VARIANTS WITH FC FRAGMENT HAVING AN INCREASED AFFINITY FOR FCRN AND AN INCREASED AFFINITY FOR AT LEAST ONE RECEPTOR OF THE FC FRAGMENT

Abstract

Disclosed is a variant of a parent polypeptide including an Fc fragment, the variant having an increased affinity for the FcRn receptor, and an increased affinity for at least one receptor of the Fc fragment (FcR) chosen from the FcRI (CD64), FcRIIIa (CD16a) and FcRIIa (CD32a) receptors, relative to that of the parent polypeptide, characterised in that it includes: (i) the four mutations 334N, 352S, 378V and 397M; and (ii) at least one mutation chosen from 434Y, 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K; the numbering being that of the EU index or the Kabat equivalent.

Claims

1. Variant of a parent polypeptide comprising an Fc fragment, said variant having an increased affinity for the FcRn receptor, and an increased affinity for at least one Fc receptor (FcR) selected from the FcRI (CD64), FcRIIIa (CD16a) and FcRIIa (CD32a), relative to that of the parent polypeptide, comprising: (i) the four mutations 334N, 352S, 378V and 397M; and (ii) at least one mutation selected from 434Y, 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K; wherein the numbering is that of the EU index or equivalent in Kabat.

2. The variant according to claim 1, further comprising at least one mutation (iii) in the Fc fragment chosen from among Y296W, K290G, 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, V262S, V263T, V264L, V264S, V264T, V266L, S267A, S267Q, S267V, K290D, K290E, K290H, K290L, K290N, K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E293Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V Q295I, Q295M, Y296H, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S3041, V305A, V305F, V3051, V305L, V305R and V305S, wherein the numbering is that of the EU index or equivalent in Kabat,

3. The variant according to claim 1, comprising: (i) the four mutations 334N, 352S, 378V and 397M; (ii) at least one mutation selected from 434Y, 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K; and (iii) at least one mutation selected from K290G and Y296W, wherein the numbering is that of the EU index or equivalent in Kabat.

4. The variant according to claim 1, having an increased affinity for the FcRn receptor, relative to that of the parent polypeptide, of a ratio at least equal to 2.

5. The variant according to claim 1, having an increased affinity for at least one Fc receptor (FcR) selected from FcRI receptors (CD64), FcRIIIa (CD16a) and FcRII (CD32a), relative to that of the parent polypeptide, of a ratio at least equal to 2.

6. The variant according to claim 1, wherein the variant is produced in mammary epithelial cells of transgenic non-human mammals.

7. The variant according to claim 1, wherein the variant is produced in transgenic non human animals.

8. The variant according to claim 7, wherein the transgenic non-human animal is a transgenic goat.

9. The variant according to claim 1, wherein the variant the parent polypeptide comprises a parent Fc fragment which is a human Fc fragment.

10. The variant according to claim 1, wherein the variant is selected from an isolated Fc fragment, a sequence derived from an isolated Fc fragment, an antibody, an antibody fragment comprising an Fc fragment, and a fusion protein comprising an Fc fragment.

11. The variant according to claim 1, directed against an antigen selected from a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, a toxin, a membrane or circulating cytokine, a membrane receptor.

12. A method for treating a patient in need thereof, comprising administering an effective amount of the variant according to claim 1 to said patient.

13. A method for treating an autoimmune or inflammatory pathology, comprising administering an effective amount of the variant according to claim 1 to a patient in need thereof.

14. Pharmaceutical composition comprising a variant according to claim 1, and at least one pharmaceutically acceptable excipient.

15. Process of producing a variant of a parent polypeptide comprising an Fc fragment, said variant having increased affinity for the FcRn receptor, and increased affinity for at least one Fc receptor (FcR) selected from FcRI receptors (CD64), FcRIIIa (CD16a) and FcRIIa (CD32a), relative to that of the parent polypeptide, comprising: (i) the four mutations 334N, 352S, 378V and 397M; and (ii) at least one mutation selected from 434Y, 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K; wherein the numbering is that of the EU index or equivalent in Kabat, said process comprising expressing said variant in mammary epithelial cells of transgenic non-human mammals, or said process comprising expressing said variant in mammalian cells in culture.

16. The process for producing a variant of a parent polypeptide comprising an Fc fragment according to claim 15, wherein said variant further comprises at least one mutation (iii) in the Fc fragment chosen from among Y296W, K290G, 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, V262S, V263T, V264L, V264S, V264T, V266L, S267A, S267Q, S267V, K290D, K290E, K290H, K290L, K290N, K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E293Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y296H, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S304T, V305A, V305F, V3051, V305L, V305R and V305S, wherein the numbering is that of the EU index or equivalent in Kabat.

17. The process of producing a variant of a polypeptide comprising an Fc fragment according to claim 15, comprising the steps of: a) preparing a DNA sequence comprising a sequence encoding the variant, a sequence encoding a mammalian casein promoter or a mammalian whey promoter, and a sequence encoding a signal peptide permitting the secretion of said variant; b) introducing the DNA sequence obtained in a) into a non-human mammalian embryo, to obtain a transgenic non-human mammal expressing the variant encoded by said DNA sequence obtained in a) in the mammary gland; and c) recovery of the variant in the milk produced by the transgenic nonhuman mammal obtained in b).

18. The process for producing a variant of a polypeptide comprising an Fc fragment according to claim 15, wherein the transgenic non-human mammal is selected from cattle, pigs, goats, sheep and rodents.

19. The process for producing a variant of a polypeptide comprising an Fc fragment according to claim 15, comprising the steps of: a) preparing a DNA sequence encoding the variant; b) introducing the DNA sequence obtained in a) into mammalian cells in transient or stable culture; c) expression of the variant from the cells obtained in b), and d) recovering the variant in the culture medium.

20. DNA sequence comprising a gene encoding a variant of a parent polypeptide comprising an Fc fragment, said variant having increased affinity for the FcRn receptor, and an increased affinity for at least one Fc receptor (FcR) selected from the receptors FcRI (CD64), FcRII1a (CD16) and FcRI1a (CD32), relative to that of the parent polypeptide, wherein said variant comprises: (i) the four mutations 334N, 352S, 378V and 397M; and (ii) at least one mutation selected from 434Y, 434S, 226G, P228L, P228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 389T and 389K; wherein the numbering is that of the EU index or equivalent in Kabat.

21. DNA sequence comprising a gene encoding a variant of a parent polypeptide comprising an Fc fragment according to claim 20, said variant further comprising at least one mutation (iii) in the Fc fragment selected from Y296W, K290G, 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, V262S, V263T, V264L, V264S, V264T, V266L, S267A, S267Q, S267V, K290D, K290E, K290H, K290L, K290N, K290Q, K290R, K290S, K290Y, P291G, P291Q, P291R, R292I, R292L, E293A, E293D, E293G, E293M, E293Q, E293S, E293T, E294A, E294G, E294P, E294Q, E294R, E294T, E294V, Q295I, Q295M, Y296H, S298A, S298R, Y300I, Y300V, Y300W, R301A, R301M, R301P, R301S, V302F, V302L, V302M, V302R, V302S, V303S, V303Y, S3041, V305A, V305F, V3051, V305L, V305R and V305S, wherein the numbering is that of the EU index or equivalent in Kabat.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0135] FIG. 1: Production of variant A3A-184AY in goat milk and mouse using the vector Bc451

A) The beta casein vector, Bc451, was digested with XhoI.
In the vector Bc451, the NotI-NotI fragment is the prokaryotic fragment. The NotI fragment (15730)-XhoI is the 3 genomic sequence that contains the polyA signal. The BamHI-XhoI fragment is the promoter region of beta casein.
B) The Sall fragment containing the Fc A3A-184AY variant coding region (i.e. FC3179 A3A-184AY 884 bp) was inserted into the vector, to generate the BC3180 FC A3A-184AY (C) gene construct.
D) The DNA fragment for microinjection was then isolated from the prokaryotic vector. To do this, BC3180 was digested with NotI and NruI. The 16.4 kb fragment, containing the Fc gene (encoding the A3A-184AY variant) under the control of the beta casein promoter, was then purified by gel elution.

[0136] FIG. 2: Results of Tests in an Orentive Model of Arthritis Induced by K/BN Mouse Serum Transfer

[0137] The disease was induced by transferring 10 ml of K/BN mouse serum intravenously on D0 to C57/BI/6J mice. The test molecules were administered once intraperitoneally at D0, 2h before injection of the K/BN mouse serum.

[0138] The clinical score is obtained by summing the four-leg index:

0=normal, 1=swelling of a joint, 2=swelling of more than one joint, and 3=severe swelling of the entire joint (arbitrary units).

[0139] FIG. 3: Results of tests in a therapeutic model of arthritis induced by the transfer of K/BN mouse serum

[0140] The disease was induced by transferring 10 ml of K/BN mouse serum intravenously on D0 to C57/BI/6J mice. The test molecules were administered once intraperitoneally at D0, 72 hours after injection of K/BN mouse serum (indicated by dotted lines).

[0141] The clinical score is obtained by summing the four-leg index:

0=normal, 1=swelling of a joint, 2=swelling of more than one joint, and 3=severe swelling of the entire joint (arbitrary units).

[0142] FIG. 4: Test results of binding Fc and IqIV to sanitary cells

[0143] IgIV or Fc variants according to the invention labeled with Alexa were incubated at 65 nM (10 g/ml for Fc in 2% CSF PBS) with target cells for 20 minutes on ice. After 2 washes in 2% CSF, the cells were suspended in 500 ml Isoflow prior to flow cytometric analysis.

[0144] The results are as follows:

A) B cells labeled with anti-CD19 (% positive B cells);
B) NK cells labeled with anti-CD56 (% positive NK cells);
C) monocytes labeled with anti-CD14, in the presence of IgIV (% positive cells+IgIV);
D) CD16+monocytes labeled with anti-CD14 and with the anti-CD16 3G8 antibody, in the presence of IgIV (% positive cells+IgIV);
E) Neutrophils labeled with anti-CD15, in the presence of IgIV (% positive cells+IgIV);
F) NK cells labeled with anti-CD56, in the presence of IgG or Fc WT (% cell positive).

[0145] FIG. 5: Results of ADCC tests, activation of Jurkat CD64 and CDC cells

A) Inhibition of activation of Jurkat CD64 cells:
Raji cells (50 ml at 510.sup.6 cells/nil) were mixed with Rituxan (50 ml to 2m9/ml), Jurkat cells expressing human CD64 (Jurkat-H-CD64) (25 ml at 510.sup.6 cells/ml), PMA (50 ml to 40 ng/ml), then incubated with IgIV or the variant according to the invention (RFC A3A-184AY) at 1950 nM.

[0146] After a night of incubation, the plates were centrifuged (125 g for 1 minute), and IL2 contained in the supernatant was evaluated by ELISA.

[0147] The results were expressed as a percentage with respect to IgIV, according to the following formula: (IL-2 IgIV/IL-2 of the sample)100.

B) Inhibition of ADCC:

[0148] Effector cells (mononuclear cells) (25 ml at 810.sup.7 cells/nil) and Rh-positive RBCs (25 ml at 410.sup.7 cells/ml final) were incubated with different concentrations (0 to 75 ng/ml) of anti-Rh-antibody D, with an Effector/Target ratio of 2/1. After 16 hours of incubation, lysis was estimated by quantifying the hemoglobin released into the supernatant using a specific substrate (DAF).

[0149] The results are expressed as a percentage of specific lysis as a function of the amount of antibody. Inhibition of ADCC was induced by IgG or Fc variant according to the invention (RFC A3A-184AY) added at 33 nM.

[0150] The results are expressed in percent, wherein 100% and 0% are the values obtained with IgIV at 650 nM and 0 nM respectively according to the following formula:

[(ADCC with 33 nM sampleADCC without IVIg)/(ADCC with IgIV at 33 nMADCC without IVIg)100].

C) Inhibition Activity of the CDC:

[0151] Raji cells were incubated for 30 minutes with a final concentration of 50 ng/ml of rituximab. A solution of young rabbit serum diluted 1/10 and previously incubated with the variant Fc according to the invention (rFc A3A-184AY) or IgIV (vol/vol) for 1 h at 37 C. was added. After 1 hour of incubation at 37 C., the plates were centrifuged (125 g for 1 minute) and the CDC was estimated by measuring the intracellular LDH released in the culture medium. The results were expressed as percent inhibition and compared to IgG and negative control (Fc without Fc function, i.e. rFc neg), 100% corresponding to a complete inhibition of lytic activity and 0% to the control value obtained without Fc or IgIV.

[0152] FIG. 6: Results of the Cell Binding Tests

[0153] IgIV, Fc-Rec (wild-type Fc), Fc MST-HN or Fc variants according to the invention (A3A-184AY CHO, A3A-184EY CHO) labeled with Alexa-Fluor were incubated at 65 nM (10 g/ml) for Fc in 2% CSF (Colony Stimulating Factor) PBS with target cells for 20 minutes on ice. After 2 washes in 2% CSF PBS, the cells were suspended in 500 l of Isoflow before flow cytometric analysis The tests are performed on the following target cells: [0154] Natural Killer (NK) cells labeled with anti-CD56; [0155] Monocytes labeled with anti-CD14; [0156] CD16+monocytes labeled with anti-CD14 and anti-CD16 3G8 antibody; [0157] Neutrophils labeled with anti-CD15.

[0158] FIG. 7: Results of tests in an in vivo model of idiopathic thrombocytopenic purpura (ITP)

[0159] The disease was induced in mice expressing humanized FcRn by injecting an anti-platelet antibody 6A6-hlgG1 (0.3 pg/g body weight) intravenously to deplete platelets, also called thrombocytes, from mice. Negative Control (CTL PBS), IgIV (1000 mg/kg), Fc-Rec (Fc-wild) fragment (380 and 750 mg/kg), Fc MST-HN fragment (190 mg/kg) and the variant of the invention Fc A3A-184AY CHO (190 mg/kg and 380 mg/kg), were administered intraperitoneally 2 hours before platelet depletion. Platelet count was determined with an Advia Hematology system (Bayer). The number of platelets before the injection of antibodies was set at 100%.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Example 1: Preparation of Variants (Mutated Fc Fragments) According to the Invention Produced in the Milk of Transgenic Animals and Characterization of Said Variants

[0160] I. Materials and Methods

[0161] Principle:

[0162] An Fc fragment according to the invention may be produced in the milk of transgenic animals, by placing the coding sequence of the Fc fragment in a milk-specific expression vector. The vector may be introduced into the genome of a transgenic mouse or goat by microinjection. Following the screening and identification of an animal with the transgene, the females are reproduced. Following the parturition, milking the females allows recovery of their milk, in which the Fc could be secreted following the expression of the specific promoter of the milk.

[0163] Protein Sequence of Fc Variant A3A-184AY (K334N/P352S/A378V/V397M/N434Y):

TABLE-US-00002 (SEQIDNO:11) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIENTISKAKGQPREPQVYTLSPSRDELTKNQVSLTCLVK GFYPSDIVVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHYHYTQKSLSLSPGK

[0164] A signal peptide (MRWSWIFLLLLSITSANA, SEQ ID NO: 12) is bound to the N-terminus of the protein sequence, so as to obtain the sequence SEQ ID NO: 13. It allows the secretion of the protein in milk, once expressed.

Optimization of the Nucleotide Sequence:

[0165] The nucleotide sequence has been optimized for expression in the goat mammary gland. For this, the sequence was optimized for the Bos taurus species by the algorithm of a synthetic gene provider (such as GeneArt).

Expression Vector:

[0166] The goat beta casein expression vector (Bc451) was used for the production of the A3A-184AY variant in mouse and goat milk (see FIG. 1).

[0167] The beta casein vector, Bc451, was digested with XhoI (FIG. 1A). The Sall fragment containing the Fc A3A-184AY variant coding region was inserted to generate the BC3180 FC A3A-184AY gene construct (FIGS. 1B and 1C).

[0168] The DNA fragment for microinjection was then isolated from the prokaryotic vector.

[0169] BC3180 was digested with NotI and NruI (FIG. 1D). The released 16.4 kb fragment containing the Fc gene under the control of the beta casein promoter was then purified by gel elution. This DNA was then used in the microinjection stage.

Production in the Mouse:

[0170] The DNA fragment was inserted by microinjection into preimplantation mouse embryos. The embryos were then implanted in pseudopregnant females. The offspring that were born were screened for the presence of the transgene by PCR analysis.

Expression in Goats:

[0171] The DNA fragment prepared for microinjection may also be used for the production of the Fc variant A3A-184AY in goat's milk.

Example 2: Preparation of Variants (Mutated Fc Fragments) According to the Invention, Produced in HEK Cells and Characterization of Said Variants

[0172] I. Materials and Methods for Production

[0173] Each mutation of interest in the Fc fragment of sequence SEQ ID NO: 14 was inserted by overlap PCR using two sets of primers adapted to integrate the targeted mutation(s) with the codon(s) encoding the desired amino acid. Advantageously, when the mutations to be inserted are close to the Fc sequence, they are added via the same oligonucleotide. The fragments thus obtained by PCR were combined and the resulting fragment was amplified by PCR using standard protocols. The PCR product was purified on 1% (w/v) agarose gel, digested with the appropriate restriction enzymes and cloned.

[0174] The recombinant Fc fragment was produced by transient transfection (by lipofection) in HEK293 cells (293-F cells, InvitroGen freestyle) in F17 medium supplemented with L-glutamine using the pCEP4 vector. After 8 days of culture, the supernatant is clarified by centrifugation and filtered through a 0.2 m filter. Fragment Fc is then purified on Hi-Trap protein A, and elution is effected with 25 mM citrate buffer pH=3.0, neutralized and dialyzed in PBS prior to filtration sterilization (0.2 m).

[0175] II. Octet Binding Tests (BLI Technology Bio-Layer Interferometry, Device: Byte RED96, Fortebio, PaII)

[0176] Protocols:

[0177] Human FcRn Binding (hFcRn):

[0178] The biotinylated hFcRn receptor is immobilized on Streptavidin Biosensors, diluted to 0.7 g/ml in run buffer (0.1 M phosphate buffer, 150 mM NaCl, 0.05% Tween 20, pH6). The variants according to the invention, WT and IgIV, were tested at 200, 100, 50, 25, 12.5, 6.25, 3.125 and 0 nM in run buffer (200 nM=10 g/ml for Fc).

[0179] Design of the Test:

[0180] Baseline 1120 s in run buffer

[0181] Loading 300 s: the receiver is loaded on the biosensors

[0182] Baseline 260 s in run buffer

[0183] Association 60 s: samples (Fc or IVIg) are added to the biosensors loaded in hFcRn

[0184] Dissociation 30 s in run buffer

[0185] Regeneration 120 s in regeneration buffer (0.1 M phosphate buffer, 150 mM NaCl, 0.05% Tween 20, pH 7.8).

[0186] Results Interpretation:

[0187] The association and dissociation curves (first 10 s) are used to calculate the kinetic constants of association (kon) and dissociation (koff) using a 1/1 association model. KD (nM) is then calculated (kon/koff).

[0188] Link to the hCD16aV and hCD32aH Receivers:

[0189] The hCD16aV (R&D System) or hCD32aH (PX therapeutics) HisTag receptor is immobilized on anti-Penta-HIS Biosensors (HIS 1K), diluted to 1 g/ml in kinetic buffer (PaII). The Fc variants according to the invention, WT and IgIV, were tested at 1000, 500, 250, 125, 62.5, 31.25, 15 and 0 nM in kinetic buffer.

[0190] Loading Before Each Sample

[0191] Design of the test: All the stages are realized in kinetic buffer (PaII)

[0192] Baseline 160 s

[0193] Loading 400 s

[0194] Baseline 260 s

[0195] Association 60 s

[0196] Dissociation 30 s

[0197] Regeneration 5 s in regeneration buffer (Glycine 10 mM pH 1.5/Neutralization: PBS).

[0198] Results Interpretation:

[0199] The association and dissociation curves (first 5 s) are used to calculate the kinetic constants of association (kon) and dissociation (koff) using a 1/1 association model. KD (nM) is then calculated (kon/koff).

[0200] Results:

[0201] The results are shown in Table 1 below:

TABLE-US-00003 TABLE 1 Molecule hCD16aV SD hFcRn SD hCD32aH SD IVIg 653.8 4.0 34.4 1.94 438.2 114.3 Fc-WT (HEK) 504.3 75.0 36.5 8.2 659.3 203.1 A3A-184AY 132.0 14.1 7.8 0 313.0 29.7 (HEK) SD = standard deviation

[0202] The results show that the variant Fc A3A 184AY (HEK) according to the invention exhibits both an increased affinity for the hFcRn receptor, and an increased affinity for the FcRIIIa (CD16a) and FcRIIa (CD32a) receptors, and this compared to Fc parent not mutated (Fc-WT) but also compared to IVIG.

[0203] III. Model-Based Arthritis Assays Induced by K/BN Mouse Serum Transfer

[0204] Protocol:

[0205] The K/BN model was generated by crossing the transgenic mice for the KRN T cell receptor to the NOD mouse strain. K/BN F1 mice spontaneously develop a disease at 3 to 5 weeks of age and share many clinical features with human rheumatoid arthritis.

[0206] The disease was induced by transferring 10 ml of K/BxN mouse serum intravenously on D0 to C57/BI/6J mice. The molecules tested were administered once intraperitoneally at D0, 2h before or 72 hours after the injection of K/BxN mouse serum.

[0207] Mice were monitored daily for signs and symptoms of arthritis to assess incidence and severity by adding the four-leg index:

[0208] 0=normal, 1=swelling of a joint, 2=swelling of more than one joint, and 3=severe swelling of the entire joint.

[0209] Results:

[0210] Mice given K/BxN serum developed arthritis in the joint. The disease was characterized by an increase in ankle size, leading to an increase in the clinical score. These mice showed a significant increase in clinical score and ankle thickness compared to control mice treated with saline.

[0211] 1Preventive Model:

[0212] Administered 2 h before the K/BxN mouse serum injection, treatment with 750 mg/kg of wild-type Fc (Fc WT) fragment significantly reduced the clinical score compared to the serum group of K/BxN mice.

[0213] Treatment with the Fc variant A3A-184AY (HEK) according to the invention significantly reduced the clinical score in a manner similar to the Fc WT fragment, but at a dose 15 times lower (50 mg/kg) (FIG. 2).

[0214] 2Therapeutic Model:

[0215] 72 hours after the injection of K/BN mouse serum, the IgG administered at 2 g/kg did not significantly reduce the clinical score compared to the group treated with K/BN mouse serum.

[0216] However, treatment with the Fc WT fragment at 750 mg/kg (molecular dose equivalent to 2 g/kg IVIG) significantly reduced the clinical score compared to the group treated with K/BN mouse serum. In addition, treatment with the variant Fc A3A-184AY (HEK) according to the invention significantly reduced the clinical score similarly to the Fc-WT fragment, but at a dose 4-fold lower (190 mg/kg) (FIG. 3).

[0217] IV. In Vitro Cell Tests

[0218] Protocols:

[0219] Evaluation of the Binding of Fc and Ig IV Fragments to Blood Cells:

[0220] IgIV or Fc variants according to the invention labeled with Alexa were incubated at 65 nM (10 g/ml for Fc in 2% CSF PBS) with target cells for 20 minutes on ice. After 2 washes in 2% CSF, the cells were suspended in 500 ml Isoflow prior to flow cytometric analysis. B cells, NK cells, monocytes and neutrophils were specifically labeled with anti-CD19, anti-CD56, anti-CD14 and anti-CD15 respectively. The FcRIII receptor (CD16) was demonstrated using the anti-CD16 3G8 antibody.

[0221] Inhibition of ADCC:

[0222] To mimic the lysis of red blood cells observed in idiopathic thrombocytopenic purpura (ITP), involving the autoantibodies of the patient with ITP, an effector cell-mediated red cell lysis in the presence of an anti-Rhesus D (RhD) monoclonal anti-body was conducted, and the ability of different amounts of polyvalent immunoglobulins (IVIg) or mutated or non-mutated recombinant Fc fragments, to inhibit this lysis, for example by competition with anti-RhD for fixation of Fc receptors on the surface of the effector cell, were evaluated.

[0223] The cytotoxicity of anti-RhD antibodies has been studied by the technique of ADCC. Briefly, effector cells (mononuclear cells) (25 to 810.sup.7 cells/nil) and Rh-positive red cells (25 to 410.sup.7 cells/ml final) were incubated with different concentrations (0 to 75 ng/ml) of anti-RhD antibodies, with an Effector/Target ratio of 2/1. After 16 hours of incubation, lysis was estimated by quantifying the hemoglobin released into the supernatant using a specific substrate (DAF).

[0224] The results are expressed as a percentage of specific lysis as a function of the amount of antibody. The inhibition of ADCC induced by IgIV or the Fc variant according to the invention (RFC A3A-184AY) added to 33 nM was evaluated.

[0225] The results are expressed in percent, wherein 100% and 0% are the values obtained with IgIV at 650 nM and 0 nM respectively, according to the following formula:

[(ADCC with 33 nM sampleADCC without IVIg)/(ADCC with IgIV at 33 nMADCC without IVIg)100].

[0226] Inhibition of Activation of Jurkat CD64 Cells:

[0227] This test estimates the ability of the Fc variants according to the invention or IVIG (total IgG), to inhibit the secretion of IL2 by Jurkat cells expressing human CD64 (Jurkat-H-CD64) induced by the Raji cell line with Rituxan.

[0228] Briefly, Raji cells (50 ml at 510.sup.6 cells/nil) were mixed with Rituxan (50 ml at 2 mg/ml), Jurkat H-CD64 cells (25 ml at 510.sup.6 cells/ml, a phorbol ester (PMA, 50 ml at 40 ng/ml), then incubated with the IgIV or the Fc variant according to the invention at 1950 nM.

[0229] After a night of incubation, the plates were centrifuged (125 g for 1 minute) and NL2 contained in the supernatant was evaluated by ELISA.

[0230] The results were expressed as a percentage with respect to IgIV, according to the following formula:

(IL-2 IgIV/IL-2 of the sample)100.

[0231] Inhibitory Activity of the CDC:

[0232] This assay estimates the ability of the Fc variant according to the invention or IVIG to inhibit rituximab-mediated CDC activity on the Raji cell line in the presence of rabbit serum as a source of complement. Briefly, Raji cells were incubated for 30 minutes with a final concentration of 50 ng/ml of rituximab. A solution of young rabbit serum diluted 1/10 and previously incubated with the variant according to the invention or IgIV (vol/vol) for 1 h at 37 C., was added. After 1 hour of incubation at 37 C., the plates were centrifuged (125 g for 1 minute) and the CDC was estimated by measuring the intracellular LDH released in the culture medium.

[0233] The results were expressed as percentage inhibition and compared to IVIG and negative control (Fc without Fc function), 100% corresponding to a complete inhibition of lytic activity and 0% to the control value obtained without Fc or IVIG.

[0234] Results:

[0235] The results are shown in FIGS. 4 and 5.

[0236] As shown in FIG. 5, the Fc variant according to the invention (A3A-184AY (HEK)) has a better inhibition of the activity of the Jurkat cells expressing CD64, of the ADCC and of the CDC, in comparison with the IVIg. These results show that a variant according to the invention such as A3A-184AY may be effective for the treatment of pathologies involving patient autoantibodies, in particular by blocking Fc receptors on the effector cells of the patient (see FIG. 4).

Example 3: Preparation of Variants (Mutated Fc Fragments) According to the Invention, Produced in CHO Cells

[0237] The recombinant Fc fragment may be obtained from SEQ ID NO: 14 in the same manner as that described in Example 2. This mutated Fc fragment may be produced by transfection into CHOS cells with the aid of lipofection such as Freestyle Max Reagent (Thermofisher) using a vector optimized for expression in this cell line. The CHOS cells are cultured in CD FortiCHO medium+8 mM Glutamine, under conditions agitated at 135 rpm in a controlled atmosphere (8% CO.sub.2) at 37 C. On the day before the day of transfection, the cells are seeded at a density of 6.10.sup.5 cells/ml.

[0238] On the day of transfection, the linearized DNA (50 g) and 50 l of transfection agent (TA) are pre-incubated separately in Opti-Pro SFM medium and then mixed and incubated for 20 minutes to allow the formation of the DNA/AT complex. The whole is then added to a cell preparation of 1.10.sup.6 cells/ml in a volume of 30 ml. After 48 hours of incubation, transfection agents are added (Neomycin 1 g/L and Methotrexate 200 nM) to the cells. The cell density and viability are determined every 3-4 days and the culture volumes adapted to maintain a cell density greater than 6.10.sup.5 cells/ml. When the viability is greater than 90%, the stable pools obtained are saved by cryostatic congelation and productions in agitated conditions are carried out in Fed-batch mode for 10 days with an addition of 4 g/l or 6 g/l of glucose during production. At the end of production, the cells and the supernatant are separated by centrifugation. The cells are removed and the supernatant is harvested, concentrated and filtered at 0.22 m.

[0239] The Fc fragment is then purified by affinity chromatography on a protein A resin (HiTrap protein A, GE Healthcare). After capture on the balanced resin PBS buffer, the Fc fragment is eluted with 25 mM citrate buffer pH=3.0, followed by rapid pH neutralization with 1M Tris and then dialysed in PBS buffer before sterilization by filtration (0.2 pm).

Example 4: Binding Tests of FcRn, CD16aH, CD16aV, CD64 and CD32a Variants Produced in CHO Cells and in Transgenic Goat Milk

[0240] Fc receptor binding assays are performed with the following molecules: [0241] Variants of the invention A3A-184AY CHO (K334N/P352S/A378V/V397M/N434Y), A3A-184EY_CHO (Y296W/K334N/P352S/A378V/V397M/N434Y) produced in CHO cells according to the process given in example 3, A3A-184AY_TGg produced in the transgenic goat according to the process described in Example 1; [0242] The Fc MST-HN fragment containing the mutations M252Y/S254T/T256E/H433K/N434F, described in the literature as having an optimized binding only to the FcRn receptor (Ulrichts et al, JCI, 2018) was produced in HEK-293 cells. (293-F cells, InvitroGen freestyle); [0243] A wild-type Fc Fc-WT or Fc-Rec fragment obtained by digesting with papain an IgG1 produced in transgenic goat milk; [0244] IVIG

[0245] Human FcRn Binding (hFcRn):

[0246] FcRn binding is studied by competitive assay using A488 labeled Rituxan (Rituxan-A488) and Jurkat cells expressing the FcRn receptor (Jurkat-FcRn).

[0247] The Jurkat-FcRn cells are seeded in a 96-well plate (V bottom) at a concentration of 2.10.sup.5 cells per well. The cells are then incubated for 20 minutes at 4 C. with the test molecules diluted in buffer at the following final concentrations: 167 g/ml; 83 g/ml; 42 g/ml; 21 g/ml; 10 g/ml; 5 g/ml; 3 g/ml; 1 g/ml; 0 g/ml, and simultaneously with 25 g/ml Rituxan-A488.

[0248] The cells are then washed by adding 100 l of PBS at pH 6 and centrifuged at 1700 rpm for 3 minutes at 4 C. The supernatant is then removed and 300 l of cold PBS is added at pH 6.

[0249] The binding of Rituxan-A488 to FcRn expressed by Jurkat-FcRn cells is evaluated by flow cytometry. The mean fluorescence intensity (MFI) observed are expressed as a percentage, wherein 100% is the value obtained with Rituxan-A488 alone, and 0% the value in the absence of Rituxan-A488. The molecular concentrations required to induce 50% inhibition of Rituxan-A488 binding to FcRn of Jurkat-FcRn cells are calculated using Prism Software.

[0250] The results are shown in Table 2 below.

TABLE-US-00004 TABLE 2 A3A- A3A- A3A- MST- 184AY_CHO 184EY_CHO 184AY_TGg HN Fc-WT IVIG Inhibition of 13 15 12 14 476 1356 binding to FcRn (IC 50%, nM)

[0251] The results show that the Fc A3A-184AY CHO, Fc A3A-184EY CHO and A3A-184AY-TGg variants show increased Rituxan-A488 binding inhibition (100 compared to IVIG). The variants of the invention show an FcRn binding affinity equivalent to that observed with the Fc MST-HN fragment described in the literature as optimized only for FcRn (Ulrichts et al, JCI, 2018).

[0252] Binding to hCD64 and hCD16aH, hCD16aV, hCD32aH, hCD32aR Receptors:

[0253] Binding to Human CD64 (hCD64)

[0254] Human CD64 binding is studied by competitive assay using Rituxan-A488 and Jurkat cells expressing the CD64 receptor (Jurkat-CD64).

[0255] Jurkat-CD64 cells are seeded in a 96-well plate (V-bottom) at a concentration of 2.10.sup.5 cells per well. The cells are then incubated for 20 minutes at 4 C. with the test molecules diluted in the buffer with the final concentrations: 167 g/ml; 83 g/ml; 42 g/ml; 21 g/ml; 10 g/ml; 5 g/ml; 3 g/ml; 1 g/ml; 0 g/ml, and simultaneously with 25 g/ml Rituxan-A488.

[0256] The cells are then washed by adding 1 l of PBS at pH 6 and centrifuged at 1700 rpm for 3 minutes at 4 C. The supernatant is then removed and 300 l of cold PBS is added at pH 6.

[0257] The binding of Rituxan-A488 to CD64 expressed by Jurkat-CD64 cells is evaluated by flow cytometry. The mean fluorescence intensities (MFI) observed are expressed as a percentage, wherein 100% is the value obtained with Rituxan-A488 alone, and 0% is the value in the absence of rituxan-A488. The molecular concentrations required to induce 50% inhibition of Rituxan-A488 binding to CD64 of Jurkat-CD64 cells are calculated using Prism Software.

[0258] Binding to CD32aH and CD32aR Human CD32 receptor binding is studied by competitive assay using Rituxan-A488 and HEK cells transfected with CD32aH and CD32aR (HEK-CD32) receptors.

[0259] The HEK-CD32 cells are seeded in a 96-well plate (V bottom) at a concentration of 2.10.sup.5 cells per well. The cells are then incubated for 20 minutes at 4 C. with the test molecules diluted in buffer at the following final concentrations: 333 g/ml; 167 g/ml, 83 g/ml; 42 g/ml; 21 g/ml; 10 g/ml; 5 g/ml; 3 g/ml; 1 g/ml; 0 g/ml, and simultaneously with 30 g/ml Rituxan-A488.

[0260] The cells are then washed by adding 100 l of PBS at pH 6 and centrifuged at 1700 rpm for 3 minutes at 4 C. The supernatant is then removed and 300 l of cold PBS is added at pH 6.

[0261] The binding of Rituxan-A488 to CD32aH and CD32aR expressed by HEK-CD32 cells is evaluated by flow cytometry. The mean fluorescence intensities (MFI) observed are expressed as a percentage, wherein 100% is the value obtained with the Rituxan-A488 alone, and 0% is the value in the absence of Rituxan-A488. The molecular concentrations required to induce 50% inhibition of Rituxan-A488 binding to CD32aH and CD32aR of HEK-CD32 cells are calculated using Prism Software.

[0262] Binding to hCD16aH

[0263] The binding to human CD16aH is studied by competitive assay using a murine anti-CD16 3G8 antibody labeled with phycoerythrin (3G8-PE) and Jurkat cells transfected with the human CD16aH receptor (Jurkat-CD16aH).

[0264] The Jurkat-CD16aH cells are seeded in a 96-well plate (V bottom) at a concentration of 2.10.sup.5 cells per well. The cells are then incubated for 20 minutes at 4 C. with the test molecules diluted in buffer at the following final concentrations: 83 g/ml; 42 g/ml; 21 g/ml; 10 g/ml; 5 g/ml; 3 g/ml; 1 g/ml; 0 g/ml, and simultaneously with 0.5 g/ml mAb 3G8-PE.

[0265] The cells are then washed by adding 1 l of PBS at pH 6 and centrifuged at 1700 rpm for 3 minutes at 4 C. The supernatant is then removed and 300 l of cold PBS is added at pH 6.

[0266] The binding of mAb 3G8-PE to CD16aH expressed by Jurkat-CD16aH cells is evaluated by flow cytometry. The average fluorescence intensities (MFI) observed are expressed as a percentage, wherein 100% is the value obtained with the mAb 3G8-PE alone, and 0% is the value in the absence of mAb 3G8-PE. The molecular concentrations required to induce 50% inhibition of mAb 3G8-PE binding to CD16aH of Jurkat-CD16aH cells, are calculated using Prism Software.

[0267] The results are shown in Table 3 below.

TABLE-US-00005 TABLE 3 A3A- A3A- A3A- MST- 184AY_CHO 184EY_CHO 184AY_TGg HN Fc-WT IVIG Inhibition of 262 123 105 >2170 282 1684 binding to the CD16a-F (IC 50%, nM) Inhibition of 135 147 170 >2170 >2170 671 binding to the CD32a-H (IC 50%, nM) Inhibition of 176 132 Not >2170 >2170 1308 binding to the determined CD32a-R (IC 50%, nM) Inhibition of 57 55 59 >2170 >2170 761 binding to the CD32b (IC 50%, nM) Inhibition of 84 70 87 494 176 880 binding to the CD64 (IC 50%, nM)

[0268] The results show that the A3A-184AY CHO Fc, A3A-184EY CHO Fc and A3A-184AY_TGg variants have an increased affinity for the FcRIIIa (CD16a), FcRI (CD64) and FcRIIa (CD32a) receptors, compared to the Fc non mutated (Fc-WT) but also compared to IVIG.

[0269] The mutants of the invention show a very increased affinity for FcRIIIa (CD16a), FcRI (CD64) and FcRIIa (CD32a) receptors compared to MST-HN.

[0270] Binding to Human CD16aV:

[0271] HisTag hCD16aV (R&D System) receptor is immobilized on anti-Penta-HIS Biosensors (HIS 1K), diluted to 1 g/ml in kinetic buffer (PaII). The molecules were tested at concentrations of 1000, 500, 250, 125, 62.5, 31, 25, 15 and 0 nM in kinetic buffer.

[0272] Loading Before Each Sample

[0273] Design of the Test: All the Steps are Realized in Kinetic Buffer (PaII)

[0274] Baseline 160 s

[0275] Loading 400 s

[0276] Baseline 260 s

[0277] Association 60 s

[0278] Dissociation 30 s

[0279] Regeneration 5 s in regeneration buffer (Glycine 10 mM pH 1.5/Neutralization: PBS).

[0280] Results Interpretation:

[0281] The association and dissociation curves (first 5 s) are used to calculate the kinetic constants of association (kon) and dissociation (koff) using a 1/1 association model. KD (nM) is then calculated (kon/koff).

[0282] The results are shown in Table 4 below.

TABLE-US-00006 TABLE 4 Molecule KD hCD16aV (nM) SD A3A-184AY_CHO 80.3 18.1 A3A-184EY_CHO 59.3 7.7 A3A-184AY_TGg 51.2 10.7 MST-HN 268.2 83.6 Fc-WT 314.1 72.7 IVIG 339.0 103.9 SD: standard deviation

[0283] The results show that the Fc A3A-184AY CHO, Fc A3A-184EY CHO and A3A-184AY_TGg variants show a binding increase for the human FcRIIIa-V receptor (CD16a-V), and this compared to the non-mutated Fc (Fc-WT) but also compared to IgM and Fc fragment MST-HN containing M252Y/S254T/T256E/H433K/N434F mutations.

Example 5: ADCC Inhibition and Jurkat Cell Activation Tests of Variants Produced in CHO Cells and in Transgenic Goat Milk

[0284] ADCC inhibition and Jurkat cell activation tests are performed with the following molecules: [0285] Variants of the invention A3A-184AY_CHO (K334N/P352S/A378V/V397M/N434Y), A3A-184EY_CHO (Y296W/K334N/P352S/A378V/V397M/N434Y) produced in CHO cells according to the process given in Example 3, [0286] The Fc MST-HN fragment containing the M252Y/S254T/T256E/H433K/N434F mutations, described in the literature as having a binding optimized only to the FcRn receptor (Ulrichts et al, JCI, 2018) was produced in HEK-293 cells (293-F cells, Freestyle InvitroGen), [0287] A wild-type Fc Fc-Rec or Fc-WT fragment, obtained by digesting with papain an IgG1 produced in transgenic goat's milk, [0288] IgIV

[0289] ADCC Inhibition Test:

[0290] To mimic the lysis of red blood cells observed in idiopathic thrombocytopenic purpura (ITP), involving the autoantibodies of the patient with ITP, an effector cell-mediated red cell lysis in the presence of a Rhesus D (RhD) anti-human monoclonal antibody was conducted, and the ability of different amounts of polyvalent immunoglobulins (IgMV) or mutated or non-mutated recombinant Fc fragments, to inhibit this lysis, for example by competition with anti-RhD for fixation Fc receptors on the surface of the effector cells, were evaluated.

[0291] The cytotoxicity of anti-RhD antibodies has been studied by the technique of ADCC. Briefly, effector cells (mononuclear cells) (25 to 810.sup.7 cells/nil) and Rh-positive red cells (25 to 410.sup.7 cells/ml final) were incubated with different concentrations (0 to 75 ng/ml) of anti-RhD antibodies, with an Effector/Target ratio of 2/1. After 16 hours of incubation, lysis was estimated by quantifying the hemoglobin released into the supernatant using a specific substrate (DAF).

[0292] The results are expressed as a percentage of specific lysis as a function of the amount of antibody. The inhibition of ADCC is induced by the molecules tested (IgM, MST-HN, Fc-WT A3A-184AY CHO, A3A-184EY CHO) at concentrations of 500, 50, 5, 0.5 g/ml. for MST-HN, Fc-WT A3A-184AY_CHO, A3A-184EY_CHO and 1500, 150, 15, 1.5 g/ml for IgIV. The molecule concentrations to induce 25% or 50% inhibition were calculated with Prism Software.

[0293] The results are shown in Table 5 below.

TABLE-US-00007 TABLE 5 A3A- A3A- MST- 184AY_CHO 184EY_CHO HN Fc-WT IVIg Inhibition of the lysis of 13.5 7.6 190.2 82 59.6 the red blood cells medited by the anti-D AD1 (IC 25%, nM) Inhibition of the lysis of 97 56 441 1500 351 the red blood cells medited by the anti-D AD1 (IC 50%, nM)

[0294] The results show that the Fc variants, A3A-184AY CHO and A3A-184EY CHO, show an inhibition of lysis of red blood cells by an increased anti-Rhesus D antibody compared to non-mutated Fc (Fc-WT) but also compared with IVIG.

[0295] In addition, the inhibition of A3A-184AY CHO or A3A-184EY CHO is greatly increased compared to the Fc fragment, MST-HN, containing the M252Y/S254T/T256E/H433K/N434F mutations.

[0296] Inhibition of Activation of Jurkat CD64 Cells:

[0297] This test estimates the ability of the Fc variants according to the invention or IVIG (total IgG) to inhibit the secretion of IL2 by Jurkat cells expressing human CD64 (Jurkat-H-CD64) induced by the Raji cell line with Rituxan.

[0298] Briefly, Raji cells (50 ml at 510.sup.6 cells/nil) were mixed with Rituxan (50 ml at 2 mg/ml), Jurkat H-CD64 cells (25 ml at 510.sup.6), a phorbol ester (PMA, 50 ml at 40 ng/ml), then incubated with the IGVI or Fc variant according to the invention at 1950 nM.

[0299] After a night of incubation, the plates were centrifuged (125 g for 1 minute) and NL2 contained in the supernatant was evaluated by ELISA.

[0300] Inhibition of IL2 secretion was induced by IVIG, Fc-WT, MST-HN or Fc variants according to the invention (A3A-184AY CHO or A3A-184EY CHO) added at 50 and 100 g/ml. for Fc-WT, MST-HN fragments or Fc variants according to the invention (A3A-184AY CHO or A3A-184EY CHO), and 150 and 300 g/ml for IGVI.

[0301] The concentrations of the molecule to induce 25% or 50% inhibition were calculated with Prism Software.

[0302] The results are shown in Table 6 below.

TABLE-US-00008 TABLE 6 A3A- A3A- MST- 184AY_CHO 184EY_CHO HN Fc-WT IVIG Inhibition of the 448 442 1455 926 1106 secretion of IL-2 of the Jurkat cells transfected with CD64 (IC 25%, nM) Inhibition of the 600 600 <1950 <1950 <1950 secretion of IL-2 of the Jurkat cells transfected with CD64 (IC 50%, nM)

[0303] The results show that the A3A-184AY-CHO and A3A-184EY-CHO Fc variants show increased inhibition of IL2 secretion compared to non-mutated Fc (Fc-WT) but also compared to IVIG.

[0304] In addition, the inhibition of RFC A3A-184AY CHO or A3A-184EY CHO is greatly increased compared to the MST-HN Fc fragment containing the M252Y/S254T/T256E/H433K/N434F mutations.

Example 6: Tests of Binding Fc Variant to Blood Cells

[0305] The blood cell binding tests are performed with the following molecules: [0306] Variants of the invention A3A-184AY CHO (K334N/P352S/A378V/V397M/N434Y), A3A-184EY_CHO (Y296W/K334N/P352S/A378V/V397M/N434Y) produced in CHO cells according to the process given in example 3, A3A-184AY_TGg produced in the transgenic goat according to the process described in Example 1, [0307] The fragment Fc MST-HN containing the mutations M252Y/S254T/T256E/H433K/N434F, described in the literature as having an optimized binding only to the FcRn receptor (Ulrichts et al, JCI, 2018), was produced in HEK-293 cells (293-F cells, Freestyle InvitroGen), [0308] A wild-type Fc Fc-Rec or Fc-WT fragment, obtained by digesting with papain an IgG1 produced in transgenic goat's milk, [0309] IgIV

[0310] The molecules labeled with the Alexa Fluor marker (highly fluorescent protein marker) were incubated at 65 nM (10 g/ml for Fc in 2% CSF PBS) with target cells for 20 minutes on ice.

[0311] After 2 washes in 2% CSF, the cells were suspended in 500 ml Isoflow prior to flow cytometric analysis. The tests are performed on the following cells: [0312] Natural Killer (NK) cells labeled with anti-CD56 (% positive NK cells); [0313] Monocytes labeled with anti-CD14 (% positive cells); [0314] CD16+ monocytes labeled with anti-CD14 and with the anti-CD16 3G8 antibody (% positive cells); [0315] Neutrophils labeled with anti-CD15 (% positive cells)

[0316] The FcRIII receptor (CD16) was demonstrated using the anti-CD16 3G8 antibody.

[0317] The results show that the variants Fc A3A-184AY CHO, A3A-184EY CHO and A3A-184AY_TGg, whatever the mode of production, offer increased binding compared to the non-mutated Fc (Fc-Rec), but also compared to the IgIV. In addition, the binding of A3A-184AY or A3A-184EY is greatly increased compared to the MST-HN fragment for NK cells, CD16+ monocytes and neutrophils (see FIG. 6).

Example 7: In Vivo Model Tests of Idiopathic Thrombocytopenic Purpura (ITP)

[0318] The disease was induced in mice expressing a humanized FcRn (mFcRn/hFcRnTg 276 heterozygous B6 gene background (The Jackson Laboratory) by injecting an anti-platelet antibody 6A6-hIgG1 (0.3 pg/g body weight) intravenously to deplete the platelets of the mice. A blood test is made (number of thrombocytes) 24 hours before the injection of 6A6-hIgG1, 4h after the induction of the disease. The IgIV (1000 mg/kg), Fc-Rec (380 and 750 mg/kg), Fc MST-HN (190 mg/kg) and Fc A3A-184AY CHO (190 mg/kg and 380 mg/kg), were administered intraperitoneally 2 hours before platelet depletion.

[0319] Platelet count was determined with an Advia Hematology system (Bayer). The number of platelets before the injection of antibodies was set at 100%.

[0320] The anti-platelet antibody 6A6-hIgG1 (0.3 g/g) makes it possible to deplete 90% of the platelets.

[0321] The administration of drug candidates 2 hours before depletion of platelets can restore (FIG. 7): [0322] 100% platelets for A3A 184AY CHO at a dose of 380 mg/kg; [0323] 106% platelets for A3A-184AY CHO at a dose of 190 mg/kg; [0324] 90% platelets for IgIV at a dose of 1000 mg/kg; [0325] 64% platelets for Fc-WT at a dose of 750 g/kg; [0326] 75% platelets for Fc-WT at a dose of 380 mg/kg; [0327] 61% of the platelets for the MST-HN variant at a dose of 190 mg/kg.