FC MUTANTS WITH MODIFIED FUNCTIONAL ACTIVITY

Description

[0001] The present invention relates to a polypeptide comprising a mutated Fc region and exhibiting functional activity mediated by the modified Fc region relative to a parent polypeptide.

[0002] 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 includes two subtypes, lambda and kappa.

[0003] IgG are soluble antibodies 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.

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

[0005] 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 antibodies. Monoclonal antibodies are obtained from a single antibody-producing cell line, which shows identical specificity for a single antigen. Therapeutic Fc fusion proteins are used or developed as drugs for autoimmune diseases, such as etanercept (Amgen's Enbrel, which is a TNF receptor linked to an Fc fragment) or Alefacept (Biogen's Amevive, which is LFA-3 linked to the Fc portion of human IgG1).

[0006] For antibodies and Fc fusion proteins to exert their effects, it is necessary that the Fc fragment exhibits the best possible activity (e.g., antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, or antibody-dependent cellular phagocytosis), i.e., that it is optimized. Such optimization would result in a protein with improved activity and / or efficacy and / or reduced side effects.

[0007] WO2010 / 106180 describes Fc variants comprising mutations.

[0008] The Applicant has now developed specific Fc fragments with improved activity. These fragments can be used in therapy, with a view to providing greater efficacy to the product containing them.

[0009] The present invention thus provides a variant of a parent polypeptide having optimized properties relating to Fc region-mediated functional activity.

[0010] The present invention relates to a method according to claim 1, and to a polypeptide comprising a mutated Fc region according to claim 6. Such a polypeptide is called "polypeptide according to the invention" in the present application.

[0011] The description discloses a polypeptide comprising a mutated Fc region and exhibiting a functional activity mediated by the modified Fc region compared to that of a parent polypeptide, said Fc region comprising at least one mutation 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, K290E, 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, Y319H, C226Y, F405L, T393I, N434S, R255W, A287T, N286Y, A231V, K274R, V308G, K414R, M428T, E345G, F243L, P247T, Q362R, S440N, Y278H, D312G, V262A, V305A, K246R, V308I, 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, the numbering being that of the EU index or equivalent in Kabat.,

[0012] Also described is a polypeptide comprising a mutated Fc region and exhibiting a functional activity mediated by the modified Fc region compared to that of a parent polypeptide, said Fc region comprising at least one combination of 2 mutations, said combination being selected from: (i) a mutation chosen from 307N, 326E, 326T, 334N, 334R, 352L, 378V, 378T, 394P, 396L, 397M and 421T; (ii) at least one mutation selected from 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 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).

[0013] Preferably, the mutation (i) is selected from 378V, 396L and 397M. Preferably, the polypeptide further comprises a mutation selected from 248E, 326T, 333G and 423Y.

[0014] Preferably, mutation (ii) is selected from 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 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.

[0015] Throughout this application, the numbering of residues 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 term "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 ).

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

[0017] "Amino acid" means any of the 20 naturally occurring amino acids or unnatural 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 the common sense. It corresponds 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, chimeric antibodies, humanized antibodies, and fully human antibodies. The amino-terminal portion of each heavy chain includes a variable region of approximately 100 to 110 amino acids responsible for antigen recognition. Within each variable region, three loops are brought together to form an antigen-binding site. Each of the loops is called a complementarity-determining region (hereinafter referred to as a "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. IgM subclasses include IgM1 and IgM2. Thus, an "isotype" refers to one of the immunoglobulin subclasses defined by the chemical and antigenic characteristics of their constant regions. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.

[0021] Full-length IgGs are tetramers and consist of two identical pairs of two immunoglobulin chains, each pair having a light chain and a heavy chain, each light chain comprising the VL and CL domains, and each heavy chain comprising the VH, Cγ1 (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 IgG heavy chain also includes an N-terminal flexible 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."Variable region" means the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the VK, Vλ, and / or VH genes that comprise the immunoglobulin kappa, lambda, and heavy chains, respectively. Variable regions include complementarity-determining regions (CDRs) and framework regions (FRs).

[0022] The term "Fc" or "Fc region" refers to the constant region of an antibody excluding the first immunoglobulin constant region domain (CH1). Thus Fc refers to the last two domains (CH2 and CH3) of the IgG1 constant region, and to the N-terminal flexible hinge of these domains. For human IgG1, the Fc region corresponds to residue C226 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 to 226 according to the EU index or equivalent in Kabat; in this case, the Fc region used corresponds to residues 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 is according to the EU index or equivalent in Kabat.Preferably in this case, the Fc region used corresponds to residues from position 216 to 447, where the numbering is according to the EU index or equivalent in Kabat.

[0023] Preferably, the Fc region used is chosen from the sequences SEQ ID NO: 1 to 10.

[0024] By "parent polypeptide" is meant 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, called "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 of a human IgG1. The parent polypeptide may comprise 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 limiting.By "sequence derived from an isolated Fc region" is meant a sequence comprising at least two isolated Fc regions linked together, such as a scFc (single chain Fc) 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" is meant a compound which 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 generally uses functional groups on the Fc region and the conjugation partner.Various linking groups are known in the prior art as being suitable for the synthesis of a conjugate; for example, homo- or heterobifunctional linking 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, but are not limited to, diphtheria toxin, exotoxin A, ricin, abrin, saporin, gelonin, calicheamicin, auristatins E and F and mertansine.

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

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

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

[0028] By "mutation" is meant a change of at least one amino acid in the sequence of a polypeptide, in particular a change of 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 polypeptide according to the invention. Such a polypeptide comprises a mutated Fc region, compared to the parent polypeptide. Preferably, the mutation is a substitution, an insertion or a 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 substitution N434S refers to a variant polypeptide, in this case a variant for which the 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. By “amino acid deletion” or “deletion” is meant the deletion of an amino acid at a particular 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”, and means that the parent polypeptide includes 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 polypeptide according to the invention exhibits a functional activity mediated by the modified Fc region compared to that of the parent polypeptide.

[0030] By "functional activity mediated by the Fc region" is meant in particular the effector functions. The functional activity mediated by the Fc region thus notably includes antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), endocytosis 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 chosen 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).

[0031] The functional activity mediated by the Fc region of the polypeptide according to the invention is increased compared to that of the parent polypeptide.

[0032] The polypeptide according to the invention has an increased Fc region-mediated functional activity compared to that of the parent polypeptide. Preferably, the polypeptide according to the invention has an increased Fc region-mediated functional activity, compared to that of the parent polypeptide, by a ratio at least equal to 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.

[0033] 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) chosen from complement C1q, FcgRIIIa (CD16a), FcgRIIa (CD32a), and FcgRIIb (CD32b). Complement C1q is indeed involved in CDC activity. The FcgRIIIa receptor (CD16a) is involved in ADCC; it has a V / F polymorphism at position 158. The FcgRIIa receptor (CD32a) is involved in platelet activation and phagocytosis; it has an H / R polymorphism at position 131. Finally, the FcgRIIb receptor (CD32b) is involved in the inhibition of cellular activity.

[0034] Preferably, said mutated Fc region has an increased affinity for at least one of the FcRs. Preferably, the affinity is increased, relative to that of the parent Fc, by a ratio at least equal to 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. Alternatively, said mutated Fc region has a decreased affinity for at least one of the FcRs. Preferably, the affinity is reduced, compared to that of the parent Fc, by a ratio at least equal to 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 lower than that of the parent polypeptide.

[0035] The affinity of a polypeptide comprising an Fc region for an FcR can be assessed by methods well known in the prior art. For example, one skilled in the art can determine the affinity (Kd) using surface plasmon resonance (SPR). Alternatively, one 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 detected signals specific to the mutated Fc and the parent Fc are compared. The binding affinity can be determined either by assessing the whole polypeptides or by assessing the isolated Fc regions thereof.

[0036] Also disclosed is a mutated Fc region of the polypeptide comprising from 1 to 20 mutations relative to the parent polypeptide, preferably from 2 to 20 mutations. By "from 1 to 20 amino acid changes" is meant 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.

[0037] Also disclosed is a mutated Fc region of the polypeptide comprising at least one combination of 2 mutations, said combination being selected from: (i) a mutation selected from 378V, 396L and 397M; (ii) at least one mutation selected from 231V, 248E, 286I, 286Y, 290E, 298N, 308A, 315D, 316D, 326E, 333G, 334N, 334R, 336T, 352S, 361H, 366A, 378T, 396L, 397M, 412M, 421T, 423Y 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).

[0038] Also disclosed is a mutated Fc region of the polypeptide comprising at least one combination of 2 mutations, said combination being selected from: (i) a mutation selected from 378V, 396L and 397M; (ii) at least one mutation selected from 248E, 316D, 326E, 333G, 378T, 396L and 421T, 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).

[0039] Also disclosed is a polypeptide comprising a mutated Fc region exhibiting increased ADCC and CDC functional activities compared to that of a parent polypeptide, characterized in that said Fc region comprises at least one combination of 2 mutations, said combination being chosen from: (i) a mutation selected from 378V, 396L and 397M; (ii) at least one mutation selected from 248E, 316D, 326E, 333G, 378T, 396L and 421T, 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).

[0040] Also disclosed is a mutated Fc region of the polypeptide comprising at least one combination of 2 mutations, said combination being selected from: (i) the 378V mutation; (ii) at least one mutation selected from 298N and 336T, 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).

[0041] Also disclosed is a mutated Fc region of the polypeptide comprising at least one combination of 2 mutations, said combination being selected from: (i) a mutation selected from 378V, 396L and 397M; (ii) at least one mutation selected from 231V, 286I, 286Y, 290E, 315D, 334N, 352S, 361H, 366A, 378T, 397M, 412M, 421T and 423Y, 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).

[0042] Also disclosed is a polypeptide comprising a mutated Fc region exhibiting increased ADCC functional activity relative to that of a parent polypeptide, characterized in that said Fc region comprises at least one combination of 2 mutations, said combination being chosen from: (i) a mutation selected from 378V, 396L and 397M; (ii) at least one mutation selected from 231V, 286I, 286Y, 298N, 290E, 315D, 334N, 336T, 352S, 361H, 366A, 378T, 397M, 412M, 421T and 423Y, 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).

[0043] Preferably, the mutated Fc region of the polypeptide according to the invention comprises at least one combination of 2 mutations, said combination being chosen from: (i) mutation 378V; (ii) at least one mutation selected from 248E, 308A, 334R, 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).

[0044] Preferably, the polypeptide according to the invention comprises a mutated Fc region exhibiting increased CDC functional activity compared to that of a parent polypeptide, characterized in that said Fc region comprises at least one combination of 2 mutations, said combination being chosen from: (i) mutation 378V; (ii) at least one mutation selected from 248E, 308A, 334R, 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).

[0045] Also disclosed is a mutated Fc region of the polypeptide comprising a combination of mutations selected from the combinations: K320E / T394P / G402D K290E / K320E / T350A / P396L T359A / S383R / V397M

[0046] Also disclosed is a mutated Fc region of the polypeptide having improved affinity for complement C1q, and comprising 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, 286I, 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).

[0047] Also disclosed is a mutated Fc region of the polypeptide having improved affinity for the FcgRIIIa receptor (CD16a), and comprising 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, 286I, 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).

[0048] Also disclosed is a mutated Fc region of the polypeptide having increased affinity for the FcgRIIa receptor (CD32a), and comprising 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 selected from: 316D, 334R, 334N, 323I, 231V, 246R, 336T, 378T, 286Y, 286I, 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).

[0049] Preferably, mutation (ii) is selected from: 316D, 334R, 334N, 323I, 231V, 246R, 336T, 378T, 286Y, 286I, 352S, 383R, 359A, 421T, 361H, 366A, 290E, 307P and 439R.

[0050] Also disclosed is a mutated Fc region of the polypeptide having increased affinity for the FcgRIIb receptor (CD32b), and comprising 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 (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).

[0051] Also disclosed is a mutated Fc region of the polypeptide having increased CDC activity, and comprising 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, 286I, 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).

[0052] Also disclosed is a mutated Fc region of the polypeptide having increased ADCC activity, and comprising 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, 286I, 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).

[0053] Also disclosed is a mutated Fc region of the polypeptide comprising at least one combination of 3 mutations, said combination comprising: (i) une mutation choisie parmi 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N et 394P ; et (ii) au moins 2 mutations choisies parmi 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 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).

[0054] Preferably, the at least 2 mutations (ii) are selected from 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 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.

[0055] More preferably, the mutated Fc region of the polypeptide comprises at least one 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).

[0056] More preferably, the mutated Fc region of the polypeptide comprises at least one combination of 5 mutations, said combination comprising at least one mutation (i) as described above, and at least 4 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).

[0057] The present invention also relates to a composition of polypeptides according to the invention, said purified polypeptides having on their Asn297 glycosylation site N-glycans whose fucosylation level is less than 65%, preferably less than 50%, more preferably less than 40%. Preferably, said purified polypeptides have on their Asn297 glycosylation site a biantennary type glycan structure, with short chains, low sialylation, having terminal mannoses and / or non-intercalated terminal N-acetylglucosamines.

[0058] More preferably, said purified polypeptides have a content greater than 60% for the G0+G1+G0F+G1F forms, the G0F+G1F forms being less than 50%. More preferably, said purified polypeptides have a content greater than 60% for the G0+G1+G0F+G1F forms, the fucose content being less than 65%. More preferably, said purified polypeptides have a content less than 40% for the G1F+G0F forms.

[0059] 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.

[0060] The present invention also relates to the polypeptide according to the invention or the composition as described previously, for its use as a medicament.

[0061] 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 chosen from a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, a toxin, a membrane or circulating cytokine and a membrane receptor.

[0062] When the antibody is directed against a tumor antigen, its use is particularly suitable in the treatment of cancers. The term "cancer" means any physiological condition characterized by abnormal proliferation of cells. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas (including liposarcomas), neuroendocrine tumors, mesotheliomas, meningiomas, adenocarcinomas, melanomas, leukemias, and lymphoid malignancies.

[0063] When the antibody is directed against a viral antigen, its use is particularly suitable for the treatment of viral infections. Viral infections include, but are not limited to, HIV, retrovirus, coxsackievirus, smallpox, influenza, yellow fever, West Nile, cytomegalovirus, rotavirus, or hepatitis B or C. When the antibody is directed against a toxin, its use is particularly suitable for the treatment of bacterial infections, for example, tetanus toxin, diphtheria toxin, anthrax toxin, and anthrax toxins. Bacillus anthracis, or in the treatment of infections by botulinum toxins, ricin toxins, shiga toxins, this list not being exhaustive.

[0064] 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 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, gouty arthritis, celiac disease, Crohn's disease, Hashimoto's chronic thyroiditis (hypothyroidism), Adisson's disease, autoimmune hepatitis, Graves' disease (hyperthyroidism), ulcerative colitis,vasculitis such as ANCA (anti-neutrophil cytoplasmic antibody)-associated systemic vasculitis, autoimmune cytopenias and other hematological complications in adults and children, such as acute or chronic autoimmune thrombocytopenias, autoimmune hemolytic anemias, hemolytic disease of the newborn (HDN), cold agglutinin disease, autoimmune acquired hemophilia; Goodpasture syndrome, extramembranous nephropathies, autoimmune bullous skin disorders, refractory myasthenia gravis, mixed cryoglobulinemia, psoriasis, juvenile chronic arthritis, inflammatory myositis, dermatomyositis and systemic autoimmune disorders in children including antiphospholipid syndrome, connective tissue disease, autoimmune pulmonary inflammation, Guillain-Barré syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), autoimmune thyroiditis,mellitis, myasthenia gravis, inflammatory autoimmune eye disease, neuromyelitis optica (Devic's disease), scleroderma, pemphigus, insulin-resistant diabetes, polymyositis, Biermer's anemia, glomerulonephritis, Wegener's disease, giant cell arteritis, 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-deposited glomerulonephritis, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmia, Fiessinger-Leroy-Reiter syndrome and uveomeningoencephalic syndrome.

[0065] 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.

[0066] In a particular embodiment of the invention, the autoimmune disease is idiopathic thrombotic purpura (ITP) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).

[0067] Also disclosed is a method for producing a polypeptide comprising an Fc region and having a 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, K290E, 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, Y319H, C226Y, F405L, T393I, N434S, R255W, A287T, N286Y, A231V, K274R, V308G, K414R, M428T, E345G, F243L, P247T, Q362R, S440N, Y278H, D312G, V262A, V305A, K246R, V308I, 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, the number is at the same time as the EU index or equivalent in the Kabat.

[0068] Also disclosed is a method for producing a polypeptide comprising an Fc region and having a 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) une mutation choisie parmi 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N et 394P ; And (ii) in the presence of a different mutation 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 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).

[0069] Preferably, mutation (ii) is selected from 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 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] Also disclosed is a method of increasing the binding of a polypeptide comprising an Fc region to at least one of the Fc region receptors (FcR) selected from the C1q receptor, FcgRIIIa (CD16a), and FcgRIIa (CD32a), and FcgRIIb (CD32b), 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, K290E, 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, Y319H, C226Y, F405L, T393I, N434S, R255W, A287T, N286Y, A231V, K274R, V308G, K414R, M428T, E345G, F243L, P247T, Q362R, S440N, Y278H, D312G, V262A, V305A, K246R, V308I, 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, the number is at the same time as the EU index or equivalent in the Kabat.

[0071] Also disclosed is a method of increasing the binding of a polypeptide comprising an Fc region to at least one of the Fc region receptors (FcR) selected from the C1q 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) une mutation choisie parmi 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N et 394P ; And (ii) in the presence of a different mutation 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267N, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 315D, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378V, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 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).

[0072] Preferably, mutation (ii) is selected from 226Y, 227S, 230S, 231V, 234P, 243I, 243L, 246R, 246E, 247T, 248E, 253F, 254F, 255W, 259A, 261R, 262A, 263A, 266M, 267G, 274E, 274R, 276S, 278H, 282A, 283G, 284L, 286I, 286Y, 287T, 288E, 288R, 290E, 298N, 302A, 305A, 307P, 308A, 308I, 308G, 309P, 312G, 316D, 319H, 320T, 320R, 320M, 322E, 323I, 325S, 333G, 334N, 334R, 336T, 339T, 340E, 343S, 345G, 349S, 349H, 350A 352S, 359A, 361H, 362R, 363I, 366A, 373D, 375R, 377T, 378T, 379A, 380G, 383R, 385R, 389S, 389T, 392R, 393A, 393I, 394P, 396L, 397I, 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.

[0073] The sequences described in this application can be summarized as follows: SEQ ID NO: Protein 1 Human IgG1 Fc region G1m1,17 (residues 226-447 according to 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 G1m1,17 with upper N-terminal hinge region (residues 216-447 according to EU index or equivalent in Kabat) 7 Human IgG2 Fc region with N-terminal upper hinge region 8 Human IgG3 Fc region with N-terminal upper hinge region 9 Human IgG4 Fc region with N-terminal upper hinge region 10 Human IgG1 Fc region G1m3 with upper N-terminal hinge region 11 and 12 Primers MG_619 and MG_621

[0074] The present invention will be better understood from the following examples. EXAMPLES EXAMPLE 1: Identification of polypeptides with a mutated Fc region according to the invention and characterization of said polypeptides I. Materials and methods 1. Constructions of the banks of the human Fc region

[0075] 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 human IgG1 heavy chain and having the allotype G1m1.17 (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. Multiple completely randomized libraries were generated using the MUTAGEN ™ method (WO02 / 038756) which uses low-fidelity human DNA polymerases (mutases) to introduce random mutations homogeneously over the entire target sequence. Three distinct mutases (pol β, pol η and pol ) 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 SeI 21:267-274, (2008)). 1.1. Mutagenesis with the MUTAGEN ™ process

[0076] The MUTAGEN ™ process has been described in application WO02 / 038756.

[0077] Briefly, the human Fc gene (Fc gene) was replicated using mutases using the 5' primer MG_619: 5'-AGTACTGACTCTACCTAGGATCCTGCCCCACCGTGC-3' (SEQ ID NO:11) and the 3' primer MG_621: 5'-ACTGCTCGATGTCCGTACTATGCGGCCGCGAATTC-3' (SEQ ID NO:12). A mixture containing 0.6µg of plasmid pMG58_Fc226 as template (wild-type Fc region or selected variants), primers MG_619 and MG_621 (250nM each) and the appropriate replication buffer (see details below) was treated for 5 min at 95°C and immediately cooled to 4°C to denature the DNA strands. For pol β, 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 η (or pol η and pol ι) 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 50 µM dATP / dCTP, 100 µM dTTP / dGTP and 1 µg pol β or 100 µM dNTP and 1 µg pol η (or pol η and pol . , 1 µg 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 columns (Millipore). 1.2. Selective amplification and cloning of mutated fragments

[0078] 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 the specific amplification of DNA fragments synthesized by mutases. A fraction of the replication products was added to a mixture containing the PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 10 pmol of the 5' and 3' primers, 200 pM dNTPs and 1.25 U of Platinum Taq DNA polymerase (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.

[0079] 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 into 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 100 µg / ml ampicillin and 1% (w / v) glucose. After growth, colony numbers were determined to estimate library sizes and at least 48 clones per library were subjected to high-throughput PCR and DNA sequencing. Cells were resuspended in 2YT medium with 15% glycerol, frozen, and stored at -80°C. 1.3. Construction of the Mut3 bank

[0080] A first library was obtained using pol β on the wild-type Fc gene and contained 3.2×10 6< clones (called Mut1.1). The DNA from this first library was used to generate the second and third libraries, using pol β (3.8×10 6< clones, Mut1.2) and pol η and i (3.0×10 6< clones, Mut1.3), respectively. This two-step cumulative replication strategy increased the mutation rate. The fourth library was made with pol η alone on the wild-type Fc gene (1.0×10 6< clones, Mut1.4). Finally, these four libraries were proportionally mixed to obtain the final library called Mut1, representing 1.1×10 7< different clones.

[0081] Two different libraries were then constructed using a DNA pool of 42 single and double mutants isolated on the FcRn receptor. A first library was obtained using pol β (1.9×10 7< clones, Mut2.1) and a second library with pol η (1×10 6< clones, Mut2.2). Finally, these two libraries were proportionally mixed to obtain the final library called Mut2, i.e. 2×10 7< different clones.

[0082] A new library was obtained using pol β on the wild-type Fc gene, with a diversity of 4.7×10 7< clones.

[0083] Finally, the Mut1, Mut2 and new libraries were proportionally mixed to obtain the final library called Mut3, i.e. 7.8×10 7< different clones. Finally, the Mut3 library contains 3×10 7< in-frame mutated clones with an average of 2.2 amino acids mutated per Fc. 1.4. Construction of the Mut4sel bank

[0084] An Fc variant library was constructed from 36 clones with improved binding for C1q and / or CD16aV selected from the Mut3 library (1.6 amino acids mutated per Fc on average). The Mut4 library was obtained using an equimolar mixture of pol β and pol η polymerases (in pol β replication buffer). The Mut4 library has a diversity of 1.3×10 7< clones, with 77% of clones in phase and on average 2.6 amino acids mutated per Fc.

[0085] This library was cloned into the pMG93 vector, a selection vector developed for Fc, which allows the selection of coding sequences (ORFs) on ampicillin medium thanks to a fusion with the □-lactamase gene. This construct was transformed into the non-suppressant bacteria HB2151, which eliminates the TAG codons that are recognized as stop codons in this strain. The clones of this library were plated on agar plates containing low concentrations of ampicillin, at a low density, in order to carry out the selection of ORFs, the viable clones being then recovered and frozen. Plating on 500 Petri dishes (120x120) allowed the entire Mut4 library to be covered. The library thus selected was noted Mut4sel and contains 92% of clones in phase, or 1×10 7< clones mutated in phase with an average of 2.5 amino acids mutated per Fc.The Mut4sel library was then subcloned into the pMG58 phagemid in XL1-Blue bacteria to enable phage display selection on the targets. 1.5. Construction of the Mut5 bank

[0086] An Fc variant library was constructed from 42 clones with improved binding for C1q and / or one of the FcgRs selected from the Mut4seI library (2.4 amino acids mutated per Fc on average). The Mut5 library was obtained using pol β polymerase alone to achieve a low mutation rate. Finally, the Mut5 library obtained contained 1.2×10 7< clones with 95% of clones in frame with 3.1 amino acids mutated per Fc on average. The good 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

[0087] During the selection steps, the Mut3, Mut4se1, and Mut5 libraries were expressed on the surface of bacteriophage M13 using standard procedures (Smith GP, Science 228: 1315 (1985)). E. coli XL1-Blue bacteria, containing the expression library cloned into the pMG58 vector, were grown in 60 ml of 2YT medium supplemented with 100 µg / ml ampicillin, 15 µg / ml tetracycline, and 1% (w / v) glucose at 30°C. Cells were then infected with M13 helper phage (M13K07, Biolabs, bacteria / phage ratio = 1 / 3) at 37°C for 20 min and phage-Fc production was continued overnight at 26°C, 230rpm 2YT / ampicillin / glucose with 0.5 mM IPTG and 50 µg kanamycin / ml. The following day, 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:

[0088] C1q supplement is commercially available (Calbiochem).

[0089] CD16a is an activating receptor that exhibits a V / F polymorphism at position 158, at the Fc binding site. Affinity is better for CD16aV.

[0090] CD16aV is commercially available (R&D system).

[0091] CD16aF was produced by PX'Therapeutics.

[0092] CD32a is an activating receptor that exhibits an H / R polymorphism at position 131, at the Fc binding site. Affinity is better for CD32aH.

[0093] CD32aR is commercially available (R&D system).

[0094] 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). 2.2. Solid phase selections:

[0095] For solid-phase selections, Fc-phages diluted in PBS / 5% skim milk / 0.1% Tween 20 were incubated in 8-well Maxisorp plates (1-4×10 11< phages / well in 100µl final) pre-coated with 500ng / well of CD16aV, biotinylated CD16aV, biotinylated CD16aF, biotinylated C1q, biotinylated CD32aR or biotinylated CD32b and blocked with 5% skim milk in PBS. After incubation for 2 hours at 37°C, the wells were washed 8 times with PBS / 0.1% Tween 20 and 2 times with PBS. For selections on CD32aR and CD32b, counter-selection rounds were also performed: before the binding step on the target immobilized on Maxisorp plates, the Fc-phages were pre-incubated in a similar manner on 8 wells with the competing 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 exponentially growing XL1-Blue bacteria (2×150µl / 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 round of selection. 2.3. Liquid phase selections:

[0096] For liquid-phase selections, 4×10 11< phages were first incubated with biotinylated CD16aV (250 nM), biotinylated CD16aF (1000 nM), or biotinylated C1q (250 nM) for 1 hour at room temperature with gentle agitation. Streptavidin-coated magnetic beads (Dynal) pre-blocked with 5% nonfat milk in 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, cells were resuspended in 2YT medium with 15% glycerol, frozen, and stored at -80°C until the next round of selection. 2.4. Selections from banks: Mut3 Bank:

[0097] For liquid-phase selections, 6 rounds were performed on 3 biotinylated targets: CD16aV, CD16aF and C1q (clones denoted DL6A, DL6B and QL6A). For solid-phase selection rounds, 6 rounds were performed on 3 targets: CD16aV, biotinylated CD16aV and biotinylated C1q (clones denoted DS6A, DS6B and QS6A). Mut4sel Bank:

[0098] The selection rounds were carried out in solid phase only: 3 rounds of selection on CD16aV-biotinylated, 3 rounds on C1q-biotinylated, 3 rounds on CD32aR-biotinylated (+ / depletion on CD32b for the 3rd round) and 3 rounds on CD32b-biotinylated (+ / - depletion on CD32aR for the 3rd round), (clones noted A3A, G3A, J3A / B and K3A / B). Mut5 Bank:

[0099] The selection rounds were performed as previously: 3 rounds of selection on CD16aF-biotinylated, 3 rounds on C1q-biotinylated, 3 rounds on CD32aR-biotinylated, 3 rounds on CD32b-biotinylated, 2 rounds on CD32aR-biotinylated and 2 rounds with depletion on CD32b-biotinylated, and 2 rounds on CD32b-biotinylated and 2 rounds of depletion on CD32aR-biotinylated (clones noted N3A, O3A, P3A, Q3A, P4B, and Q4B).

[0100] The selection is then made as previously described. 2.5. Pool cloning into the pMGM05 vector:

[0101] The clones selected at the end of the selection rounds were then directly transferred in a mixture (approximately 10 4< 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 VH variable chain of the anti-CD20 antibody. This construction 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 at the C-terminal of the Fc, but allows very rapid testing of a very large number of clones. Initially, it was verified that these mutations did not modify the binding of IgG-WT to the different receptors. Subsequently, the positive controls were cloned into this system in order to validate it: IgG1-S239D, I332E, from Xencor anti-CD19 antibody XmAb5574 (C1): positive control for CD16a; IgG1-G236A, from Xencor (C4): positive control for CD32aH / R; IgG1-K326W, E333S, from Abgenix / Genentech (C3): positive control for C1q; and IgG1-S267E, L328F, from Xencor anti-CD19 antibody XmAb5574 (C5): positive control for CD32b.

[0102] DNA from approximately 100 clones isolated per selection round was sequenced by colony PCR. After bioinformatic analyses, clones containing novel mutations were frozen at -80°C in XL1-Blue bacteria and the sequences 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:

[0103] The anti-CD20 light chain was inserted into a pCEP4 vector identical to the vector used for the heavy chain, denoted pMGM01-CDC20 (pCEP4 InvitroGen). HEK293-F Freestyle™ cells (Invitrogen), grown in 24-well plates, were co-transfected with pMGM01-CD20 and pMGM05-CD20 (Fc-WT and variants) vectors in equimolar amounts (250 ng / ml) with Freestyle™ MAX reagent (1 µl / ml) using standard protocols (Invitrogen). Cells were grown in suspension in serum-free medium for 7 days post-transfection and supernatants (1 ml) containing IgG were harvested after centrifugation of the cells at 100 g for 10 min. Secreted IgG in the supernatants were quantified using a recombinant protein L ELISA (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% skim 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 goat anti-human IgG HRP F(ab')2 fragment (y-chain specific) (Sigma). Produced IgG variants were quantified (1-4 µg / ml) using the standard curve. 2.7. ELISA tests of IgG variants produced in 293-F cell supernatants:

[0104] IgG variants were tested for binding to human complement C1q and several human FcRs by ELISA. Maxisorp immunoplates were coated with 0.5 µg complement C1q / well, 0.05 µg CD32aH / well, 0.2 µg CD16aF / well, or 0.1 µg CD16aV / well in PBS. Immobilizing nickel chelating plates (Nunc) were coated with 0.1 µg CD32aR / well or 0.4 µg CD32b / well in 0.01 M KCl. After coating overnight at 4°C, 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 / ml and mixed with goat anti-human IgG HRP F(ab')2 at the same concentration for 2 hours at room temperature. The F(ab')2-aggregated IgG were then incubated with gentle shaking for 1 hour at 30°C on the saturated ELISA plates without dilution for C1q, CD16aF, CD32aR and CD32b (i.e.IgG at 0.5 µg / ml), diluted in PBS at 0.25 µg / ml for CD16aV and CD32aH. The plaques are then revealed with TMB (Pierce) and the absorbance read at 450 nm. Selections on the Mut3 bank:

[0105] Using this ELISA, the phage display-selected variants were tested for their binding to complement C1q and different receptors. They were tested in comparison with wild-type Fc (Fc-WT) and positive controls. 36 CD16aV and / or C1q-positive clones were thus selected from the Mut3 library and used to construct the Mut4 library. Selections on the Mut4sel bank:

[0106] The ELISA tests carried out on the 371 isolated clones made it possible to identify 116 clones with a ratio greater than 2 for at least one FcyR and 17 clones with a ratio greater than 3 for C1q only, which therefore corresponds to 133 clones of interest (Table 1). Table 1: 133 isolated clones of interest Mutant Name Mutations ELISA test results C1q CD16aV CD32aR CD32 b 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, N325S, 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 K290E, S375R, F405V 1,89 1,64 3,93 3,12 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 1,56 2,31 2,35 3,10 A3A-91 K340E, A378V, N389S, N421T 2,39 1,88 2,67 3,03 DS3A-93 F243I, 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 N286I, 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 (>30) 1,63 2,96 2,76 A3A-164 A378V, K447N 1,64 2,41 2,82 2,71 G3A-118 N286I, 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 DL3 A-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 2,29 G3A-154 A378T, V397M, F423P, S442P 2,51 2,11 4,14 2,28 A3A-12 V363I, V397M 3,02 1,56 2,99 2,27 J3A-14 K326T 1,83 1,43 4,25 2,26 G3A-05 V323I, 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 I253F, 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, V323I 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 K290E, V308I, 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 V323I, T393I 1,33 1,52 4,15 2,04 J3B-135 K248E, S383R, V397M, N434S 6,36 1,51 3,99 2,04 A3A-137 K290E, V323I, 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 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, V323I 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 V323I, 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 V323I 1,41 1,31 2,03 1,68 K3B-91 V308I, 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 K290E, E380G 1,64 2,02 1,91 1,59 J3B-16 S267G, A378T, V397M 0,85 0,57 4,95 1,58 J3B-23 N286I, 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, K290E, 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 V323I, 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, V323I 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 G3A-95 I336T, 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 K290E, 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 V323I, 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 V240I, K246E, P353L 3,90 0,95 0,51 0,64

[0107] 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 components and comparison options for reference NA = Non Déterminé Nom du mutant Mutations Résultats tests ELISA C1q CD16aF CD16aV CD32aH CD32aR CD32b 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 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 I336T, A378V 1,05 1,29 2,25 1,17 1,82 1,31 G3A-95A I336T, A378V, T394P NA 1,50 1,90 NA NA NA G3A-95B I336T, A378V, P396L NA 2,00 2,54 NA NA NA G3A-95C I336T, 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

[0108] Finally, from these 151 tested variants, 26 variants of interest were selected to be produced on a larger scale and studied more precisely (Table 3). Tableau 3: 26 new variants selections NA = Non Déterminé Nom du mutant Mutations Résultats tests ELISA C1q CD16 aF CD16a V CD32a H CD32a R CD32 b 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 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, V323I 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 I336T, 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 N286I, 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:

[0109] ELISA tests performed on the 171 isolated clones identified 87 clones with a ratio greater than 2 for at least one of the FcyRs tested or the C1q complement (Table 4). Tableau 4: 87 clones with superior ratio to 2 Nom du mutant Mutations Résultats tests ELISA C1q CD16a F CD16aV CD32a H CD32aR CD32 b P3A-01 K334N, S383R 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 7 0,94 1,62 1,07 1,24 1,13 P3A-23 N361H, P396L 2,70 1,75 2,49 2,53 3,33 1,76 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, 5,95 1,15 1,28 1,68 3,22 1,68 T394P 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 1,77 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 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 O3A-04 A231V, T359A, S383R, V397M 1,00 1,16 1,21 1,69 2,35 1,09 O3A-05 K290E, T366A, A378V 0,99 2,79 3,12 2,07 3,34 1,34 O3A-10 Y300H, T394P 0,98 0,76 0,90 1,81 2,25 1,17 O3A-11 R255W, A287T, P352S, A378V, N421T 1,85 1,01 2,10 2,20 3,15 1,55 O3A-16 K334R, A378V, K447N 4,30 1,71 2,51 2,43 3,98 1,59 O3A-17 I336V, T359A, S383R, V397M 0,85 1,79 2,70 2,30 1,73 0,93 O3A-22 K334R, P396L, H435R 23,5 5 1,68 2,16 2,75 4,79 1,62 O3A-24 K326T, K447N 2,12 1,23 2,27 1,55 2,94 1,40 O3A-25 V308A, K334R, A378T, V397M 11,1 2 1,40 2,07 2,04 4,16 1,96 O3A-34 A231V, Y349S, V397M 0,73 1,40 1,90 2,34 2,62 1,28 O3A-38 K248E, N286Y, Q418P 2,17 1,59 1,16 0,99 0,69 0,57 O3A-40 V308A, A378T, V397M, V412M 0,82 1,65 2,15 2,09 2,64 1,03 O3A-42 S304N, A378V 1,04 2,16 0,53 0,87 0,87 1,77 O3A-44 A378V, K439R 1,95 2,44 4,40 3,20 3,13 1,81 O3A-45 D270N, K334R, L410R 3,32 1,57 0,61 0,56 1,19 1,36 O3A-46 V302A, A378V 0,94 1,15 1,75 2,36 2,91 1,37 O3A-50 K334R, A378V, N421T 3,62 1,28 2,49 2,15 4,02 2,30 O3A-57 T359A, V397M 1,50 1,18 2,27 2,23 2,78 1,59 O3A-66 A378T, T394P 1,10 1,42 2,42 2,36 2,45 1,76 O3A-67 K248E, N361H 2,05 0,89 1,36 1,06 1,07 1,76 O3A-69 K290E, A378V 1,44 2,64 4,04 2,62 4,11 2,06 O3A-80 K274R, T394P, G402D, K447N 1,26 1,26 1,81 2,16 2,32 1,28 O3A-81 K274R, A378T, V397M 1,36 1,20 2,39 2,06 3,08 1,58 O3A-86 K248E, K290E, N361H, P396L 24,3 4 2,49 4,89 2,86 4,19 2,57 O3A-93 K290E, T394P 2,07 1,45 3,53 2,54 3,36 1,78 P4B-89 T307P, T366A, 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, V397I 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 O3A-103 K290E, K320T, A378V 2,11 1,22 2,08 2,76 2,41 1,50 O3A-117 K334R, T394P, N421S 0,98 1,05 1,71 2,29 3,05 1,37 O3A-119 K334N, V397M 1,53 1,04 1,97 1,45 2,28 1,18 O3A-126 P227S, V284L, A378V 4,58 1,31 2,93 2,85 4,47 1,29 O3A-127 V302A, K334R, T366A, S383R, V397M 1,53 1,00 1,24 2,85 3,56 1,37 O3A-131 C261R, A378T, V397M, V412M 2,25 1,03 1,72 2,00 2,27 1,15 O3A-137 K248E, T350A 0,84 0,66 0,43 0,68 2,06 0,97 O3A-172 K320E, T394P 8,19 0,88 1,46 2,01 2,52 1,06 O3A-179 R255Q, G385R 0,81 0,74 0,83 0,57 2,86 1,30 O3A-186 T307P, V397M 1,09 1,41 2,32 2,17 3,97 1,48 O3A-99 N276S, N286I, T359A, S383R, V397M 2,14 1,32 2,27 2,84 3,09 1,42

[0110] Among these variants, 10 variants of interest were selected (Table 5). Tableau 5: 10 variants Nom du mutant Mutations Résultats tests ELISA C1 q CD16a F CD16a V CD32aH CD32a R CD32b O3A-05 K290E, T366A, A378V 0,9 9 2,79 3,12 2,07 3,34 1,34 P4B-89 T307P, T366A, A378V 0,6 3 2,99 4,15 0,70 1,19 0,87 O3A-86 K248E, K290E, N361H, P396L 24, 34 2,49 4,89 2,86 4,19 2,57 N3A-32 K326T, K334N, P352S, N421T 1,6 1 2,47 4,35 1,59 3,24 2,04 O3A-44 A378V, K439R 1,9 5 2,44 4,40 3,20 3,13 1,81 N3A-27 K290E, A378V, K392R, S440G 1,6 7 2,25 3,77 2,05 2,59 1,49 N3A-87 K290E, K320E, T350A, P396L 0,9 1 2,15 2,85 3,12 5,94 4,63 Q4B-68 T307P, A378V, T394P 3,3 3 1,53 4,12 4,01 3,69 1,03 N3A-58 V284L, K334R, A378V, K447N 5,3 7 1,02 1,67 2,01 4,00 1,67 Q3A-39 N286I, P396L, N421T 2,8 0 1,63 1,99 3,52 3,09 1,02 3. Production and purification of internal variants

[0111] IgG variants are obtained by direct mutagenesis in pCEP4-WT-H-CD20. IgG controls, i.e. C1, C3, C4, C5 and wild type (WT), are produced with the G1m3 allotype (comprising 3 mutations compared to G1m1,17: (K214R / )D356E / L358M).

[0112] The 26 IgG variants from the Mut4sel library, as well as the wild type, were produced with the G1m1,17 allotype. They are produced by incubation for 6-7 days in batches (250-300ml) of 293-E cells (Freestyle Invitrogen) in F17 medium.

[0113] Centrifugation and filtration are then carried out.

[0114] Purification is carried out on Protein A Hi-Trap, and elution is carried out with a 25mM citrate buffer pH = 3.0, neutralization and dialysis in TBS or PBS before sterilization.

[0115] 10mg of each IgG control, i.e. C1, C3, C4, C5 and wild type (WTG1m3 and WTGlm1,17) are obtained, as well as 2-3mg of 26 IgG variants.

[0116] Their characterization shows that the molecular weight is conserved, and that the glycosylation profiles are similar for all variants. 4. Tests of variants used 4.1. FcRn binding assays

[0117] Jurkat-FcRn cells were incubated at pH = 6.0 with IgG variants at different concentrations (0 to 1000µg / ml) and Rituximab-Alexa.

[0118] Flow cytometry is performed on the bound Rituximab-Alexa.

[0119] The results show no loss of FcRn binding for all IgG variants. 4.2 Antigen binding tests

[0120] Raji cells are incubated with IgG variants at 1µg / ml for 15 minutes at 4°C.

[0121] Bound IgG is revealed by binding with an anti-human IgG-PE secondary antibody (for 15 minutes at 4°C).

[0122] The results show that antigen recognition is not altered by the different mutations present on the Fc.

[0123] All IgG variants bind to CD20 on cells, similar to the IgG-WT control. 4.3 ELISA tests for binding to CD16aV / F

[0124] The purified antibodies were tested by ELISA for their binding to CD16F and CD16aV according to a protocol identical to that described in 2.6, by diluting the antibodies to different concentrations. 4.4. ADCC Activity Tests

[0125] NK cells are incubated with Raji target cells expressing CD20, in the presence of different concentrations of IgG variants (0.005 to 5000 ng / ml).

[0126] The level of intracellular LDH released by lysed target cells is measured.

[0127] Human NK cells were purified from peripheral blood of healthy volunteer donors using the negative depletion technique developed by Miltenyi. The ADCC assay involves the incubation of 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, the cytotoxicity induced by anti-CD20 antibodies was measured by quantifying intracellular lactate dehydrogenase (LDH) released by lysed target cells in cell supernatants. Specific lysis results are expressed as percentage lysis as a function of antibody concentration. EC50 (antibody concentration inducing 50% of the maximal lysis induced by IgG-WT) and Emax (percentage of maximal lysis) values ​​were calculated using GraphPad PRISM software.

[0128] The results are presented in Tables 6 and 7. Tableau 6: Results from ADCC tests Mutant G3A4 3 G3A4 5 J3B-118 J3B16 A3A-184 WT EC50: Antibody concentration (ng / ml) giving 50% lysis of an internal control 1,03 4,38 1,27 > 5545,36 2,35 12,34 Ratio [WT] / [antibody] 11.98 2.82 9.72 <0.0022 5.25 1 Table 7a: ADCC test results Mutant EC50: Antibody concentration (ng / ml) giving 50% maximum lysis of an internal control Ratio [WT] / [antibody] 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 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 Table 7b: ADCC test results Mutant EC50: Antibody concentration (ng / ml) giving 50% maximum lysis of an internal control Ratio [WT] / [antibody] WT 0,45 1 A3A-173 1,38 0,33 K3B-90 0,75 0,60 A3A-105 1,374 0,33 A3A-31 1,839 0,24 J3B-115 0,57 0,79 Painting 7c : ADCC test results (EC45 instead of EC50) Mutant EC45: Antibody concentration (ng / ml) giving 45% maximum lysis of an internal control Ratio [WT] / [antibody] WT 1,60 1 O3A-44 2,6 0,62 N3A-27 18,7 0,86 N3A-58 4,4 0,36 O3A-86 4,4 0,36 N3A-87 1,0 1 P4B-89 3,6 0,44 O3A-05 0,2 8 N3A-32 2,9 0,55 Q3A-39 0,3 5,33 Q4B-68 1,7 0,94

[0129] The best variants are A3A-184A and G3A-103. 4.5. CDC Activity Tests

[0130] 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 measured chromogenically (Roche Applied Sciences Cytotoxicity Detection Kit) and used to quantify antibody-mediated complement-dependent cytotoxicity. Results are expressed as percentage lysis. EC50 (amount of antibody that induces 50% maximal lysis) and Emax (percentage of maximal lysis) were calculated using GraphPad PRISM software.

[0131] The results are presented in Tables 8 and 9. Table 8: CDC test results Antibody Antibody concentration (ng / ml) giving 50% lysis of an internal control Ratio [WT] / [antibody] A3A-173 16,01 16.36 K3B-90 23,12 11.33 J3B-118 38,69 6.77 A3A-105 64,76 4.05 A3A-31 108,37 2.42 J3B-115 145,44 1.80 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 Tableau 9a: Results from CDC tests Anticorps Concentration of anticorpores (ng / ml) less than 50% of internal control Ratio [WT] / [anticorps] J3B-118A 21,564 10.98 J3A-28 31,004 7.64 G3A-103 33,339 7.10 A3A-105D 38,550 6.14 QL2A-16 51,544 4.59 K3B-01 59,600 3.97 J3A-06 64,089 6.70 J3A-14 64,089 6.70 A3A-184A 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 Tableau 9b: Results from CDC tests Anticorps Concentration of anticorpores (ng / ml) less than 50% of internal control Ratio [WT] / [anticorps] WT 133 1 O3A-44 129 1,03 N3A-27 179 0,74 N3A-58 36 3,69 O3A-86 44 3,02 N3A-87 106 1,25 P4B-89 115 1,16 O3A-05 91 1,46 N3A-32 66 2,02 Q3A-39 61 2,18 Q4B-68 126 1,06

[0132] The following Table 10 shows the results obtained with some variants, classified by subgroup. Tableau 10 Ratio ELISA (Ac purifiés) Nom Mutations ADCC CDC CD32aH CD32aR CD32b CD64 G3A-103 K248E, A378V 33,9 7,1 0,5 0,9 1,2 1,2 J3A-28 E333G, A378T, V397M 18,4 7,6 1,1 1,8 1,6 1,3 J3B-118A P396L, N421T, A378V 10,8 11,0 2,7 1,8 3,7 1,4 J3B-118 P396L, N421T 9,7 12,2 1,0 1,1 1,0 1,6 A3A-105D G316D, K326E, A378V 8,3 6,1 1,4 2,6 2,0 1,5 A3A-14 5298N,A378V 12,8 0,7 1,1 0,8 0,9 1,6 G3A-95 I336T, A378V 10,8 1,3 0,7 0,9 1,0 1,3 A3A-184A K334N, P352S, V397M, A378V 30,9 3,2 1,8 4,3 3,1 1,5 J3B-23 N286I, A378V, F423Y 18,4 2,4 ND ND ND ND K3B-01 N315D, N361H, P396L 15,2 4,0 5,7 1,0 1,0 2,3 G3A-43 A231V, A378V 12,0 2,8 5,3 3,6 2,7 1,4 J3A-06 A378T, V397M, V412M 8,3 3,7 0,84 2,0 2,4 1,3 J3A-16 N286Y, P352S, A378V 8,3 1,8 2,61 1,7 1,7 1,3 O3A-05 K290E, T366A, A378V 8,0 1,5 4,02 3,3 4,5 1,2 Q3A-39 N286I, P396L, N421T 5,3 2,2 4,83 2,3 3,8 1,5 A3A-184 K334N, P352S, V397M 5,3 2,2 1,33 3,2 3,3 1,4 K3B-90 V308A, K334R, A378V, K447N 0,6 20,4 1,20 1,2 1,1 1,5 A3A-173 K248E, K334R, A378V 0,3 29,5 0,95 1,3 1,3 1,2 J3A-14 K326T 1,4 3,7 1,86 1,3 1,5 2,3 G3A-88 K320E, T394P, G402D 1,3 0,9 2,94 3,7 3,9 2,4 N3A-87 K290E, K320E, T350A, P396L 1,0 1,3 3,92 3,7 9,0 1,5 J3B-115 T359A, S383R, V397M 0,8 3,2 4,65 5,1 4,2 1,5

[0133] The following Table 11 shows some possible variants according to the invention: Tableau 11 Nom Mutations Variant de départ Mutation ajoutée G3A-95D S298N, I336T, A378V G3A-95 S298N K3B-90A K248E, V308A, K334R, A378V, K447N K3B-90 K248E G3A-88A K320E, K326T, T394P, G402D G3A-88 K326T N3A-87A K290E, K320E, K326T, T350A, P396L N3A-87 K326T A3A-105E K248E, G316D, K326E, A378V A3A-105D K248E A3A-14A K248E, S298N, A378V A3A-14 K248E A3A-184B K248E, K334N, P352S, V397M, A378V A3A-184 K248E G3A-43D K248E, A231V, A378V G3A-43 K248E G3A-95E K248E, I336T, A378V G3A-95 K248E J3A-28A K248E, E333G, A378T, V397M J3A-28 K248E J3B-118B K248E, P396L, N421T, A378V J3B-118A K248E J3B-23A N286I, A378V, F423Y J3B-23 K248E K3B-01A N315D, N361H, P396L K3B-01 K248E A3A-105F G316D, K326E, E333G, A378V A3A-105 E333G A3A-14B S298N, E333G, A378V A3A-14 E333G A3A-184C E333G, K334N, P352S, V397M, A378V A3A-184 E333G G3A-103A K248E, E333G, A378V G3A-103 E333G G3A-43E A231V, E333G, A378V G3A-43 E333G G3A-95F E333G, I336T, A378V G3A-95 E333G J3B-118C E333G, P396L, N421T, A378V J3B-118 E333G J3B-23B N286I, E333G, A378V, F423Y J3B-23 E333G K3B-01B N315D, E333G, N361H, P396L K3B-01 E333G A3A-105G G316D, K326E, A378V, F423Y A3A-105 F423Y A3A-14C E333G, S298N, A378V, F423Y A3A-14 F423Y A3A-184D K334N, P352S, V397M, A378V, F423Y A3A-184 F423Y G3A-103B K248E, A378V, F423Y G3A-103 F423Y G3A-43F A231V, A378V, F423Y G3A-43 F423Y G3A-95G I336T, A378V, F423Y G3A-95 F423Y J3A-28B E333G, A378T, V397M, F423Y J3A-28 F423Y J3B-118D P396L, N421T, A378V, F423Y J3B-118 F423Y K3B-01C N315D, N361H, P396L,F423Y K3B-01 F423Y

Claims

1. Method to produce a polypeptide comprising a human IgG1 Fc region that is mutated as compared to a parent polypeptide and having functional activity mediated by the Fc region that is selected from among antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), that is increased compared with that of the parent polypeptide, the parent polypeptide comprising a parent Fc region chosen from sequences SEQ ID NO:1, 5, 6 and 10, said method comprising a step to introduce a combination of mutations into the parent Fc region selected from among: 248E / 378V, 396L / 421T / 378V, 316D / 326E / 378V, 298N / 378V, 336T / 378V, 334N / 352S / 397M / 378V, 286I / 378V / 423Y, 231V / 378V, 286Y / 352S / 378V, 290E / 366A / 378V, 308A / 334R / 378V / 447N, 248E / 334R / 378V, 298N / 336T / 378V, 248E / 308A / 334R / 378V / 447N, 248E / 316D / 326E / 378V, 248E / 298N / 378V, 248E / 334N / 352S / 397M / 378V, 248E / 231V / 378V, 248E / 336T / 378V, 248E / 396L / 421T / 378V, 316D / 326E / 333G / 378V, 298N / 333G / 378V, 333G / 334N / 352S / 397M / 378V, 248E / 333G / 378V, 231V / 333G / 378V, 333G / 336T / 378V, 333G / 396L / 421T / 378V, 286I / 333G / 378V / 423Y, 316D / 326E / 378V / 423Y, 333G / 298N / 378V / 423Y, 334N / 352S / 397M / 378V / 423Y, 248E / 378V / 423Y, 231V / 378V / 423Y, 336T / 378V / 423Y, or 396L / 421T / 378V / 423Y, the numbering being that of the EU Index.

2. Method according to claim 1, comprising a step to introduce a combination of mutations into the parent Fc region selected from among: 248E / 378V, 396L / 421T / 378V, 316D / 326E / 378V, 298N / 378V, 336T / 378V, 334N / 352S / 397M / 378V, 286I / 378V / 423Y, 231V / 378V, 286Y / 352S / 378V, 290E / 366A / 378V, 308A / 334R / 378V / 447N, or 248E / 334R / 378V.

3. Method according to claim 1 or 2, characterized in that the parent polypeptide comprises a parent Fc region that is sequence SEQ ID NO:1.

4. Method according to one of claims 1 to 3, characterized in that the polypeptide is selected from among an isolated Fc region, a sequence derived from an isolated Fc region, an antibody and a fusion protein comprising an Fc region.

5. Method according to one of claims 1 to 4, characterized in that the polypeptide is a polypeptide produced in the milk of transgenic animals.

6. Polypeptide consisting in sequence SEQ ID NO:1 that is mutated according to one of the following combinations: 248E / 378V, 396L / 421T / 378V, 316D / 326E / 378V, 298N / 378V, 336T / 378V, 334N / 352S / 397M / 378V, 286I / 378V / 423Y, 231V / 378V, 286Y / 352S / 378V, 290E / 366A / 378V, 308A / 334R / 378V / 447N, 248E / 334R / 378V, 298N / 336T / 378V, 248E / 308A / 334R / 378V / 447N, 248E / 316D / 326E / 378V, 248E / 298N / 378V, 248E / 334N / 352S / 397M / 378V, 248E / 231V / 378V, 248E / 336T / 378V, 248E / 396L / 421T / 378V, 316D / 326E / 333G / 378V, 298N / 333G / 378V, 333G / 334N / 352S / 397M / 378V, 248E / 333G / 378V, 231V / 333G / 378V, 333G / 336T / 378V, 333G / 396L / 421T / 378V, 286I / 333G / 378V / 423Y, 316D / 326E / 378V / 423Y, 333G / 298N / 378V / 423Y, 334N / 352S / 397M / 378V / 423Y, 248E / 378V / 423Y, 231V / 378V / 423Y, 336T / 378V / 423Y, or 396L / 421T / 378V / 423Y, the numbering being that of the EU Index.

7. Pharmaceutical composition comprising (i) a polypeptide according to claim 6, and (ii) at least one pharmaceutically acceptable excipient.

8. Polypeptide according to claim 6, for use as medicinal product.