the Mut5 bank: ELISA tests carried out on the 171 isolated clones made it possible to identify 87 clones with a ratio greater than 2 for at least one of the Fe Rs tested or the complement Clq 5 (Table 4). Table 4: 87 clones with a ratio greater than 2 Name of Mutation ELISA Test Results Mutant C lq CD16a CD16aV CD32a H CD32aR CD32 F b P3A-01 K334N, 5383R 0.93 1.34 2.05 1.10 2.00 1.57 P3A-17 K334N, Y373D, A378V 0.88 1.46 1.63 1.35 2.17 1.55 P3A-18 K248E, N286Y, K334R, A378V, K447N 24.7 0.94 1.62 1.07 1.24 1.13 7 P3A-23 N361H, P396L 2.70 1.75 2.49 2.53 3.33 1.76 3035879 32 P3A-30 K288E, K334N, P352S, V397M 1.18 1.35 4.33 2.14 1.79 1.41 P3A-31 K248E, T359A, S383R, V397M 5.80 0.80 2.05 1.80 1.62 1.51 P3A-50 T307P, A378V 1.11 1.55 2.89 2.20 1.85 1.44 P3A-56 G316D 1.74 1.07 1.90 2.02 1.45 1.39 P3A-70 K274R, A378V, V397M 1.13 1.93 4.05 3.54 4.89 3.20 P3A-74 V302A, V397M 0.92 0.72 0.63 1.47 2.43 1.32 P3A-94 L309P, T359A, S383R, V397M 1.02 1.32 2.11 2.54 1.79 1,32 N3A-111 V302A, K334N, S375R 1.36 0.82 1.18 1.20 2.73 2.33 N3A-113 E283G, Y349S, P396L 6.55 1.30 1.73 2.17 5.19 2.94 N3A-114 K320E, S375R, F405V 0.69 1.03 1.12 0.92 2.11 1.60 N3A-117 K334N 1.32 1.61 2.28 1.31 1.93 1.98 N3A-123 K326E 3.15 1.03 2.21 1.27 7.71 3.53 N3A-132 S375R, N389S 1.71 0.99 1.10 1.41 3.50 2.35 N3A-133 K334N, P352S, K447N 0.77 1.02 2.23 0.88 1.25 1.40 N3A-138 S298T, K334R, K370E 3.56 0.90 0.68 0.59 0.73 1.17 N3A-14 T307N, A378V, K414R 1.00 1.70 2.83 2.50 2.49 1.74 N3A-141 T350A, V379A, G385R, P396L 2.82 1.30 1.79 2.36 5.60 5.70 N3A-145 S267G, K290E, E293G, E380G, V397M 0.99 0.70 0.38 0.31 2.45 1.33 N3A-150 S267G, K334N, P352S, V397M 0.86 0.76 0.68 0.33 4.82 1.78 N3A-161 K248E, N315D, A378V 8.59 1.28 2.49 1.12 1.23 1.32 N3A-175 S375R, F405V 0.94 1.16 2.47 1.99 1.30 1.10 N3A-177 V282A, K334R, T394P 5.95 1.15 1.28 1.68 3.22 1.68 N3A-190 T307P, T394P 2.46 1.35 2.46 2.93 3.33 1.94 N3A-27 K290E, A378V, K392R, S440G 1.67 2.25 3.77 2.05 2.59 1.49 N3A-28 T366A, A378V 1.04 177 2.89 2.26 1.84 1.36 N3A-32 K326T, K334N, P352S, N421T 1.61 2.47 4.35 1.59 3.24 2.04 N3A-52 K326T, S383R, V397M 3.70 1.11 2.07 1.92 5.40 2.41 N3A-58 V284L, K334R, A378V, K447N 5.37 1.02 1.67 2.01 4.00 1.67 3035879 33 N3A-59 N315D, T366A, G385R, P396L 1.08 1.25 1.66 1.75 2.73 1.56 N3A-61 V308A, K334R, A378V 2.07 1.10 1.57 1.11 2.87 2.67 N3A-74 K288R, T394P 1.38 1.22 1.51 1.58 2.15 1.73 N3A-85 K334R, V397M 4.96 1.11 1.45 1.89 3.75 2.77 N3A-87 K290E, K320E, T350A, P396L 0.91 2.15 2.85 3.12 5.94 4.63 N3A-93 A378T, P396L 1.63 1.84 2.21 2.55 3.86 3.06 03A-04 A231V, T359A, S383R, V397M 1.00 1.16 1.21 1.69 2.35 1.09 03A-05 K290E, T366A, A378V 0.99 2.79 3.12 2.07 3.34 1.34 03A-10 Y300H, T394P 0.98 0.76 0.90 1.81 2.25 1.17 03A-11 R255W, A287T, P352S, A378V, N421T 1.85 1.01 2.10 2.20 3.15 1.55 03A-16 K334R, A378V, K447N 4.30 1.71 2.51 2.43 3.98 1.59 03A-17 1336V, T359A, S383R, V397M 0.85 1.79 2.70 2.30 1.73 0.93 03A-22 K334R, P396L, H435R 23.5 1.68 2.16 2.75 4.79 1.62 5 03A-24 K326T, K447N 212 1.23 2.27 1.55 2.94 1.40 03A-25 V308A, K334R, A378T, V397M 11.1 1.40 2.07 2.04 4.16 1.96 2 03A-34 A231V, Y349S, V397M 0.73 1.40 1.90 2.34 2.62 1.28 03A-38 K248E, N286Y, Q418P 2.17 1.59 1.16 0.99 0.69 0.57 03A-40 V308A, A378T, V397M, V412M 0.82 1.65 2.15 2.09 2.64 1.03 03A-42 S304N, A378V 1.04 2.16 0.53 0.87 0.87 1.77 03A-44 A378V, K439R 1.95 2.44 4.40 3.20 3.13 1.81 03A-45 D270N, K334R, L41OR 3.32 1.57 0.61 0.56 1.19 1.36 03A-46 V302A, A378V 0.94 1.15 1.75 2.36 2.91 1.37 03A-50 K334R, A378V, N421T 3.62 1.28 2.49 2.15 4.02 2.30 03A-57 T359A, V397M 1.50 1.18 2.27 2.23 2.78 1.59 03A-66 A378T, T394P 1.10 1.42 2.42 2.36 2.45 1.76 03A-67 K248E, N361H 2.05 0.89 1.36 1.06 1.07 1.76 03A-69 1(290E, A378V) 1.44 2.64 4.04 2.62 4.11 2.06 03A-80 K274R, T394P, G402D, K447N 1.26 1.26 1.81 2.16 2.32 1.28 03A-81 K274R, A378T, V397M 1.36 1.20 2.39 2.06 3.08 1.58 03A-86 K248E, 1(290E, 24.3 2.49 4.89 2.86 4.19 2.57 3035879 34 N361H, P396L 4 03A-93 1(290E, T394P 2.07 1.45 3.53 2.54 3.36 1.78 P4B-89 T307PT366A, A378V 0.63 2.99 4.15 0.70 1.19 0.87 Q4B-08 V284L, K290E, A378V 2.43 1.75 3.47 2.69 3.58 1.63 Q4B-15 K274R, A378V 1.26 1.04 2.69 2.61 1.75 1.03 Q4B-18 I377T, A378V, F423Y 1.21 1.08 2.82 1.96 1.37 1.07 Q4B-34 K248E, K290M, V308A, P352S 4.47 0.63 0.56 0.38 0.70 0.75 Q4B-59 T350A, A378T, V3971 1.32 2.22 2.01 2.76 2.52 1.51 Q4B-61 K320E, T394P, V397M 0.87 1.10 0.98 1.60 2.43 1.08 Q4B-68 T307P, A378V, T394P 3.33 1.53 4.12 4.01 3.69 1.03 Q4B-91 K334R, A378V 2.81 1.14 2.35 2.61 2.46 0.94 Q3A-01 P352L, A378V 1.13 1.14 1.53 2.04 1.81 0.87 Q3A-39 N286I, P396L, N421T 2.80 1.63 1.99 3.52 3.09 1.02 Q3A-58 S267G, V397M 0.73 1.30 0.34 0.40 2.87 1.02 Q3A-85 P396L, N421T, K447N 3.80 1.65 2.17 3.67 3.50 1.13 03A-103 K290E, K320T, A378V 2.11 1.22 2.08 2.76 2.41 1.50 03A-117 K334R T394P, N421S 0.98 1.05 1.71 2.29 3.05 1.37 03A-119 K334N, V397M 1.53 1.04 1.97 1.45 2.28 1.18 03A-126 P227S, V284L, A378V 4.58 1.31 2.93 2.85 4.47 1.29 03A-127 V302A, K334R, T366A, S383R, V397M 1.53 1.00 1.24 2.85 356 1.37 03A-131 C261R, A378T, V397M, V412M 2.25 1.03 1.72 2.00 2.27 1.15 03A-137 K248E, T350A 0.84 0.66 0.43 0.68 2.06 0.97 03A-172 K320E, T394P 8.19 0.88 1.46 2.01 2.52 1.06 03A-179 R255Q, G385R 0.81 0.74 0.83 0.57 2.86 1.30 03A-186 T307P, V397M 1.09 1.41 2.32 2.17 3.97 1.48 03A-99 N276S, N286I, T359A, S383R, V397M 2.14 1.32 2.27 2.84 3.09 1.42 Among these variants, 10 variants of interest were selected (Table 5). Table 5: 10 variants 3035879 Name of Mutations ELISA test results mutant Cl g CD16a CD16a CD32 aH CD32a R CD321) FV 03A-05 K290E, T366A, A378V 0.9 2.79 3.12 2.07 3.34 1.34 9 P4B-89 T307P, T366A, A378V 0.6 2.99 4.15 0.70 1.19 0.87 3 03A-86 K248E, K290E, N361H, P396L 24, 2.49 4.89 2.86 4.19 2.57 34 N3A-32 K326T, K334N, P352S, N421T 1.6 2.47 4.35 1.59 3.24 2.04 1 03A-44 A378V, K439R 1.9 2.44 4.40 3.20 3.13 1.81 5 N3A-27 1(290E, A378V, K392R, S440G 1.6 2.25 3.77 2.05 2.59 1.49 7 N3A-87 K290E, K320E, T350A, P396L 0.9 2.15 2.85 3.12 5.94 4.63 1 Q4B-68 T307P, A378V,T394P 3.3 1.53 4.12 4.01 3.69 1.03 3 N3A-58 V284L, K334R, A378V, K447N 5.3 1.02 1.67 2.01 4.00 1.67 7 Q3A-39 N2861, P396L, N421T 2.8 1.63 1.99 3.52 3.09 1.02 0 3. Production and purification of variants of interest IgG variants are obtained by direct mutagenesis in pCEP4-WT-H-CD20. The IgG controls, i.e., Cl, C3, C4, C5, and wild-type (WT), were produced with the G1m3 5 allotype (containing 3 mutations compared to Glm1,17: (K214R / )D356E / L358M). The 26 IgG variants from the Mut4sel library, as well as the wild-type, were produced with the Glm1,17 allotype. They were produced by incubation for 6–7 days in batches (250–300 mIU) of 293-E cells (Freestyle Invitrogen) in F17 medium. Centrifugation and filtration were then performed. Purification was carried out using Protein A Hi-Trap, and elution was performed with 25 mM citrate buffer at pH 3.0, followed by neutralization and dialysis in TBS or PBS before sterilization. 10mg of each IgG control, i.e. Cl, C3, C4,C5 and wild-type (WTG1m3 and WTG1m1,17) are obtained, as well as 2-3 mg of 26 IgG variants. 15 Their characterization shows that the molecular weight is conserved,and that the glycosylation profiles are similar for all variants. 4. Variant Tests of Interest 3035879 36 4.1. FcRn Binding Assays Jurkat-FcRn cells are incubated at pH 6.0 with the IgG variants at different concentrations (0 to 10001 µtg / m¹) and Rituximab-Alexa. Flow cytometry is performed on the bound Rituximab-Alexa. 5 The results show no loss of FcRn binding for any of the IgG variants. 4.2 Antigen Binding Assays Raji cells are incubated with the IgG variants at 1 µtg / m¹ for 15 minutes at 4°C. Bound IgG is detected by binding with an anti-human IgG-PE secondary antibody (for 15 minutes at 4°C). The results show that antigen recognition is not altered by the 15 different mutations present on the Fc. All IgG variants bind to CD20 on cells.similarly to the IgG-WT control. 4.3 CD16aV / F Binding ELISA Assays 20 Purified antibodies were tested by ELISA for their binding to CD16F and CD16aV according to a protocol identical to that described in 2.6, diluting the antibodies to different concentrations. 4.4 ADCC Activity Assays 25 NK cells were incubated with Raji target cells expressing CD20, in the presence of different concentrations of IgG variants (0.005 to 5000 ng / ml). The level of intracellular LDH released by the lysed target cells was measured. Human NK cells were purified from peripheral blood of healthy volunteer donors using the negative depletion technique developed by Miltenyi. The ADCC assay 30 involves incubating NK cells with Raji target cells expressing the CD20 antigen, in the presence of different concentrations of anti-CD20 antibodies. After 16 hours of incubation,Anti-CD20 antibody-induced cytotoxicity was measured by quantifying intracellular lactate dehydrogenase (LDH) released by lysed target cells in cell supernatants. Specific lysis results are expressed as a percentage of lysis as a function of antibody concentration. EC50 (antibody concentration inducing 50% of the maximum lysis induced by IgG-WT) and Emax (percentage of maximum lysis) values ​​were calculated using GraphPad PRISM software. The results are presented in Tables 6 and 7. Table 6: ADCC assay results Mutant G3A4 G3A4 J3B-118 J3B16 A3A-184 WT 3 5 EC50: 1.03 4.38 1.27 > 5545.36 2.35 12,Table 7: ADCC Test Results EC50 Mutant: Antibody Concentration Ratio [WT] / [antibody] (ng / ml) giving 50% lysis of an internal control 11.98 2.82 9.72 <0.0022 5.25 Table 7: ADCC Test Results EC50 Mutant: Antibody Concentration Ratio [WT] / [antibody] (ng / ml) giving 50% maximum lysis of an internal control G3A-103 0.31 33.90 A3A-184A 0.34 30.91 J3B-23 0.57 18.44 J3A-28 0.57 18.44 K3B-01 0.69 15.23 A3A-14 0.82 12.82 G3A-95 0.97 10.84 J3B-118A 0.97 10.84 J3A-16 1.27 8.28 J3A-06 1.27 8.28 A3A-105D 1.27 8.28 G3A-139 2.80 3.75 3035879 38 QL2A-16 4.36 2.41 J3A-14 7.39 1.42 G3A-88 8.07 1.30 WT 10.51 1 K3B-87 16.33 0.64 DL4A-54 >5000 <0.002 A3A-90 >5000 <0.002 A3A-34 >5000 <0.002 The best variants are A3A-184A and G3A-103. 4.5. CDC Activity Assays 5 Raji target cells expressing the CD20 antigen were incubated with different concentrations of anti-CD20 antibodies (0 to 5000 ng / ml) in the presence of rabbit serum as a complement source (Cedarlane,(1 / 10 dilution). After 1 hour of incubation at 37°C, the level of LDH released into the supernatant by the lysed target cells was chromogenically measured (Roche Applied 10 Sciences cytotoxicity detection kit) and used to quantify antibody-mediated complement-dependent cytotoxicity. The results are expressed as a percentage of lysis. EC50 (quantity of antibody that induces 50% of maximum lysis) and Emax (percentage of maximum lysis) were calculated using GraphPad PRISM software. 15 The results are presented in Tables 8 and 9. Table 8: CDC test results Antibody Concentration Ratio [WT] / [antibody] of antibodies (ng / ml) giving 50% lysis of an internal control A3A-173 16.01 16.36 K3B-90 23.12 11.33 J3B-118 38.69 6.77 A3-105 64.76 4.05 A3A-31 108.37 2.42 J3B-115 145.44 1.80 3035879 39 G3A-43 168.49 1.55 G3A-45 195.20 1.34 A3A-184 210.10 1.25 J3B-16 210.10 1.25 WT 261,97 1 Table 9: CDC test results Anticorporeal Concentration of Ratio [WT] / [anticorps] Anticorpores (ng / ml) less than 50% of internal control J3B-118A 21,564 10.98 J3A-28 31,004 7.64 G3A-103 A3A-184A 33,339 7.10 A3A-105D 38,550 6.14 QL2A-16 51,544 4.59 K3B-01 59,600 3.97 74,107 3.20 G3A-139 85,691 2.76 J3B-23 99,085 2.39 K3B-87 106,548 2.22 A3A-34 123,202 1.92 J3A-16 132,481 1.79 DL4A-54 164,727 1.44 G3A-95 177,134 1.34 WT 236,837 1 G3A-88 273,856 0.86 A3A-14 340,513 0.70 A3A-90 566,098 0.42

Claims

CLAIMS 1. Polypeptide comprising a mutated Fc region and exhibiting functional activity mediated by the modified Fc region compared to that of a parent polypeptide, characterized in that said Fc region comprises at least one combination of 2 mutations, said combination being selected from: (i) a mutation selected from 307N, 326E, 326T, 334N, 334R, 352L, 378V, 378T, 394P, 396L, 397M, 421T;and (ii) at least one mutation selected from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 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 mutation (i) does not take place on the same amino acid as mutation (ii).; 2. Polypeptide according to claim 1, characterized in that said Fc region-mediated functional activity is selected from antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell phagocytosis (ADCP), and a combination of at least two of these activities.

3. Polypeptide according to claim 1 or 2, characterized in that it exhibits increased Fc region-mediated functional activity compared to that of the parent polypeptide.

4. Polypeptide according to any one of claims 1 to 3, characterized in that it has an increased Fc region-mediated functional activity, compared to that of the parent polypeptide, of a ratio of at least 2, preferably greater than 5, of 3035879 41 preferably greater than 10, preferably greater than 15, preferably greater than 20, preferably greater than 25, preferably greater than 30.

5. Polypeptide according to claim 1 or 2, characterized in that it has decreased Fc region-mediated functional activity compared to that of the parent polypeptide.

6. Polypeptide according to claim 1, 2 or 5, characterized in that it has a decreased Fc region-mediated functional activity, compared 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.

7. Polypeptide according to any one of claims 1 to 6, characterized in that said mutated Fc region has a modified affinity for at least one of the Fc region receptors (FcR) selected from complement C1q and the FcgRIIIa (CD16a), FcgRIIa (CD32a), and FcgRIIb (CD32b) receptors.

8. Polypeptide according to claim 7, characterized in that said mutated Fc region has an enhanced affinity for at least one of the Fc region receptors (FcR), selected from complement C1q and the FcgRIIIa (CD16a), FcgRIIa (CD32a), and FcgRIIb (CD32b) receptors.

9. Polypeptide according to claim 7, characterized in that said mutated Fc region has a decreased affinity for at least one of the Fc region receptors (FcR), selected from complement C1q and the FcgRIIIa (CD16a), FcgRIIa (CD32a), and FcgRIIb (CD32b) receptors.

10. Polypeptide according to any one of claims 1 to 9, characterized in that said mutated Fc region comprises from 2 to 20 mutations relative to said parent polypeptide, preferably from 2 to 10 mutations. 3035879 42 11 Polypeptide according to any one of claims 1 to 8 or 10, characterized in that said mutated Fc region has an improved affinity for complement C1q, and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 378V, 378T, 396L, 421T, 334R and 326E; and ii) at least one mutation selected from 361H, 290E, 316D, 248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T, 396L, 2861, 315D and 397M, the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii).

12. Polypeptide according to any one of claims 1 to 8 or 10, characterized in that said mutated Fc region has an enhanced affinity for the FcgRIIIa receptor (CD16a),and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 378V, 326E, 397M, 334N and 396L; and ii) at least one mutation selected from 316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P, 307P, 378V, 378T, 2861, 286Y and 298N, the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii).

13. Polypeptide according to any one of claims 1 to 8 or 10, characterized in that said mutated Fc region has an increased affinity for the FcgRIIa receptor (CD32a), and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 378V, 326E, 397M, 307N, 394P, 326T, 396L and 334N; and ii) at least one mutation chosen from: 316D, 334R, 334N, 3231, 231V, 246R, 336T, 378T, 286Y, 2861, 352S, 383R, 359A, 421T, 361H, 315D, 366A, 290E, 307P and 439R,the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii).

14. Polypeptide according to any one of claims 1 to 8 or 10, characterized in that said mutated Fc region has an increased affinity for the FcgRIIb receptor (CD32b), and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 326E, 326T, 378V, 397M, 352L, 394P, 396L and 421T; and 3035879 43 ii) at least one mutation selected from 316D, 334R, 248E, 334N, 418P, 231V, 320E, 402D, 359A, 383R, 421T and 361H, the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii).

15. Polypeptide according to any one of claims 1 to 4 or 10, characterized in that said mutated Fc region has increased CDC activity, and comprises at least a combination of 2 mutations,said combination comprising: i) a mutation selected from 378V, 378T, 396L, 421T, 334R and 326E; and ii) at least one mutation selected from 361H, 290E, 316D, 248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T, 396L, 2861, 315D and 397M, the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii).

16. Polypeptide according to any one of claims 1 to 4 or 10, characterized in that said mutated Fc region has increased ADCC activity, and comprises at least one combination of 2 mutations, said combination comprising: i) a mutation selected from 378V, 326E, 397M, 334N and 396L; and ii) at least one mutation selected from 316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P, 307P, 378V, 378T, 2861, 286Y and 298N,the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii).

17. Polypeptide according to any one of claims 1 to 16, characterized in that said mutated Fc region comprises at least one combination of 3 mutations, said combination comprising: (i) a mutation selected from 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and (ii) at least 2 mutations chosen from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 266M, 287T, 312G, 334R, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 259A, 261R, 262A, 283G, 284L, 2861, 308A, 3081, 308G, 3231, 325S, 333G, 350A 352S, 359A, 263A, 286Y, 309P, 334N, 361H, 3035879 44 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, 43611, 439R, 440G, 440N, 442F, 442P, and 447N, numbering being that of the EU index or equivalent in Kabat, and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii), preferably said mutated Fc region comprises a combination of 4 mutations, said combination comprising at least one mutation (i), and at least 3 mutations (ii), and preferably a combination of 5 mutations, said combination comprising at least one mutation (i), and at least 4 mutations (ii).

18. Polypeptide according to any one of claims 1 to 17, characterized in that the parent polypeptide comprises a parent Fc region which is a human Fc region, preferably an Fc region of a human IgG1 or a human IgG2.

19. Polypeptide according to any one of claims 1 to 18, characterized in that it is selected from an isolated Fc region,20. A sequence derived from an isolated Fc region, an antibody, and a fusion protein comprising an Fc region.

21. A polypeptide according to any one of claims 1 to 19, characterized in that it consists of an Fc region.

22. A polypeptide according to any one of claims 1 to 19, characterized in that it is an antibody.

23. A polypeptide according to any one of claims 1 to 21, characterized in that it is a polypeptide produced in the milk of transgenic animals.

24. A polypeptide composition according to any one of claims 1 to 22, characterized in that the purified polypeptides of said composition possess, at their Asn297 glycosylation site, N-glycans having a fucosylation rate of less than 65%, preferably less than 50%, and preferably even less than 40%. 3035879 45 24. Composition of polypeptides according to claim 23,characterized in that the purified polypeptides of said composition possess at their Asn297 glycosylation site a biantene-type glycan structure, with short chains, low sialylation, exhibiting terminal mannose and / or non-intercalated terminal N-acetylglucosamines.

25. Polypeptide composition according to claim 24, characterized in that the purified polypeptides of said composition have a content greater than 60% for the G0+G1+G0F+G1F forms, the G0F+G1F forms being less than 50%.

26. Polypeptide composition according to claim 24, characterized in that the purified polypeptides of said composition have a content greater than 60% for the G0+G1+G0F+G1F forms, the fucose content being less than 65%.

27. Polypeptide composition according to claim 24,characterized in that the purified polypeptides of said composition have a content of less than 40% for the G1F+GOF forms.

28. Pharmaceutical composition comprising (i) a polypeptide according to any one of claims 1 to 22 or a composition according to any one of claims 23 to 27, and (ii) at least one pharmaceutically acceptable excipient.

29. Polypeptide according to any one of claims 1 to 22 or composition according to any one of claims 23 to 27, for use as a medicament.

30. Antibody according to claim 21, 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.

31. Antibody according to claim 30 directed against a tumor antigen for use in the treatment of cancers.

32. Antibody according to claim 30 directed against a viral antigen for use in the treatment of viral infections.

33. Antibody according to claim 30 directed against a bacterial toxin for use in the treatment of bacterial infections.

34. Antibody according to claim 30 directed against a cytokine for use in the treatment of inflammatory and / or autoimmune diseases.

35. Method for producing a polypeptide comprising an Fc region and having functional activity mediated by the modified Fc region compared to that of a parent polypeptide, said method comprising a step of introducing at least one combination of 2 mutations, said combination being selected from: (i) a mutation selected from 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T,334N, 334R, 307N et 394P; And there are a number of different mutations: 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 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, et 447N,the numbering being that of the EU index or equivalent in Kabat and with the condition that mutation (i) does not occur on the same amino acid as mutation (ii).

36. Method for increasing the binding of a polypeptide comprising an Fc region to at least one of the Fc region receptors (FcR) selected from the Clq receptor, FcgRIIIa (CD16a), and FcgRIIa (CD32a), and FcgRIIb (CD32b), said method comprising a step of introducing at least one combination of 2 mutations, said combination being selected from: (i) a mutation selected from 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and 3035879 47 (ii) at least one mutation selected from 226Y, 227S, 230S, 231V, 234P, 2431, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 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 mutation (i) does not occur on the same amino acid as mutation (ii).