Anti-Fc-gamma receptor IIB antibodies and uses thereof

Abstract

The present invention provides an anti-FcyRIIB antibodies which, in comparison to prior art antibodies, markedly increase ITIM phosphorylation of FcyRIIB and can thus be used for the treatment or prophylaxis of autoimmune diseases.

Claims

1. An anti-FcRIIB antibody which: (a) comprises in its heavy chain variable region H-CDR1, H-CDR2 and H-CDR3 as shown in SEQ ID NOs. 14, 15 and 16 and in its light chain variable region L-CDR1, L-CDR2 and L-CDR3 shown in SEQ ID NOs. 17, 18 and 19; or (b) comprises in its heavy chain variable region H-CDR1, H-CDR2 and H-CDR3 as shown in SEQ ID NOs. 20, 21 and 22 and in its light chain variable region L-CDR1, L-CDR2 and L-CDR3 shown in SEQ ID NOs. 23, 24 and 25; wherein said antibody increases ITIM phosphorylation of FcRIIB of Daudi cells about 4 to 10-fold in comparison to Daudi cells not treated with said antibody.

2. The antibody of claim 1, wherein said antibody is chimeric or humanized.

3. The antibody of claim 1, comprising in its heavy chain variable region H-CDR1, H-CDR2 and H-CDR3 as shown in SEQ ID NOs. 20, 21 and 22 and in its light chain variable region L-CDR1, L-CDR2 and L-CDR3 shown in SEQ ID NOs. 23, 24 and 25, wherein said antibody comprises the heavy chain variable region shown in SEQ ID NO. 3.

4. The antibody of claim 1, wherein said antibody specifically binds to amino acids No. 20-40 of human FcRIIB according to SEQ ID NO. 5.

5. The antibody of claim 1, wherein said antibody in vitro binds to human FcRIIb with an affinity having an off-rate constant of at least 4.910.sup.4 s.sup.1.

6. The antibody according to claim 1, wherein said antibody comprises in its heavy chain constant region the amino acid sequence shown in SEQ ID NO. 6.

7. The antibody according to claim 1, wherein said antibody comprises in its light chain constant region the amino acid sequence shown in SEQ ID NO. 7.

8. An anti-FcRIIB antibody wherein said antibody comprises the heavy chain variable region shown in SEQ ID NO. 1 or 3 and/or the light chain variable region shown in SEQ ID NO. 2 or 4.

9. The antibody of claim 8, wherein said antibody comprises in its heavy chain constant region the amino acid sequence shown in SEQ ID NO. 6.

10. The antibody of claim 8, wherein said antibody comprises in its light chain constant region the amino acid sequence shown in SEQ ID NO. 7.

11. A pharmaceutical composition comprising as an active ingredient an antibody of claim 1.

12. A method of treating an autoimmune disease, the autoimmune disease being characterized by the production of auto-antibodies, comprising administering the antibody of claim 1 to a subject in need thereof, wherein the autoimmune disease is Immune Thrombocytopenia, Systemic Lupus Erythematosus, Pernicious Anemia, Addison's disease, Diabetes type 1, Rheumatoid Arthritis, Sjogren's syndrome, Dermato-myositis, Multiple Sclerosis, Myasthenia gravis, Reiter's syndrome, Graves disease, Pemphigus vulgaris and bullosus, autoimmune Hepatitis, ulcerative Colitis, cold agglutinin disease, and Autoimmune peripheral neuropathy.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Surface Plasmon Resonance analysis of humanized 8A6 according to SEQ. ID. No. 3 and 4 in either wildtype or N297A format, ch8A6_N297A (according to SEQ. ID. NO. 14 and 15 and chGB3_N297A.

(2) FIG. 2: Sequences of hu8A6_wt and hu8A6_N297A depicting position of N to A amino acid exchange in N297A format.

(3) FIG. 3: Non-blocking characteristic of ch8A6_N297A. Raji cells were incubated with a set amount of aggregated human IgG and varying amounts of ch8A6_N297A, chGB3_N297A or blocking antibodies 2B6 or R&D Ab. The antibodies according to the invention are non-blocking.

(4) FIG. 4: Binding from 15 g/ml to 0.005 g/ml of Protein A purified antibody (hu8A6 VL+hu8A6_VH and ch8A6_N297A) to native FcRIIB expressed on Raji cells. Humanized 8A6 variants bind with high avidity to FcRIIB expressed on Raji cells.

(5) FIG. 5: Binding of 15 g/ml antibody (hu8A6_VH+hu8A6_VL and ch8A6_N297A) to native FcRIIA expressed on K562 cells. Antibodies according to the invention do not bind to FcRIIA on K-562.

(6) FIG. 6a: ITIM-Phosphorylation increased by chimeric 8A6 (ch8A6_N297A) in PBMC from healthy donor. PBMC from healthy donor were isolated using Ficoll separation and subsequently left untreated or incubated for 25 minutes with an antibody mix containing anti-IgM (anti-human, mouse) and anti-mouse IgG (rabbit). Subsequently the cells were either treated 20 minutes with 5 g/mL ch8A6_N297A or buffer as control (w/o). Cells were harvested after incubation and lysed according to protocol. Lysates were subjected to WB-analysis. -Actin=loading control.

(7) FIG. 6b: Control experiment for ITIM-Phosphorylation. Daudi cells were left untreated or treated for 25 minutes with an isotype control antibody, polyclonal anti-human anti-IgM (polycl. anti-hIgM), monoclonal anti human IgM (anti-hIgM), anti-hIgM+5 g/mL ch8A6_N297A, anti-mouse IgG from rabbit (-mouseIgG), -mouse IgG+5 g/mL ch8A6_N297A, mix of anti-hIgM and -mouseIgG (Ab mix) or Ab mix+5 g/mL ch8A6_N297A). -Actin=loading control.

(8) FIG. 6c: Antibodies of the present invention enhance ITIM phosphorylation in the presence or absence of crosslinking/colligation of BCR and Fcgamma RIIB in primary PBMCs.

(9) FIG. 7: Comparison of ch8A6_N297A with antibody from the state of the art (chGB3_N297A). Human Daudi cells were incubated for 25 minutes with an antibody mix containing anti-IgM (anti-human, mouse) and anti-mouse IgG (rabbit) or left untreated. Subsequently the cells were either treated 20 minutes with varying amounts of chGB3_N297A or ch8A6_N297A or buffer as control (w/o). Cells were harvested after incubation and lysed according to protocol. Lysates were subjected to WB-analysis. -Actin=loading control.

(10) FIG. 8: Comparison of the effect of the humanized variant hu8A6_N297A and ch8A6_N297A and chGB3_N297A on ITIM phosphorylation in primary PBMCs. After crosslinking of BCR and FcRIIB by the antibody mix, the different antibodies were added at 5 g/ml and Western Blot analysis for ITIM phosphorylation was conducted. -Actin=loading control.

(11) FIG. 9: Co-Immunoprecipitation of phosphorylated SHIP-1 with FcRIIB ITIM. After stimulation of Daudi cells with the antibody mix and either ch8A6_N297A, blocking anti-FcRIIB antibody 2B6 or chGB3_N297A (5 g/mL), FcRIIB was precipitated from cell lysates and Western Blot analysis was performed for the phosphatase SHIP-1. Anti-CD32 using pan anti-CD32 antibody (AF1330)=loading control.

(12) FIG. 10: Comparison of humanized 8A6 variants (hu8A6-VH10+VL2/VH10+VL6/VH10+VL7/VH12+VL2/VH12+VL6/VH12+VL7) to ch8A6_N297A. Daudi cells were left incubated for 25 minutes with buffer (untreated) or a mix (antibody mix) containing anti-IgM (anti-human, mouse) and anti-mouse IgG (rabbit). Subsequently the cells were either treated 20 minutes with 0.25 g/mL of ch8A6_N297A or humanized 8A6 variants (hu8A6-VH10+VL2/VH10+VL6/VH10+VL7/VH12+VL2/VH12+VL6/VH12+VL7). Cells were harvested after incubation and lysed according to protocol. Lysates were subjected to WB-analysis. -Actin=loading control.

(13) FIG. 11: Comparison of humanized 8A6 variants (hu8A6-VH10+VL2/VH10+VL6/VH10+VL7/VH12+VL2/VH12+VL6/VH12+VL7) to ch8A6_N297A, blocking anti-FcRIIB and chGB3_N297A. Daudi cells were left incubated for 25 minutes with buffer (untreated) or a mix (antibody mix) containing anti-IgM (anti-human, mouse) and anti-mouse IgG (rabbit). Subsequently the cells were either treated 20 minutes with 0.25 g/mL of ch8A6_N297A, humanized 8A6 variants (hu8A6-VH10+VL2/VH10+VL6/VH10+VL7/VH12+VL2/VH12+VL6/VH12+VL7), blocking anti-FcRIIB antibody 2B6 or chGB3_N297A. Cells were harvested after incubation, lysed according to protocol and analyzed in a Western Blot assay. Lysates were subjected to WB-analysis. -Actin=loading control.

(14) FIG. 12: Experimental setup for SLE-PBL mouse model. PBL from human SLE patients are transfered into immuno-compromised mice. PBL cells are engrafted and mice are subsequently treated with control (PBS) or anti-FcRIIB ch8A6_N297A antibody according to the invention.

(15) FIG. 13: Total human IgG level [g/mL] in mice grafted with PBL from human donors suffering from SLE. Depicted are mice treated with control (#2, PBS) or ch8A6_N297A (#3 and #4).

(16) FIG. 14: Reduction in disease specific human anti-DNA IgG in ch8A6_N297A treated mice starting in week 4 post SLE-PBL transfer/grafting using PBL from human donors suffering from SLE. Depicted are anti-DNA IgG titers in two different mice, #3 and #4 (treated with ch8A6_N297A), #2 shows PBS control

EXAMPLES

Preparation of the Monoclonal Antibody 8A6

(17) The monoclonal antibody clone 8A6 was produced by immunizing Long-Evans rats with recombinant soluble human FcRIIB receptor. Hybridoma cell lines from rat spleen cells were produced and screened for antibodies that specifically bind to human FcRIIB with greater affinity than to FcRIIA. Secondly the antibodies produced by the aforementioned Hybridomas were screened for non-blocking characteristics, i.e. these antibodies still allow binding of IgG or ICs to membrane-bound FcRIIB, using techniques known in the art. 50 g of the purified recombinant soluble human FcRIIB (sFcRIIB, SEQ ID NO. 5) were injected intraperitoneally (i.p.) and subcutaneously (s.c.) into LOU/C rats using incomplete Freund's adjuvant supplemented with 5 nmol CpG 2006 (TIB MOLBIOL, Berlin, Germany). After a six weeks interval a final boost with 50 g sFcRIIB and CpG 2006 was given i.p. and s.c. three days before fusion. Fusion of the myeloma cell line with the rat immune spleen cells was performed according to standard procedures. Hybridoma supernatants were tested in a solid-phase immunoassay with sFcRIIB or the irrelevant sFcRIIA (SEQ ID NO. 12) protein coated to ELISA plates. MAbs from tissue culture supernatant bound to the proteins were detected with HRP conjugated mAbs against the rat IgG isotypes (TIB173 anti-IgG2a, TIB174 anti-IgG2b, TIB170 anti-IgG1 all from ATCC, R-2c anti-IgG2c homemade), thus avoiding mAbs of IgM class. The mAb 8A6 (rat IgG2a) recognized FcRIIB and did not bind to FcRIIA using the antigen-specific ELISA assay. A FACS based assay was used to screen the antibodies for specific binding of the native antigen). Additionally the antibodies were screened for non-blocking characteristics, i.e. these antibodies still allow binding of IgG or immune complexes to membrane-bound FcRIIB.

(18) To produce sufficient amount of antibody for characterization of the chimeric and humanized constructs, FreeStyle CHO-S cells were transiently transfected.

(19) The day before transfection cells were seeded at 0.510.sup.6 cells/ml. Cells were centrifuged and resuspended in medium to obtain a final cell concentration of 2010.sup.6 cells/ml. For each transfection 3 ml of the cell suspension was transferred to a 6-well plate. The plasmid DNA for transfection was isolated using the HiSpeed Plasmid Maxi Kit (Qiagen) according to the manufacturer's instruction and eluted in endotoxin-free H.sub.2O.

(20) For each transfection 75 g plasmid DNA encoding for the light chain and 75 g plasmid DNA encoding for the heavy chain were added to the cell suspension and mixed gently. Afterwards PEI (Polyplus) was added and mixed gently. The cells were incubated for 3 h at 37 C. and 5% CO.sub.2 under continuous shaking (100 rpm). The cell suspension was filled up with medium to a final volume of 15 ml to achieve a final cell concentration of 410.sup.6 cells/ml and transferred into a 125 ml flask. The cells were incubated at 37 C. and 5% CO.sub.2 under continuous shaking (100 rpm) and after 6 d the supernatant was harvested by centrifuging it 2 times (4000 rpm, 5 min). The supernatant was filtered through 0.2 m filter and antibody titer was determined (see below).

(21) The antibody-titer determination was conducted via two different HPLC methods, Reverse-phase (RP) HPLC and Protein A-HPLC. The RP-HPLC analysis was conducted with an Agilent 1200 Series HPLC system. Data were analyzed with the software Chem Station for LC Systems Rev. B.04.02. The solvent components were: Isopropanol (HPLC Grade; Roth), Acetonitril (HPLC Gradient Grade; Roth), H20 (0.2 m filtered) and Tetrafluoracetat (TFA) (for peptide synthesis; Sigma). Solvent A: H2O, 0.1% TFA; Solvent B: 60% Isopropanol, 30% Acetonitril, 9.9% H2O, and 0.1% TFA. A Phenomenex Jupiter column (#525166-6) with porosity of 300 and separation material with a particle diameter of 5 m was used. Bound antibody was eluted with a linear gradient from 30% to 43% solvent B within 10 min. Detection occurred at =210 nm with a UV/VIS detector.

(22) The Protein A-HPLC analysis was conducted with an Agilent 1200 Series HPLC system. Data were analyzed with the software Chemstation version Rev. B.04.02. The following solvents were used, solvent A: 0.05M Tris/HCl, 0.1M Glycine, 0.15M NaCl, pH 8.0; solvent B: 0.05M Tris/HCl, 0.1M Glycine, 0.15M NaCl, pH 3.0. For analysis, an Upchurch 220 mm analytical guard column was packed with 120 l of Applied Biosystems Poros 20A perfusion chromatography media (Life technologies). Bound antibody was eluted with 100% solvent B. In case purified antibody was collected, fractions were neutralized with 56 mM Tris pH 8.0. The expressed antibodies were purified with a 1 ml HiTrap rProtein A FF column (GE Healthcare) via fast protein liquid chromatography (kta Explorer). The running buffer contained 10 mM Tris-HCl pH 8.0, 150 mM NaCl, the elution buffer was composed of 100 mM glycine, 150 mM NaCl, pH 2.7. Eluted antibodies were neutralized with 60 mM Tris-HCl pH 8.0, concentrated, sterile filtered and stored at 80 C.

(23) Antibody Screening and Characterization of 8A6

(24) Screening of hybridoma supernatants is performed using techniques as known in the state of the art, e.g. ELISA-Binding assay, Biacore assay or FACS-binding analysis. To test antigen specific binding of chimeric 8A6 or the humanized variants, ELISA plates (Nunc-Immuno Plate, F96 Maxisorp) were coated with 1 g/ml sFcRIIB, sFcRIIA (SEQ ID NO. 12) or sFcRIIAmut (SEQ ID NO. 13) in PBS (100 l/well) overnight at 4 C. After 3 washing steps in 0.01% Tween in PBS, blocking with 2% BSA in PBS (300 l/well) was conducted for 2 hours at room temperature. After 3 washing steps, serial dilutions of the purified antibody or supernatant were applied (100 l/well) and incubated for 1 hour at room temperature. Purified antibodies were diluted in PBS, 2% BSA. Supernatants were supplemented with 10 times PBS and 20% BSA to obtain a final concentration of 2% BSA in PBS. As positive control for FcRIIA the goat-anti-human FcRIIA (R&D Systems, AF1875) was used. After 3 washing steps in 0.01% Tween in PBS, the respective secondary antibody donkey-anti-goat-HRP (F(ab).sub.2, Jackson-Immuno-Research) or goat-anti-human-HRP (F(ab).sub.2, Dianova) was incubated for 1 hour at room temperature (100 l/well). Wells were washed 6 times in 0.01% Tween in PBS. Substrate (OPD containing 0.03% H.sub.2O.sub.2) was added (100 l/well) and the reaction was stopped with 4 M H.sub.2SO.sub.4 (50 l/well). Afterwards the absorbance was measured in a spectrometer at 492 nm.

(25) Analysis of the specific binding to the native antigen of the chimeric 8A6 or humanized variants were conducted via cell binding on Raji (ATCC CCL-86) and K-562 cells (ATCC CCL-243).

(26) Cells were pelletized by centrifugation (400 g, 5 min) and washed in FACS-buffer (Hanks balanced salt solution, 1% FCS, 0.01% NaN.sub.3). After an additional centrifugation step, cells were resuspended in FACS-buffer to obtain a final cell concentration of 210.sup.6 cells/ml and 50 l of the cell suspension was aliquoted in a 96-well U-bottom plate. Serial dilutions of the humanized variants and ch8A6 were prepared in FACS-buffer.

(27) To verify the expression of FcRIIA on K-562 cells the mouse-anti-human CD32 antibody (Stem Cell Technologies Inc., Clone VI.3) was diluted in FACS-buffer. 50 l of the diluted antibodies was added to the cells and incubated for 30 min at 4 C. Cells were washed 2 times in FACS-buffer. Afterwards, 50 l goat-anti-human IgG-PE conjugated (F(ab).sub.2, Dianova) or goat-anti-mouse IgG-PE conjugated (F(ab).sub.2, Dianova) secondary antibody was diluted in FACS-buffer, added to the cells and incubated for 30 min at 4 C. After 2 washing steps in FACS-buffer, cells were resuspended in 300 l FACS-buffer and measured in BD FACSCanto II (Software: BD FACSDiva).

(28) To determine whether FcRIIB antibodies still allow binding of IgG or immune complexes to membrane-bound FcRIIB a FACS based assay was conducted. Cells were pelletized by centrifugation (400 g, 5 min) and washed in FACS-buffer (Hanks balanced salt solution, 1 FCS, 0.01% NaN.sub.3). After an additional centrifugation step, cells were resuspended in FACS-buffer to obtain a final cell concentration of 210.sup.6 cells/ml. Serial dilutions of the antibodies (ch8A6_N297A, chGB3_N297A, R&D Ab mab1875) were prepared in FACS-buffer. 25 l of the diluted antibodies were mixed in a 96-well U-bottom plate with 25 l Alexa488-labeled aggregated Beriglobin (2.5 l/well). Aggregated human IgG was isolated by size-exclusion chromatography on a Superdex-200 (16/60) from a commercially available pooled IgG product (Beriglobin).

(29) 50 l of the cell suspension was added to the antibody-Beriglobin mixture and incubated for 1 hour at 4 C. Cells were washed 2 times in FACS-buffer. Afterwards, 50 l goat-anti-human IgG-PE conjugated (F(ab).sub.2, Dianova) or anti-rat-PE secondary antibody was diluted in FACS-buffer, added to the cells and incubated for 30 min at 4 C. After 2 washing steps in FACS-buffer, cells were resuspended in 300 l FACS-buffer and measured in BD FACSCanto II (Software: BD FACSDiva) (FIG. 3).

(30) FACS Binding Assay

(31) Analysis of the specific binding to the native antigen of the chimeric 8A6 or humanized variants were conducted via cell binding on Raji and K-562 cells.

(32) Cells were pelletized by centrifugation (400 g, 5 min) and washed in FACS-buffer (Hanks balanced salt solution, 1% FCS, 0.01% NaN3). After an additional centrifugation step, cells were resuspended in FACS-buffer to obtain a final cell concentration of 2106 cells/ml and 50 l of the cell suspension was aliquoted in a 96-well U-bottom plate. Serial dilutions of the humanized variants and ch8A6 were prepared in FACS-buffer.

(33) Raji and K562 cells were incubated with increasing concentrations of humanized antibodies and the chimeric antibody as control. Raji cells were used to test binding on FcRIIB (FIG. 4), K562 cells to analyze unspecific binding to FcRIIA (FIG. 5). Cell bound antibodies were detected with PE-conjugated secondary antibody. All humanized variants bind to FcRIIB with a comparable affinity as ch8A6_N297A and all humanized variants still bind FcRIIB with greater avidity than FcRIIA.

(34) Antibody binding to sFcRIIB and sFcRIIA was analysed by surface plasmon resonance using a Biacore T200 biosensor (GE Healthcare/Biacore). Experiments were conducted at LMU, Department of Biology and Microbiology, Service Unit Bioanalytic. Analysed antibodies were captured on a Series S Sensor Chip CM5 sensor chip using the Human Antibody Capture Kit according to the manufacturer's protocol. Hu8A6 variants or ch8A6 were captured at a concentration of 10 nM for 1 min. The analyte sFcRIIB was injected in various concentrations for 3 min. Measurements were performed at 25 C. and continuous flow (10 l/min). Data was evaluated using the Biacore T200 Evaluation Software (version 1.0) assuming 1:1 binding.

(35) Chimerization of 8A6 Rat Antibody

(36) The anti-FcRIIB chimeric monoclonal antibody 8A6 was constructed by fusing the rat 8A6 VH region to a signal peptide and a human IgG1 constant region. Additionally a deglycosylated variant of the heavy chain was generated using an IgG1 constant domain containing a N297A mutation. To construct the 8A6 light chain gene, the rat 8A6 VL region was likewise fused to a signal sequence and the sequence for a human kappa constant region. DNA synthesis of heavy and light chain were performed at Geneart/Life Technologies followed by sub-cloning into a mammalian expression vector.

(37) In Vitro Assays

(38) Cells, Reagents and Antibodies

(39) The human Burkitt lymphoma cell lines Daudi and Ramos were purchased from DSMZ (ACC 78 and ACC 603) and maintained in RPMI 1640 (Gibco/Invitrogen) supplemented with 10% FBS (Gibco/Invitrogen), MEM NEAA (Gibco/Invitrogen), 1 mM sodium pyruvate (Gibco/Invitrogen) and 2 mM L-Glutamine (Gibco/Invitrogen) at 37 C. and 5% CO.sub.2. Primary human B cells were purified from heparinized blood of healthy donors using Ficoll density gradients (Leucosep, Greiner Bio-One, Biocoll Separating Solution, Biochrom) and negative magnetic isolation (Dynabeads Untouched Human B Cells, Invitrogen). Purity of the enriched B cells was studied by FACS analysis by staining with anti-hCD19-APC (BD Pharmingen #555415), anti-hCD3-PerCP-Cy5.5 (BD Biosciences #332771) and anti-hCD56-PE (BD Pharmingen #555515). Primary B cells were directly used for the experiments without further culturing. Blocking anti-FcRIIB antibody 2B6 according to EP1534335.

(40) Stimulation Protocol Using Soluble Antibody Stimulation Mix

(41) For simultaneous stimulation of BCR and FcRIIB an antibody system was set up using an antibody mix of 2 g/ml monoclonal mouse anti-hIgM (Southern Biotech #9022-01, clone UHB) and 20 g/ml monoclonal rabbit anti-mIgG(1,2a,3) (Epitomics #3020-1, clone M111-2) of which the Fc part cross-reacts with the human FcRIIB receptor. Controls were conducted with 20 g/ml polyclonal rabbit anti-hIgM (antibodies online #ABIN117299) or a mix containing 2 g/ml anti-hIgM and isotype control mIgG2b (clone MPC-11, BD Pharmingen #557351).

(42) 310.sup.5 cells of the lymphoma cell lines Daudi or Ramos and primary B cells were harvested by centrifugation and incubated with the different stimulation mixes in Assay medium (RPMI 1640+1% FBS) for 20 min at 37 C.

(43) Subsequently 5 g/ml anti-FcRIIB antibodies ch8A6 (0.8 l of a 1:10 dilution), 2B6 (1.5 l of a 1:10 dilution) or chGB3_N297A (1.1 l of a 1:10 dilution) were added to the samples and cells were further incubated for 25-30 min. Lysis was performed as described separately.

(44) Western Blot Analysis of Phosphorylation Patterns

(45) Cell Lysis

(46) Cells were pelleted at 4 C., washed with ice-cold PBS and incubated in 10 L lysis buffer (RIPA buffer (Cell Signaling) supplemented with phosphatase inhibitors (PhosStop, Roche), protease inhibitors (Complete Ultra Mini, EDTA-free, Roche) and 1 mM PMSF (Fluka Biochemica) for 30-45 min on ice.

(47) SDS-PAGE

(48) After centrifugation, supernatants were loaded with sample buffer (NuPAGE LDS Sample Buffer, NuPAGE Sample Reducing Agent, Invitrogen) applied to SDS PAGE (NuPAGE Novex Bis-Tris Mini Gels, MES SDS Running Buffer (Invitrogen)). For SDS-PAGE, LDS sample buffer and Reducing Agent were added and samples were heated at 95 C. for 5 min. Samples were stored at 20 C. or directly analyzed by SDS-PAGE and Western Blot.

(49) Protein Transfer to PVDF Membranes and Detection

(50) Subsequently, proteins were transferred to PVDF membranes (Roti-PVDF, Roth, Transfer buffer 10 mM Tris, 100 mM Glycin, 10% Methanol, transfer conditions 240 mA const., 90 min at 4 C.). Membranes were blocked with 5% BSA in TBS-T (10 mM Tris, 150 mM NaCl, 0.1% Tween20) and stained with anti-FcRIIB/CD32 Phospho (pY292) (Cell Epitomics #2308-1, 1:50000, 4 C. overnight) or anti-phosphoSHIP (1:1000, Cell Signaling #3941) and anti-rabbit-HRP (Jackson ImmunoResearch #111-036-045, 1:50,000 in TBS-T, 1 h RT). Chemiluminescence (developed with WesternLightning Plus, Perkin Elmer) was detected on X-ray films.

(51) Stripping

(52) For subsequent analyses with antibodies directed against other phosphorylated proteins, membranes were stripped (Re-Blot Plus, Millipore) for 10 min, washed and blocked before staining with anti--Actin antibody (Sigma-Aldrich # A1978, 1:50,000 and anti-mouse IgG-HRP, Sigma-Aldrich # A9044) or antibodies for other signaling proteins.

(53) FcRIIB-ITIM Phosphorylation in PBMC from Healthy Donor Markedly Increased by Inventive Antibodies

(54) PBMC from a healthy donor were isolated and either left untreated or incubated for 25 minutes with the stimulation mix (monoclonal mouse anti-hIgM and monoclonal rabbit anti-mIgG). Subsequently, cells were treated either with ch8A6 or buffer as control. Cell lysates were subjected to Western Blot analysis using appropriate detection antibodies as outlined above. A markedly increase in the phosphorylation of the FcRIIB-IITIM motif of cells (PBMC, B cells) was detected (FIG. 6a). Control experiments with stimulation of cells with stimulation mix alone, or only monoclonal mouse anti-hIgM, monoclonal rabbit anti-mIgG in combination with ch8A6 did not show an increased FcRIIB-ITIM-phosphorylation (FIG. 6b). The antibodies of the invention thus show a markedly effect on the ITIM-phosphorylation of human cells with crosslinked BCR and membrane-bound (endogenously expressed) FcRIIB and not on unstimulated cells, i.e. cells without crosslinked BCR and membrane-bound FcRIIB. During an auto-immune disease, BCR and membrane-bound FcRIIB will be crosslinked by auto-antigens or immune complexes (ICs). The inventive antibodies are able to inhibit pathogenic autoreactive B cells in an auto-immune disease by increasing FcRIIB-ITIM-phosphorylation. However, antibodies of the present invention are also able to increase ITIM-phosphorylation without crosslinked BCR (FIG. 6c).

(55) Comparison of the Effects of ch8A6 with Antibody from the State of the Art (chGB3_N297A)

(56) Comparison of the effect of clone ch8A6_N297A and clone chGB3_N297A on ITIM phosphorylation. Human Daudi cells were treated with an antibody mix and, subsequently, ch8A6_N297A, chGB3_N297A or 2B6 as described above. The antibody chGB3_N297A like ch8A6_N297A is a non-blocking anti-FcRIIB antibody and recognizes a similar epitope.

(57) Addition of ch8A6_N297A to the antibody-mix treated cells showed an increase of FcRIIB-ITIM phosphorylation already at a concentrations of 0.05 g/ml. Though increasing concentrations of the chGB3_N297A showed a dose-dependent stimulation of phosphorylation of the inhibitory motif, surprisingly this antibody clone was not able to reach phosphorylation levels comparable to 8A6. Densitometric quantitation of the X-ray film with the software ImageJ calculated values of a maximum of 2.8-fold phospho-signals, whereas hu8A6_N297A lead to a 9.8-fold increase compared to untreated cells (FIG. 7). Thus the inventive antibodies clearly and surprisingly show an increased FcRIIB-ITIM-phosphorylation in comparison to antibodies in the state of the art.

(58) Comparison of the Effect of the Humanized Variant hu8A6, Chimeric 8A6_N297A and chGB3_N297A on ITIM Phosphorylation in Primary PBMC

(59) Antibody chGB3_N297A, ch8A6_N297A and humanized 8A6 were compared in their influence on FcRIIB-ITIM phosphorylation of primary human PBMC. After crosslinking of BCR and FcRIIB by the antibody mix, the different antibodies were added at 5 g/ml and Western Blot analysis for ITIM phosphorylation was conducted. Again the inventive antibodies surprisingly have a markedly increased effect on FcRUB-ITIM-phosphorylation compared to the antibody in the state of the art (FIG. 8).

(60) Co-Immunoprecipitation of the Phosphorylated FcRIIB-ITIM Motif and SHIP-1

(61) Subsequent to the crosslinking of receptors, the phosphatase SHIP is recruited to the membrane via binding of its SH2 domain to the phospho-tyrosine in the FcRIIB-ITIM motif, followed by tyrosine phosphorylation at the NPXY motif in the C-terminal domain of SHIP-1. The relocalization in the membrane and subsequent phosphorylation of the NPXY motif is essential for the regulatory function of SHIP-1. Its effect on calcium flux, cell survival, growth, cell cycle arrest and apoptosis is mediated through the PI3K and Akt pathways. Tyr1021 is located in one of the NPXY motifs in SHIP-1, and its phosphorylation is important for SHIP-1 function (Nimmerjahn, Ravetch, 2008).

(62) Human Daudi cells were stimulated with the antibody mix as defined in section above and after lysis in a mild lysis buffer (CoIP lysis buffer), the samples were incubated with 2B6 for capturing FcRIIB. Complexes were bound to ProteinG-coupled ferromagnetic beads and isolated with a magnetic rack.

(63) Lysates of 110.sup.7 cells/sample were prepared in 500 l CoIP lysis buffer, incubating the cells for 30 min on ice and vortexing every 10 min. Cell debris was spun down at 13,000 rpm for 10 min at 4 C. and the supernatants were transferred to new tubes. 500 l of the lysates were incubated with 10 g 2B6 for 2-3 h at 4 C. end over end. Magnetic Protein G-coupled beads were washed twice with 500 l lysis buffer and 50 l beads (1.5 mg) were added to the lysate-antibody-complexes over night at 4 C. (rotating wheel). Complexes were eluted from the beads by washing twice with 200 l lysis buffer and heating the beads for 5 min in 25 l 1LDS sample buffer containing reducing agent. After centrifugation at 4000g for 30 sec, 10 l of the supernatant were applied to SDS-PAGE for Western Blot analysis.

(64) Western Blot analyses of the lysates show significantly elevated levels of phospho-SHIP-1 in the samples of cells treated with antibody mix and ch8A6_N297A. As the precipitation was performed with the FcRIIB-specific antibody 2B6, only isolated SHIP-1 was co-precipitated that had bound to FcRIIB. Membranes after stripping and restaining showed enhanced phosphorylation of the FcRIIB-ITIM motif in samples treated with ch8A6_N297A, correlating with the phospho-SHIP1 signals. A second restaining with -hFcRIIB a, b, c showed equal amounts of the precipitated receptor FcRIIB in all samples, serving as a loading control for SDS-PAGE (FIG. 9).

(65) Humanization of ch8A6

(66) ch8A6 was humanized by grafting the complementarity-determining region sequences from the rat antibody onto human frameworks. To select human frameworks the V.sub.H and V.sub.L sequences were compared with those of human Ig variable and joining region germline segments, obtained from publicly available databases (IMGT; V-BASE). VH_3_30 plus IGHJ4 human germline sequences and the VK3_15 plus IGKJ2 human germ-line sequences were chosen for the heavy and light chains, respectively.

(67) Several variants for humanized heavy and light chains were generated. The genes coding for the designed sequences of the humanized V.sub.H and V.sub.L were synthesized at Life Science Technologies/Geneart, followed by sub-cloning into a mammalian expression vector. The screening procedure of the antibody variants were performed directly from the supernatant of transfected CHO-S cells (Invitrogen). The chimeric 8A6 antibody served as a transfection control and standard during the screening of the humanized variants. Hu8A6 variants were analysed for binding on sFcRIIB and sFcRIIA via ELISA and on native FcRIIB via FACS on Raji cells (see above). Additionally a kinetic characterization of the antibody variants was performed with surface plasmon resonance.

(68) Test of Humanized 8A6 Variants

(69) To test the phosphorylation activities of 8A6 humanization variants, Daudi cells were stimulated with the antibody mix, treated with 0.5 or 5 g/ml of the various 8A6 variants, and Western Blot analysis for ITIM phosphorylation was conducted.

(70) Comparison of Humanized 8A6 Variants to ch8A6_N297A

(71) All tested humanized variants of 8A6 were able to induce phosphorylation of the receptor and phosphorylation levels were comparable to that induced by ch8A6_N297A from the same purification batch. Thus, no loss of activity was detected after the second humanization round. Although Biacore data suggested different affinities for the different combinations of heavy and light chain, those differences were not detectable by Western Blot analyses (FIG. 10)

(72) Comparison of Humanized 8A6 Variants to ch8A6 N297A, Blocking Anti-FcRIIB (2B6) and chGB3 N297A

(73) After the final humanized chain combination was chosen, this variant, combining the heavy chain V.sub.H10 with the light chain V.sub.L6, was finally compared to the antibodies ch8A6_N297A, 2B6 and chGB3_N297A (FIG. 11).

(74) In Vivo Assays

(75) SLE-PBL-Model

(76) Rag2/gamma-c/Fc/ mice were irradiated at a dosage of 6 Gy and injected intraperitoneally with varying amounts of human peripheral blood leucocytes in 500 l PBS.

(77) Treatment of mice was started 2 weeks following injection of cells after grafting of human SLE-patient PBL in mice was verified by the presence of human immunoglobulin M or G. Mice were treated with 200 l buffer (PBS) or 20 g antibody (ch8A6_N297A) in 200 l PBS intraperitoneally twice weekly for 4 weeks. Mice were weighed and bled to obtain serum once weekly. Serum samples were frozen at 80 C. until further use (FIG. 12).

(78) ELISA

(79) Serum samples were analyzed by ELISA for the presence of total human IgG, IgM and anti-DNA IgM and IgG.

(80) For quantification of total serum IgM and IgG in serum samples, the Bethyl Human IgM ELISA Quantitation Kit and the Human IgG ELISA Quantitation Kit (Biomol) were used according to the manufacturer's instructions. OD was measured with VersaMax tuneable microplate reader (Molecular Devices) at 450 and 650 nm.

(81) For the detection of anti-DNA antibodies, ELISA plates were coated with 10 g/mL methylated BSA (Sigma) in PBS for 2 h at room temperature. After washing, the plates were coated with 50 g/mL calf thymus DNA (Sigma) in PBS at 4 C. overnight. Blocking of unspecific binding was performed with PBS/0.1% Gelatin/3% BSA/1 mM EDTA for 2 h at room temperature. Sera were diluted 1:100 in the blocking solution and incubated for 1 h at room temperature. As a detection antibody, the HRP-conjugated antibody of the human IgM Quantitation Kit (Bethyl) was used and diluted 1:10,000 in blocking solution followed by incubation for 1 h at room temperature. PBS was used for all washing steps. For detection, TMB Solution was added and the reaction was stopped with 6% orthophosphoric acid.

(82) SLE PBL Model, Total Human Serum Immunoglobulin

(83) Total human IgG levels [g/mL] were analyzed in mice grafted with PBL from human donors suffering from SLE. No significant difference in total human IgG between PBS or anti-FcRIIB was detected. The antibody according to the invention does not significantly influence total human IgG (FIG. 13).

(84) SLE PBL Model, Influence on Anti-DNA Antibodies (Disease Specific IgG)

(85) A markedly reduction in disease specific human anti-DNA IgG in anti-FcRIIB mice starting in week 4 post

(86) SLE-PBL transfer/grafting was observed. The inventive antibodies specifically reduce the amount of disease relevant anti-DNA antibodies (FIG. 14).

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