Abstract
The present invention relates to a compound that specifically binds to the region between amino acid residues 58 and 201 of human STIM1 (SEQ ID NO: 1). The present invention also relates to a composition comprising a therapeutically effective amount of the compound, a host cell that produces an isolated antibody, an isolated nucleic acid sequence encoding the isolated antibody and an expression vector comprising the nucleic acid. The present invention additionally relates to a method of producing the isolated antibody, to the isolated antibody for its use as a drug, especially for its use in treating a condition or a disorder in which the STIM1 protein localized to the plasma membrane of the cells is overexpressed, and to an isolated protein fragment consisting of the region between amino acid residues 58 and 201 for developing modulators of the STIM1 amino acid sequence SEQ ID NO: 1.
Claims
1-27. (canceled)
28. A compound that specifically binds to the region between amino acid residues 58 and 201 of the STIM1 amino acid sequence SEQ ID NO: 1 and modulates STIM1 activity.
29. The compound according to claim 28, wherein said compound specifically binds to the region between amino acid residues 128 and 168 of the STIM1 amino acid sequence SEQ ID NO: 1 and modulates STIM1 activity.
30. The compound according to claim 28, wherein said compound specifically binds to the region between amino acid residues 145 and 153 of the STIM1 amino acid sequence SEQ ID NO: 1 and modulates STIM1 activity.
31. The compound according to claim 28, wherein said compound is an activator or an inhibitor of STIM1 activity.
32. The compound according to claim 28, wherein said compound is selected from the group consisting of isolated antibodies or fragments thereof, proteins, peptides, chemical compounds, aptamers or any biological compound.
33. The compound according to claim 32, wherein said compound is a peptide, the sequence of which corresponding to amino acid residues 58 to 201 of the STIM1 amino acid sequence SEQ ID NO: 1.
34. The compound according to claim 32, wherein said compound is an isolated antibody.
35. The compound according to claim 34, wherein said isolated antibody comprises at least one sequence selected from the group consisting of: a variable heavy (V.sub.H) chain complementary determining region (CDR) 1 having the amino acid sequence SEQ ID NO: 3; a variable heavy (V.sub.H) chain CDR2 having the amino acid sequence SEQ ID NO: 4; a variable heavy (V.sub.H) chain CDR3 having the amino acid sequence SEQ ID NO: 5; a variable light (V.sub.L) chain CDR1 having the amino acid sequence SEQ ID NO: 6; a variable light (V.sub.L) chain CDR2 having the amino acid sequence SEQ ID NO: 7; and a variable light (V.sub.L) chain CDR3 having the amino acid sequence SEQ ID NO: 8.
36. The compound according to claim 34, wherein said isolated antibody comprises: a variable heavy (V.sub.H) chain CDR1 having the amino acid sequence SEQ ID NO: 3; a variable heavy (V.sub.H) chain CDR2 having the amino acid sequence SEQ ID NO: 4; a variable heavy (V.sub.H) chain CDR3 having the amino acid sequence SEQ ID NO: 5; a variable light (V.sub.L) chain CDR1 having the amino acid sequence SEQ ID NO: 6; a variable light (V.sub.L) chain CDR2 having the amino acid sequence SEQ ID NO: 7; and a variable light (V.sub.L) chain CDR3 having the amino acid sequence SEQ ID NO: 8.
37. The compound according to claim 34, wherein said isolated antibody comprises: a) a variable light chain comprising the sequence SEQ ID NO: 9; and b) a variable heavy chain comprising the sequence SEQ ID NO: 10.
38. The compound according to claim 34, wherein said isolated antibody is a monoclonal antibody.
39. The compound according to claim 34, wherein said isolated antibody is a chimeric, human or humanized antibody.
40. The compound according to claim 34, wherein said isolated antibody is modified with a drug, or with another antibody, or with a fluorophore, or with any other molecules or ligand selected from nanoparticles, metals or radioelements.
41. A composition comprising a therapeutically effective amount of a compound according to claim 28.
42. A host cell that produces an isolated antibody according to claim 34.
43. An isolated nucleic acid sequence encoding an antibody and comprising a nucleic acid encoding at least one sequence of the group consisting of: a variable heavy (V.sub.H) chain CDR1 having the amino acid sequence SEQ ID NO: 3; a variable heavy (V.sub.H) chain CDR2 having the amino acid sequence SEQ ID NO: 4; a variable heavy (V.sub.H) chain CDR3 having the amino acid sequence SEQ ID NO: 5; a variable light (V.sub.L) chain CDR1 having the amino acid sequence SEQ ID NO: 6; a variable light (V.sub.L) chain CDR2 having the amino acid sequence SEQ ID NO: 7; and a variable light (V.sub.L) chain CDR3 having the amino acid sequence SEQ ID NO: 8.
44. An expression vector comprising a nucleic acid according to claim 43.
45. A method of producing an antibody comprising culturing the host cell according to claim 42 under conditions that result in production of said antibody, and isolating said antibody from the host cell or culture medium of the host cell.
46. A method of treating a condition or a disorder in which the STIM1 protein localized to the plasma membrane of the cells is overexpressed comprising the administration of a compound according to claim 28 to a subject in need of treatment.
47. The method according to claim 46, wherein the condition or disorder is selected from a pathology with an increase in specific cells of mSTIM1 expression, Systemic Lupus Erythematous, Chronic Lymphocytic Leukemia, an autoimmune disease, immunological, cancer, cardiovascular, muscular, neurological, hematological, inflammatory, respiratory, infectious endocrine, cutaneous, gastrointestinal, metabolic, allergic diseases, in transplantation with an increase in specific expression cells of mSTIM1, myasthenia, cutaneous lupus, and extra-membranous glomerulonephritis.
48. An isolated protein fragment consisting of all or part of the region between amino acid residues 58 and 201 of the STIM1 amino acid sequence SEQ ID NO: 1 or consisting of the region between amino acid residues 128 and 168 of the STIM1 amino acid sequence SEQ ID NO: 1; or consisting of the region between amino acid residues 145 and 153 of the STIM1 amino acid sequence SEQ ID NO: 1.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0108] FIG. 1 presents the localization within the STIM1 protein of the peptides designed for mice immunization realized to obtain the monoclonal anti-STIM1 antibody clone B-Y12. Footpad immunization was realized for raising B-Y12 mAb in mice. Four balb/c mice were immunized 5 times in footpad with 1 μg/mouse of mixed peptide 1A and peptide 1B. Three successive cloning steps were done to obtain a monoclonal hybridoma.
[0109] FIG. 2 illustrates the validation of monoclonal antibody anti-STIM1 clone B-Y12 purity and specificity by Western Blot. Panel A: Lane 1 migration profile in denaturing conditions of anti-STIM1 antibody clone B-Y12: band at 50 kDa and 25 kDa corresponds respectively to the heavy chain and to the light chain of the denaturated mAb. Lane 2 is the reference ladder. Lane 3: A single band at 150 kDa is observed in native condition corresponding to the full length B-Y12 protein. Panel B: Confirmation of STIM1 protein recognition by B-Y12 antibody in pancreatic cell line PANC-1—Polyacrylamide gel (4-7.5%) loaded with 75 μg protein deposit—B-Y12 mAb was diluted to 1/1000 (initial concentration: 1.9 mg/ml) and an anti-mouse IgG HRP (Abcam) diluted at 1/10 000 was used. Panel C: Confirmation of STIM1 EF/Hand peptide recognition by the B-Y12 antibody—Polyacrylamide gel (4-7.5%) loaded with 0.5 μg to 2 μg STIM1 EF/Hand peptides were loaded. B-Y12 mab diluted to 1/1000 (initial concentration: 2 mg/ml) and an anti-mouse IgG HRP (Abcam) 1/10 000). Panel D: Confirmation of STIM1 protein recognition by B-Y12 in B cells from CLL patients or B cells from Tonsils of healthy controls. Polyacrylamide gel (4-7.5%) loaded with 75 μg protein deposit—$B-Y12 mab diluted to 1/1000 (initial concentration: 2 mg/ml) and an anti-mouse IgG HRP (Abcam) 1/10 000 was used.
[0110] FIG. 3 illustrated the validation by Elisa of the monoclonal anti-STIM1 antibody clone B-Y12. Panel A: Specificity of B-Y12 antibody for the STIM1 peptide 1A is demonstrated by ELISA using the biotinylated STIM1 peptides used for immunization. Anti-STIM1 mAb clone B-Y12 mAb (10 μg/ml) was diluted to 1/10 and antigens STIM1 biotinylated peptides used at concentrations of 50 ng/well for peptides 1A and 1B and 100 ng/well for peptide 2A and 10 ng/well for peptide 2B). Panel B: Specificity of the B-Y12 antibody for STIM1 is confirmed by ELISA using the peptide 1A and a recombinant STIM1 EF/SAM peptide (1 μg/mL). B-Y12 mAb used at 100 μg/mL diluted to 1/10. Panel C: Specificity for the B-Y12 antibody for the peptide 1A over the other peptides (1A, 1B, 2A, 2B, C2, C3, C4, C5, C6, C7 and C8. Peptides are used at a concentration of 1 μg/mL. Histograms represents mean optical density (OD) measured using an Elisa approach.
[0111] FIG. 4 shows the indirect detection of intra-cytoplasmic STIM1 and plasma membrane STIM1 (mSTIM1) in different cell lines by flow cytometry using the monoclonal Antibody anti-STIM1 clone B-Y12 and a secondary antibody. B-Y12 labeling was tested in parallel for membrane and intracytoplasmic staining on different cell lines. An indirect staining was realized with a secondary antibody (GAM FITC). Labeling in cell lines show an intra-cytoplasmic labeling as well as a surface staining. In each condition, cells are incubated with 50 μl of purified antibody at 0.5 μg at +4° C. during 30 min and then incubated with a secondary antibody (GAM FITC 30 min at +4° C.) to reveal the staining.
[0112] FIG. 5 presents the direct detection by flow cytometry of the intra-cytoplasmic and the plasma membrane STIM1 (mSTIM1) fractions of STIM1 in two different cell lines using the anti-STIM1 monoclonal antibody clone B-Y12 coupled to Phycoerythrine (PE). PE coupled B-Y12 antibody was used at different concentrations and was tested in parallel for membrane and intra-cytoplasmic staining on Nalm6 and Hacat cell lines. Cells are incubated with 50 μl of purified PE coupled antibody (at indicated dilution) at +4° C. during 30 min.
[0113] FIG. 6 represents the direct detection by flow cytometry of intra-cytoplasmic and plasma membrane fractions (mSTIM1) of STIM1 using the anti-STIM1 monoclonal antibody clone B-Y12 coupled to Phycoerythrine (PE) in primary B cells from Systemic Erythematous Lupus (SLE) and Chronic Lymphocytic Leukemia (CLL) patients. Panel A: PE coupled B-Y12 antibody was tested in parallel for membrane and intra-cytoplasmic staining in B and T cells from SLE or CLL patients and from healthy donors. Left side of panel A presents mSTIM1 in B and T cells from SLE patients. Levels of mSTIM1 CLL patients or healthy donors are presented on the right side of this panel A. 100 μl of whole blood cells (PBMC cells) were incubated with 100 μl of purified PE coupled antibody (2 μg/ml) at room temperature for 30 min during 30 min. Panel B presents the Mean Fluorescence Intensity (MFI) of mSTIM1 labeling obtained with the PE coupled B-Y12 antibody in CLL (left side of panel B) and compares MFI from cells of healthy donor and SLE patients (right side of panel B). Data are analyzed by non-parametric Wilcoxon matched-pairs analysis, ***p<0.001 for control and SLE patient. Data are analyzed by non-parametric Mann Whitney analysis, ***p<0.001 for CLL.
[0114] FIG. 7 represents the validation by flow-cytometry of the anti-STIM1 antibody clone B-Y12 specificity for the STIM1 protein. Plasma membrane STIM1 (mSTIM1) is detected in B and T cells isolated from spleen of mice direct detection by flow cytometry using Monoclonal Antibody anti-STIM1 clone B-Y12 coupled to Phycoerythrine (PE). Panel A: Labelling of mSTIM1 by PE coupled B-Y12 mAb in B and T cells from MRL/Lpr mice (n=4). Mean Fluorescence Intensity (MFI) of mSTIM1 labeling with PE coupled B-Y12 is compared to labeling with an isotype control. Panel B: Labeling of mSTIM1 by PE coupled B-Y12 mAb in B and T cells from C57Bl/6 mice (n=3). Mean Fluorescence Intensity (MFI) of mSTIM1 labeling with PE coupled B-Y12 is compared to labeling obtained with an isotype control.
[0115] FIG. 8 represents the direct detection by flow cytometry of the intra-cytoplasmic and plasma membrane (mSTIM1) fractions of STIM1 in cells line using the anti-STIM1 monoclonal antibody (mAb) clone B-Y12 coupled to Phycoerythrine (PE). Panel A: Labeling of STIM1 using the PE coupled B-Y12 mAb in DAUDI, RAMOS and JOK B cell lines and in endothelial cell line HUVEC. Cells were either left intact to labeled mSTIM1 or permeabilized to label total STIM1 (mSTIM1+intracellular STIM1). 100 μl of cells were incubated with the PE coupled B-Y12 antibody (2 μg and 4 μg for membrane and intracellular staining respectively), at 4° C. during 30 min. Panel B presents the Mean Fluorescence Intensity (MFI) of mSTIM1 labeling with the PE coupled B-Y12 antibody in HEK293 cells over-expressing STIM1 (++) compared to cells transfected with an empty vector (+). Cells are incubated with PE coupled B-Y12 antibody (2 μg) 4° C. during 30 min.
[0116] FIG. 9 represents the sequences corresponding to complete variables light (SEQ ID NOs: 17 and 9, respectively) and heavy chains (SEQ ID NOs: 18 and 10, respectively) of the anti-STIM1 antibody clone B-Y12. Total RNA is extracted and reverse transcription of the RNA (5′CDS primer) was done. Amplification of variable chains by RACE-PCR (various reverse primer) and cloning of the amplicons in shuttle vector were done. The figure shows the sequences after analysis of the complementarity determining region (CDR) in heavy chain variable domain (nucleotide and amino acid sequence) and light chain variable domain (nucleotide and amino acid sequence).
[0117] FIG. 10 represents the determination of the anti-STIM1 mAb clone B-Y12 affinity. Evaluation of the B-Y12 mAb affinity for the soluble recombinant protein corresponding to the STIM1 EF-Hand domain (amino-acids 59 to 201) was determined using the Octet technology. The KD (kdis/kon) was determined using a 1 to 1 fitting model. EF-SAM peptide (STIM1 aa 58 to 201) was tested at 200, 133.5 and 88.9 nM. High affinity interaction is characterized by a low K, a fast recognizing (high kon) and a stable formation of complexes (low kdis). kdis: dissociation rate constant. kon: complex formation rate.
[0118] FIG. 11 represents the epitope Identification for the anti-STIM1 antibody clone B-Y12. Epitope mapping was realized by mass spectrometry analysis using a MALDI-TOF/TOF approach to analyze STIM1 peptides/antibody complexes following enzymatic digestion of the antibody/antigen complex. After digestion, eluates containing peptides were analyzed by a liquid chromatography coupled tandem mass spectrometry (LC-MS/MS). Peptides were separated by liquid chromatography, and analyzed by Electrospray Ionization (ESI). The peptide of interest was fragmented and the resulting fragment ions were measured to produce the MS/MS spectrum in order to determine the epitope sequence (Panel A). This analysis shows that B-Y12 recognizes the linear sequence VELPQYEET. The localization of this sequence is represented within the N terminal extracellular part of the STIM1 protein in panel B. The projection of this sequence (white line) is highlighted in the 3D structure of N terminal extracellular part of STIM1 (Panel C).
[0119] FIG. 12 represents the inhibition of Constitutive Ca.sup.2+ entry (CCE) by anti-STIM1 mAb clone B-Y12 in B cells. Panel A illustrates the Inhibition by the B-Y12 anti-STIM1 mAb of Constitutive Ca.sup.2+ entry from purified B lymphocytes isolated from CLL patients with either high or low CCE. Cells are incubated for 1H with 10 μg/ml of the B-Y12 mAb. CCE was revealed by changing external Ca.sup.2+ concentration from 5 mM to 0.5 mM and the amplitude of CCE evaluated by the difference in normalized fluorescence ratio when changing external Ca.sup.2+ concentration. Representative curves and average amplitudes of constitutive entry (CCE) are presented. Panel B: Inhibition of CCE from HEK cell line by the B-Y12 anti-STIM1 mAb. Cells are incubated for 1 H with 10 μg/of the B-Y12 mAb. The percentage (%) of CCE inhibition compared to the isotype treatment is presented. Panel C: Store Operated Ca.sup.2+ entry measured in a B cell line (JOK cells) cells is not modulated by the anti-STIM1 mAb clone B-Y12. SOCE is recorded after endoplasmic reticulum Ca.sup.2+ store depletion with Thapsigargin (2 μM) in 0 mM external Ca.sup.2+ and re-addition of 1.8 mM extracellular Ca.sup.2+. JOK cells were incubated with an isotype or with the anti-STIM1 antibody. Histograms display the individual values of SOCE amplitude mean values+/−SEM.
[0120] FIG. 13 represents the modulation signals by anti-STIM1 mAb clone B-Y12 of the BCR induced Ca.sup.2+ signal. Panel A illustrates that the Anti-IgM induced Ca.sup.2+ response are enhanced by the B-Y12 antibody in B-cells from CLL patients with reduced BCR induced Ca.sup.2+ responses but not in B cells from healthy donors (left side of panel A). Amplitude of Ca.sup.2+ responses is measured after Anti-IgM stimulation of B-cells incubated or not for 1 h with B-Y12 antibody. The Amplitude of the Ca.sup.2+ transient is compared to what measured in B cells from healthy donors. Panel B displays the amplitude of Ca.sup.2+ responses measured after Anti-IgM stimulation of Jok cell line incubated for 1 h with B-Y12 mAb or its isotype. IgM stimulation is realized with 10 μM of polyclonal goat anti-human IgM. Data are analyzed by non-parametric Wilcoxon matched-pairs analysis, *p<0.05.
[0121] FIG. 14 represents the demonstration of the beneficial effect of anti-STIM1 mAb clone B-Y12 treatment on the survival of lupus prone mice MRL/Lpr. Survival is greatly increased in mice injected twice a week with the anti-STIM1 mAb B-Y12 (2.5 mg/Kg; n=15 mice) compared to mice injected with mAb anti-CD20 (2.5 mg/Kg; n=15 mice) and mice injected with Isotype (IgG2b—2.5 mg/Kg; n=15 mice). Data are analyzed by non-parametric Log-rank (Mantel-Cox) Wilcoxon matched-pairs analysis, *p<0.05.
[0122] FIG. 15 represents the beneficial effect on a clinical score of lupus prone mice (MRL/Lpr) treatment with the anti-STIM1 mAb clone B-Y12. The clinical score is defined by addition of the lymph node hypertrophic score (normal=0, moderate=1, severe=2), the cutaneous score (alopecia=1, ulceration=2) and the score of mice pain (moderate=2 and severe=4). The clinical score is significantly reduced in mice injected twice a week with the anti-STIM1 mAb clone B-Y12 (10 μg) compared to mice injected with mAb isotype (IgG2a, 10 μg). Histograms represents the average of the area under the curve (AUC) of the clinical score evolution over time. Data are analyzed by Welch's unpaired t analysis ****p<0.0001.
[0123] FIG. 16 represents the demonstration of the beneficial effect on the proteinuria of lupus prone mice MRL/Lpr mice of the anti-STIM1 mAb clone B-Y12 treatment. Panel A shows that the proteinuria score is reduced in mice injected twice a week with the anti-STIM1 mAb B-Y12 (0.3 mg/kg) compared to mice injected with the mAb isotype (IgG2a, 0.3 mg/kg). Histogram represents the average of the area under the curve (AUC) of proteinuria evolution over time. Panel B illustrates that proteinuria score is significantly reduced in mice injected twice a week with anti-STIM1 mAb clone B-Y12 (2.5 mg/kg) compared to mice injected with anti-CD20 mAb (2.5 mg/kg) and to non-treated mice. Histogram represents the average of the area under the curve (AUC) for proteinuria evolution over time. Urine samples were tested for proteinuria using Multistix 10 SG on a 0-4+ scale, corresponding to the following approximate protein concentrations: 0, negative or trace; 1+, 30 mg/dl; 2+, 100 mg/dl; 3+, 300 mg/dl; and 4+, 2000 mg/dl. Mice were considered to have severe nephritis if two consecutive urine samples scored 3+. Data are analyzed by Welch's unpaired t analysis ****p<0.0001; *p<0.05.
[0124] FIG. 17 illustrates that the treatment of lupus prone mice with the anti-STIM1 mAb clone B-Y12 reduces renal injuries. C3 deposit and interstitial injuries in kidney are reduced in mice treated with anti-STIM1 B-Y12 mAb. Kidneys were fixed overnight in 4% paraformaldehyde and then embedded in paraffin. Paraffin sections (5 μm) were stained with H&E. IHC was performed on paraffin sections using Abs against the C3 an appropriate secondary Ab coupled to Rodhamin. Panel A shows the clear reduction of C3 complement deposit detected by immunofluorescence in MRL/Lpr mice kidneys treated with the anti-STIM1 mAb. MRL/Lpr mice were injected with anti-STIM1 mAb B-Y12 (10 μg) twice a week. Panel B: illustrates the reduction of kidney interstitial lesions in MRL/Lpr mice injected with the anti-STIM1 mAb B-Y12 (10 μg) twice a week compared to mice injected with mAb isotype (IgG2a, 10 μg). Data are analyzed by non-parametric Wilcoxon matched-pairs analysis, ****p<0.0001.
[0125] FIG. 18 represents the beneficial effects of the treatment of lupus prone mice MRL/Lpr mice with the monoclonal antibody anti-STIM1 clone B-Y12 on lymphoproliferation. Panel A shows that injection of MRL/Lpr mice with anti-STIM1 mAb B-Y12 (10 μg) twice a week reduces lymph node size and weight compared to the injection of mice with mAb isotype (IgG2a, 10 μg). The number of plasma cells in lymph node (Panel B) and in the blood (Panel C) is reduced. Lymph node cells and blood leukocytes were incubated with 5 μg/ml of rat anti-mouse CD16/CD32 and incubated with the appropriate Abs CD138. Data are analyzed by non-parametric Mann Whitney analysis, *p<0.05.
[0126] FIG. 19 represents the beneficial effects of monoclonal antibody anti-STIM1 clone B-Y12 treatment on the auto-immune symptoms of lupus prone mice MRL/Lpr. Anti-STIM1 clone B-Y12 treatment reduces auto-antibody production in lupus injected mice. Panel A illustrates that injection twice a week of MRL/Lpr mice with the anti-STIM1 mAb clone B-Y12 (10 μg) reduces the amount of anti-phospholipid (cardiolipin) auto-antibodies detected in the blood of injected mice. Autoantibodies were detected by Elisa with Cardiolipin in serum diluted to 1/100. Panel B shows that Injection of MRL/Lpr mice with anti-STIM1 mAb B-Y12 (2.5 mg/Kg) or anti-CD20mAb (2.5 mg/Kg) twice a week reduces the amount of anti-DNA autoantibodies were detected by Elisa with salmon sperm and in serum diluted to 1/1000.
[0127] FIG. 20 illustrates the in vitro inhibition effect of the monoclonal antibody anti-STIM1 clone B-Y12 on B-cell differentiation to auto-reactive plasma cells. MRL/Lpr mice B cell differentiation into plasma cell was performed by stimulating B cells for 4 days with LPS (10 μg/mL) Cells were treated or not for these 4 days with the anti-STIM1 mAb clone B-Y12 (10 μg/mL). Cells were stained with specific antibodies to evaluate plasma cell number (CD138+) by flow cytometry (Panel A) and the number of secreted auto-DNA cell were evaluated by Elispot (Panel B).
[0128] FIG. 21 illustrates that the In vitro migration of B cells is inhibited by the anti-STIM1 mAb clone B-Y12. B cell migration experiments were realized with Boyden Chambers and migration was stimulated by a CCL12 chemokine gradient. Anti-STIM1 clone B-Y12 mAb (10 μg/ml) inhibits both the trans-endothelial migration through an endothelial cell layer (HUVEC cells) of JOK B cell line (Panel A) and the migration through a filter of DAUDI cell line and B cells from CLL patients (Panel B).
[0129] FIG. 22 shows that the anti-STIM1 mAb clone B-Y12 affects B cell survival in vitro. Treatment with B-Y12 mAb (100 μg/mL) of isolated B cells from CLL patients displaying a high level of plasma membrane STIM1 (mSTIM1) reduces B cells survival after 48 h (Panel A) whereas B cells with low amount of mSTIM1 are not affected (Panel B). Membrane staining with PE coupled B-Y12 was performed to evaluate mSTIM1 expression in B cells isolated from CLL patients. Cell survival (annexin V-/PI-) was evaluated by Anexin/PI staining in flow cytometry.
[0130] FIG. 23 illustrates the In vitro inhibition of B cell trans endothelial migration by using a peptide with the amino acid sequence of the EF/SAM (aa 58-201) domain of the protein STIM1. Trans-endothelial migration of B cells (JOK cell line) across a monolayer of endothelial cell (HUVEC cell line ATCC) was induced with a CXCL12 chemokine gradient (200 ng/ml). The number of cells that migrated across the endothelial monolayer was evaluated by flow cytometry. B cells (panel A), HUVEC endothelial cells (panel B) or both cell types (panel C) were incubated during 1 h with the STIM1 EF/SAM peptide (STIM1 aa 58-201). For each experimental condition, the percentage of migrated cells is normalized to what measured in the untreated control condition. Data are expressed as mean+/−SEM of n observations, N=1 (panels A and B) or (N=3 panel C). Data are analyzed with non parametric Mann Whitney analysis, *P<0.05, **P<0.01 and ***P<0.001.
[0131] FIG. 24 (A) illustrates the In vitro inhibition of B cell trans endothelial migration (%) by using a STIM1 peptide (peptide 1A) made of the STIM1 protein amino acids 128-168. Trans-endothelial migration of B cells (JOK cell line) across a monolayer of endothelial cell (HUVEC cell line ATCC) was induced with a CXCL12 chemokine gradient (200 ng/ml). B cells were incubated for 1 H with 10 μg/ml of the peptide or with a control isotype used at the same concentration. The percentage of migrated cells is normalized to what measured in the untreated control condition. FIG. 24 (B) illustrates the effects of the STIM1 EF/SAM peptide (STIM1 aa 58-201) on the amplitude of the constitutive calcium entry measured in B cells incubated for 1 H with 10 μg/ml of the peptide by single cell fluorescence imaging. CCE was revealed by changing external Ca.sup.2+ concentration from 5 mM to 0.5 mM and the amplitude of CCE evaluated by the difference in normalized fluorescence ratio when changing external Ca.sup.2+ concentration. Average amplitudes of constitutive entry (CCE) are presented.
[0132] FIG. 25 illustrates the superiority of anti-STIM1 mAb clone BY-12 treatment compared to anti-STIM1 mAb clone GOK treatment in MRL/Lpr mice. Treatment of lupic prone mice with BY-12 mAb has a greater impact on renal injuries reduction than what observed in mice treated with anti-STIM1 clone GoK. Proteinuria (FIG. 25A) and IgG deposit in kidney (FIG. 25B) are significantly more reduced in mice treated with BY-12 mAb than animals treated with a control IgG or with an anti-STIM mAb clone GoK. Proteinuria score was evaluated over time in treated animals using dedicated strips and histograms represents the average of the area under the curve (AUC) for proteinuria evolution over time. IgG deposit was detected by immunofluorescence in MRL/Lpr mice kidneys and fluorescence values were normalized to control conditions. As illustrated in FIG. 25C, the number of plasmocytes in the spleen of mice treated with BY-12 mAb was also significantly more reduced compared to what observed in mice treated with a control IgG or with an anti-STIM mAb clone GoK. The number of plasmocytes was detected by cytometry using specific markers and values are normalized to control conditions. FIG. 25D illustrates survival enhancement (percent) observed in mice injected twice a week with anti-STIM1 mAb BY-12 compared to mice injected with anti-STIM1 mAb clone GoK respective to non-treated mice.
[0133] FIG. 26 illustrates the superiority of anti-STIM1 mAb clone BY-12 treatment compared to anti-STIM1 mAb clone GOK treatment on the inhibition of Constitutive Calcium Entry (CCE). As observed in FIGS. 26A and 26B, anti-STIM1 mAb clone BY-12 has a superior effect on CCE inhibition measured in the Ramos B cell line than what observed for anti-STIM1 mAb clone GoK. A similar observation is made when CCE is measured on B cells from Systemic Lupus Erythematosus patients (FIG. 26C). In each experimental condition, cells are incubated for 1 hour with the appropriate antibody at a concentration ranging from 10 to 100 μg/ml or with a control isotype used at the same concentration. CCE was revealed by changing external Ca.sup.2+ concentration from 5 mM to 0.5 mM and the amplitude of CCE evaluated by the difference in normalized fluorescence ratio when changing external Ca.sup.2+ concentration. Average amplitudes of constitutive entry (CCE) normalized to control values are presented.
EXAMPLES
Example 1
Process of Preparation of the Monoclonal Antibody B-Y12
[0134] An antibody of the invention (named “B-Y12”) was obtained by immunization of mice injected with two peptides corresponding to the SAM domain of the STIM1 protein: peptides 1A (SEQ ID NO: 2) and 1B (SEQ ID NO: 19, VTNTTMTGTVLKMTDRSHRQKLQLKALDT corresponding to amino acids 169 to 197 of STIM1) (see FIG. 1). Footpad simultaneous immunization with both peptides was realized for raising anti-STIM1 antibody clone B-Y12 mAb in mice. Four balb/c mice were immunized 5 times in footpads with 1 μg/mouse of mixed peptide 1A and peptide 1B. Three successive cloning steps were done to obtain a monoclonal hybridoma.
[0135] By Western blot and ELISA approaches, it has been demonstrated that the antibody B-Y12 is a specific antibody recognizing STIM1 and more specifically the region of this protein corresponding to amino acids (aa) 128 to 168 of the SAM domain of STIM1 protein (see FIG. 2 and FIG. 3).
[0136] Protein extraction was performed on 10.sup.7 B cells for 30 on ice with a lysis buffer containing: 20 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X100, 2.5 mM Na+ pyrosodium tetraphosphate, 1 mM glycerophosphate, 1 mM Na+ orthovanadate, 1 μg/ml leupeptin and a protease inhibitor cocktail. Protein extracts were sonicated and centrifuged for 12 min at 16,000 g. Protein concentration of cell lysates were determined using the Folin method. 75 μg of proteins were run on SDS-PAGE 7.5% polyacrylamide gels in denaturing conditions, and then transferred onto PVDF (PolyVinyliDene Fluoride) membrane sheets. Unspecific blocking was done by incubation with 5% fat milk in PBS, 0.1% tween 20 for 1 hour. Blots were incubated overnight with 5% fat milk in PBS, 0.1% tween 20, containing mouse monoclonal anti-STIM1 (clone: BY-12, 1:1,000 dilution) or mouse monoclonal anti-GAPDH antibody (6C5 clone Abcam; 1:10,000 dilution,). Blots were incubated with Horseradish Peroxydase (HRP)-conjugated goat anti-mouse after washing with PBS, 0.1% tween 20 and revealed with the Luminata Forte reagent. All results were normalized upon GAPDH quantification.
[0137] For the ELISA measurements, 5×10.sup.6 cells were loaded for 1 hour on 96 wells pre-coated CellTak plates. Cells were fixed using PFA 4% for 10 min at room temperature (RT). Cells were then washed with Phosphate Buffer Solution (PBS) and next incubated with PBS supplemented with 5% of fat milk for 30 minutes. Cells were next incubated with the anti-STIM1 antibody directed against the N terminus (clone: B-Y12, 1 μg/ml) for 1 h 30 at RT. After 3 washes with PBS, cells were incubated with in PBS+5% of fat milk containing the peroxidase conjugated secondary antibody for 30 min at RT. After 3 washes, the substrate for peroxidase conjugated secondary antibody (SIGMAFAST™ OPD tablets, Sigma-Aldrich) was added for 20 min at 37° C. and the reaction was stopped using H.sub.2SO.sub.4 solution. ELISA plate was read at 392 nm in absorbance.
[0138] By Flow cytometry, direct or indirect recognition by monoclonal antibody B-Y12 of the plasma membrane-bound STIM1 protein (mSTIM1) or the endoplasmic reticulum membrane has been demonstrated in different cell lines (see FIG. 4 and FIG. 5) as well as in B lymphocytes of patients with SLE or CLL (see FIG. 6) or B cells from mice (see FIG. 7). The specificity of mSTIM1 labeling by clone B-Y12 was confirmed in flow cytometry by labeling STIM1 in cells expressing different levels of this protein (see FIGS. 4, 5, 6 and 8). This antibody makes it possible to specifically detect, in flow cytometry, the membrane fraction of STIM1 (mSTIM1) as well as the majority fraction of STIM1 located at the reticulum membrane.
[0139] 5×10.sup.6 B cells were used per condition. Cells were either left intact or permeabilized to labeled mSTIM1 or total STIM1. B cells were centrifuged for 5 min at 1500 rpm and incubated with 100 μL of PBS containing anti-STIM1 antibody directed against the N terminus (clone: PE coupled B-Y12; 2 μg for mSTIM1 and 4 μg for total STIM1 or 0.5 μL of an isotype control for 30 min on ice. After 3 washes, cells were read in PBS using a Flow cytometer (Navios, Beckman Coulter Life Sciences).
[0140] The variable domains of the heavy and light chains of the B-Y12 antibody have been sequenced (see FIG. 9). The isotype of clone B-Y12 is IgG2b/Kappa.
[0141] Total RNA is extracted and reverse transcription of the RNA (5′CDS primer) was done. Amplification of variable chains by RACE-PCR (various reverse primer) and cloning of the amplicons in shuttle vector were done. Sequences of the complementarity determining region (CDR) in heavy chain variable domain (nucleotide and amino acid sequence) and light chain variable domain (nucleotide and amino acid sequence) were analysis.
[0142] The affinity of this B-Y12 mAb for the STIM1 protein was determined by the octet technology using a peptide corresponding to the EF-SAM domain of this protein (aa: 58 to 201). In the conditions used, B-Y12 has a KD of 1.Math.5.Math.10.sup.−8 (see FIG. 10).
[0143] Evaluation of the anti-STIM1 mAb affinity for the soluble recombinant protein corresponding to the STIM1 EF-Hand domain (amino-acids 59 to 201) was determined using the Octet technology (Octet™). The KD (kdis/kon) was determined using a 1 to 1 fitting model. EF-SAM peptide (STIM1 aa 58 to 201) was tested at 200, 133.5 and 88.9 nM. Affinity constants (Kon, Kdis) were calculated.
[0144] The epitope of the antibody anti-STIM1 clone B-Y12 was identified as linear epitope with the sequence “VELPQYEET”.
[0145] The epitope mapping was realized by mass spectrometry analysis using a MALDI-TOF/TOF approach of STIM1 peptides/antibody complexes following enzymatic digestion of the antibody/antigen complex. After digestion, eluates containing peptides were analyzed by a liquid chromatography coupled tandem mass spectrometry (LC-MS/MS). Peptides were separated by liquid chromatography, and analyzed by Electrospray Ionization (ESI). The peptide of interest was fragmented and the resulting fragment ions were measured to produce the MS/MS spectrum in order to determine the epitope sequence (see FIG. 11).
Example 2
Effect of the Monoclonal Antibody B-Y12 on the Constitutive Entry of Ca.SUP.2+ of B Lymphocytes from Patients Suffering from CLL or SLE
[0146] The antibody B-Y12 inhibits the constitutive entry of Ca.sup.2+ of B lymphocytes from patients suffering from CLL or SLE. This blockage of constitutive entry is also observed in cell lines (HEK293, B-Cell Lines) (see FIG. 12).
[0147] For constitutive Ca.sup.2+ entry (CCE) measurements, 5×10.sup.6 B cells were loaded with 2 μM of the Fura-2/AM fluorescent dye in the presence of 2 μM Pluronic acid for 30 min at 37° C. in a medium containing: 135 mM NaCl, 5 mM KCl, 1 mM MgCl.sub.2, 10 mM HEPES, 10 mM Glucose with an 7.4-adjusted pH supplemented with 5 mM CaCl.sub.2. Cells were washed and left to attach in the same buffer on 12 mm CellTaK precoated coverslides for 20 min. Fura-2 was excited alternatively at 340 and 380 nm using a monochromator, and fluorescence emission was recorded at 510 nm using a fluorescence microscope equipped with a dichroic mirror and a 14-bit CCD camera. After the stabilization of basal fluorescence, the extracellular medium was replaced by Buffer A supplemented with 0.5 mM CaCl.sub.2 for 100 s and again with the original 5 mM CaCl.sub.2-containing Buffer A after curve stabilization. Values of the ratio of fluorescence measured at 340 and 380 nm are collected over time and normalized.
[0148] This antibody has no effect on activated Ca.sup.2+ entry by the effect of thapsigargin on the release of intracellular calcium stores (SOCE: Store Operated Ca.sup.2+ Entry) (see FIG. 12). Anti-STIM1 mAb clone B-Y12 does not modulate BCR induced Ca.sup.2+ signals but increase this signal in CLL cells with high level of mSTIM1 (see FIG. 13).
[0149] For SOCE measurement 5×10.sup.5 cells were seeded in precoated CellTak 96 wells. Cells are loaded with Fura-2 acetoxymethyl ester (Fura-2 QBT™, Molecular Probes) fluorochrome according to the manufacturer's protocol. The Fura-2 QBT™ was aspirated and replaced by an equal volume of free Ca.sup.2+ Hepes-buffered solution containing (in mM): 135 NaCl, 5 KCl, 1 MgCl.sub.2, 1 EGTA, 10 Hepes, 10 glucose, pH adjusted at 7.45 with NaOH. Intracellular calcium level variations were monitored by using the FlexStation 3™ (Molecular Devices, Berkshire, UK), Dual excitation wavelength capability permits ratiometric measurements of Fura-2AM peak emissions (510 nm) after excitations at 340 nm (bound to Ca.sup.2+) and 380 nm (unbound to Ca.sup.2+). Modifications in the 340/380 ratio reflect changes in intracellular-free Ca.sup.2+ concentrations. The SOCE was elicited by releasing the Ca.sup.2+ stores from the endoplasmic reticulum with thapsigargin (2 μM) solution under Ca.sup.2+-free conditions to determine the magnitude of intracellular Ca.sup.2+ release (Hepes-buffered solution). Next, cells were returned to a Ca.sup.2+-containing Hepes-buffered solution to measure SOCE. The magnitude and speed of SOCE were estimated. Igm stimulation to stimulate BCR induced Ca.sup.2+ signals is realized with 10 μM of a polyclonal goat anti-human IgM in the presence of in 2 mM external Ca.sup.2+ and in a solution containing (in mM): 135 NaCl, 5 KCl, 1 MgCl.sub.2, 1 EGTA, 10 Hepes, 10 glucose, pH adjusted at 7.45 with NaOH.
Example 3
Biological Effect of the Monoclonal Antibody B-Y12 on MRL/Lpr Mice
[0150] The anti-STIM1 clone B-Y12 antibody treatment increases MRL/Lpr lupus prone mice survival compared to mice injected with antibody isotype or a reference treatment such as anti-CD20 antibody (see FIG. 14).
[0151] Anti-STIM1 clone B-Y12 antibody treatment decreases the clinical score indicative of the general condition of the mice injected with this antibody compared to the mice injected with an isotype (see FIG. 15).
[0152] Only MRL/Lpr female mice were used in this work. Mrl/Lpr lupus prone mice were injected twice a week with anti-STIM1 mAb B-Y12 (2.5 mg/Kg) compared to mice injected with mAb anti-CD20 (2.5 mg/Kg mice) and mice injected with Isotype (IgG2b) (2.5 mg/Kg).
[0153] Clinical score is defined by addition of lymph node hypertrophy score (normal=0, moderate=1, severe=2) and cutaneous score (alopecia=1, ulceration=2) and the score of pain of mice (moderate=2 and severe=4). Mrl/Lpr lupus prone mice were injected twice a week with anti-STIM1 mAb B-Y12 (10 μg) compared to mice injected with Isotype (IgG2b 10 μg).
[0154] Anti-STIM1 B-Y12 antibody decreases renal damage in MRL/Lpr mice. These disorders result from the exacerbated production of autoantibodies in these mice (autoimmune symptoms). The treatment of the MRL/Lpr mice with the anti-STIM clone B-Y12 antibody induces in particular a reduction in the increase of the proteinuria in these mice compared to the mice injected with a control isotype (see FIG. 16). The injection of the mice with the B-Y12 antibody leads to a reduction of the renal lesions and in particular to a reduction of the C3 complement deposition and to a reduction of the interstitial lesions (see FIG. 17).
[0155] MRL/Lpr mice were injected with anti-STIM1 mAb B-Y12 (10 μg) with or mAb isotype (IgG2a, 10 μg) twice a week. Urine samples were tested for proteinuria using Multistix 10 SG (Bayer Diagnostics, Puteaux, France) on a 0-4+ scale, corresponding to the following approximate protein concentrations: 0, negative or trace; 1+, 30 mg/dl; 2+, 100 mg/dl; 3+, 300 mg/dl; and 4+, 2000 mg/dl. Kidneys were fixed overnight in 4% paraformaldehyde and then embedded in paraffin. Paraffin sections (5 μm) were stained with H&E and then scored for interstitial injuries. IHC was performed on paraffin sections using Abs against the C3 an appropriate secondary Ab coupled to Rodhamin and C3 complement deposit was detected by immunofluorescence.
[0156] The treatment of lupus mice with the B-Y12 antibody decreases the lymphoproliferation observed in these MRL/Lpr mice. Injection of the B-Y12 antibody reduced the size and weight of the ganglia of these mice compared to mice injected with a control isotype. The injection with the B-Y12 antibody greatly reduces the number of lymph node infiltrating plasmocytes as well as the number of plasma cells present in the blood (see FIG. 18). Anti-STIM1 B-Y12 antibody reduces the production of autoantibodies (anti-cardiolipin) in mice MRL/Lpr (see FIG. 19).
[0157] MRL/Lpr mice were injected with anti-STIM1 mAb B-Y12 (10 μg) with or the mAb isotype (IgG2a, 10 μg) twice a week. Lymph node size was measured and their weight was evaluated for each mice. The number of plasma cells in lymph node and in the blood was evaluated. Lymph node cells and blood leukocytes were incubated with 5 μg/ml of rat anti-mouse CD16/CD32 and incubated with the appropriate Abs CD138 to identify and count plasma cells by flow cytometry. Autoantibodies were detected in mice sera diluted to 1/100 by Elisa with Cardiolipin coated on place. Autoantibodies against DNA were detected using mice serum diluted to 1/1000 by Elisa with salmon sperm coated on plates.
Example 4
Effect of the Monoclonal Antibody B-Y12 on Differentiation of B-Lymphocytes and Antibodies Secretion by Autoreactive Plasmocytes
[0158] Anti-STIM1 B-Y12 antibody inhibits the in vitro differentiation of MRL/Lpr B-lymphocytes into auto reactive plasmocytes (see FIG. 20). The anti-STIM1 B-Y12 antibody inhibits autoantibodies secretion (see FIG. 20).
[0159] B cells were positively sorted from murine splenocytes by using a CD19 isolation kit. B cells were cultured at a concentration of 1×10.sup.6 /mL in RPMI containing 10% FCS, L-glutamine, penicillin/streptomycin, B cells were stimulated with LPS (lipopolysaccharides) and incubated or not with 10 μg/mL of B-Y12 antibody. After 3 days in culture, 2×10.sup.5 cells were cultured in Elispot plate and cells were staining with the appropriate CD138 antibody to count the number plasma cells by flow cytometry The number of anti DNA IgG secreting cells was evaluated using the classical Elispot protocol.
[0160] Anti-STIM1 B-Y12 antibody reduces in vitro migration of B-cells (see FIG. 21). Very interestingly, migration of B cells is inhibited by incubating B cells or endothelial cells with a STIM1 EF/SAM peptide (STIM1 aa 58-201) (see FIG. 23).
[0161] B cell migration experiments were realized with Boyden Chambers and migration was stimulated by a CCL12 chemokine gradient. Trans-endothelial migration of JOK B cell line was evaluated by measuring the migration of these cells through an endothelial cell monolayer of HUVEC cells cultured on a 5 μM pore filter. Simple migration of B cells was evaluated by measuring the migration of DAUDI B cells and B cells from CLL patients through a 5 μM pore filter.
[0162] B cells were treated all along the 24 hours of migration time with 10 μg/ml of the anti-STIM1 clone B-Y12 mAb. The number of migrating cells was evaluating by counting the number of B cells in the lower chamber of the boyden chamber after 24 hours using flow cytometry.
[0163] In experiments with EF/SAM STIM1 peptide, either HUVEC endothelial cells or B cells or both cell types were incubated during 1 h with 10 μg/ml of the STIM1 EF/SAM peptide (STIM1 aa 58-201). For each experimental condition, the percentage of migrated cells is normalized to what measured in the untreated control condition.
[0164] Anti-STIM1 B-Y12 antibody reduces in vitro survival of B lymphocytes (see FIG. 22).
[0165] B cells were incubated with 10 μg/ml of the anti-STIM1 clone B-Y12 mAb for 48h and cell survival was evaluated at the end of 48 hours by Anexin/PI staining in flow cytometry. For mSTIM1 detection and quantification in B cells isolated from CLL patients, membrane staining of B cells was realized with PE coupled B-Y12 mAb in flow cytometry.
REFERENCE LIST
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