NEW ANTIBODY BLOCKING HUMAN FCGRIIIA AND FCGRIIIB

Abstract

The present invention relates to novel antibodies, in particular murine monoclonal antibodies, chimeric and humanized, that are able to block specifically the human IgG receptors FcγRIIIA (CD16A) and FcγRIIIB (CD16B) as well as the amino and nucleic acid sequences coding for such antibodies. The invention also comprises the use of such antibodies or of fragments thereof as a medicament for the preventive and/or therapeutic treatment of diseases involving CD16, like autoimmune diseases, inflammatory disorders, allergies and infections, without inducing any adverse effects. In particular, these antibodies and fragments can prevent or treat anti-drug idiopathic thrombocytopenic purpura (ITP), rheumatoid arthritis (RA) and autoimmune hemolytic anemia (ANA).

Claims

1-31. (canceled)

32. An antagonistic antibody against CD16 or an antigen-binding fragment thereof, that binds specifically to the extracellular domain of CD16A and CD16B on monocytes, macrophages, NK cells and neutrophils, without inducing intracellular signal events in said cells.

33. The anti-CD16 antibody or antigen-binding fragment according to claim 32, comprising: a) a light chain comprising three CDRs of the sequences SEQ ID NO:1, 2 or 3, or having a sequence of at least 80% identity with sequences SEQ ID NO:1, 2 or 3 and b) a heavy chain comprising three CDRs of the sequences SEQ ID NO: 4, 5 or 6, or having a sequence of at least 80% identity with sequences SEQ ID NO: 4, 5 or 6.

34. The antibody or fragment of claim 32, comprising: a) a light chain variable domain (V.sub.L) of sequence SEQ ID NO: 7, or an amino acid sequence having at least 80% identity with SEQ ID NO: 7 and b) a heavy chain variable domain (V.sub.H) of sequence SEQ ID NO: 8, or an amino acid sequence having at least 80% identity with SEQ ID NO:8.

35. The antibody or fragment of claim 32, wherein the dissociation constant (K.sub.D) of said antibody or fragment with CD16AV158 is comprised between 5 nM and 20 nM measured by Surface Plasmon Resonance.

36. The antibody or fragment of claim 32, wherein it is a full-human antibody comprising a light chain comprising the CDR-L1, CDR-L2 and CDR-L3 having respectively the amino acid sequences SEQ ID NO: 1, 2 and 3; and a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 having respectively the amino acid sequences SEQ ID NO: 4, 5 and 6.

37. The antibody or fragment of claim 32, wherein it is a chimeric, a humanized or a full-human antibody that has been recombinantly modified by introducing a N297A mutation in the human IgG1 heavy chain.

38. An isolated nucleic acid or a vector comprising same, said nucleic acid being chosen from the following nucleic acids: a) a nucleic acid, DNA or RNA, coding for an antibody, or one of its functional fragments or derivatives as defined in claim 32; b) a nucleic acid comprising a DNA sequence selected from the group of sequences consisting of SEQ ID NO: 9 and 13, said nucleic acid encoding an antibody, or one of its functional fragments or derivatives as defined in claim 32; c) a nucleic acid comprising a DNA sequence selected from the group of sequences consisting of SEQ ID NO: 10, 14 and 16; said nucleic acid encoding an antibody, or one of its functional fragments or derivatives as defined in claim 32; d) a nucleic acid present in the pUC plasmids contained in the E. coli cells deposited at the Collection Nationale de Cultures de Microorganismes from Institut Pasteur, on Nov. 25, 2019, under the number 1-5458, 1-5459, or I-5460; said nucleic acid encoding the fragments of the invention; or e) a nucleic acid whose sequence exhibits a percentage identity of at least 80%, after optimal alignment with any of the sequence referred to in b) and c).

39. A pharmaceutical composition comprising the antibody or fragment according to claim 32, and a pharmaceutically-acceptable carrier.

40. A method for preventing and/or treating CD16-related disorders in which CD16 is engaged, or a disease selected from the list consisting of: auto-immune disease, inflammatory disease, infectious disease and allergy, said method comprising the administration of the pharmaceutical composition according to claim 39 in a subject in need thereof.

41. The method of claim 40, wherein said subject suffers from an autoimmune disorder selected in the group consisting of: idiopathic thrombocytopenic purpura (ITP), rheumatoid arthritis (RA), autoimmune hemolytic anemia (AIHA), multiple sclerosis (MS), psoriasis, psoriatic arthritis, Reiter's syndrome, type 1 or immune mediated diabetes mellitus, inflammatory-bowel disease (IBD), chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis.

42. The method of claim 40, wherein said subject suffers from: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Bechet's disease, bullous pemphigoid, cardiomyopathy, celiac disease, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatics, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteritis giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis. Examples of inflammatory disorders include, but are not limited to, asthma, encephalitis, inflammatory-bowel disease (IBD), chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacteria infections.

43. The method according to claim 40, wherein said subject suffers from asthma, encephalitis, inflammatory bowel disease (IBD), chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacteria infections.

44. An in vitro method to stain cells expressing CD16A or CD16B in a biological sample, or for determining the level of CD16A or CD16B in a tissue or in cells derived from a tissue, or for detecting NK-cell-binding molecule-additional antigen complexes, or for isolating CD16-expressing cells from a biological sample, or for diagnosing a CD16-related disorder in which CD16 is engaged, said method comprising the use of the antibody or fragment according to claim 32.

45. In vitro method according to claim 44, wherein said antibody or fragment is conjugated to a labelling molecule.

46. In vitro method according to claim 44, for detecting and/or quantifying tissue-infiltrating NK cells, monocytes, macrophages or neutrophils expressing CD16A or CD16B, in a biological sample.

47. In vitro method according to claim 44, wherein said antibody or fragment is conjugated to a binding molecule that exhibits specificity for an additional antigen.

48. A kit comprising the antibody or fragment according to claim 32 and means for detecting said antibody or fragment when bound to CD16A or CD16B.

49. The kit of claim 48, wherein said antibody or fragment is conjugated to a labelling molecule, and said means comprise film sensitive to the radio- or chemi-luminescent label, or wherein said antibody or fragment is conjugated to a labelling tag molecule such as histidine or c-myc tag, and said means comprise an antibody which recognizes said tag molecule.

50. The antibody or fragment according to claim 32, further comprising the functional domain of an enzyme that is capable of converting a pro-drug to an active drug.

51. The antibody or fragment according to claim 32, wherein it is conjugated to a toxin molecule such as a ribosyl transferase, serine protease, guanyl cyclase activator, calmodulin-dependent adenyl cyclase, ribonuclease, DNA alkylating agent or mitosis inhibitor (e.g. doxorubicin).

Description

FIGURE LEGENDS

[0183] FIG. 1 shows the binding of the antibodies of the invention (the chimeric modified 3G4NA) on members of the hFcγR family, assessed by FACS. The member of the hFcγR family is indicated on top of the panels, as well as their potential polymorphism found in the human population. Grey histogram represents secondary antibody alone (background), whereas the black line represents primary and secondary antibody (binding of the antibody).

[0184] FIG. 2 shows the blocking activity of the antibodies of the invention (the chimeric modified 3G4NA) towards IgG (the ligand) on members of the hFcγR family, assessed by FACS. The member of the hFcγR family is indicated on top of the panels, as well as their potential polymorphism found in the human population. Grey histogram represents no staining (background), dotted line represents the binding of fluorescently-labelled human IgG-immune complexes and the black line represents the binding of fluorescently-labelled human IgG-immune complexes in the presence of the chimeric mAb 3G4 N297A.

[0185] FIG. 3 discloses the variation of body central temperature observed after the injection of the antibodies of the invention (the chimeric modified 3G4NA-A), or after the injection of the antibodies of the prior art (chimeric 3G8-B or chimeric 3G8N297A-C) or controls (hIgG1 Herceptin) in hFcγR.sup.KI or mFcγR.sup.null mice. Statistical test: Two Way ANOVA (n=4 mice per group)

[0186] FIG. 4 discloses the variation of platelet numbers observed after the injection of the antibodies of the invention (the chimeric modified 3G4NA-A), or after the injection of the antibodies of the prior art (chimeric 3G8-B or chimeric 3G8N297A-C) or controls (hIgG1 Herceptin) in hFcγR.sup.KI or mFcγR.sup.null mice. Statistical test: t test with Mann-Whitney post-test (n=4 mice)

[0187] FIG. 5 discloses the variation of neutrophil numbers (CD11b+Ly6G+ cells) observed after the injection of the antibodies of the invention (the chimeric modified 3G4NA-A), or after the injection of the antibodies of the prior art (chimeric 3G8-B or chimeric 3G8N297A-C) or controls (hIgG1 Herceptin) in hFcγR.sup.KI or mFcγR.sup.null mice.

[0188] FIG. 6 discloses the variation of monocyte numbers (CD115+Ly6C+ cells) observed after the injection of the antibodies of the invention (the chimeric modified 3G4NA-A), or after the injection of the antibodies of the prior art (chimeric 3G8-B or chimeric 3G8N297A-C) or controls (hIgG1 Herceptin) in hFcγR.sup.KI or mFcγR.sup.null mice.

[0189] FIG. 7 shows the effect of the injection of the antibodies of the invention (the chimeric modified 3G4NA (9 mg/kg) on the platelet numbers in hFcγR.sup.KI mice when acute thrombocytopenia is induced subsequently (prophylactic treatment). Statistical test: t test with Mann-Whitney post-test.

[0190] FIG. 8 shows the effect of the injection of the antibodies of the invention (the chimeric modified 3G4NA, (9 mg/kg) on the platelet numbers in hFcγR.sup.KI mice A) when chronic thrombocytopenia has been induced previously (therapeutic treatment) with either an antibody that engages FcγR but not C1q (antibody 6A6KA), or B) with an antibody that engages both FcγR and C1q (antibody 6A6WT). Statistical test: Two Way ANOVA (n=4 mice per group).

EXAMPLES

[0191] 1. Material and Methods

[0192] Antibodies and reagents. Recombinant hFcγRIIIA/CD16a variant V158 was purchased from R&D Systems, anti-mouse IgG Fc fragment HRP conjugated from Bethyl, anti-FLAG mAb, anti-human CD64 (clone 10.1), anti-human CD32B (clone AT10), anti-human CD16 (clone MEM-154), anti-mouse CD11b from BD Pharmingen, anti-human CD32A (clone IV.3) from StemCell Technologies, anti-mouse CD115 from Biolegend, anti-mouse Ly6G, anti-mouse Ly6C and anti-mouse CD45 from Miltenyi Biotec, PE-labelled F(ab′).sub.2 Fragment Donkey Anti-Human IgG from Jackson Immuno Research.

[0193] Mice. BiozzyABH mice were purchased from Harlan laboratories. hFcγR.sup.KI (expressing hFcγRI, hFcγRIIA.sub.H131, hFcγRIIB.sub.I232, hFcγRIIC.sub.stop13, hFcγRIIIA.sub.V158 and hFcγRIIIB.sub.NA2 polymorphic variants) and FcγR.sup.null mice (expressing no endogenous FcγR) were generated by Regeneron Pharmaceuticals Inc. as described previously (Beutier H. et al, 2018). All mice were bred at Institut Pasteur and used for experiments at 9-13 weeks of age, and starting 10-15 weeks for immunizations. All mice demonstrated normal development and breeding patterns. All animal care and experimentation were conducted in compliance with the guidelines and specific approval of the Animal Ethics committee CETEA (Institut Pasteur, Paris, France) registered under #2013-0103, and by the French Ministry of Research under agreement #00513.02.

[0194] Immunization of mice. BiozzyABH mice were injected intraperitoneally with 10 μg of recombinant hFcγRIIIA/CD16A variant V158 first in complete Freund adjuvant (CFA; Sigma Aldrich), then three times in incomplete Freund adjuvant (IFA) at 3-week intervals. Three weeks after the last immunization, a boost was performed by a intraperitoneal injection of 10 ug recombinant hFcγRIIIA/CD16A without adjuvant. Three days later the spleen was removed and splenocytes used for fusion and hybridoma generation using the ClonaCell™-HY Hybridoma kit (StemCell Technologies).

[0195] Screening of hybridomas. Specificity against hFcγRIIIA was tested for hybridomas by ELISA. 96-well plates (Costar) were coated with recombinant hFcγRIIIA/CD16A variant V158 at 1 μg/well in coupling buffer (50 nM carbonate-bicarbonate buffer at pH 9.6) at 4° C. for 16 h. Plates were washed 3 times with PBS Tween 0.01% (PBST), and blocked for 2 h at room temperature in PBST containing 3% BSA. Plates were washed 3 times before addition of 100 μl of supernatant from each hybridoma. After 2 hours, plates were washed with PBST and incubated with 1:4,000 of HRP-conjugated anti-mouse IgG Fc fragment for 1 h. Plates were washed 3 times with PBST before addition of 100 μL/well OPD peroxidase (Sigma). Reaction was stopped by addition of 100 μL 2M H2504 and absorbance was recorded at 492 nm and corrected at 620 nm.

[0196] Cloning. Sequencing of V.sub.H and V.sub.L DNA fragments, codon optimization for expression in human cells and synthesize were done by Eurofins. V.sub.H sequences were cloned into a human pUC19-Igγ1-expressing vector using Sall and Agel restriction sites, and V.sub.L sequences were cloned into a human Igκ-expressing vector using Agel and BsiWI restriction sites (a kind gift of Hugo Mouquet, Institut Pasteur, Paris). For the generation of an Fc-engineered mAb, a point mutation in the Igγ1-expressing vector was introduced at position 297 (N297A) to exchange an asparagine for an alanine using the QuickChange Site-Directed Mutagenesis Kit (Agilent Technologies). All vectors were sequenced before being used for antibody production.

[0197] Production of mAbs. The cDNA encoding the heavy chain (variable region; VH) of mAb 3G4 anti-human CD16 (human FcgammaRIII) fused to the cDNA encoding the heavy chain (constant regions; CH1-CH2-CH3) of human IgG1 harboring or not the N297A mutation leading to aglycosylation was inserted into a pUC expression vector (plasmid). These plasmids can be obtained by standard alkaline lysis followed by plasmid DNA precipitation and solubilization from the E. coli bacteria deposited on Nov. 25, 2019 at the Collection Nationale de Cultures de Microorganismes from Institut Pasteur under the numbers CNCM I-5459 (wild-type N297) or CNCM I-5460 (mutated N297A).

[0198] Similarly, the cDNA encoding the light chain (variable region; VL) of mAb 3G4 anti-human CD16 (human FcgammaRIII) fused to the cDNA encoding the human kappa light chain (kappa constant regions) was inserted into a pUC expression vector (plasmid). This plasmid can be obtained by standard alkaline lysis followed by plasmid DNA precipitation and solubilization from the E. coli bacteria deposited on Nov. 25, 2019 at the Collection Nationale de Cultures de Microorganismes from Institut Pasteur under the numbers CNCM I-5458.

[0199] Antibodies were produced by transient co-transfection of WT or N297A Fc-engineered mAb 3G4 heavy chain and mAb 3G4 light chain expression plasmids into exponentially growing Freestyle™ HEK 293-F that were cultured in serum-free Freestyle™ 293 Expression Medium (Life Technologies) in suspension at 37° C. in a humidified 8% CO.sub.2 incubator on a shaker platform rotating at 110 rpm. Twenty-four hours before transfection, cells were harvested by centrifugation at 300×g for 5 min, and resuspended in Freestyle™ 293 expression medium at a density of 1×10.sup.6 cells/ml, and cultured overnight in the same conditions as mentioned above. For the production of mAbs, 40 μg of each V.sub.H and V.sub.L expressing plasmids were diluted in 80 μl of FectoPRO reagent (Polyplus) at a final DNA concentration of 0.8 μg/ml, incubated for 10 minutes at RT before addition to the cells. Twenty-four hours post-transfection, cells were diluted 1:1 with Freestyle™ 293 expression medium. Cells were cultured for 6 days after transfection, supernatants were harvested, centrifuged at 1800×g for 40 min and filtered (0.2 μm). Antibodies were purified by affinity chromatography using an AKTA pure FPLC instrument (GE Healthcare) on a HiTrap Protein G Column (GE Healthcare) and desalted on a HiTrap Desalting Column (GE Healthcare).

[0200] Assessment of Binding Specificity and Antagonistic Properties of Anti-hFcγRs Antibodies.

[0201] Specificity. A collection of Chinese Hamster Ovarian (CHO) cells expressing FLAG tagged human FcγR (Bruhns P. et al, 2009) was used to analyze the specificity of the antibody against the hFcγR family. Cells were incubated on ice for 30 min with mAbs at 1 μg/ml, washed 3 time in PBS containing 0.5% BSA and 2 mM EDTA (MACS buffer) and then incubated on ice for 30 min with 5 μg/ml PE-labelled F(ab′).sub.2 Donkey Anti-Human IgG (Jackson ImmunoResearch). Data acquisition was performed on a MACSQuant flow cytometer (Miltenyi Biotec), and data analyzed using the Flowjo Software (FlowJo).

[0202] Antagonistic properties. hFcγR ligands, i.e. human IgG-immune complexes, were formed by 5 incubating hIgG1 anti-dinitrophenyl (DNP) with BSA coupled to Trinitrophenyl (TNP) and to VT680 (BSA-TNP-VT680) at a 5:3 ratio for 30 min at 3TC in borate buffer saline. hFcγR-expressing CHO cells were pre-incubated or not on ice for 10 min with 10 μg/ml anti-hFcγRIII mAbs, then diluted 1:2 with human IgG-immune complexes (final concentration of 5 μg/ml hIgG1 anti-DNT and 3 μg/ml BSA-TNP-VT680) and incubated 30 min on ice. After 3 washes in MACS buffer, Data acquisition was performed on a MACSQuant flow cytometer and data analyzed using the Flowjo Software.

[0203] Surface Plasmon Resonance analysis. His-tagged ectodomains of hFcγRIIIA variant V158 were covalently immobilized on a His-tag capture sensor chip for a ProteON instrument (BioRad). A range of dilutions of anti-hFcγRIII mAbs were injected onto the chip. Background binding was measured on an empty HTE sensor chip channel and subtracted from the binding values observed on coated channels. The resulting sensorgrams were fitted using a “1:1 binding with mass transfer” model, and association (K.sub.on), dissociation (K.sub.off) constants and K.sub.D were calculated as the K.sub.on/K.sub.off ratio using BIAevaluation 4.1 software.

[0204] Experimental immune thrombocytopenia. Acute immune thrombocytopenia was induced by injecting hFcγR.sup.KI or FcγR.sup.null mice with the depleting anti-platelet mAb 6A6, in a human IgG1 format (termed herein 6A6WT; 10 μg/mouse) or in a human IgG1 format, containing a Lysine to Alanine mutation at position 322 of the heavy chain to prevent complement component Clq binding (termed herein 6A6KA; 20 μg/mouse). Injection of 200 μg of irrelevant human IgG1 was used as control. Blood was drawn in EDTA and platelets counts were acquired with an ABC Vet automatic blood analyzer (HoribaABX). Baseline platelet counts were performed 3-5 days before the experiment. For prophylaxis experiments, mice were pretreated by intravenous injection of 9 mg/kg of anti-hFcγRIII mAbs 30 min before injection of the depleting anti-platelet mAb.

[0205] For therapeutic experiments, severe chronic thrombocytopenia was induced by repeated, daily injections of depleting anti-platelet mAbs, followed by concomitant injections of depleting anti-platelet mAbs and 9 mg/kg of mouse of anti-hFcγRIII mAbs.

[0206] Assessment of central body temperature. hFcγR.sup.KI and FcγR.sup.null mice were injected intravenously with indicated quantities of mAbs in 100 μL saline, or saline only as a control. Body temperature measurements were performed using a digital thermometer (YSI) with a rectal probe, 30 min before and at indicated timepoints for up to 120 min.

[0207] Analysis of neutrophil and monocyte populations. Four hours after intravenous injection of mAbs, blood samples were drawn in heparin and lysed using Lysis buffer (BD Pharmingen). Leucocytes were stained on ice 30 min with the following panel: CD45, CD115, Ly6G, Ly6C, CD11b and propidium iodure solution to characterize neutrophil (CD45+ CD11 b+ Ly6G+) and monocyte (CD45+ CD115+ Ly6C+) populations. After 3 washes in MACS buffer, cells were analyzed on MACSQuant flow cytometer, and data were analyzed using the Flowjo Software.

[0208] Statistical analyses. Data are presented as mean±SD. Central body temperature experiments were analyzed with Two Way ANOVA-multiple comparisons with Sidak test. Platelets numbers (FIG. 4), and prophylactic immune thrombocytopenia experiments were analyzed with t test with Mann-Whitney post-test (FIG. 7). Therapeutic immune thrombocytopenia experiments were analyzed with two way ANOVA-multiple comparisons with Sidak test (FIGS. 8.a and 8.b) Statistical analyses were performed using the Prism Software (GraphPad Software). P values<0.05 were considered statistically significant.

[0209] 2. Results

[0210] 2.1. Generation of the Hybridoma

[0211] The spleens of three mice immunized with recombinant ectodomains of CD16A V158 variant were used to generate hybridomas using standard fusion protocols. Hybridoma supernatants (>600 hybridomas tested) containing potential anti-CD16A antibodies were screened using an anti-CD16A V158 variant ELISA, followed by a flow cytometry screen using a collection of transfectant cells (deposited at CNCM) expressing the entire family of hFcγRs.

[0212] 2.2. Sequencing of the CDRs and Important Regions of the Antibodies of the Invention

[0213] 2.2.1. Murine Antibody of the Invention (3G4)

[0214] The CDRs expressed in the murine antibody produced by the hybridoma 3G4 have been characterized.

[0215] They have the following peptide sequences:

[0216] For the light chain:

TABLE-US-00003 CDR1 V.sub.L: QDIIKN = SEQ ID NO: 1 CDR2 V.sub.L: YAT = SEQ ID NO: 2 CDR3 V.sub.L: LQFYEFPYT = SEQ ID NO: 3

[0217] For the heavy chain:

TABLE-US-00004 CDR1 V.sub.H: GYTFIRNW = SEQ ID NO: 4 CDR2 V.sub.H: IDPSDGES = SEQ ID NO: 5 CDR3 V.sub.H: TRSRYYGGDWDWYFDV = SEQ ID NO :6

[0218] The 3G4 light chain variable domain amino acid sequence is depicted in SEQ ID NO:7 (CDRs underlined+Framework sequences):

TABLE-US-00005 DIVLTQSPSSISASLGDRITITCQATQDIIKNLNWYQQKPGKPPSFLI YYATEVAEGVPSRFSGSGSGSDYSLTISNLESEDFADYYCLQFYEFPY TFGGGTKLEIK

[0219] The 3G4 heavy chain variable domain amino acid sequence is depicted in SEQ ID NO:8 (CDRs underlined+Framework sequences):

TABLE-US-00006 GVQLQESGAELVRPGSSVKLSCKPSGYTFIRNWIHWVKQRPIQGLEWIG AIDPSDGESHYNHKFTDKATLTVDKSSSTGYMQLNSLTSEDSAVYYCTR SRYYGGDWDWYFDVWGTGTTVTVSS

[0220] Nucleotide sequences encoding variable domains of the light chain and the heavy chain of said murine antibody are respectively SEQ ID NO:9 (3G4 Light chain DNA sequence, CDRs+Framework sequences) and SEQ ID NO: 10 (3G4 Heavy chain DNA sequence, CDRs+Framework sequences).

[0221] 2.2.2. Chimeric Mouse-Human Antibody of the Invention (Mouse-Human 3G4)

[0222] The V.sub.H mouse sequence of SEQ ID NO:10 of the anti-hFcγRIII monoclonal antibody has been inserted into a human IgG1 framework and the mouse V.sub.L sequence of SEQ ID NO:9 has been inserted into the human kappa light chain sequence.

[0223] The chimeric 3G4 of the invention is therefore a chimeric mouse-human IgG1 kappa antibody containing the mouse V.sub.H and mouse V.sub.L sequences of the anti-hFcγRIII mAb mouse clone 3G4.

[0224] The light chain of said chimeric antibody is of SEQ ID NO:11:

TABLE-US-00007 MGWSCIILFLVATATGVHSDIVLTQSPSSISASLGDRITITCQATQDII KNLNWYQQKPGKPPSFLIYYATEVAEGVPSRFSGSGSGSDYSLTISNLE SEDFADYYCLQFYEFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

[0225] The heavy chain of said chimeric antibody is of SEQ ID NO:12 (the N297 position is underlined):

TABLE-US-00008 MGWSCIILFLVATATGVHSEVQLQESGAELVRPGSSVKLSCKPSGYTFI RNWIHWVKQRPIQGLEWIGAIDPSDGESHYNHKFTDKATLTVDKSSSTG YMQLNSLTSEDSAVYYCTRSRYYGGDWDWYFDVWGTGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0226] Nucleotide sequences encoding the light chain and the heavy chain of said chimeric antibody are respectively SEQ ID NO:13 (chimeric 3G4 Light chain DNA sequence, CDRs+Framework sequences) and SEQ ID NO: 14 (chimeric 3G4 Heavy chain DNA sequence, CDRs+Framework sequences).

[0227] 2.2.3. Chimeric Modified Mouse-Human Antibody of the Invention (Mouse-Human 3G4NA)

[0228] The chimeric anti-hFcγRIII mAb clone 3G4 has been generated as a modified format under the name “3G4NA” or “3G4N297A”. 3G4NA is expressed as a chimeric mouse-human IgG1, kappa antibody mutated at position 297 of the heavy chain into an alanine (N297A mutation) and containing the mouse V.sub.H and mouse V.sub.L sequences of anti-hFcγRIII mAb mouse clone 3G4.

[0229] The light chain of said chimeric modified antibody 3G4NA is of SEQ ID NO:11:

TABLE-US-00009 MGWSCIILFLVATATGVHSDIVLTQSPSSISASLGDRITITCQATQDII KNLNWYQQKPGKPPSFLIYYATEVAEGVPSRFSGSGSGSDYSLTISNLE SEDFADYYCLQFYEFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

[0230] The heavy chain of said chimeric modified antibody 3G4NA is of SEQ ID NO:15: (the N297A mutation is underlined)

TABLE-US-00010 MGWSCIILFLVATATGVHSEVQLQESGAELVRPGSSVKLSCKPSGYTFI RNWIHWVKQRPIQGLEWIGAIDPSDGESHYNHKFTDKATLTVDKSSSTG YMQLNSLTSEDSAVYYCTRSRYYGGDWDWYFDVWGTGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0231] Nucleotide sequences encoding the light chain and the heavy chain of said chimeric antibody are respectively SEQ ID NO:13 (chimeric 3G4NA Light chain DNA sequence, CDRs+Framework sequences) and SEQ ID NO: 16 (chimeric 3G4NA Heavy chain DNA sequence, CDRs+Framework sequences).

[0232] 2.3. In Vitro Characterization of mAb 3G4 Using a Collection of CHO-K1 Transfectant Cells Expressing Each a Different Human FcγR

[0233] 2.3.1. Specificity of the Chimeric Modified Antibody 3G4NA to its Targets

[0234] The specificity of the chimeric modified antibody 3G4NA towards the members of the hFcγR family has been assessed as exposed in the Material & Methods part, in CHO cells expressing FLAG tagged human FcγR. The cells were incubated with 3G4NA mAbs at 1 μg/ml, washed and then incubated on ice for 30 min with 5 μg/ml PE-labelled F(ab′).sub.2 Donkey Anti-Human IgG. The FACS results are disclosed on FIG. 1.

[0235] It is important to note that the antibody is specific for his target, with only an expected unspecific binding of mAb 3G4 N.sub.297A to hFcγRI in the family, as observed with all human IgG1 antibodies in the N.sub.297A format. This binding is due to the affinity of hFcγRI for the Fc portion of mAb 3G4 N.sub.297A. This binding to hFcγRI will however not induce any side effects in vivo, since human sera contain only 8-15 mg/mL of IgG (The 3G4NA antibody will compete with circulating human IgGs of the patient to bind hFcγRI but hFcγRI will be completely or totally occupied, saturated with endogenous IgGs. The 3G4 antibody of the invention can therefore not be “captured” by hFcγRI, which is much less expressed relative to hFcγRIII).

[0236] 2.3.2. Blocking Ability of the Antibody to Prevent Binding of IgG (the Ligand) to FcγRIII (the Receptors)

[0237] The blocking properties of the chimeric modified antibody 3G4NA towards the members of the hFcγR family has also been assessed as detailed above. This time, the cells were incubated with 3G4NA mAbs at 10 μg/ml.

[0238] The FACS results are disclosed on FIG. 2.

[0239] It shows a very efficient blockade of human IgG immune complex binding to the hFcγRIII family in the presence of the chimeric mAb 3G4 N.sub.297A. The blockade is less efficient on hFcγRIIIA V158, the polymorphic variant of hFcγRIIIA with higher affinity binding to human IgG1.

[0240] 2.3.3. Affinities of the Antibody Formats for their Respective Targets by Surface Plasmon Resonance Analysis.

[0241] The affinity of the antibodies of the invention for the hFcγRIIIA (V158 variant) has been assessed by SPR as detailed above. The results are provided in Table 3:

TABLE-US-00011 TABLE 3 affinity of the antibodies of the invention towards hFcγRIIIA Chimeric Chimeric Chimeric Chimeric 3G4 3G4 hlgG1 3G8 3G8 hlgG1 hlgG1 N.sub.297A format hlgG1 N.sub.297A format K.sub.D (nM) 7.9 ± 1.2 16.0 ± 0.9 2.1 ± 1.6 2.88 ± 1.2 K.sub.on (1/Ms) 1.3 × 10.sup.5 1.9 × 10.sup.4 3.0 × 10.sup.5 1.1 × 10.sup.5 K.sub.off (1/s) .sup.  1.0 × 10.sup.−3 .sup.  3.4 × 10.sup.−4 .sup.  2.1 × 10.sup.−4 .sup.  3.0 × 10.sup.−4

[0242] This Table shows that both mAb 3G4 expressed as a human IgG1 format (chimeric antibody) or as a human IgG1 format bearing a N.sub.297A mutation (chimeric and modified antibody) bind hFcγRIIIA (V158 variant) with an affinity close to 10 nM. These high affinities are compatible with a therapeutic use of these antibodies in vivo in humans or animals.

[0243] 2.4. In Vivo Evaluation of the Antibodies of the Invention

[0244] 2.4.1. Assessment of the Body Temperature

[0245] Mice expressing human IgG receptors (hFcγRI, hFcγRIIA(H131), hFcγRIIB, hFcγRIIIA(V158), hFcγRIIIB(NA2)) in place of their endogenous IgG receptors (hFcγR.sup.KI mice) (Beutier H, et al. Science Immunol. 2018) were treated by injection of 9 mg/kg of one of the antibodies listed below. As a negative control, mice expressing no endogenous FcγRs (FcγR.sup.null) were also injected: [0246] Chimeric and modified 3G4 (hIgG1 N.sub.297A format) [0247] Chimeric and modified 3G8 (hIgG1 N.sub.297A format) [0248] Chimeric WT 3G8 hIgG1 [0249] hIgG1 Herceptin (negative control=irrelevant antibody)

[0250] Read out corresponding to potential adverse effects: [0251] Body temperature: every 10 minutes after injection and up to 120 minutes [0252] Platelets number: 4 h after injection [0253] Blood neutrophil and monocyte numbers: 4 h after injection

[0254] The results are disclosed on FIG. 3.

[0255] As shown on FIG. 3, no significant variation in central temperature observed after the chimeric modified mAb 3G4 hIgG1 N.sub.297A injection, whereas a significant drop occurred in central body temperature after mAb 3G8 hIgG1 or after mAb 3G8 hIgG1 N.sub.297A injection. This temperature drop is reminiscent of anaphylactic reactions or adverse drug reactions

[0256] 2.4.2. Assessment of the Platelets Number

[0257] Mice expressing human IgG receptors (hFcγRI, hFcγRIIA(H131), hFcγRIIB, hFcγRIIIA(V158), hFcγRIIIB(NA2)) in place of their endogenous IgG receptors (hFcγR.sup.KI mice) (Beutier H, et al. Science Immunol. 2018) were treated by injection of 9 mg/kg of one of the antibodies listed below. As a negative control, mice expressing no endogenous FcγRs (FcγR.sup.null) were also injected: [0258] Chimeric and modified 3G4 (hIgG1 N.sub.297A format) [0259] Chimeric and modified 3G8 (hIgG1 N.sub.297A format) [0260] Chimeric WT 3G8 hIgG1 [0261] hIgG1 Herceptin (negative control=irrelevant antibody) The results are disclosed on FIG. 4.

[0262] As disclosed on FIG. 4, no significant variation in platelet numbers observed after the chimeric and modified mAb 3G4 hIgG1 N.sub.297A injection, but significant thrombocytopenia was observed after administration of mAb 3G8 hIgG1 both in hFcγR.sup.KI mice or in FcγR.sup.null mice. These adverse effects are prevented by using the mAb 3G8 hIgG1 N.sub.297A mutant, suggestive that FcγR and complement may be responsible for platelet removal/destruction.

[0263] 2.4.3. Neutrophil and Monocyte Populations

[0264] Four hours after intravenous injection of the mAbs of the invention and control antibodies, blood samples were collected. Leucocytes were stained to characterize neutrophil (CD45+CD11b+Ly6G+—FIG. 5) and monocyte (CD45+CD115+Ly6C+—FIG. 6) populations. After 3 washes, the cells were analyzed by FACS.

[0265] As disclosed on FIGS. 5 and 6, no significant variation in neutrophil or monocyte numbers was observed after injection of the chimeric and modified mAb 3G4 hIgG1 N.sub.297A of the invention, of the mAb 3G8 hIgG1 or of the mAb 3G8 hIgG1 N.sub.297A.

[0266] 2.5. In Vivo Characterization of mAb 3G4 Blocking Antibody on IgG-Dependent Preclinical Disease Models

[0267] 2.5.1. Injection of the Blocking Chimeric Modified mAb 3G4 (hIgG1 N.sub.297A Format) can Prevent the Induction of Thrombocytopenia.

[0268] Immune thrombocytopenia was induced by injecting hFcγR.sup.KI or FcγR.sup.null mice with the depleting anti-platelet mAb 6A6, in a human IgG1 format (termed herein 6A6WT; 10 μg/mouse) or in a human IgG1 format, containing a Lysine to Alanine mutation at position 322 of the heavy chain to prevent complement component Clq binding (termed herein 6A6KA; 20 μg/mouse). Injection of 200 μg of irrelevant human IgG1 was used as control.

[0269] For prophylaxis experiments, mice were pretreated by intravenous injection of 9 mg/kg of mouse of anti-hFcγRIII mAbs 30 min before the injection of the depleting anti-platelet mAb.

[0270] The FIG. 7 shows the results obtained on eight hFcγR.sup.KI mice (n=4 per group).

[0271] A significant protection from IgG-induced thrombocytopenia was observed when using the chimeric modified mAb 3G4 of the invention (IgG1 N.sub.297A format).

[0272] 2.5.2. Injection of the Blocking Chimeric Modified mAb 3G4 (IgG1 N.sub.297A Format) can Treat Thrombocytopenia.

[0273] For therapeutic experiments, severe chronic thrombocytopenia was induced in the above-mentioned mice by repeated daily injections of depleting anti-platelet mAbs followed by concomitant injections of depleting anti-platelet mAbs 6A6KA (20 μg/mouse) and 9 mg/kg of mouse of anti-hFcγRIII mAbs.

[0274] In a first experiment, the anti-platelet mAb used (6A6KA) engages FcγR but not C1q.

[0275] The FIG. 8A shows that, under these chronic thrombocytopenia conditions, injections of the chimeric modified 3G4 (hIgG1N.sub.297A format) allow the restauration of normal circulating platelet numbers, 48 h after the start of therapeutic treatment.

[0276] In a second experiment, the anti-platelet mAb 6A6WT engages both FcγR and C1q (chronic injections of 6A6WT (10 μg/mouse).

[0277] The FIG. 8B shows that, also under these chronic thrombocytopenia conditions, injections of the chimeric modified 3G4 (hIgG1 N.sub.297A format) allow restauration of normal circulating platelet numbers, 48 h after the start of therapeutic treatment.

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