Application of modified Fc fragments in immunotherapy

Abstract

The present invention relates to the use of an antibody Fc fragment in the treatment of autoimmune diseases and / or inflammatory diseases, wherein the Fc fragment is an isolated recombinant Fc fragment having a modified affinity for at least one Fc receptor (FcR), in particular having an increased affinity for FcRn.

Description

[0001] This application is a divisional application of PCT application PCT / FR2016 / 051708, filed on July 6, 2016, with the invention name “Application of modified Fc fragments in immunotherapy”. The date on which the PCT application entered the Chinese national phase is March 5, 2018, and the application number is 201680051282.5. Technical field:

[0002] The present invention relates to immunotherapy for autoimmune and / or inflammatory diseases. Background technology:

[0003] Immunotherapy involves the administration of exogenous antibodies to a patient and is currently widely used to treat various pathologies, in particular those of autoimmune and inflammatory diseases.

[0004] Two immunoglobulin-based therapies are typically proposed for the treatment of these diseases: i) intravenous immunoglobulin (IVIg)-based therapies, which consist of the intravenous administration to the patient of immunoglobulins (most commonly IgG) derived from human plasma pools and ii) therapies based on the use of recombinant antibodies (i.e. antibodies obtained by genetic engineering). The latter have allowed real advances in the management of patients with inflammatory and autoimmune diseases, primarily because they offer the possibility of avoiding the drawbacks associated with the use of immunoglobulins from plasma, in particular the risks of supply shortages and the risks of transmitting pathogens that may be present in plasma.

[0005] The presence of Fab fragments in immunoglobulins may be responsible for significant adverse reactions in treated patients. In order to avoid these side effects, patent application WO 2004 / 099374 discloses the use of isolated recombinant Fc fragments for the treatment of patients, in particular for patients suffering from autoimmune diseases.

[0006] However, there remains a need to optimize these Fc fragments, in particular in order to increase their half-life and / or therapeutic efficacy. Summary of the invention:

[0007] The inventors now propose to use Fc fragments having a modified affinity for at least one Fc receptor (FcR) compared to the parent Fc fragment. According to the invention, the Fc fragments are isolated, i.e. they are not associated with Fab fragments or conjugated or fused to any other protein, polypeptide or peptide. In particular, they are not complete immunoglobulins.

[0008] The present invention relates to a composition comprising an antibody Fc fragment for use in treating autoimmune and / or inflammatory diseases, wherein the Fc fragment is an isolated Fc fragment having improved affinity for Fc-gamma receptor III (FcγRIII) compared to a parent Fc fragment.

[0009] In a preferred aspect, they are

[0010] - An Fc fragment having a combination of mutations 315D / 330V / 361D / 378V / 434Y compared to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragment consists of the sequence SEQ ID NO: 11

[0011] - An Fc fragment having a combination of mutations T260A and 315D / 330V / 361D / 378V / 434Y compared to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragment consists of sequence SEQ ID NO: 12

[0012] - An Fc fragment having a combination of mutations E258I and 315D / 330V / 361D / 378V / 434Y compared to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragment consists of sequence SEQ ID NO: 13

[0013] - An Fc fragment having a combination of mutations K290Y and 315D / 330V / 361D / 378V / 434Y compared to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragment consists of sequence SEQ ID NO: 14

[0014] - An Fc fragment having a combination of mutations E294A and 315D / 330V / 361D / 378V / 434Y compared to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragment consists of sequence SEQ ID NO: 15

[0015] - An Fc fragment having a combination of mutations Y296W and 315D / 330V / 361D / 378V / 434Y compared to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragment consists of sequence SEQ ID NO: 16

[0016] - An Fc fragment having the combination of mutations K334N / P352S / A378V / V397M compared to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragment consists of the sequence SEQ ID NO: 19

[0017] - An Fc fragment having a combination of mutations G316D / K326E / A378V compared to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragment consists of sequence SEQ ID NO: 20

[0018] - An Fc fragment having a combination of mutations A378V / P396L / N421T compared to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragment consists of the sequence SEQ ID NO: 21

[0019] - An Fc fragment having the mutation T260A compared to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragment consists of the sequence SEQ ID NO: 17

[0020] - An Fc fragment having the mutation K290Y compared to a parent Fc fragment of wild-type IgG1, preferably wherein the Fc fragment consists of the sequence SEQ ID NO:18.

[0021] The invention also more generally provides a composition comprising an antibody Fc fragment, an isolated Fc fragment having a modified affinity for at least one Fc receptor (FcR) compared to a parent Fc fragment, for use in treating autoimmune and / or inflammatory diseases.

[0022] In a specific embodiment, the composition comprises an antibody Fc fragment having at least one mutation in one or more amino acids and / or having an N-glycan at its glycosylation site (Asn 297), wherein the N-glycan of the Fc fragment has a fucosylation degree of less than 65%. Sequence Listing <110> French Blood Fractionation and Biochemical Products Laboratory (Laboratoire Francais duFractionnement et des Biotechnologies) <120> Application of modified Fc fragments in immunotherapy <130> B2057PC <160> twenty one <170> BiSSAP 1.3 <210> 1 <211> 222 <212> PRT <213> Artificial sequence <220> <223> Fc de l'IgG1 G1m1,17 <400> 1 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 35 40 45 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 50 55 60 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 65 70 75 80 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 85 90 95 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 115 120 125 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 165 170 175 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 180 185 190 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 195 200 205 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 2 <211> 221 <212> PRT <213> Artificial Sequence <220> <223> Fc of IgG2 <400> 2 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 1 5 10 15 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 20 25 30 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 35 40 45 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 50 55 60 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 65 70 75 80 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 85 90 95 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 100 105 110 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115 120 125 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 130 135 140 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 145 150 155 160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 165 170 175 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 180 185 190 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 195 200 205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 3 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Fc d' IgG3 <400> 3 Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 35 40 45 Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 50 55 60 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val 65 70 75 80 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 85 90 95 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 100 105 110 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 115 120 125 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp 165 170 175 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 180 185 190 Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His 195 200 205 Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 4 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Fc of IgG4 <400> 4 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 35 40 45 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 50 55 60 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 65 70 75 80 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 85 90 95 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 115 120 125 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 165 170 175 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 180 185 190 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 195 200 205 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 220 <210> 5 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Fc of IgG1 G1m3 <400> 5 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 1 5 10 15 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 20 25 30 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 35 40 45 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 50 55 60 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 65 70 75 80 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 85 90 95 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 100 105 110 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 115 120 125 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 130 135 140 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 145 150 155 160 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 165 170 175 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 180 185 190 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 195 200 205 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 6 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Fc of IgG1 G1m1,17 <400> 6 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 7 <211> 228 <212> PRT <213> Artificial sequence <220> <223> Fc of IgG2 <400> 7 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 65 70 75 80 Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys 225 <210> 8 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> Fc of IgG3 <400> 8 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys 1 5 10 15 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 20 25 30 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 35 40 45 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro 50 55 60 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 65 70 75 80 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 85 90 95 Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val Asp 100 105 110 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 115 120 125 Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 130 135 140 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 145 150 155 160 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg 165 170 175 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 180 185 190 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 195 200 205 Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn 210 215 220 Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 225 230 235 240 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser 245 250 255 Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser 260 265 270 Leu Ser Leu Ser Pro Gly Lys 275 <210> 9 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> Fc of IgG4 <400> 9 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 10 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Fc of IgG1 G1m3 <400> 10 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 11 <211> 227 <212> PRT <213> Synthetic Sequence <220> <223> Mutated Fc Fragment T5A-74 <400> 11 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asp Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asp Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 12 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Mutated Fc Fragment T5A-74I <400> 12 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Ala Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asp Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asp Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 13 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Mutated Fc fragment T5A - 74J <400> 13 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Ile Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asp Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asp Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 14 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Mutated Fc fragment T5A-74K <400> 14 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Tyr Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asp Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asp Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 15 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Mutated Fc Fragment T5A-74L <400> 15 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Ala Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asp Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asp Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 16 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Mutated Fc fragment T5A - 74M <400> 16 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Trp Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asp Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asp Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 17 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Mutated Fc Fragment ZAC2-85 <400> 17 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Ala Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 18 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Mutated Fc fragment ZAC3 - 172 <400> 18 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Tyr Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 19 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Mutated Fc Fragment A3A - 184A <400> 19 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Asn Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Ser Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 20 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Mutated Fc Fragment A3A - 105D <400> 20 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Asp 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Glu Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 21 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Mutated Fc Fragment J3B - 118A <400> 21 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Leu 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Thr Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 Brief description of the drawings:

[0023] Figure 1 The sequence alignment of native human IgG1 with respect to position 216 to 447 (according to the EU index) and the corresponding sequences of human IgG2 (SEQ ID NO: 7), human IgG3 (SEQ ID NO: 8) and human IgG4 (SEQ ID NO: 9) is shown. The IgG1 sequence refers to the G1m1, 17 allotype (SEQ ID NO: 6) and the G1m3 allotype (SEQ ID NO: 10). The "CH2-CH3 lower hinge" domain of IgG1 starts at position 226 (see arrow). The CH2 domain is highlighted in gray and the CH3 domain is shown in italics.

[0024] Figure 2 Schematic diagrams of IgG1 Fc (A) and scFc (B) are shown. CH2 is represented in grey and CH3 is represented in white. Disulfide bridges are represented by thin grey dashed lines. In scFc, two CH2-CH3 polypeptides are connected by a peptide linker represented by a thick dashed line.

[0025] Figure 3 The G0, G0F, G1 and G1F forms of the glycan structure that can be present on the Fc fragment of the present invention are shown.

[0026] Figure 4Presented are the percentages of effector cell-mediated lysis of Rhesus D+ red blood cells observed in the presence of monoclonal anti-Rhesus D (RhD) antibodies after the addition of varying amounts of multivalent immunoglobulin (IVIg) or recombinant Fc fragments, either unmutated or mutated (Fc fragment containing mutations 315D / 330V / 361D / 378V / 434Y).

[0027] Figure 5 The table presents the percentage of effector cell-mediated lysis of rhesus D+ red blood cells in the presence of monoclonal anti-rhesus D (RhD) antibodies observed after the addition of different amounts of multivalent immunoglobulin (IVIg) or recombinant Fc fragments, either unmutated or mutated (Fc fragments containing mutations according to Table 1). "rFc WT" indicates wild-type recombinant Fc fragment. Positive controls are represented as: IVIG (i.e., a mixture of plasma antibodies extracted from human plasma - IVIG is the reference treatment for idiopathic thrombocytopenic purpura (ITP)); and "IgG Mabs starting" corresponding to the Herceptin Fc fragment (antibodies transgenically produced in mammals followed by papain digestion to produce the Fc fragment).

[0028] Figure 6 Represents an in vitro binding assay measuring the affinity (Kd) of WT (wild type) and mutant Fc fragments to human FcRn (A) and CD16aV (V polymorphism at position 158 of CD16) assessed by surface plasmon resonance (SPR) using Biacore.

[0029] Figure 7 Shown are a map of the pCEP4 vector for expressing variant T5A-74 defined below and a map of the OptiCHO vector for expressing variant T5A-74. Detailed description of the invention:

[0030] definition

[0031] Throughout this specification, the numbering of residues in the Fc fragment is according to the numbering of immunoglobulin heavy chains according to the EU index or as described in Kabat et al., Sequences of proteins of immunological interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is expressly incorporated herein by reference. Herein, the "EU index" or "EU index or Kabat equivalent" refers to the numbering of residues in human IgG1 antibodies.

[0032] The term "immunoglobulin" refers to the structure that constitutes the natural biological form of an antibody, including constant regions and variable regions (also called fragments). An immunoglobulin molecule is a molecule whose basic unit is a heterotetramer consisting of two heavy chains (H) of about 50-70 kDa each and two light chains (L) of about 25 kDa each, linked together by intra-chain and inter-chain disulfide bonds.

[0033] Each chain is composed of: at the N-terminal position, a variable domain or region (called VL in the case of a light chain and VH in the case of a heavy chain); at the C-terminal position, a constant region (which is composed of a single domain called CL in the case of a light chain and three or four domains called CH1, CH2, CH3 and CH4 in the case of a heavy chain).

[0034] Only IgM and IgE have a CH4 domain.

[0035] Each domain contains about 110 amino acids and is structured in a comparable manner. The two heavy chains are linked by disulfide bonds at the CH2 domain, and each heavy chain is linked to a light chain by a disulfide bond between CH1 and CL. The region that determines the specificity of the antibody for the antigen is carried by the variable part, while the constant part can interact with effector cells or molecules such as Fc receptors (FcR) of complement proteins to induce various functional properties. The assembly of the chains that make up the antibody makes it possible to define a characteristic Y-shaped three-dimensional structure, in which,

[0036] - the bases of Y correspond to the constant Fc region (or Fc fragment) recognized by complement and Fc receptors to mediate the effector functions of the molecule, and

[0037] The ends of the arms of the -Y correspond to the respective assemblies of the light chain variable region and the heavy chain variable region, which constitute the Fab fragment and determine the specificity of the antibody for the antigen.

[0038] More specifically, there are five heavy chain isotypes (gamma, alpha, mu, delta, and epsilon) and two light chain isotypes (kappa and lambda, the lambda chain itself being divided into two types: lambda 1 and lambda 2). The heavy chain determines the immunoglobulin class. Thus, there are five classes of Ig: IgG (gamma isotype), IgA (alpha isotype), IgM (mu isotype), IgD (delta isotype), and IgE (epsilon isotype).

[0039] Kappa and lambda light chains are shared by all classes and subclasses. In humans, kappa and lambda are produced in a 2 to 1 ratio.

[0040] IgG is the most abundant immunoglobulin in serum (75% to 80% of circulating antibodies). In monomeric form, they have the longest serum half-life of all immunoglobulins (approximately 21 days).

[0041] There are four types of gamma heavy chains, which determine the four IgG subclasses (IgG1 for gamma 1, IgG2 for gamma 2, IgG3 for gamma 3, and IgG4 for gamma 4). These four subclasses differ in the variable number and position of disulfide bridges (Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6).

[0042] Despite their highly homologous structures (more than 95% sequence homology in the Fc region), the four human IgG subclasses differ in their biological activities.

[0043] The term "biological activity" refers in particular to the ability of the IgG constant region to bind to complement proteins, in particular (e.g. protein C1q) [Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.)]) and / or to the IgG receptors: FcγR (FcγRI, FcγRII, FcγRIII; Ravetch and Kinet, Annual Review of Immunology, Vol. 9: 457-492 (1991)).

[0044] Depending on the type of binding, various mechanisms of action may be activated: for example, opsonization, phagocytosis, antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). See Uananue and Benacerraf, Textbook of Immunology, 2nd edition, Williams & Wilkins, p. 218 (1984)) for further details.

[0045] The term "Fc fragment" refers to the constant region of a full-length immunoglobulin excluding the first immunoglobulin constant region domain. Therefore, "Fc fragment" refers to the last two constant domains (CH2-CH3) of IgA, IgD, IgG and the last three constant domains (CH2-CH3-CH4) of IgE and IgM, as well as the N-terminal flexible hinge of these domains. For IgA and IgM, the Fc fragment may include a J chain. For IgG, the Fc fragment includes CH2, CH3 domains and the lower hinge region between CH1 and CH2. In other words, the Fc region of IgG1 consists of the CH2-CH3 lower hinge, i.e., the portion from amino acid C226 to the carboxyl terminus, and the numbering is expressed according to the EU index or Kabat equivalents. Similar domains of other IgG subclasses can be determined by aligning the amino acid sequence of the heavy chain or heavy chain fragment of the IgG subclass with the amino acid sequence of human IgG1 (see Figure 1 ). The Fc fragment used according to the present invention may also include a portion of the upper hinge region upstream of position 226 (according to the EU index). In this case, it is preferred to use an Fc fragment of human IgG1 that includes a portion of the region between positions 216 and 226. In this case, "Fc fragment of human IgG1" refers to the portion from amino acids 216, 217, 218, 219, 220, 221, 222, 223, 224 or 225 to the carboxyl terminus. The term "Fc fragment" also refers to a single-chain Fc (scFc) fragment. The term "scFc fragment" refers to a single-chain Fc fragment obtained by genetic fusion of two Fc monomers connected by a polypeptide linker. scFc naturally folds into a functional dimer Fc region.

[0046] As used herein, the term "parent Fc fragment" or "parent polypeptide" refers to a reference polypeptide in a wild-type Fc region or a variant optionally containing mutations other than those under consideration. The term "wild-type" or "WT" herein refers to an amino acid sequence or nucleotide sequence found in nature, i.e., it is of natural origin (including allelic variations) and has not been intentionally modified by molecular biology techniques such as mutagenesis. For example, a "wild-type" Fc region specifically refers to an IgG1 Fc region (G1m1, 17 allotype) having a sequence of SEQ ID NO: 1, an IgG2 Fc region having a sequence of SEQ ID NO: 2, an IgG3 Fc region having a sequence of SEQ ID NO: 3, an IgG4 Fc region having a sequence of SEQ ID NO: 4, and an IgG1 Fc region having a sequence of SEQ ID NO: 5 (G1m3 allotype).

[0047] The Fc fragments used in the present invention are in monomeric form, ie, they are not fused or conjugated to each other.

[0048] The term "modified affinity" refers to decreased or increased affinity compared to the parent Fc fragment.

[0049] As used herein, the term "neonatal Fc receptor" or "FcRn" refers to a protein that binds to the IgG Fc region and is at least partially encoded by the FcRn gene. FcRn can be from any organism, including but not limited to humans, mice, rats, rabbits and monkeys. As known in the prior art, a functional FcRn protein comprises two polypeptides, usually named after a heavy chain and a light chain. The light chain is β2-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, the term "FcRn" or "FcRn protein" refers to a complex of an α chain and β2-microglobulin. In humans, the gene encoding FcRn is called FCGRT.

[0050] As used herein, the term "increased FcRn binding" refers to an increase in the in vivo or in vitro binding affinity of the mutant Fc fragment of the present invention to FcRn compared to the parent polypeptide. As described in, for example, patent application WO2010 / 106180, the ability of the mutant Fc fragment of the present invention to bind to FcRn can be assessed in vitro by ELISA.

[0051] In the present invention, the term "half-life" refers to the amount of time it takes for an Fc fragment, once present in the serum of a patient to whom it is administered, to be eliminated by half from the circulation or from other tissues.

[0052] The term "Fcγ receptor" or "FcγR" refers to the IgG type immunoglobulin receptors called CD64 (FcγRI), CD32 (FcγRII) and CD16 (FcγRIII), specifically five expressed receptors (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb). Except for human FcγRIIb (which is a receptor that inhibits immune cell activation), they are all effector cell activation receptors (Muta T et al., Nature, 1994, 368: 70-73).

[0053] The term "effector cell" refers to any cell that bears an Fc receptor, such as lymphocytes, monocytes, neutrophils, natural killer (NK) cells, eosinophils, basophils, mast cells, dendritic cells, Langerhans cells, and platelets.

[0054] In the context of the present invention, the term "glycosylation" refers to the addition of one or more carbohydrates to the sequence of a recombinant Fc fragment by an enzymatic reaction.

[0055] In the context of the present invention, the term "hypersialylation" refers to the addition of one or more sialic acid groups to the sequence of the Fc fragment. The addition of one or more sialic acid groups can be performed by an enzymatic reaction, by a cellular reaction, or by directed mutagenesis of the Fc targeting one or more amino acids involved in Fc sialylation.

[0056] The term "patient" refers to any human or animal subject, preferably a mammal. In a preferred embodiment, the patient is a human, regardless of age and gender.

[0057] The term "treat" or "treating" refers to ameliorating or preventing or reversing a disease or condition, or at least a symptom distinguishable therefrom, or ameliorating or preventing or reversing at least one measurable physical parameter associated with the disease or condition being treated, which is not necessarily distinguishable in or by the subject being treated. The term "treat" or "treating" further includes inhibiting or slowing the progression of a disease or condition, physically, such as stabilizing a distinguishable symptom, physiologically, such as stabilizing a physical parameter, or both.

[0058] Composition of the Fc fragment according to the present invention

[0059] The antibody Fc fragment used in the present invention is preferably an Fc fragment of an IgG1, IgG2, IgG3 or IgG4 immunoglobulin, which has a modified affinity for at least one Fc receptor (FcR) compared to the parent Fc fragment.

[0060] The Fc fragment according to the invention has a decreased affinity for at least one Fc receptor and / or an increased affinity for at least one Fc receptor compared to a parent Fc fragment.

[0061] Preferably, the affinity is increased 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 and preferably higher than 30 compared to the parent Fc. In other words, the affinity of the mutated Fc region for the FcR is higher than the affinity of the parent polypeptide. Alternatively, the mutated Fc region has a reduced affinity for at least one FcR. Preferably, the affinity is reduced 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 and preferably higher than 30 compared to the parent Fc. In other words, the affinity of the mutated Fc region for the FcR is lower than the affinity of the parent polypeptide.

[0062] The affinity of a polypeptide comprising an Fc region to an FcR can be assessed by methods known in the prior art. For example, one skilled in the art can determine affinity (Kd) by using surface plasmon resonance (SPR) that can be measured by a Biacore system. Alternatively, one skilled in the art can perform a suitable ELISA. A suitable ELISA can be used to compare the binding power of a parent Fc and a mutant Fc. Compare the specific signals from the mutant Fc and from the parent Fc. Binding affinity can be determined equally by assessing the entire polypeptide or by assessing the Fc region separated therefrom.

[0063] In particular, the Fc fragment used according to the present invention has a decreased affinity for at least one Fc receptor selected from the group consisting of FcRn, Fcγ receptor and complement C1q, and / or an increased affinity for at least one Fc receptor selected from the group consisting of FcRn, Fcγ receptor and complement C1q, compared to the parent Fc fragment.

[0064] According to a specific embodiment, the Fc fragment used according to the invention has a modified affinity for FcRn, advantageously an increased affinity. This increase in FcRn binding leads to an improvement in serum retention in vivo and thus to an increase in half-life.

[0065] The Fc fragments can be used alone or in mixture; for example, several Fc fragments having different mutations can be administered as a mixture or co-administered.

[0066] The Fc fragments of the present invention can also be used in a composition comprising a single type of mutated Fc fragment. In other words, the composition comprises molecules of Fc fragments of the same sequence.

[0067] According to one aspect of the invention, an Fc fragment having a modified affinity for at least one Fc receptor comprises a mutation of at least one amino acid compared to a parent Fc fragment. The mutations involved are not natural variations that define immunoglobulin isotypes, but rather artificial mutations, i.e., a production method that produces an Fc fragment containing the desired mutation.

[0068] Preferably, the mutation is a substitution, deletion or insertion of one or more amino acids.The mutant Fc fragment may have several mutations affecting several amino acids, preferably from two to ten.

[0069] In a preferred embodiment, the Fc fragment has a mutation selected from the group consisting of: at amino acids 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 90, 291, 292, 293, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 351 52, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403, 404 04, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 or 447, the numbering of the Fc fragment amino acids is according to the EU index or the Kabat equivalent.

[0070] Certain amino acid positions from the above list, namely 226, 230, 241, 256, 259, 264, 307, 315, 330, 342, 361, 362, 378, 382, ​​383, 389, 396, 397, 421, 428 and 434 are preferred. Specifically, the mutant Fc fragment with high binding affinity to FcRn may comprise at least one amino acid change at said amino acid position. Among them, positions 230, 264, 307, 315, 330, 378 and 434 are preferred, and positions 264, 315, 378 and 434 are more preferred.

[0071] In a specific embodiment, at least two, or even three, four or five amino acid mutations can significantly improve the binding affinity to FcRn compared to the parent Fc.

[0072] In specific embodiments, the mutation is:

[0073] (i) one or two mutations selected from positions 226, 230, 241, 264, 307, 315, 330, 342, 362, 378, 382, ​​389, 396, 397, 421 and 434; preferably 230, 264, 307, 315, 330, 378 and 434, more preferably 264, 315, 378 and 434; and

[0074] (ii) selected from positions 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314 94, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, ​​383, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403, 404, 408, 411, 412, 414, 415, 416, 417 8, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447, at least one other, different mutation, preferably 226, 230, 241, 264, 307, 315, 330, 342, 362, 378, 382, ​​389, 396, 397, 421 and 434.

[0075] In a preferred embodiment, at least one of positions 378 and 434 is mutated; and optionally at least one other position selected from 226, 230, 241, 264, 307, 315, 330, 342, 362, 378, 382, ​​389, 396, 397, 421 and 434.

[0076] Preferably, the mutation is 226G, 226Y, 227S, 227L, 228R, 228L, 230S, 230T, 230L, 230A, 230Q, 231T, 231V, 233D, 234R, 239A, 241L, 241Y, 241R, 243L, 246R, 250A, 252L, 256N, 259I, 264A, 264E, 264M, 265G, 265N, 267N, 267R, 269D, 269G, 270N, 270E, 276S, 284L, 285Y, 288R, 289I, 290R, 290E, 291S, 291Q 292W, 293del, 294del, 297D, 298G, 298N, 299M, 299A, 299K, 301C, 302A, 303A, 303I, 305A, 307P, 307A, 30 7N, 3081, 309P, 311R, 315D, 317R, 320T, 320E, 322R, 325S, 327V, 327T, 330V, 330T, 332V, 334E, 334R, 335A , 338R, 340E, 342R, 342E, 342K, 343S, 345Q, 345G, 347R, 350A, 352S, 354P, 355Q, 355G, 356N, 359A, 360N, 360R, 361D, 361S, 362R, 362E, 369A, 370R, 371D, 375A, 375G, 378V, 378T, 378S, 380Q, 382V, 382G, 383R, 38 3N, 384I, 384T, 385R, 386R, 386K, 387S, 387T, 389T, 389K, 389R, 390S, 392E, 392R, 393N, 394A, 395A, 395 S, 396S, 396L, 397A, 397M, 398P, 399N, 400P, 401A, 401G, 403T, 404L, 408T, 411A, 412A, 414R, 415D, 415N, 416K, 416G, 418R, 418K, 418E, 419H, 420R, 421T, 421S, 421D, 422A, 424L, 426T, 428L, 433R, 433P, 434Y, 434S, 434H, 438R, 439R, 440R, 440N, 443R, 444F, 444P, 445S, 446A, 447N and 447E, and are also described in patent application WO2010 / 106180.

[0077] In another embodiment, the Fc fragment includes 226G, 227L, 230S, 230T, 230L, 231T, 241L, 243L, 250A, 256N, 259I, 264E, 265G, 267R, 290E, 293del, 294del, 303A, 305A, 307P, 307A, 308I, 315D, 322R, 325S, 327V, 330V, 342R, 347R, 352S, 361D, 362R, 362E, 370R, 378V, 378T, 382V, 383N, 386R , 386K, 387T, 389T, 389K, 392R, 395A, 396L, 397M, 403T, 404L, 415N, 416K, 421T, 426T, 428L, 433R, 434Y, 434S and 439R, preferably at least one mutation of 226G, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 342R, 362R, 362E, 378V, 378T, 382V, 389T, 389K, 396L, 397M, 421T, 434Y and 434S.

[0078] Examples of specific mutation combinations are presented below:

[0079] 226G / 330V, 230L / 264E, 230L / 378V, 230S / 315D, 230S / 434Y, 230T / 378V, 241L / 434S, 250A / 434Y, 264E / 378T, 305A / 315D, 305A / 33 0V, 305A / 434Y, 307P / 434Y, 315D / 389T, 330V / 382V, 330V / 389T, 378V / 421T, 389K / 434Y, 389T / 434Y, 396L / 434S, 230T / 264E, 230T / 315D, 230T / 434S, 230T / 434Y, 241L / 307P, 264E / 307P, 264E / 396L, 315D / 362R, 315D / 382V, 362R / 434Y, 378V / 434Y, 382V / 434Y, 226G / 315D, 226G / 434Y, 241L / 378V, 307P / 378V, 241L / 264E, 378V / 434S, 264E / 378V, 264E / 434S, 315D / 330V, 330V / 434Y and 315D / 434Y; or

[0080] 226G / 315D / 330V, 226G / 315D / 434Y, 226G / 330V / 434Y, 230L / 264E / 378V, 230T / 26 4E / 378V, 230T / 264E / 434S, 230S / 315D / 434Y, 230T / 315D / 434Y, 230T / 389T / 434S, 241L / 264E / 434S, 241L / 264E / 378V, 241L / 264E / 307P, 241L / 307P / 378V, 250A / 389 K / 434Y, 256N / 378V / 434Y, 259I / 315D / 434Y, 264E / 378T / 396L, 264E / 378V / 416K, 2 94del / 307P / 434Y, 264E / 307P / 378V, 264E / 396L / 434S, 264E / 378V / 434S, 305A / 3 15D / 330V, 305A / 315D / 434Y, 305A / 330V / 434Y, 307P / 378V / 434Y, 315D / 330V / 382V , 315D / 330V / 389T, 315D / 378V / 434Y, 315D / 389T / 434Y, 315D / 362R / 434Y, 315D / 38 2V / 434Y, 315D / 330V / 434Y, 330V / 382V / 434Y, 330V / 389T / 434Y and 378V / 383N / 434Y.

[0081] In a particularly advantageous embodiment, the Fc fragment has a combination of mutations selected from 315D / 330V / 361D / 378V / 434Y (a combination of mutations also called "T5A-74"; an Fc fragment having this combination of mutations is therefore also called "rFc T5A-74"), 230S / 315D / 428L / 434Y, 307A / 315D / 330V / 382V / 389T / 434Y, 259I / 315D / 434Y, 256N / 378V / 383N / 434Y, E294del / T307P / N434Y (a combination of mutations also called "C6A_66"; an Fc fragment having this combination of mutations is therefore also called "rFc C6A_66").

[0082] In another embodiment, the Fc fragment has V240H, V240I, V240M, V240N, V240S, F241H, F241Y, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, F243L, F243S, E258G, E258I, E258R, E258M, E258Q, E258Y, V259C, V259I, V259L, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, K290H, K290L, K 290N, K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E 293Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y296H, Y296W, S2 at least one mutation of 98A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R and V305S; the numbering is that of the EU index or Kabat equivalent.

[0083] In one embodiment, the variant according to the invention has an increased affinity for FcγRIIIa (CD16a). In this specific embodiment, the variant comprises at least one mutation selected from i) S298A, S298R, F243S, F243L, L242A, L242F, L242G, L242I, L242K, L242S, L242V, V240I, V240M, V240N, V240S, E258I, T260A, K290D, K290E, K290G, K290H, K290Q, K290S, K290Y, Y296H, Y296W of the Fc fragment;

[0084] Numbering is that of the EU index or Kabat equivalent.

[0085] The specific Fc fragment is fragment ZAC2-85(T260A) of SEQ ID NO:17:

[0086] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVACVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0087] or ZAC3–172(K290Y) of SEQ ID NO:18:

[0088] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTYPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0089] In another embodiment, the variant according to the invention has an increased affinity for FcγRIIa (CD32a). In this specific embodiment, the variant comprises i) a polypeptide selected from the group consisting of F241H, F241Y, F243L, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, V240H, V240I, V240M, V240S, E258G, E258I, E258R, E258M, E258Q, E2 58Y, S267A, S267Q, S267V, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V259C, V 259I, V259L, V262A, V262S, V263T, V264L, V264S, V264T, V266L, V266M, E293A, E293D, E293G, E293M, E293Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, K290D, K290E , K290G, K290H, K290L, K290N, K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, Q295I, Q295 M, R292I, R292L, R301A, R301P, R301S, S304T, V302A, V302F, V302L, V302M, V302R, V302S, V303Y, V305A, V305F, V305L, V305R, V305S, Y300I, Y300V or Y300W; the numbering is that of the EU index or Kabat equivalent.

[0090] In another embodiment, the variant according to the invention has an increased affinity for FcγRIIb (CD32b). In this specific embodiment, the variant comprises at least one mutation selected from i) E258R, E258Y, V262A, S267A, S267Q, S267V, V264S, V266L, V266M, K290R, R301A, R301M, S304T, V302A, V302L, V302R, V303S, V305A, V305F, V305I, V305R, Y300V of the Fc fragment; the numbering is that of the EU index or Kabat equivalent.

[0091] Preferably, the variant according to the invention is characterized in that the Fc fragment of the parent polypeptide comprises at least:

[0092] (i) V240H, V240I, V240M, V240N, V240S, F241H, F241Y, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, F243L, F243S, E258G, E258I, E258R, E258M, E258Q, E258Y, V25 9C, V259I, V259L, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V262A, V262S, V263T, V 264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, K290H, K290L, K290N , K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E2 93Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y296H, Y296W,

[0136] S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R, and V305S; the numbering is that of the EU index or Kabat equivalent.

[0093] (ii) at least one mutation selected from the group consisting of 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 361D, 362R, 378V, 378T, 389T, 389K, 434Y and 434S,

[0094] The numbering is that of the EU index or Kabat equivalent, with the proviso that mutations (ii) and (iii) are not in the same amino acid.

[0095] In a particularly advantageous embodiment, the Fc fragment has a combination of mutations selected from the group consisting of:

[0096] (i) T260A and 315D / 330V / 361D / 378V / 434Y (the combination of mutations is also referred to as "T5A-74"). The fragment designated "T5A-74I" is an Fc fragment that differs from fragment T5A-74 only by the addition of the mutation T260A.

[0097] or

[0098] (ii) E258I and 315D / 330V / 361D / 378V / 434Y (the combination of mutations is also referred to as "T5A-74"). The fragment designated "T5A-74J" is an Fc fragment that differs from fragment T5A-74 only by the addition of the mutation E258I.

[0099] or

[0100] (iii) K290Y and 315D / 330V / 361D / 378V / 434Y (the combination of mutations is also referred to as "T5A-74"). The fragment designated "T5A-74K" is an Fc fragment that differs from fragment T5A-74 only by the addition of the mutation K290Y.

[0101] or

[0102] (iv) E294A and 315D / 330V / 361D / 378V / 434Y (the combination of mutations is also referred to as "T5A-74"). The fragment designated "T5A-74L" is an Fc fragment that differs from fragment T5A-74 only by the addition of the mutation E294A.

[0103] or

[0104] (v) Y296W and 315D / 330V / 361D / 378V / 434Y (the combination of mutations is also referred to as "T5A-74"). The fragment designated "T5A-74M" is an Fc fragment that differs from fragment T5A-74 only by the addition of the mutation Y296W.

[0105] The sequences of Fc fragments T5A–74, T5A–74I, T5A–74J, T5A–74K, T5A–74L, T5A–74M are presented in the attached sequence listing.

[0106] T5A–74(N315D / A330V / N361D / A378V / N434Y):

[0107] SEQ ID NO:11

[0108] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLDGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSRDELTKDQVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK

[0109] T5A–74I(T260A / N315D / A330V / N361D / A378V / N434Y)

[0110] SEQ ID NO:12

[0111] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVACVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLDGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSRDELTKDQVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK

[0112] T5A–74J(E258I / N315D / A330V / N361D / A378V / N434Y)

[0113] SEQ ID NO:13

[0114] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPIVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLDGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSRDELTKDQVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK

[0115] T5A–74K(K290Y / N315D / A330V / N361D / A378V / N434Y)

[0116] SEQ ID NO:14

[0117] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTYPREEQYNSTYRVVSVLTVLHQDWLDGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSRDELTKDQVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK

[0118] T5A–74L(E294A / N315D / A330V / N361D / A378V / N434Y)

[0119] SEQ ID NO:15

[0120] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREAQYNSTYRVVSVLTVLHQDWLDGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSRDELTKDQVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK

[0121] T5A–74M(Y296W / N315D / A330V / N361D / A378V / N434Y)

[0122] SEQ ID NO:16

[0123] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQWNSTYRVVSVLTVLHQDWLDGKEYKCKVSNKALPVPIE KTISKAKGQPREPQVYTLPPSRDELTKDQVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK

[0124] Preferably, the variant according to the invention is characterized in that the Fc fragment of the parent polypeptide comprises at least:

[0125] (i) V240H, V240I, V240M, V240N, V240S, F241H, F241Y, L242A, L242F, L242G, L242H, L242I, L242K, L242P, L242S, L242T, L242V, F243L, F243S, E258G, E258I, E258R, E258M, E258Q, E258Y, V25 9C, V259I, V259L, T260A, T260H, T260I, T260M, T260N, T260R, T260S, T260W, V262A, V262S, V263T, V 264L, V264S, V264T, V266L, V266M, S267A, S267Q, S267V, K290D, K290E, K290G, K290H, K290L, K290N , K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E2 93Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y296H, Y296W,

[0136] S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302A, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V305I, V305L, V305R, and V305S; the numbering is that of the EU index or Kabat equivalent.

[0126] (ii) a mutation selected from 378V, 378T, 434Y and 434S.

[0127] In another embodiment, the Fc fragment has at least one mutation selected from the group consisting of 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, V2 59A, T393A, P352L, Q418P, V302A, L398P, F423P, S442P, V363I, S383N, S254F, K320E, G402D, I253F, V284A, A431T, N315 H, 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, K3 20M, 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,

[0128] Numbering is that of the EU index or Kabat equivalent.

[0129] In a specific embodiment, the Fc fragment has a combination of at least 2 mutations selected from:

[0130] (i) a mutation selected from 307N, 326E, 326T, 334N, 334R, 352L, 378V, 378T, 394P, 396L, 397M and 421T, and;

[0131] (ii) at least one mutation selected from the group consisting of 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, 3 20T, 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,

[0132] The numbering is that of the EU index or Kabat equivalent, with the proviso that mutation (i) and mutation (ii) are not in the same amino acid.

[0133] Preferably, the mutated Fc fragment has an increased affinity for complement C1q and comprises a combination of at least two mutations, the combination comprising:

[0134] i) a mutation selected from the group consisting of 378V, 378T, 396L, 421T, 334R and 326E; and

[0135] ii) at least one mutation selected from the group consisting of 361H, 290E, 316D, 248E, 410R, 421T, 334R, 394P, 307P, 447N, 378V, 284L, 421T, 396L, 286I, 315D and 397M,

[0136] The numbering is that of the EU index or Kabat equivalent, with the proviso that mutation (i) and mutation (ii) are not in the same amino acid.

[0137] Preferably, the mutated Fc fragment has an increased affinity for FcγRIIIa (CD16a) and comprises a combination of at least 2 mutations, the combination comprising:

[0138] i) a mutation selected from 378V, 326E, 397M, 334N and 396L; and

[0139] ii) at least one mutation selected from the group consisting of: 316D, 397M, 334N, 248E, 231V, 246R, 336T, 421T, 361H, 366A, 439R, 290E, 394P, 307P, 378V, 378T, 286I, 286Y and 298N,

[0140] The numbering is that of the EU index or Kabat equivalent, with the proviso that mutation (i) and mutation (ii) are not in the same amino acid.

[0141] Preferably, the mutated Fc fragment has an increased affinity for FcγRIIa (CD32a) and comprises a combination of at least 2 mutations, the combination comprising:

[0142] i) a mutation selected from 378V, 326E, 397M, 307N, 394P, 326T, 396L and 334N; and

[0143] ii) at least one mutation selected from the group consisting of 316D, 334R, 334N, 323I, 231V, 246R, 336T, 378T, 286Y, 286I, 352S, 383R, 359A, 421T, 361H, 315D, 366A, 290E, 307P and 439R,

[0144] The numbering is that of the EU index or Kabat equivalent, with the proviso that mutation (i) and mutation (ii) are not in the same amino acid.

[0145] Preferably, the mutated Fc fragment has an increased affinity for FcγRIIIb (CD32b) and comprises a combination of at least 2 mutations, the combination comprising:

[0146] i) a mutation selected from 326E, 326T, 378V, 397M, 352L, 394P, 396L and 421T; and

[0147] ii) at least one mutation selected from the group consisting of: 316D, 334R, 248E, 334N, 418P, 231V, 320E, 402D, 359A, 383R, 421T and 361H,

[0148] The numbering is that of the EU index or Kabat equivalent, with the proviso that mutation (i) and mutation (ii) are not in the same amino acid.

[0149] Preferably, the mutant Fc fragment comprises a combination of at least 3 mutations, wherein the combination comprises:

[0150] (i) a mutation selected from the group consisting of 326E, 326T, 352L, 378V, 378T, 396L, 397M, 421T, 334N, 334R, 307N and 394P; and

[0151] (ii) at least two mutations selected from the group consisting of 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, 3 20T, 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,

[0152] The numbering is that of the EU index or Kabat equivalent, with the proviso that mutation (i) and mutation (ii) are not in the same amino acid.

[0153] In a particularly advantageous embodiment, the Fc fragment has a combination of mutations selected from K334N / P352S / V397M / A378V (a combination of mutations also called "A3A-184A"; an Fc fragment having this combination of mutations is therefore also called "A3A-184A"), G316D / K326E / A378V (a combination of mutations also called "A3A-105D"; an Fc fragment having this combination of mutations is therefore also called "A3A-105D"), P396L / N421T / A378V (a combination of mutations also called "J3B-118A"; an Fc fragment having this combination of mutations is therefore also called "J3B-118A").

[0154] The sequence of fragment A3A-184A (K334N / P352S / A378V / V397M) is presented in the sequence listing as SEQ ID NO: 19:

[0155] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE NTISKAKGQPREPQVYTLSPSRDELTKNQVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0156] The sequence of fragment A3A-105D (G316D / K326E / A378V) is presented in the sequence listing as SEQ ID NO: 20:

[0157] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNDKEYKCKVSNEALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0158] The sequence of fragment J3B-118A (A378V / P396L / N421T) is presented in the sequence listing as SEQ ID NO: 21:

[0159] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGTVFSCSVMHEALHNHYTQKSLSLSPGK

[0160] According to another specific embodiment, the mutant Fc fragment used in the present invention has an amino acid deletion (DEL293 or DEL294) at position 293 or 294, the numbering of the Fc fragment amino acids referring to the EU index or the numbering of the Kabat equivalent. This deletion can be the only mutation of the Fc fragment, or it can be accompanied by other mutations, particularly mutations among those listed above.

[0161] This single mutation results in a specific glycosylation of the fragment, namely hypersialylation, which is particularly advantageous with regard to glycoprotein half-life and inflammatory processes.

[0162] According to another more specific embodiment, the mutant Fc fragment used in the present invention has an amino acid deletion at position 293 or 294, and also has one or more mutations at a position selected from the following list: 226, 230, 241, 256, 259, 264, 307, 315, 330, 342, 361, 362, 378, 382, ​​383, 389, 396, 397, 421, 428 and 434.

[0163] For example, a preferred Fc fragment has a combination of mutations 307P, 434Y and deletion DEL294 or DEL293.

[0164] According to another aspect of the invention, a composition comprising a plurality of Fc fragments is used, said Fc fragments all having substantially the same sequence and having a specific glycosylation pattern as a whole.

[0165] According to a specific aspect, the composition for use in the context of the present invention comprises an Fc fragment having an N-glycan at its glycosylation site (Asn 297), characterized in that said N-glycan of said Fc fragment has a degree of fucosylation of less than 65%, preferably less than 60%, preferably less than 55%, preferably less than 50%, more preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%.

[0166] According to yet another aspect, the composition for use in the context of the present invention comprises an Fc fragment having an N-glycan at its glycosylation site (Asn 297), characterized in that the N-glycan of the Fc fragment has a biantennary glycan structure with a short chain, low sialylation, with a terminal mannose and / or a non-inserting terminal N-acetylglucosamine.

[0167] According to a more specific aspect, the composition for use in the context of the present invention comprises an Fc fragment having N-glycans at its glycosylation site (Asn 297), characterized in that greater than 60% of the N-glycans of the Fc fragment are in the form of G0+G1+G0F+G1F, and less than 50% are in the form of G0F+G1F.

[0168] According to another more specific aspect, the composition for use in the context of the present invention comprises an Fc fragment having N-glycans at its glycosylation site (Asn 297), characterized in that greater than 60% of the N-glycans of the Fc fragment are in the form of G0+G1+G0F+G1F and the fucose content is less than 65%.

[0169] According to another more specific aspect, the composition for use in the context of the present invention comprises an Fc fragment having N-glycans at its glycosylation site (Asn 297), characterized in that less than 40% of said N-glycans of the Fc fragment are in the form of G1F+G0F.

[0170] According to a more specific aspect, the composition used in the context of the present invention comprises an Fc fragment having an N-glycan at its glycosylation site (Asn297), said N-glycan of the Fc fragment having a degree of fucosylation equal to 0%. Therefore, the present invention provides a composition comprising an Fc fragment having an N-glycan at its glycosylation site Asn297, characterized in that said N-glycan of said Fc fragment does not contain fucose.

[0171] Moreover, according to a specific aspect, the composition used in the context of the present invention comprises an Fc fragment having an N-glycan at its glycosylation site Asn297, characterized in that said N-glycan of the Fc fragment has a degree of fucosylation in the range between 20% and 55%. Specifically, the present invention provides a composition comprising an Fc fragment having an N-glycan at its glycosylation site Asn297, characterized in that said N-glycan of the Fc fragment has a degree of fucosylation in the range between 20% and 50%, between 25% and 55%, between 25% and 50%, between 20% and 45% or between 25% and 45%.

[0172] According to a more specific aspect, a useful composition according to the present invention comprises an Fc fragment having an N-glycan at its glycosylation site Asn297, characterized in that greater than 60%, preferably greater than 80% of the N-glycans of the Fc fragment are in the form of G0+G1+G0F+G1F, and less than 50%, preferably less than 40% or 30% are in the form of G0F+G1F.

[0173] According to another more specific aspect, more than 60% of the N-glycans of the Fc fragment in the composition are in the form of G0+G1+G0F+G1F, and the fucose content is less than 65%.

[0174] According to yet another more specific aspect, less than 50%, preferably less than 40% or 30% of the N-glycans of the Fc fragment in the composition are in the form of G1F+G0F.

[0175] The G0, G0F, G1 and G1F forms are selected from Figure 3 The form shown.

[0176] Advantageously, the N-glycans of the Fc fragments in the composition have an average sialic acid content of less than 25%, 20%, 15% or 10%, preferably 5%, 4%, 3% or 2%.

[0177] The composition useful in the context of the present invention comprises an Fc fragment having an N-glycan at its glycosylation site (Asn 297), wherein the N-glycan of the Fc fragment has a biantennary glycan structure, is short chain, poorly sialylated, has terminal mannose and / or non-inserted terminal N-acetylglucosamine, for example, greater than 60% of the N-glycans consist of the G0+G1+G0F+G1F form and is poorly fucosylated, less than 50% of the N-glycans consist of the G0F+G1F form.

[0178] In a specific embodiment, the Fc fragment according to the invention has a glycan structure as described in patent application WO01 / 77181.

[0179] According to an advantageous embodiment, the Fc fragment used in the present invention comprises at least one amino acid mutation compared to the parent Fc fragment and has an N-glycan at its glycosylation site (Asn 297), the N-glycan of said Fc fragment having a degree of fucosylation of less than 65%, preferably less than 60%, preferably less than 55%, preferably less than 50%, more preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%. Preferably, the Fc fragment has one or more mutations at positions selected from those listed above, in particular at positions selected from the following list: 226, 230, 241, 256, 259, 264, 307, 315, 330, 342, 361, 362, 378, 382, ​​383, 389, 396, 397, 421, 428 and 434; and further has N-glycans with a degree of fucosylation at its glycosylation site (Asn 297) of less than 55%, preferably less than 50%, more preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%.

[0180] More preferably, the Fc fragment of the composition according to the present invention has a combination of mutations selected from 315D / 330V / 361D / 378V / 434Y, 230S / 315D / 428L / 434Y, 307A / 315D / 330V / 382V / 389T / 434Y, 259I / 315D / 434Y, 256N / 378V / 383N / 434Y and DEL294 / 307P / 434Y; and furthermore has N-glycans with a degree of fucosylation at its glycosylation site (Asn 297) of less than 55%, preferably less than 50%, more preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%.

[0181] Advantageously, an Fc fragment having modified glycosylation, particularly hypofucosylation, at glycosylation site 297 has increased binding to Fc-gamma receptor (FcγR), particularly FcγRIIIa (CD16a).

[0182] Preferably, the Fc fragment has a binding affinity to CD16a of at least 2×10 6 M -1 , at least equal to 2×10 7 M -1 , 2×10 8 M -1 or 2×10 9 M -1The affinity of the binding site is determined by Scatchard analysis or BIAcore technology (a label-free surface plasmon resonance-based technology).

[0183] Particularly advantageously, the mutant Fc fragments of the present invention can be used in combination with different mutant Fc fragments having different mutations. For example, a mixture of mutant Fc Fc-Del294, mutant Fc T5A-74 (also targeting FcRn), and mutant Fc (type A3A-184A) improved for binding to FcγR can be advantageously used.

[0184] Production of the Fc fragment according to the present invention

[0185] The Fc fragment used in the present invention can be produced by any method known to those skilled in the art, such as by chemical synthesis or by recombination.

[0186] In a preferred embodiment, the Fc fragments used in the present invention are referred to as "recombinant", ie they are obtained by recombination.

[0187] When the Fc fragment according to the present invention has one or more amino acid mutations, the mutations can be optionally introduced by known techniques such as gene synthesis, directed mutagenesis (particularly obtained by PCR using specific primers that introduce the desired mutations) or random mutagenesis. Preferably, random mutagenesis as described in application WO02 / 038756, i.e., MutaGen technology, is used. This technology uses human DNA mutase, particularly selected from DNA polymerase β, η and ι. Conventional recombinant techniques involve reorganization in host cells, which are transformed with one or more vectors, which enable expression with or without secretion of the Fc fragment sequence into the extracellular medium. The vector generally comprises a promoter, translation initiation and termination signals, and an appropriate region for regulating transcription. It can be stably maintained in the host cell and can optionally have a specific signal for secretion of a protein specified for translation. Depending on the host cell used, those skilled in the art select and optimize these various elements.

[0188] These vectors are prepared by methods generally used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods such as lipofection, electroporation, use of polycationic agents, heat shock or chemical methods.

[0189] The host cell may be selected from prokaryotic or eukaryotic systems, such as bacterial cells, yeast cells or animal cells, in particular mammalian cells. Insect cells or plant cells may also be used.

[0190] The Fc fragment used in the present invention can be produced by culturing a host cell expressing the Fc fragment in an appropriate medium and culture conditions; and recovering the fragment thus produced from the medium or the cultured cells.

[0191] Preferred mammalian cells expressing Fc fragments are the rat cell line YB2 / 0, the cell line Vero, the hamster cell line CHO, in particular the cell lines CHO dhfr- and CHO Lec13, CHO-lec10, CHO-lec1, CHOK1SV (Lonza, Switzerland), CHOGnTIII (Glycart, Switzerland), PER.C6 TM (Crucell), HEK293, T1080, EB66, K562, NS0, SP2 / 0, BHK or COS.

[0192] Another production mode is the expression of Fc fragments in transgenic organisms, such as in plants (Ayala M, Gavilondo J, Rodríguez M, Fuentes A, Enríquez G, Perez L, Cremata J, Pujol M. Production of plantibodies in Nicotiana plants. Methods Mol Biol. 2009; 483: 103–34.), or in the milk of transgenic animals such as rabbits, goats, rats or pigs (Pollock, DP, JP Kutzko, E. Birck–Wilson, JL Williams, Y. Echelard and HM Meade. (1999). Transgenic milk as a method for the production of recombinant antibodies. Journal of Immunological Methods. 231: 147–157). See also document WO200748077 in this regard.

[0193] In an alternative embodiment, the Fc fragment is obtained by proteolytic processing of a mutated immunoglobulin itself.

[0194] The glycosylation of the Fc fragment can be modified by known techniques. The glycosylated Fc fragment according to the present invention can be produced notably by cleaving an antibody produced according to the technique described in WO01 / 77181, in particular by papain. Slightly fucosylated Fc fragments can also be obtained by, for example, producing in cells cultured in the presence of kifunensine as described in document US7700321, or producing in cells in which the GDP-fucose production pathway is inhibited (for example by inhibiting at least one enzyme of the fucose production cycle) (see in particular documents US 2010291628 or US20090228994, EP 1500698, EP 1792987 or US 7846725). Interfering RNA (RNAi) that inhibits 1,6-fucosyltransferase can also be used as described in document US7393683 or document WO2006133148. This may be a problem of the preparation method in yeast, as described, for example, in document WO 0200879.

[0195] If the Fc fragment has 100% non-fucosylated oligosaccharides, i.e. when the Fc fragment is completely fucose-free, a preparation method known to a person skilled in the art can be used, such as those disclosed in documents EP1176195, US 7214775, US 6994292, US7425449, US2010223686, WO2007099988, EP 1705251 (this list is non-limiting). This can be, for example, a method using a host cell expressing at least one nucleic acid encoding an Fc fragment, wherein the glycosylation is modified by deleting a gene encoding α1,6-fucosyltransferase or by adding a mutation of said gene to eliminate α1,6-fucosyltransferase activity, thereby expressing an antibody fragment without fucose.

[0196] Therapeutic applications

[0197] Due to their many advantages in terms of efficacy and optimized effector function or reduced side effects, the Fc fragments described herein can be used to treat autoimmune and / or inflammatory diseases.

[0198] Described herein are methods for treating an autoimmune and / or inflammatory disease in a patient comprising administering to the patient a therapeutically effective amount of an Fc fragment having altered affinity for at least one Fc receptor described herein.

[0199] In the present invention, the expression "autoimmune and / or inflammatory disease" refers to organ-specific or systemic, primary or secondary autoimmune and / or inflammatory diseases which are optionally associated with pathogenic autoantibodies.

[0200] For example, the disease may be selected from thrombotic thrombotic purpura (TTP), idiopathic thrombotic purpura (ITP), organ or transplant rejection, graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, different types of sclerosis, primary Sjögren's syndrome (or syndrome), autoimmune polyneuropathies such as multiple sclerosis, type 1 diabetes, autoimmune hepatitis, ankylosing spondylitis, Reiter syndrome, gouty arthritis, celiac disease, Crohn's disease, Hashimoto's thyroiditis (hypothyroidism), Addison's disease, autoimmune hepatitis, Basedow's disease (hyperthyroidism), ulcerative colitis, vasculitis such as antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, autoimmune cytopenia in adults or children Oligocytopenia and other hematological complications such as acute or chronic autoimmune thrombocytopenia, autoimmune hemolytic anemia, hemolytic disease of the newborn (HDN), cold agglutinin disease, thrombocytopenic purpura and acquired autoimmune hemophilia; Goodpasture syndrome, extramembranous nephropathy, autoimmune bullous dermatosis, refractory myasthenia, mixed cryoglobulinemia, psoriasis, juvenile chronic arthritis, inflammatory myositis, childhood dermatomyositis and systemic autoimmune diseases, including antiphospholipid syndrome, Connective tissue diseases, different types of sclerosis, autoimmune inflammatory diseases of the lung, Guillain-Barré syndrome, Kawasaki disease, multiple motor neuropathy (MMN), chronic demyelinating inflammatory polyneuropathy (CDIP), autoimmune thyroiditis, diabetes, myasthenia gravis, inflammatory autoimmune diseases of the eye, neuromyelitis optica (Devic's disease), scleroderma, pemphigus, diabetes due to insulin resistance, polymyositis, Biermer's anemia, glomerulonephritis, Wegener's disease, giant cell arterioles arthritis, nodular periarteritis and Churg-Strauss syndrome, Still's disease, polychondritis atrophicans, Behcet's disease, monoclonal gammopathy, Wegener's granulomatosis, lupus, ulcerative colitis, psoriatic rheumatism, sarcoidosis, collagenous colitis, dermatitis herpetiformis, familial Mediterranean fever, glomerulonephritis with IgA deposition, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmia, Fiessinger-Leroy-Reiter syndrome, and meningoencephalitis syndrome.

[0201] Also included are other inflammatory diseases, such as acute respiratory distress syndrome (ARDS), acute septic arthritis, adjuvant arthritis, allergic encephalomyelitis, allergic rhinitis, allergic vasculitis, allergies, asthma, atherosclerosis, chronic inflammation caused by chronic bacterial or viral infections, chronic obstructive pulmonary disease (COPD), coronary heart disease, encephalitis, inflammatory bowel disease, inflammatory osteolysis, inflammation associated with acute and delayed allergic reactions, inflammation associated with tumors, peripheral nerve damage or demyelinating diseases, inflammation associated with tissue trauma such as burns and ischemia, inflammation caused by meningitis, multiple organ dysfunction syndrome (MODS), pulmonary fibrosis, sepsis and septic shock, Stevens-Johnson syndrome, undifferentiated arthritis and undifferentiated spondyloarthropathy.

[0202] In a specific embodiment of the invention, the autoimmune disease is idiopathic thrombotic purpura (ITP) and chronic demyelinating inflammatory polyradiculoneuropathy (CDIP).

[0203] Among the effects observed were in particular the limitation or reduction of platelet destruction observed in ITP pathology and the limitation or reduction of myelin loss in peripheral nerves in CDIP.

[0204] In another specific embodiment, the disease is an inflammatory disease, such as, in particular, graft-versus-host disease. In this case, an Fc fragment with a mutation that induces hypersialylation, such as a deletion of the amino acid at position 294 (DEL294) or 293 (DEL293), is particularly advantageous.

[0205] Any route of administration is conceivable, in particular the parenteral route, such as intravenous, intramuscular, subcutaneous, intradermal or topical route, or via a mucosal route, such as by inhalation. Enteral (oral, rectal) and intrathecal routes are also possible. The intravenous route is preferably used.

[0206] The Fc fragment according to the present invention is usually formulated in a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

[0207] The pharmaceutical composition may be in any pharmaceutical form suitable for the chosen route of administration.

[0208] Useful pharmaceutical compositions according to the present invention advantageously comprise one or more pharmaceutically acceptable excipients or vehicles.For example, saline compatible with drug use and known to those skilled in the art, physiological, isotonic or buffered solutions etc. can be mentioned.Compositions can contain one or more materials or vehicles selected from dispersants, solubilizers, stabilizers, preservatives etc.Material or vehicles that can be used for preparations (liquid and / or injectable and / or solid) are particularly methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oil, gum arabic etc.Compositions can optionally be prepared by providing a pharmaceutical form or device for extended release and / or delayed release.For this type of preparation, advantageously use materials such as cellulose, carbonate or starch.

[0209] The dose administered can vary according to, for example, the weight and age of the patient, and the severity of the disease as assessed by those skilled in the art.

[0210] In a preferred embodiment, the dosage of the Fc fragment according to the present invention is about 0.05 mg / kg to about 1 g / kg body weight per day, i.e. about 20 mg to about 100 g for an adult. Preferably, the dosage is about 330 mg / kg to about 660 mg / kg per day.

[0211] The following examples illustrate the invention without limiting its scope.

[0212] Construction of expression vectors of recombinant Fc (WT and mutant):

[0213] The sequence of recombinant Fc (aa221-447) was cloned into a universal eukaryotic expression vector derived from pCEP4 (Invitrogen) for expression in HEK cells and into the OptiCHO vector for expression in YB2 / 0 cells using a standard PCR protocol.

[0214] All mutations of interest in the Fc fragment were inserted into the expression vector by overlapping PCR using two primers containing the mutations. The fragments obtained by PCR were then combined and amplified by PCR using a standard protocol. The PCR products were purified on a 1% (w / v) agarose gel, digested with appropriate restriction endonucleases and cloned into an expression vector for recombinant Fc.

[0215] Figure 7 Shown are maps of the pCEP4 vector used to express variant T5A-74 in HEK cells and a map of the OptiCHO vector used to express variant T5A-74 in YB2 / 0 cells.

[0216] Recombinant Fc production in HEK cells

[0217] HEK293 cells were transfected with pCEP4 expression vector for recombinant Fc (WT or mutant) according to standard protocols (Invitrogen). Cells were cultured for transient production of antibodies. The antibodies produced can be isolated and purified according to standard techniques in the art, given their characteristics. The production rates obtained were in the order of 150-500 μg / mL. The purity and quality of the product were verified by SDS-PAGE and SEC.

[0218] Recombinant Fc production in YB2 / 0 cells

[0219] YB2 / 0 cells were stably transfected with OptiCHO expression vectors for recombinant Fc (WT or mutant) by electroporation following standard protocols. Production was performed in stable pools or after cloning YB2 / 0 cells.

[0220] The steps of producing antibodies and purifying antibodies by cell culture are carried out according to standard techniques in the art and in view of their characteristics. The production rate obtained is on the order of 3 to 30 μg / mL. The purity and quality of the product are verified by SDS-PAGE and SEC.

[0221] Example 1: Efficacy test (inhibition of red blood cell lysis):

[0222] To mimic the situation observed in idiopathic thrombocytopenic purpura (ITP) involving red blood cell lysis by autoantibodies of ITP patients, effector cell-mediated red blood cell lysis was performed in the presence of monoclonal anti-rhesus D (RhD) antibodies, and the ability of various amounts of multivalent immunoglobulin (IVIg) or mutated (containing a recombinant Fc fragment mutated according to Table 1 below) and non-mutated recombinant Fc fragments to inhibit said lysis, e.g., by competing with anti-RhD for binding to Fc receptors on the surface of effector cells was evaluated.

[0223] Table 1: Construction of preferred mutant Fc variants of the present invention

[0224] The table below represents the different variants of the mutant Fc fragments tested in experiments for efficacy testing (inhibition of red blood cell lysis) as well as testing for binding to human FcRn and CD16aV by surface plasmon resonance (SPR) testing.

[0225]

[0226]

[0227] For this purpose, as effector cells, peripheral blood mononuclear cells (PBMC) were purified from peripheral blood by Ficoll gradient. Rh-D+ red blood cells obtained from healthy Rh-D+ blood donors were mixed with anti-RhD monoclonal antibodies. PBMC were incubated with opsonized Rh-D+ red blood cells (effector / target ratio of 2:1).

[0228] To evaluate the ability of the candidates (IVIg and recombinant Fc fragments, mutated and unmutated as defined in Table 1) to inhibit the cytotoxicity induced by anti-RhD antibodies by competing and / or saturating Fc receptors, various concentrations of candidates (0 to 9.75 μM) were added to each well. After 16 hours, the percentage of lysed red blood cells was evaluated by color development by measuring the amount of hemoglobin released into the supernatant (measurement of optical density (OD)). The specific lysis percentage was calculated according to the following formula:

[0229] OD sample – OD control 0% / OD control 100% – OD control 0% × 100 = % lysis

[0230] in:

[0231] "OD control 100%" corresponds to complete lysis of red blood cells (e.g. NH4Cl)

[0232] "OD control 0%" corresponds to the lysis observed with the reaction mixture without antibody

[0233] Results are expressed as percentage of specific lysis (see Figure 4 and 5 ).

[0234] Example 2: Testing of binding to human FcRn and CD16aV by surface plasmon resonance (SPR) on Biacore X100:

[0235] Protein used:

[0236] Recombinant human FcRn (FcRnα and β2-microglobulin) was produced in baculovirus cells by GTP Technology (Labège, France) as described previously (Popov et al., Mol. Immunol. 33:521-530 (1996)). Recombinant human CD16aV is commercially available (R&D Systems).

[0237] Testing on FcRn:

[0238] FC1 and FC2 cells on a CM5 chip (Biacore, GE Healthcare) were activated with a 1:1 mixture of 0.1M N-hydroxysuccinimide and 0.1M 3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide at 30 μL / min for 3 minutes. Recombinant FcRn was then immobilized on FC2 cells at 32.6 μg / mL in 10mM sodium acetate buffer (pH 5) (fixation for 100 seconds, final fixation level 350RU). FC1 cells were used as negative controls, prepared in the same manner as FC2, but without the presence of recombinant FcRn. The recombinant Fc to be tested was injected on FC1 and FC2 cells at 6 different concentrations (300nM, 150nM, 75nM, 30nM, 15nM and 0) in 50mM sodium phosphate, 150mM NaCl, 0.05% Tween 20 buffer (pH 6) at 10 μL / min for 8 minutes. FC1 and FC2 cells were regenerated between each sample concentration by injecting 50 mM sodium phosphate, 150 mM NaCl, 0.05% Tween 20 buffer (pH 7.8) for 1 min. The resulting data were analyzed using BIAevaluation version 3.1 software (Biacore), and the control signal obtained on FC1 was subtracted from the test signal obtained on FC2.

[0239] Tested on CD16aV:

[0240] FC1 and FC2 cells of CM5 chips (Biacore, GE Healthcare) were prepared using the His Capture kit (GE Healthcare, item 28-9950-56) to immobilize anti-histidine antibodies at 50 μg / mL (5 μL / min flow rate, 7 minutes of EDC / NHS activation, 7 minutes of immobilization, final immobilization level of 12,000RU on FC1 and FC2). Recombinant human CD16aV was immobilized on FC2 cells (1 μg / mL in HBS-EP+, Biacore, GE Healthcare) at 5 μL / min for 60 seconds. Under single cycle kinetic (SCK) conditions, the recombinant Fc to be tested was injected onto FC1 and FC2 cells at 5 different concentrations (1000 nM, 500 nM, 250 nM, 125 nM and 25 nM) in HBS-EP+ buffer, with a contact time of 60 seconds, a dissociation of 300 seconds and a flow rate of 30 μL / min, without regeneration between each concentration. The final regeneration between each recombinant Fc was performed in 10 mM glycine buffer (pH 1.5) at 30 μL / min for 60 seconds in FC1 and FC2 cells. The generated data were analyzed using BIAevaluationversion 3.1 software (Biacore), and the control signal obtained on FC1 was subtracted from the test signal obtained on FC2.

Claims

1. Use of an antibody Fc fragment for preparing a composition for treating an autoimmune disease selected from idiopathic thrombotic purpura, chronic demyelinating inflammatory polyradiculoneuropathy (CDIP), organ or transplant rejection and myasthenia gravis, wherein the Fc fragment is an isolated Fc fragment having improved affinity for the FcγRIII receptor compared to a parent Fc fragment, wherein the difference between the Fc fragment and the parent Fc fragment of wild-type IgG1 is the combination of mutations K334N / P352S / A378V / V397M, wherein the Fc fragment is an Fc fragment of an IgG1 immunoglobulin, and the numbering of the residues in the Fc fragment is that of an immunoglobulin heavy chain according to the EU index.

2. The use according to claim 1, wherein the Fc fragment consists of the sequence SEQ ID NO:

19.

3. The use according to claim 1 or 2, wherein the Fc fragment is recombinantly obtained.

4. The use according to claim 1 or 2, wherein the Fc fragment has N-glycans at its glycosylation site Asn 297, characterized in that The N-glycans of the Fc fragment have a fucosylation degree of less than 65%.

5. The use according to claim 4, characterized in that The N-glycans of the Fc fragment have a fucosylation degree of less than 60%.

6. The use according to claim 4, characterized in that The N-glycans of the Fc fragment have a fucosylation degree of less than 55%.

7. The use according to claim 4, characterized in that The N-glycans of the Fc fragment have a fucosylation degree of less than 50%.

8. The use according to claim 4, characterized in that The N-glycans of the Fc fragment have a fucosylation degree of less than 45%.

9. The use according to claim 4, characterized in that The N-glycans of the Fc fragment have a fucosylation degree of less than 40%.

10. The use according to claim 4, characterized in that The N-glycans of the Fc fragment have a fucosylation degree of less than 35%.

11. The use according to claim 4, characterized in that The N-glycans of the Fc fragment have a fucosylation degree of less than 30%.

12. The use according to claim 4, characterized in that The N-glycans of the Fc fragment have a fucosylation degree of less than 25%.

13. The use according to claim 4, characterized in that The N-glycans of the Fc fragment have a fucosylation degree of less than 20%.

14. The use according to claim 1 or 2, wherein the Fc fragment has N-glycans at its glycosylation site Asn 297, characterized in that The N-glycans of the Fc fragment have a biantennary glycan structure with a short chain, low sialylation, terminal mannose and / or non-inserting terminal N-acetylglucosamine.

15. The use according to claim 1 or 2, wherein the Fc fragment has N-glycans at its glycosylation site Asn 297, characterized in that More than 60% of the N-glycans of the Fc fragment are in the form of G0+G1+G0F+G1F, and less than 50% are in the form of G0F+G1F.

16. The use according to claim 1 or 2, wherein the Fc fragment has N-glycans at its glycosylation site Asn 297, characterized in that More than 60% of the N-glycans of the Fc fragment are in the form of G0+G1+G0F+G1F, and the fucose content is less than 65%.

17. The use according to claim 15, wherein the Fc fragment has N-glycans at its glycosylation site Asn 297, characterized in that Less than 40% of the N-glycans of the Fc fragment were in the form of G1F+G0F.

18. The use according to claim 16, wherein the Fc fragment has N-glycans at its glycosylation site Asn 297, characterized in that Less than 40% of the N-glycans of the Fc fragment were in the form of G1F+G0F.

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