FC MUTANTS WITH MODIFIED FUNCTIONAL ACTIVITY

Abstract

Disclosed is a polypeptide including a mutated Fc region and having functional activity, mediated by the Fc region, that is modified compared with that of a parent polypeptide. The Fc region includes at least one combination of 2 mutations, the combination being selected from among one mutation selected from among a first set of mutations, and at least one mutation selected from among a second set of mutations, and provided that mutation (i) does not take place on the same amino acid as mutation (ii). Also disclosed are use of the polypeptide, compositions including the same, and methods for preparing the polypeptide.

Claims

1. Polypeptide comprising a mutated Fc region and having functional activity. mediated by the Fc region, that is modified compared with that of a parent polypeptide, wherein said Fc region comprises at least one combination of 2 mutations, said combination being selected from among: (i) one mutation selected from among 307N, 326E, 326T, 334N, 334R, 352L, 378V, 378T, 394P, 396L, 397M, 421T; and (ii) at least one mutation selected from among 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 Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).

2. Polypeptide according to claim 1, wherein the mutation (i) is selected from among 378V, 396L and 397M.

3. Polypeptide according to claim 2, further comprising a mutation selected from among 248E, 326T, 333G and 423Y.

4. Polypeptide according to claim 1, wherein said functional activity mediated by the Fc region is selected from among antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell phagocytosis (ADCP), and a combination of at least two of these activities.

5. Polypeptide according to claim 1, having functional activity mediated by the Fc region that is increased compared with that of the parent polypeptide, preferably by a ratio of at least 2, preferably higher than 5, preferably higher than 10, preferably higher than 15, preferably higher than 20, preferably higher than 25, preferably higher than 30.

6. Polypeptide according to claim 1, having functional activity mediated by the Fc region that is reduced compared with that of the parent polypeptide, preferably by a ratio of at least 2, preferably higher than 5, preferably higher than 10, preferably higher than 15, preferably higher than 20, preferably higher than 25, preferably higher than 30.

7. Polypeptide according to claim 1, wherein said mutated Fc region has modified affinity for at least one of the receptors (FcRs) of the Fc region selected from among the C1q complement and FcgRIIIa (CD16a), FcgRIIa (CD32a) and FcgRIIb (CD32b) receptors.

8. Polypeptide according to claim 1, wherein said mutated Fc region comprises 2 to mutations compared with the parent polypeptide, preferably 2 to 10 mutations.

9. Polypeptide according to claim 1, wherein said mutated Fc region has improved affinity for the C1q complement, and comprises at least one combination of 2 mutations, said combination comprising: i) one mutation selected from among 378V, 378T, 396L, 421T, 334R and 326E; and ii) at least one mutation selected from among 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 Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).

10. Polypeptide according to claim 1, wherein said mutated Fc region has improved affinity for the FcgRIIIa receptor (CD16a), and comprises at least one combination of 2 mutations, said combination comprising: i) one mutation selected from among 378V, 326E, 397M, 334N and 396L; and ii) at least one mutation selected from among 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 Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).

11. Polypeptide according to claim 1, wherein said mutated Fc region has increased affinity for the FcgRIIa receptor (CD32a), and comprises at least one combination of 2 mutations, said combination comprising: i) one mutation selected from among 378V, 326E, 397M, 307N, 394P, 326T, 396L and 334N; and ii) at least one mutation selected from among: 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 Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).

12. Polypeptide according to claim 1, wherein said mutated Fc region has increased affinity for the FcgRIIb receptor (CD32b), and comprises at least one combination of 2 mutations, said combination comprising: i) one mutation selected from among 326E, 326T, 378V, 397M, 352L, 394P, 396L and 421T; and ii) at least one mutation selected from among 316D, 334R, 248E, 334N, 418P, 231V, 320E, 402D, 359A, 383R, 421T and 361H, the numbering being that of the EU Index or Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).

13. Polypeptide according to claim 1, wherein said mutated Fc region has increased CDC activity, and comprises at least one combination of 2 mutations, said combination comprising: i) one mutation selected from among 378V, 378T, 396L, 421T, 334R and 326E; and ii) at least one mutation selected from among 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 Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).

14. Polypeptide according to claim 1, wherein said mutated Fc region has increased ADCC activity, and comprises at least one combination of 2 mutations, said combination comprising: i) one mutation selected from among 378V, 326E, 397M, 334N and 396L; and ii) at least one mutation selected from among 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 Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).

15. Polypeptide according to claim 1, wherein said mutated Fc region comprises at least one combination of 3 mutations, said combination comprising: (i) one mutation selected from among 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and (ii) at least 2 mutations selected from among 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 Kabat equivalent, and provided that mutation (i) does not take place 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).

16. Polypeptide according to claim 1, wherein the parent polypeptide comprises a parent Fc region that is a human Fc region, preferably an Fc region of a human IgG1 or human IgG2.

17. Polypeptide according to claim 1, wherein 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.

18. Polypeptide according to claim 1, wherein the polypeptide consists of an Fc region or it is an antibody.

19. Polypeptide according to claim 1, wherein the polypeptide is produced in the milk of transgenic animals.

20. Composition of polypeptides according to claim 1, wherein the purified polypeptides of said composition, on their Asn297 glycosylation site, have N-glycans with a fucosylation rate lower than 65%, preferably lower than 50%, more preferably lower than 40%.

21. Composition of polypeptides according to claim 20, wherein the purified polypeptides of said composition, on their Asn297 glycosylation site, have a glycan structure of biantennary type with short chains, low sialylation, having non-intercalary terminal mannoses and/or terminal N-acetylglucosamines.

22. Composition of polypeptides according to claim 21, wherein the purified polypeptides of said composition have a content higher than 60% for the G0+G1+G0F+G1F forms, the G0F+G1F forms being lower than 50%, preferably lower than 40%.

23. Composition of polypeptides according to claim 21, wherein the purified polypeptides of said composition have a content higher than 60% for the G0+G1+G0F+G1F forms, the fucose content being lower than 65%.

24. Pharmaceutical composition comprising (i) a polypeptide according to claim 1, and (ii) at least one pharmaceutically acceptable excipient.

25. A method for treating a subject in need thereof, comprising the step of administering to said subject a polypeptide according to claim 1.

26. Antibody according to claim 18 directed against an antigen selected from among a tumour antigen, viral antigen, bacterial antigen, fungal antigen, a toxin, membrane-bound or circulating membrane, a membrane receptor.

27. Method to produce a polypeptide comprising an Fc region and having functional activity, mediated by the Fc region, that is modified compared with that of a parent polypeptide, said method comprising a step to introduce a least one combination of 2 mutations, said combination being selected from among: (i) one mutation selected from among 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and (ii) at least one mutation selected from among 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 Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).

28. Method to increase the binding of a polypeptide comprising an Fc region to at least one of the receptors of (FcR) of the Fc region, selected from among the receptors C1q, FcgRIIIa (CD16a), FcgRIIa (CD32a) and FcgRIIb (CD32b), said method comprising a step to introduce at least one combination of 2 mutations, said combination being selected from among: (i) one mutation selected from among 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and (ii) at least one mutation selected from among 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 Kabat equivalent, and provided that mutation (i) does not take place on the same amino acid as mutation (ii).

Description

EXAMPLES

Example 1: Identification of Polypeptides with Mutated Fc Region of the Invention and Characterization of Said Polypeptides

[0118] I. Material and Methods

[0119] 1. Construction of Banks of the Human Fc Region

[0120] The human Fc gene encoding the amino acids 226-447 (EU Index or Kabat equivalent) i.e. a polypeptide comprising an Fc region, derived from a heavy chain of human IgG1 and having the allotype G1 m1.17 (SEQ ID NO: 1), (Poul M A et al, Eur J. Immunol 25 (7): 2005-2009, 1995) was cloned in the phagemid vector pMG58 (pMG58_Fc226) as BamHI/EcoRI fragment following standard PCR protocols. Several fully randomized banks were generated applying the MUTAGEN™ method (WO02/038756) which uses low-fidelity human DNA-polymerases (mutases) to introduce random mutations homogeneously in the entire target sequence. Three different mutases (pol R, pol n and pol i) 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 21: 76-82 (2007), Emond et al. Protein Eng Des Sel 21:267-274, (2008)).

[0121] 1.1. Mutagenesis with the MUTAGEN™ Method

[0122] The MUTAGEN™ method was described in application WO02/038756.

[0123] In brief, the human Fc gene (Fc gene) was replicated with mutases using the 5′ primer MG_619: 5 ′-AGTACTGACTCTACCTAGGATCCTGCCCACCGTGC-3′ (SEQ ID NO: 11) and the 3′ primer MG_621: 5 ′-ACTGCTCGATGTCCGTACTATGCGGCCGCGAATTC-3′ (SEQ ID NO: 12). A mixture containing 0.6 μg of the pMG58_Fc226 plasmid as model (wild-type Fc region or selected variants), the primers MG_619 and MG_621 (250 nM each) and suitable 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 R, 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 .Math.) 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 denaturing step, mutagenic replications were obtained by adding 50 μM ATP/dCTP, 100 μM dTTP/dGTP and 1 μg of pol β or 100 μM of dNTP and 1 μg of pol η (or pol η and pol .Math., 1 μg of each mutase). The replication reaction was conducted at 37° C. for two hours. The replication products were subsequently concentrated and desalted on Microcon columns (Millipore).

[0124] 1.2. Selective Amplification and Cloning of the Mutated Fragments

[0125] 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 was non-complementary to the model allowing specific amplification of the DNA fragments synthesized by the mutases. One 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 μM of dNTPs and 1.25 U of Platinum Taq DNA polymerase (Invitrogen). The PCR cycles were: first cycle 2 min. at 94° C., 10 sec. at 64° C., 30 sec. at 75° C., 1 min. at 94° C., followed by 30 selective cycles: 20 sec. at 94° C. and 30 sec. at 75° C.

[0126] The amplified replication products were purified on 1% (w/v) of agarose gels digested with BamHI and EcoRI and cloned in the pMG58 vector. The ligation mixtures were transformed in E. Coli XL1-Blue electrocompetent cells and spread over a solid 2YT medium (16 g/l peptone, 10 g/l yeast extract, 5 g/l NaCl, 15 g/l agar) to which were added 100 μg/ml of ampicillin and 1% (w/v) glucose. After growth, the number of colonies was determined to estimate the size of the banks and at least 48 clones per bank were subjected to PCR and fast DNA sequencing. The cells were re-suspended in the 2YT medium with 15% glycerol, frozen and stored at −80° C.

[0127] 1.3. Construction of the Mut3 Bank

[0128] A first bank was obtained using pol β on the wild-type Fc gene and contained 3.2×10.sup.6 clones (called Mut1.1). The DNA of this first bank was used to generate the second and third banks, respectively using pol β (3.8×10.sup.6 clones, Mut1.2) and pol η and i (3.0×10.sup.6 clones, Mut1.3).

[0129] This strategy with two accumulated replication steps allowed an increase in the mutation rate. The fourth bank was obtained with pol η alone on the wild-type Fc gene (1.0×10.sup.6 clones, Mut1.4). Finally, these four banks were proportionately mixed to obtain the final bank called Mut1, representing 1.1×10.sup.7 different clones.

[0130] Two different banks were then constructed using a DNA pool of single and double mutants isolated on the FcRn receptor. A first bank was obtained using pol β (1.9×10.sup.7 clones, Mut2.1) and a second bank with pol η (1×10.sup.6 clones, Mut2.2). Finally, these two banks were proportionately mixed to obtain the final bank called Mut2, i.e. 2×10.sup.7 different clones.

[0131] A new bank was obtained using pol β on the wild-type Fc gene, with a diversity of 4.7×10.sup.7 clones.

[0132] Finally, the banks Mut1, Mut2 and the new bank were proportionately mixed to obtain the final bank called Mut3, i.e. 7.8×10.sup.7 different clones.

[0133] The Mut3 bank finally contained 3×10.sup.7 mutated clones in phase with a mean of 2.2 mutated amino acids per Fc.

[0134] 1.4. Construction of the Mut4sel Bank

[0135] A bank of Fc variants was constructed from 36 clones having improved binding with C1q and/or CD16aV, selected from the Mut3 bank Mut3 (1.6 mutated amino acids per Fc on average). The Mut4 bank was obtained using an equimolar mixture of pol β and pol polymerases (in the replication buffer of pol β). The Mut4 bank has a diversity of 1.3×10.sup.7 clones, with 77%, of clones in phase and on average 2.6 mutated amino acids per Fc.

[0136] This bank was cloned in the pMG93 vector, a selection vector developed for Fc which allows selection of the coding sequences (ORFs) on an ampicillin medium through fusion with the gene of □-lactamase. This construction was transformed in the non-suppressive bacteria HB2151, which allowed removal of the TAGs codons that are known to be stop codons in this strain. The clones of this bank were spread plated in low density in dishes of agar medium containing small amounts of ampicillin, to carry out selection of the ORFs, the viable clones then being recovered and frozen. Spread plating in 500 Petri dishes (120×120) allowed covering of the entire bank Mut4. The bank thus selected was denoted Mut4sel and contained 92%, clones in phase, i.e. 1×10.sup.7 mutated clones in phase, with an average of 2.5 mutated amino acids per Fc. The Mut4sel bank was then sub-cloned in the phagemid pMG58, in XL1-Blue bacteria, to allow selection by «phage display» on the targets.

[0137] 1.5. Construction of the Mut5 Bank

[0138] A bank of Fc variants was constructed from 42 clones having improved binding with C1q and/or one of the FcgR receptors, selected from the Mut4sel bank (2.4 mutated amino acids per Fc on average). The Mut5 bank was obtained using the polymerase pol β alone so as to obtain a low mutation rate. Finally, the Mut5 bank obtained contained 1.2×10.sup.7 clones with 95% clones in phase and on average 3.1 mutated amino acids per Fc. The good quality of this bank did not require the selection of ORFs as was required for the Mut4 bank.

[0139] 2. Phage Display Expression of the Fc Banks and Selection of the Improved Variants

[0140] At the selection steps, the banks Mut3, Mut4sel and Mut5 were expressed on the surface of the M13 bacteriophage following standard procedures (Smith G P, Science 228: 1315 (1985)). E. coli XL1-Blue bacteria, containing the bank to be expressed cloned in the pMG58 vector, were cultured in 60 ml of 2YT medium to which were added 100 μg/ml ampicillin, 15 μg/ml tetracycline and 1% (w/v) glucose at 30° C. The cells were then infected with the auxiliary phage M13 (M13K07, Biolabs, bacteria/phage ratio=1:3) at 37° C. for 20 min and the production of Fc-phage was continued overnight at 26° C., at 230 rpm 2YT/ampicillin/glucose with 0.5 mM IPTG and 50 μg kanamycin/ml. The following day, the phages were precipitated with PEG6000 following standard procedures, re-suspended in 1 ml of PBS buffer at pH 7.4 and titrated by infecting XL1-Blue cells.

[0141] 2.1 Recombinant Proteins Used:

[0142] The C1q complement is commercially available (Calbiochem).

[0143] CD16a is an activator receptor having V/F polymorphism at position 158, on the binding side to Fc. Affinity is improved for CD16aV.

[0144] CD16aV is commercially available (R&D system).

[0145] CD16aF was produced by PX′Therapeutics.

[0146] CD32a is an activator receptor having H/R polymorphism at position 131, on the binding site to Fc. Affinity is improved for CD32aH.

[0147] CD32aR is commercially available (R&D system).

[0148] CD32aH was produced by PX′Therapeutics. CD32b is an inhibitor receptor having lesser affinity for IgG1s than CD32aR. It is commercially available (R&D system).

[0149] 2.2. Solid Phase Selections:

[0150] For solid phase selections, the Fc-phages diluted in PBS/5% skim milk/0.1% Tween 20 were incubated in 8 wells of Maxisorp plates (1-4×10.sup.11 phages/well in final 100 μl) previously coated with 500 ng/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 carried out: before the binding step on the target immobilised on a Maxisorp plate, the Fc-phages were pre-incubated in similar manner on 8 wells with the competitive receptor. Only the non-bonded Fc-phages on the first target were then transferred into wells containing the second target as previously described. The selected phages were eluted by infection with XL1-Blue bacteria in exponential growth phase (2×150 μl/well, 20 min. at 37° C. without agitation). The infected bacteria were spread plated on a solid 2YT/ampicillin/glucose medium. The following day, the cells were re-suspended in 2YT medium with 15% glycerol, frozen and stored at −80° C. until the following selection round.

[0151] 2.3. Liquid Phase Selections:

[0152] For liquid phase selections, 4×10.sup.11 phages were first incubated with biotinylated CD16aV (250 nM), biotinylated CD16aF (1000 nM), or with biotinylated C1q (250 nM) for 1 hour at ambient temperature under light agitation. Magnetic beads coated with streptavidin (Dynal) previously blocked with 5% skim milk in PBS were then added to the phages for 30 minutes at ambient temperature. The phage-bead complexes were washed 10 times with PBS/0.1 Tween 20 using a magnet. The phage-bead complexes were used to infect 5 ml of XL1-Blue bacteria in exponential growth that were spread plated on a solid 2YT/ampicillin/glucose medium. The following day, the cells were re-suspended in 2YT medium with 15% glycerol, frozen and stored at −80° C. until the following selection round.

[0153] 2.4. Selections from Banks:

[0154] Mut3 Bank:

[0155] For liquid phase selections, 6 rounds were performed on 3 biotinylated targets: CD16aV, CD16aF and C1q (clones denoted DL6A, DL6B and QL6A). For the solid phase selection rounds, 6 rounds were performed on 3 targets: CD16aV, biotinylated CD16aV and biotinylated C1q (clones denoted DS6A, DS6B and QS6A).

[0156] Mut4sel Bank:

[0157] The selection rounds were solely conducted in solid phase: 3 selection rounds on biotinylated CD16aV, 3 rounds on biotinylated C1q, 3 rounds on biotinylated CD32aR (+/−depletion on CD32b for the 3.sup.rd round) and 3 rounds on biotinylated CD32b (+/−depletion on CD32aR for the 3.sup.rd round), (clones denoted A3A, G3A, J3A/B and K3A/B).

[0158] Mut5 Bank:

[0159] The selection rounds were conducted as previously: 3 selection rounds on biotinylated CD16aF, 3 rounds on biotinylated C1q, 3 rounds on biotinylated CD32aR, 3 rounds on biotinylated CD32b, 2 rounds on biotinylated CD32aR and 2 rounds with depletion on biotinylated CD32b, and 2 rounds on biotinylated CD32b and 2 depletion rounds on biotinylated CD32aR (clones denoted N3A, 03A, P3A, Q3A, P4B and Q4B). Selection was then performed as previously described.

[0160] 2.5. Pool Cloning in the pMGM05 Vector:

[0161] The clones selected after the selection rounds were directly transferred in a mixture (about 10.sup.4 clones per condition) into the eukaryote vector pMGM05-CD20 (pCEP4 InvitroGen), which contains the same cloning sites as the pMG58 phagemid for the Fc fragment (BamHI and NotI) and the VH variable chain of the anti-CD20 antibody. This construction leads to mutation of two amino acids in Fc (aa224 and 225, HT changed to GS) and addition of the EFAAA sequence at the C-terminal of Fc, but allows rapid testing of a very large number of clones. It was initially verified that these mutations do not modify the binding of IgG-WT to the different receptors. Thereafter, the positive controls were cloned in this system for validation thereof: [0162] IgG1-5239D, 1332E, derived from the anti-CD19 XmAb5574 antibody by Xencor (C1): positive control for CD16a; [0163] IgG1-G236A, derived from Xencor (C4): positive control for CD32aH/R; [0164] IgG1-K326W, E333S, derived from Abgenix/Genentech (C3): positive control for C1q; and [0165] IgG1-5267E, L328F, derived from the anti-CD19 XmAb5574 antibody by Xencor (C5): positive control for CD32b.

[0166] The DNA of about one hundred clones, isolated by selection rounds, were sequenced by PCR on colonies. After bioinformatic analyses, the clones comprising new mutations were frozen at −80° C. in XL1-Blue bacteria and the sequences included in our database. As a result, 158 clones were isolated from the Mut3 bank, 371 clones from the Mut4sel bank and 171 clones from the Mut5 bank.

[0167] 2.6. Production of Variants in HEK293 Cells:

[0168] The light chain of anti-CD20 was inserted in a pCEP4 vector, identical to the vector used for the heavy chain, denoted pMGM01-CDC20 (pCEP4 InvitroGen). HEK293-F Freestyle™ cells (Invitrogen), cultured in 24-well plates were co-transfected with the vectors pMGM01-CD20 and pMGM05-CD20 (Fc-WT and variants) in equimolar amounts (250 ng/ml) with a Freestyle MAX reagent (1 μl/ml) following standard procedures (Invitrogen). The cells were cultured in suspension in a serum-free medium for 7 post-transfection days and the supernatants (1 ml) containing IgGs were harvested after centrifugation of the cells at 100 g for 10 min. The IgGs secreted in the supernatants were quantified using an ELISA assay on recombinant protein L (Pierce), with a purified anti-CD20 antibody produced in 293-F cells used as standard. The supernatants and standard antibodies diluted in series in PBS/0.05% Tween-20, were assayed on Maxisorp immunoplates (Nunc) previously coated with 0.25 μg protein L/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. Binding of IgG variants was detected with a F(ab′)2 fragment of goat anti-human IgG HRP (specific to the γ chain) (Sigma). The IgG variants produced were quantified (1-4 μg/ml) using the standard curve.

[0169] 2.7. ELISA Assays on IgG Variants Produced in the Supernatants of 293-F Cells:

[0170] The IgG variants were assayed by ELISA for their binding to the human C1q complement and several human FcRs. Maxisorp immunoplates were coated with 0.5 μg C1q complement/well, 0.05 μg CD32aH/well, 0.2 μg CD16aF/well or 0.1 μg CD16aV/well in PBS. Immobilising 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., 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/m1 and mixed with F(ab′)2 fragments of goat anti-human IgG HRP at the same concentration for 2 hours at ambient temperature. The IgGs aggregated to F(ab′)2 were then incubated under gentle agitation for 1 hour at 30° C. on 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 plates were then with detected with TMB (Pierce) and absorbance read at 450 nm.

[0171] Selections on the Mut3 Bank:

[0172] By means of this ELISA assay, the variants selected by phage display were assayed for their binding to the C1q complement and to the different receptors. They were assayed by comparison with the wild-type Fc (Fc-WT) and positive controls. 36 positive clones on CD16aV and/or C1q were thus selected on the Mut3 bank and used to construct the Mut4 bank.

[0173] Selections on the Mut4sel Bank:

[0174] ELISA assays performed on the 371 isolated clones allowed identification of 116 clones having a ratio higher than 2 for at least one FcγR and 17 clones with a ratio higher than 3 for C1q alone, which therefore corresponds to 133 clones of interest (Table 1).

TABLE-US-00002 TABLE 1 133 clones of interest isolated Name of Results of ELISA assays mutant Mutations C1q CD16aV CD32aR CD32b 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, 4.32 2.52 13.17 6.12 A378V, Y436H 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, 3.30 2.51 5.38 3.43 A431V 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, 2.39 1.88 2.67 3.03 N421T 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, 3.17 1.74 2.39 2.91 S442F 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 1.63 2.96 2.76 (>30) 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, 1.05 1.68 2.10 2.61 N421T 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 2.29 G3A-154 A378T, V397M, F423P, 2.51 2.11 4.14 2.28 S442P 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, 7.34 1.58 5.99 2.20 A431V 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, V3081, A327T, 0.50 0.14 0.30 2.05 S383N 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, 6.36 1.51 3.99 2.04 N434S A3A-137 K290E, V323I, F405L 1.91 1.65 3.02 2.03 K3B-94 R255W, A287T, P352S, 4.66 1.52 5.08 2.02 A378V 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, 0.74 1.65 2.88 1.80 G402D, S440N 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 V3081, K326T, F423L, 4.84 1.13 2.71 1.65 A431V 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, 1.41 1.36 2.02 1.53 F423L 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, 2.10 1.28 2.89 1.37 S440N 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, 1.73 1.65 2.00 1.33 N389T 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, 1.32 1.30 3.72 1.27 K447N 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, 0.62 1.18 2.25 1.02 K414N 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, 3.00 0.91 1.13 0.98 Q438DEL 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, 3.11 0.95 0.62 0.94 Y436N 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, 4.32 1.00 0.56 0.88 P396L G3A-33 K248E, S267N, 1336M, 5.67 0.79 0.39 0.76 P352L, P396L G3A-51 V240I, K246E, P353L 3.90 0.95 0.51 0.64

[0175] Among these clones, 47 improved clones were used to construct the Mut5 bank.

[0176] From these results, 18 new variants were constructed by directed mutagenesis so as to accumulate mutations of interest (Table 2).

TABLE-US-00003 TABLE 2 18 new variants constructed and compared with reference variants NA = Non-Determined Name of Results of ELISA assays mutant Mutations 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, 15.33 1.09 1.45 0.66 5.60 1.21 T394P A3A-105B G316D, K326E, 14.30 2.25 3.52 3.92 12.64 4.16 P396L A3A-105C G316D, K326E, 9.61 2.90 1.86 1.81 11.00 2.22 V397M A3A-105D G316D, K326E, 13.32 2.46 3.68 1.39 8.20 5.96 A378V A3A-184 K334N, P352S, 1.44 1.71 2.90 1.34 3.06 2.91 V397M A3A-184A K334N, P352S, 1.75 3.40 4.74 3.25 5.03 2.32 A378V, V397M 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, NA 1.13 1.65 NA NA NA T394P G3A-43B A231V, A378V, NA 1.85 2.42 NA NA NA P396L G3A-43C A231V, A378V, NA 1.81 2.68 NA NA NA V397M G3A-45 K246R, A378V 1.15 1.92 2.46 1.38 2.76 1.68 G3A-45A K246R, A378V, NA 1.34 2.34 NA NA NA T394P G3A-45B K246R, A378V, NA 1.37 1.75 NA NA NA P396L G3A-45C K246R, A378V, NA 1.51 2.21 NA NA NA V397M G3A-95 I336T, A378V 1.05 1.29 2.25 1.17 1.82 1.31 G3A-95A I336T, A378V, NA 1.50 1.90 NA NA NA T394P G3A-95B I336T, A378V, NA 2.00 2.54 NA NA NA P396L G3A-95C I336T, A378V, NA 1.55 2.01 NA NA NA V397M J3B-118 P396L, N421T 3.88 1.26 2.58 NA 9.00 4.99 J3B-118A A378V, P396L, NA 1.76 3.37 NA NA NA N421T 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

[0177] Finally, from these 151 assayed variants, 26 variants of interest were selected for production on a larger scale and more specific study (Table 3).

TABLE-US-00004 TABLE 3 26 selected new variants NA = Non-Determined Name of Results of ELISA assays mutant Mutations C1q CD16aF CD16aV CD32aH CD32aR CD32b A3A-105 G316D, K326E 5.18 1.31 2.48 1.50 4.50 7.13 A3A-105D G316D, K326E, 13.32 2.46 3.68 1.39 8.20 5.96 A378V A3A-14 S298N, A378V 0.53 0.96 0.85 0.22 0.35 0.88 A3A-173 K248E, K334R, NA NA 1.63 NA 2.96 2.76 A378V A3A-184 K334N, P352S, 1.44 1.71 2.90 1.34 3.06 2.91 V397M A3A-184A K334N, P352S, 1.75 3.40 4.74 3.25 5.03 2.32 A378V, V397M 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, 1.32 NA 1.88 NA 4.94 2.21 G402D G3A-95 I336T, A378V 1.05 1.29 2.25 1.17 1.82 1.31 J3A-06 A378T, V397M, 1.60 1.24 1.65 1.58 2.74 1.41 V412M J3A-14 K326T 1.83 0.52 1.43 0.99 4.25 2.26 J3A-16 N286Y, P352S, 1.46 1.35 1.83 1.77 3.54 1.98 A378V J3A-28 E333G, A378T, 1.89 1.41 1.83 1.39 1.73 1.18 V397M J3B-115 T359A, S383R, 1.72 1.32 1.69 1.42 5.17 2.77 V397M J3B-118 P396L, N421T 3.88 1.26 2.58 NA 9.00 4.99 J3B-118A A378V, P396L, NA 1.76 3.37 NA NA NA N421T J3B-16 S267G, A378T, 0.85 0.59 0.57 0.42 4.95 1.58 V397M J3B-23 N286I, A378V, 1.13 1.91 1.81 1.57 3.33 1.57 F423Y K3B-01 N315D, N361H, 3.46 0.98 2.01 2.10 7.65 7.30 P396L K3B-90 V308A, K334R, 1.32 1.06 1.30 1.40 3.72 1.27 A378V, K447N QL2A-16 K248E, N421T 5.30 0.69 0.79 0.33 0.77 1.19

[0178] Selections on the Mut5 Bank:

[0179] The ELISA assays performed on the 171 isolated clones allowed the identification of 87 clones with a ratio higher than 2 for at least one of the assayed FcγRs or C1q complement (Table 4).

TABLE-US-00005 TABLE 4 87 clones with a ratio higher than 2 Name of Results of ELISA assays mutant Mutations C1q CD16aF CD16aV CD32aH CD32aR CD32b P3A-01 K334N, S383R 0.93 1.34 2.05 1.10 2.00 1.57 P3A-17 K334N, Y373D, 0.88 1.46 1.63 1.35 2.17 1.55 A378V P3A-18 K248E, N286Y, 24.77 0.94 1.62 1.07 1.24 1.13 K334R, A378V, K447N P3A-23 N361H, P396L 2.70 1.75 2.49 2.53 3.33 1.76 P3A-30 K288E, K334N, 1.18 1.35 4.33 2.14 1.79 1.41 P352S, V397M P3A-31 K248E, T359A, 5.80 0.80 2.05 1.80 1.62 1.51 S383R, V397M 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, 1.13 1.93 4.05 3.54 4.89 3.20 V397M P3A-74 V302A, V397M 0.92 0.72 0.63 1.47 2.43 1.32 P3A-94 L309P, T359A, 1.02 1.32 2.11 2.54 1.79 1.32 S383R, V397M N3A-111 V302A, K334N, 1.36 0.82 1.18 1.20 2.73 2.33 S375R N3A-113 E283G, Y349S, 6.55 1.30 1.73 2.17 5.19 2.94 P396L N3A-114 K320E, S375R, 0.69 1.03 1.12 0.92 2.11 1.60 F405V 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, 0.77 1.02 2.23 0.88 1.25 1.40 K447N N3A-138 S298T, K334R, 3.56 0.90 0.68 0.59 0.73 1.17 K370E N3A-14 T307N, A378V, 1.00 1.70 2.83 2.50 2.49 1.74 K414R N3A-141 T350A, V379A, 2.82 1.30 1.79 2.36 5.60 5.70 G385R, P396L N3A-145 S267G, K290E, 0.99 0.70 0.38 0.31 2.45 1.33 E293G, E380G, V397M N3A-150 S267G, K334N, 0.86 0.76 0.68 0.33 4.82 1.78 P352S, V397M N3A-161 K248E, N315D, 8.59 1.28 2.49 1.12 1.23 1.32 A378V 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, 1.67 2.25 3.77 2.05 2.59 1.49 K392R, S440G N3A-28 T366A, A378V 1.04 1.77 2.89 2.26 1.84 1.36 N3A-32 K326T, K334N, 1.61 2.47 4.35 1.59 3.24 2.04 P352S, N421T N3A-52 K326T, S383R, 3.70 1.11 2.07 1.92 5.40 2.41 V397M N3A-58 V284L, K334R, 5.37 1.02 1.67 2.01 4.00 1.67 A378V, K447N N3A-59 N315D, T366A, 1.08 1.25 1.66 1.75 2.73 1.56 G385R, P396L N3A-61 V308A, K334R, 2.07 1.10 1.57 1.11 2.87 2.67 A378V 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, 0.91 2.15 2.85 3.12 5.94 4.63 T350A, P396L N3A-93 A378T, P396L 1.63 1.84 2.21 2.55 3.86 3.06 O3A-04 A231V, T359A, 1.00 1.16 1.21 1.69 2.35 1.09 S383R, V397M O3A-05 K290E, T366A, 0.99 2.79 3.12 2.07 3.34 1.34 A378V O3A-10 Y300H, T394P 0.98 0.76 0.90 1.81 2.25 1.17 O3A-11 R255W, A287T, 1.85 1.01 2.10 2.20 3.15 1.5 P352S, A378V, N421T O3A-16 K334R, A378V, 4.30 1.71 2.51 2.43 3.98 1.59 K447N O3A-17 I336V, T359A, 0.85 1.79 2.70 2.30 1.73 0.93 S383R, V397M O3A-22 K334R, P396L, 23.55 1.68 2.16 2.75 4.79 1.62 H435R O3A-24 K326T, K447N 2.12 1.23 2.27 1.55 2.94 1.40 O3A-25 V308A, K334R, 11.12 1.40 2.07 2.04 4.16 1.96 A378T, V397M O3A-34 A231V, Y349S, 0.73 1.40 1.90 2.34 2.62 1.28 V397M O3A-38 K248E, N286Y, 2.17 1.59 1.16 0.99 0.69 0.57 Q418P O3A-40 V308A, A378T, 0.82 1.65 2.15 2.09 2.64 1.03 V397M, V412M 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, 3.32 1.57 0.61 0.56 1.19 1.36 L410R O3A-46 V302A, A378V 0.94 1.15 1.75 2.36 2.91 1.37 O3A-50 K334R, A378V, 3.62 1.28 2.49 2.15 4.02 2.30 N421T 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, 1.26 1.26 1.81 2.16 2.32 1.28 G402D, K447N O3A-81 K274R, A378T, 1.36 1.20 2.39 2.06 3.08 1.58 V397M O3A-86 K248E, K290E, 24.34 2.49 4.89 2.86 4.19 2.57 N361H, P396L O3A-93 K290E, T394P 2.07 1.45 3.53 2.54 3.36 1.78 P4B-89 T307P, T366A, 0.63 2.99 4.15 0.70 1.19 0.87 A378V Q4B-08 V284L, K290E, 2.43 1.75 3.47 2.69 3.58 1.63 A378V Q4B-15 K274R, A378V 1.26 1.04 2.69 2.61 1.75 1.03 Q4B-18 I377T, A378V, 1.21 1.08 2.82 1.96 1.37 1.07 F423Y Q4B-34 K248E, K290M, 4.47 0.63 0.56 0.38 0.70 0.75 V308A, P352S Q4B-59 T350A, A378T, 1.32 2.22 2.01 2.76 2.52 1.51 V397I Q4B-61 K320E, T394P, 0.87 1.10 0.98 1.60 2.43 1.08 V397M Q4B-68 T307P, A378V, 3.33 1.53 4.12 4.01 3.69 1.03 T394P 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, 2.80 1.63 1.99 3.52 3.09 1.02 N421T Q3A-58 S267G, V397M 0.73 1.30 0.34 0.40 2.87 1.02 Q3A-85 P396L, N421T, 3.80 1.65 2.17 3.67 3.50 1.13 K447N O3A-103 K290E, K320T, 2.11 1.22 2.08 2.76 2.41 1.50 A378V O3A-117 K334R, T394P, 0.98 1.05 1.71 2.29 3.05 1.37 N421S O3A-119 K334N, V397M 1.53 1.04 1.97 1.45 2.28 1.18 O3A-126 P227S, V284L, 4.58 1.31 2.93 2.85 4.47 1.29 A378V O3A-127 V302A, K334R, 1.53 1.00 1.24 2.85 3.56 1.37 T366A, S383R, V397M O3A-131 C261R, A378T, 2.25 1.03 1.72 2.00 2.27 1.15 V397M, V412M 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, 2.14 1.32 2.27 2.84 3.09 1.42 T359A, S383R, V397M

[0180] Among these variants, 10 variants of interest were selected (Table 5).

TABLE-US-00006 TABLE 5 10 variants Name of Results of ELISA assays mutant Mutations C1q CD16aF CD16aV CD32aH CD32aR CD32b O3A-05 K290E, T366A, 0.99 2.79 3.12 2.07 3.34 1.34 A378V P4B-89 T307P, T366A, 0.63 2.99 4.15 0.70 1.19 0.87 A378V O3A-86 K248E, K290E, 24.34 2.49 4.89 2.86 4.19 2.57 N361H, P396L N3A-32 K326T, K334N, 1.61 2.47 4.35 1.59 3.24 2.04 P352S, N421T O3A-44 A378V, K439R 1.95 2.44 4.40 3.20 3.13 1.81 N3A-27 K290E, A378V, 1.67 2.25 3.77 2.05 2.59 1.49 K392R, S440G N3A-87 K290E, K320E, 0.91 2.15 2.85 3.12 5.94 4.63 T350A, P396L Q4B-68 T307P, A378V, 3.33 1.53 4.12 4.01 3.69 1.03 T394P N3A-58 V284L, K334R, 5.37 1.02 1.67 2.01 4.00 1.67 A378V, K447N Q3A-39 N286I, P396L, 2.80 1.63 1.99 3.52 3.09 1.02 N421T

[0181] 3. Production and Purification of Variants of Interest

[0182] The IgG variants were obtained by direct mutagenesis in pCEP4-WT-H-CD20. The IgG controls, i.e. C1, C3, C4, C5 and wild-type (WT), were produced with the G1m3 allotype (comprising 3 mutations compared with G1 m1,17: (K214R/)D356E/L358M).

[0183] The 26 IgG variants derived from the Mut4sel bank, and the wild-type, were produced with the G1 m1,17 allotype. They were produced by incubation for 6-7 days in batches (250-300 ml) of 293-E cells (Freestyle Invitrogen) in F17 medium.

[0184] Centrifugation and filtration were then carried out.

[0185] Purification was performed on Protein A Hi-Trap, and elution with a 25 mM citrate buffer, pH=3.0, neutralisation and dialysis in TBS or PBS before sterilisation.

[0186] 10 mg of each IgG control, i.e. C1, C3, C4, C5 and wild-type (WTG1m3 and WTG1 m1,17) were obtained, and 2-3 mg of 26 IgG variants.

[0187] Their characterization shows that the molecular weight is maintained and that glycosylation profiles are similar for all variants.

[0188] 4. Assays of Variants of Interest

[0189] 4.1. Binding Assays on FcRn

[0190] Jurkat-FcRn cells were incubated at pH=6.0 with the IgG variants at different concentrations (0 to 1000 μg/ml) and with Rituximab-Alexa.

[0191] Flow cytometry was conducted on the bound Rituximab-Alexa.

[0192] The results do not show any loss of binding to FcRn for all IgG variants.

[0193] 4.2 Binding Assays to the Antigen

[0194] Raji cells were incubated with the IgG variants at 1 μg/ml for 15 minutes at 4° C.

[0195] The bound IgGs were detected by binding with a PE anti-human IgG secondary antibody (for 15 minutes at 4° C.).

[0196] The results show that recognition of the antigen is not deteriorated by the different mutations on the Fc.

[0197] All the IgG variants were bound to CD20 on the cells, similar to the IgG-WT control.

[0198] 4.3 ELISA Binding Assays to CD16aV/F

[0199] The purified antibodies were assayed with ELISA for binding to CD16F and CD16aV following the same protocol as described under item 2.6, diluting the antibodies to different concentrations.

[0200] 4.4. ADCC Activity Assays

[0201] NK cells were incubated with target Raji cells expressing CD20, in the presence of different concentrations of IgG variants (0.005 to 5000 ng/ml).

[0202] The level of intracellular LDH released by the lysed target cells was measured.

[0203] Human NK cells were purified from the peripheral blood of healthy volunteer donors using the negative depletion technique developed by Miltenyi. The ADCC assay comprised the incubation of NK cells with target Raji cells expressing the CD20 antigen, in the presence of different concentrations of anti-CD20 antibodies. After an incubation time of 16 hours, the cytotoxicity induced by the anti-CD20 antibodies was measured by quantifying intracellular lactate dehydrogenase (LDH) in the cell supernatants. The results of specific lysis are expressed as a lysis percentage as a function of antibody concentration. The EC50 value (antibody concentration inducing 50% of the maximum lysis induced by IgG-WT) and Emax value (percentage maximum lysis) were calculated using the software GraphPad PRISM.

[0204] The results are given in Tables 6 and 7.

TABLE-US-00007 TABLE 6 Results of ADCC assays Mutant G3A43 G3A45 J3B-118 J3B16 A3A-184 WT EC50: 1.03 4.38 1.27 >5545.36 2.35 12.34 Antibody concentration (ng/ml) giving 50% lysis of an internal control Ratio [WT]/[antibody] 11.98 2.82 9.72 <0.0022 5.25 1

TABLE-US-00008 TABLE 7a Results of ADCC assays EC50: Antibody concentration (ng/ml) giving 50% maximum lysis of Ratio Mutant an internal control [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-US-00009 TABLE 7b Results of ADCC assays EC50: Antibody concentration (ng/ml) giving 50% maximum lysis of an Mutant 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

TABLE-US-00010 TABLE 7c Results of ADCC assays (EC45 instead of EC50) EC45: Antibody concentration (ng/ml) giving 45% maximum lysis of an Mutant 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 The best variants are A3A-184A et G3A-103.

[0205] 4.5. CDC Activity Assays

[0206] Target Raji 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 complement source (Cedarlane, 1/10 dilution). After an incubation time of 1 hour at 37° C., the level of LDH released into the supernatant by the lysed target cells was measured chromogenically (cytotoxicity detection kit by Roche Applied Sciences) and used to quantify antibody-mediated complement-dependent cytotoxicity. The results are expressed as percent lysis. EC50 (number of antibodies inducing 50% maximum lysis) and Emax (percent maximum lysis) were calculated using the software GraphPad PRISM.

[0207] The results are given in Tables 8 and 9.

TABLE-US-00011 TABLE 8 Results of CDC assays Antibody concentration (ng/ml) giving 50% lysis Antibody 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

TABLE-US-00012 TABLE 9a Results of CDC assays Antibody concentration (ng/ml) giving 50% lysis of an Antibody internal control Ratio [WT]/[antibody] 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

TABLE-US-00013 TABLE 9b Results of CDC assays Antibody concentration (ng/ml) giving 50% lysis of an Antibody internal control Ratio [WT]/[antibody] 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

[0208] Table 10 below gives the results obtained with some variants classified into sub-groups.

TABLE-US-00014 TABLE 10 ELISA ratio (purified antibodies) Name 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 1.3 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 S298N, 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 1.3 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

[0209] Following Table 11 gives some possible variants of the invention:

TABLE-US-00015 TABLE 11 Starting Added Name Mutations variant mutation G3A-95D S298N, I336T, A378V G3A-95 S298N K3B-90A K248E, V308A, K334R, A378V, K3B-90 K248E K447N G3A-88A K320E, K326T, T394P, G402D G3A-88 K326T N3A-87A K290E, K320E, K326T, T350A, N3A-87 K326T P396L A3A-105E K248E, G316D, K326E, A378V A3A-105D K248E A3A-14A K248E, S298N, A378V A3A-14 K248E A3A-184B K248E, K334N, P352S, V397M, A3A-184 K248E A378V 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, A3A-184 E333G A378V G3A-103A K248E, E333G, A378V G3A-103 E333G G3A-43E A231V, E333G, A378V G3A-43 E333G G3A-95F E333G, 1336T, 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, A3A-184 F423Y F423Y G3A-103B K248E, A378V, F423Y G3A-103 F423Y G3A-43F A231V, A378V, F423Y G3A-43 F423Y G3A-95G 1336T, 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