ANTI-CD117 ANTIBODIES AND METHODS OF USE THEREOF

Abstract

The invention relates to the fields of immunology, oncology, and more specifically, to antibodies that can be used in the treatment of various cancers, more particularly cancers expressing CD117 antigen. These antibodies or antigen-binding molecules may be monoclonal antibodies or binding fragments thereof, they may be in particular chimeric or humanized. These antibodies may be antagonistic. The invention also relates to anticancerous or pharmaceutical compositions containing an antibody according to this invention and methods of use of these, either for laboratory work or for medical treatment.

Claims

1. A method of treatment of a subject having a cancer expressing CD117, comprising administering to said subject in need thereof a sufficient amount of a monoclonal antibody, or an antigen-binding fragment thereof, said antibody or fragment thereof specifically binding to CD117 and comprising (1) a VH chain comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 1, a H-CDR2 of sequence SEQ ID NO: 2, a H-CDR3 of sequence SEQ ID NO: 3; and a VL chain comprising the following three CDRs: a L-CDR1 of sequence SEQ ID NO: 4, a L-CDR2 of sequence STS, a L-CDR3 of sequence SEQ ID NO: 5; (2) a VH chain comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 33, a H-CDR2 of sequence SEQ ID NO: 2, a H-CDR3 of sequence SEQ ID NO: 3; and a VL chain comprising the following three CDRs: a L-CDR1 of sequence SEQ ID NO: 36, a L-CDR2 of sequence STS, a L-CDR3 of sequence SEQ ID NO: 5; (3) a VH chain comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 6, a H-CDR2 of sequence SEQ ID NO: 7, a H-CDR3 of sequence SEQ ID NO: 8; and a VL chain comprising the following three CDRs: a L-CDR1 of sequence SEQ ID NO: 9, a L-CDR2 of sequence SEQ ID NO: 10, a L-CDR3 of sequence SEQ ID NO: 5; (4) a VH chain comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 34, a H-CDR2 of sequence SEQ ID NO: 7, a H-CDR3 of sequence SEQ ID NO: 8; and a VL chain comprising the following three CDRs: a L-CDR1 of sequence SEQ ID NO: 37, a L-CDR2 of sequence SEQ ID NO: 10, a L-CDR3 of sequence SEQ ID NO: 5; (5) a VH chain comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 11, a H-CDR2 of sequence SEQ ID NO: 12, a H-CDR3 of sequence SEQ ID NO: 8; and a VL chain comprising the following three CDRs: a L-CDR1 of sequence SEQ ID NO: 9, a L-CDR2 of sequence SEQ ID NO: 10, a L-CDR3 of sequence SEQ ID NO: 5; (6) a VH chain comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 35, a H-CDR2 of sequence SEQ ID NO: 12, a H-CDR3 of sequence SEQ ID NO: 8; and a VL chain comprising the following three CDRs: a L-CDR1 of sequence SEQ ID NO: 37, a L-CDR2 of sequence SEQ ID NO: 10, a L-CDR3 of sequence SEQ ID NO: 5; (7) a VH chain comprising the following three CDRs: a H-CDR1 of sequence RYW, a H-CDR2 of sequence SEQ ID NO: 2, a H-CDR3 of sequence SEQ ID NO: 8; and a VL chain comprising the following three CDRs: a L-CDR1 of sequence SEQ ID NO: 4, a L-CDR2 of sequence STS, a L-CDR3 of sequence SEQ ID NO: 5; or (8) a VH chain comprising the following three CDRs: a H-CDR1 of sequence SYW, a H-CDR2 of sequence SEQ ID NO: 2, a H-CDR3 of sequence SEQ ID NO: 8; and a VL chain comprising the following three CDRs: a L-CDR1 of sequence SEQ ID NO: 36, a L-CDR2 of sequence STS, a L-CDR3 of sequence SEQ ID NO: 5.

2. The method of claim 1, wherein said monoclonal antibody, or antigen-binding fragment thereof comprises: (i) a VH of sequence SEQ ID NO: 25 and a VL of sequence SEQ ID NO: 27; (ii) a VH of sequence SEQ ID NO: 29 and a VL of sequence SEQ ID NO: 27; (iii) a VH of sequence SEQ ID NO: 31 and a VL of sequence SEQ ID NO: 27; or (iv) a VH of sequence SEQ ID NO: 13 and a VL of sequence SEQ ID NO: 15.

3. The method of claim 1, wherein said monoclonal comprises (1) a Heavy Chain comprising a VH region comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 1, a H-CDR2 of sequence SEQ ID NO: 2, a H-CDR3 of sequence SEQ ID NO: 3, and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising the following three CDRs: a VL region comprising a L-CDR1 of sequence SEQ ID NO: 4, a L-CDR2 of sequence STS, a L-CDR3 of sequence SEQ ID NO: 5, and a human Kappa region; (2) a Heavy Chain comprising a VH region comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 33, a H-CDR2 of sequence SEQ ID NO: 2, a H-CDR3 of sequence SEQ ID NO: 3, and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising the following three CDRs: a VL region comprising a L-CDR1 of sequence SEQ ID NO: 36, a L-CDR2 of sequence STS, a L-CDR3 of sequence SEQ ID NO: 5, and a human Kappa region; (3) a Heavy Chain comprising a VH region comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 6, a H-CDR2 of sequence SEQ ID NO: 7, a H-CDR3 of sequence SEQ ID NO: 8, and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising the following three CDRs: a VL region comprising a L-CDR1 of sequence SEQ ID NO: 9, a L-CDR2 of sequence SEQ ID NO: 10, a L-CDR3 of sequence SEQ ID NO: 5, and a human Kappa region; (4) a Heavy Chain comprising a VH region comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 34, a H-CDR2 of sequence SEQ ID NO: 7, a H-CDR3 of sequence SEQ ID NO: 8, and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising the following three CDRs: a VL region comprising a L-CDR1 of sequence SEQ ID NO: 37, a L-CDR2 of sequence SEQ ID NO: 10, a L-CDR3 of sequence SEQ ID NO: 5, and a human Kappa region; (5) a Heavy Chain comprising a VH region comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 11, a H-CDR2 of sequence SEQ ID NO: 12, a H-CDR3 of sequence SEQ ID NO: 8, and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising the following three CDRs: a VL region comprising a L-CDR1 of sequence SEQ ID NO: 9, a L-CDR2 of sequence SEQ ID NO: 10, a L-CDR3 of sequence SEQ ID NO: 5, and a human Kappa region; (6) a Heavy Chain comprising a VH region comprising the following three CDRs: a H-CDR1 of sequence SEQ ID NO: 35, a H-CDR2 of sequence SEQ ID NO: 12, a H-CDR3 of sequence SEQ ID NO: 8, and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising the following three CDRs: a VL region comprising a L-CDR1 of sequence SEQ ID NO: 37, a L-CDR2 of sequence SEQ ID NO: 10, a L-CDR3 of sequence SEQ ID NO: 5, and a human Kappa region; (7) a Heavy Chain comprising a VH region comprising the following three CDRs: a H-CDR1 of sequence RYW, a H-CDR2 of sequence SEQ ID NO: 2, a H-CDR3 of sequence SEQ ID NO: 8, and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising the following three CDRs: a VL region comprising a L-CDR1 of sequence SEQ ID NO: 4, a L-CDR2 of sequence STS, a L-CDR3 of sequence SEQ ID NO: 5, and a human Kappa region; or (8) a Heavy Chain comprising a VH region comprising the following three CDRs: a H-CDR1 of sequence SYW, a H-CDR2 of sequence SEQ ID NO: 2, a H-CDR3 of sequence SEQ ID NO: 8, and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising the following three CDRs: a VL region comprising a L-CDR1 of sequence SEQ ID NO: 36, a L-CDR2 of sequence STS, a L-CDR3 of sequence SEQ ID NO: 5, and a human Kappa region.

4. The method of claim 1, wherein said monoclonal antibody comprises a chimeric Heavy Chain of sequence SEQ ID NO: 21; a chimeric Light Chain of sequence SEQ ID NO: 23.

5. The method of claim 1, wherein said monoclonal antibody, or a antigen-binding fragment thereof is a humanized monoclonal antibody or fragment thereof, specifically binding to CD117.

6. The method of claim 3, wherein said monoclonal antibody, or antigen-binding fragment thereof comprises: (i) a VH of sequence SEQ ID NO: 25 and a VL of sequence SEQ ID NO: 27; (ii) a VH of sequence SEQ ID NO: 29 and a VL of sequence SEQ ID NO: 27; (iii) a VH of sequence SEQ ID NO: 31 and a VL of sequence SEQ ID NO: 27; (iv) a VH of sequence SEQ ID NO: 13 and a VL of sequence SEQ ID NO: 15.

7. The method of claim 1, wherein said monoclonal antibody comprises: (i) a Heavy Chain comprising a VH region of sequence SEQ ID NO: 25 and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising a VL region of sequence SEQ ID NO: 27 and a human Kappa region; (ii) a Heavy Chain comprising a VH region of sequence SEQ ID NO: 29 and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising a VL region of sequence SEQ ID NO: 27 and a human Kappa region; (iii) a Heavy Chain comprising a VH region of sequence SEQ ID NO: 31 and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising a VL region of sequence SEQ ID NO: 27 and a human Kappa region.

8.-18. (canceled)

19. The method of claim 1, wherein the antigen-binding fragment is a F(ab′)2, a Fab, a Fab′, a dsFv, a (dsFv)2, a scFv, a sc(Fv)2, a diabody, a triabody, a minibody, a scFv-Fc, and a multimeric scFv.

20. The method of claim 1, wherein said cancer is leukemia, lymphoma or solid cancer expressing CD117.

21. The method of claim 1, wherein said cancer is selected from the group consisting of myeloid leukemia, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, renal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and head-and-neck cancer, wherein the cancer expresses CD117.

22. A humanized monoclonal antibody or an antigen-binding fragment thereof, said humanized antibody or fragment thereof specifically binding to CD117 and comprising (i) a VH of sequence SEQ ID NO: 25 and a VL of sequence SEQ ID NO: 27; (ii) a VH of sequence SEQ ID NO: 29 and a VL of sequence SEQ ID NO: 27; or (iii) a VH of sequence SEQ ID NO: 31 and a VL of sequence SEQ ID NO: 27.

23. The humanized monoclonal antibody of claim 22, further comprising a human Fc region, preferably an IgG1 human Fc region, and/or a human Kappa region.

24. The humanized monoclonal antibody of claim 22, comprising: (i) a Heavy Chain comprising a VH region of sequence SEQ ID NO: 25 and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising a VL region of sequence SEQ ID NO: 27 and a human Kappa region; (ii) a Heavy Chain comprising a VH region of sequence SEQ ID NO: 29 and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising a VL region of sequence SEQ ID NO: 27 and a human Kappa region; or (iii) a Heavy Chain comprising a VH region of sequence SEQ ID NO: 31 and a human Fc region, preferably an IgG1 human Fc region; and a Light Chain comprising a VL region of sequence SEQ ID NO: 27 and a human Kappa region.

25. An anticancerous composition comprising a monoclonal antibody or an antigen-binding fragment thereof according to claim 22, and a pharmaceutically acceptable vehicle, carrier or excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0167] FIG. 1 is a bar diagram showing percent (%) of the inhibition of SCF binding (100 ng/mL, FACS analysis) in the presence of antibody tested at different concentrations using HEL cells (5.10.sup.5 cells/mL), (mean+/−SD, n=2), in the presence of 10 ng/ml of SCF.

[0168] FIG. 2 is a bar diagram showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) of antibody tested at different concentrations using HEL cells (5.10.sup.4 cells/mL), (mean+/−SD, n=2) in the presence of 10 ng/ml of SCF.

[0169] FIG. 3 is bar diagram showing western blot data related to the level of native c-kit detected by the polyclonal detecting no Phospho-CD117/c-Kit (PY719) following TF1 cell incubation in the presence of SCF with different anti-CD117 monoclonal antibodies.

[0170] FIG. 4 is bar diagram showing western blot data related to the level of native c-kit detected by the polyclonal detecting Phospho-CD117/c-Kit (PY730) following TF1 cell incubation in the presence of SCF with different anti-CD117 monoclonal antibodies.

[0171] FIG. 5 Amino acid and nucleic acid sequence for murine or chimeric VH R036-05 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3 defined according IMGT.

[0172] FIG. 6: Amino acid and nucleic acid sequence for murine or chimeric VL R036-05 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3, defined according IMGT.

[0173] FIG. 7: Amino acid and nucleic acid sequence for humanized VH 36.5Hz27 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3, defined according IMGT.

[0174] FIG. 8: Amino acid and nucleic acid sequence for humanized VL Hz 36.5Hz31 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3, defined according IMGT.

[0175] FIG. 9: Amino acid and nucleic acid sequence for humanized VH 36.5Hz30 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3, defined according IMGT.

[0176] FIG. 10: Amino acid and nucleic acid sequence for humanized VH Hz 36.5Hz31 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3, defining according IMGT.

EXAMPLES

[0177] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1: Preparation of Murine Anti-CD117 Antibody

[0178] This example illustrates the preparation of hybridoma cell lines secreting anti-CD117 antibodies.

[0179] The murine monoclonal antibodies specific for CD117 were produced using standard hybridoma techniques (Zola et al., Aust J. Exp Biol Med Sci. 1981; 59:303-6). After hybridoma cloning, one murine Mab was obtained called 36-05 (also named B-K15, Diaclone, France). The clone was injected into the peritoneum of nude mice. Protein A chromatography from murine ascitic fluid. The murine ascitic fluid is adjusted at pH 8.3 with the equilibration buffer 0.1 M Tris and 1.5 M Sulfate Ammonium and then loaded onto the rProtein A Sepharose Fast Flow column (GE Healthcare, Saint Cyr au Mont d'or, France). The non-binding proteins are flowed through and removed by several washings with equilibration buffer. The anti-CD117 monoclonal antibody (Mab anti-CD117) is eluted off the Protein A column using the elution buffer 0.1 M Citrate Sodium at pH 3.5. After concentration, the PBS solution containing IgG was filtered and the Mab concentration was determined at 280 nm.

[0180] A chimeric version of this 36-05 was produced as disclosed herein to get the ch36-05 monoclonal antibody made of Heavy chain of SEQ ID NO: 21 and of Light chain of SEQ ID NO: 23.

Example 2: Preparation of Comparative Anti-CD117 Monoclonal Antibodies

[0181] Comparative monoclonal antibodies were prepared on the following basis: [0182] 36-NOR, based on sequences H-NEH085 and L-NEH0855 disclosed in WO2016020791; [0183] 36-KOR, based on sequences H-37M and L-37M disclosed in WO2015050959; and WO2015112822; [0184] 36-IMR, based on sequences HCVR and LCVR disclosed in U.S. Pat. No. 8,552,157.

Example 3: Cell Culture

[0185] Various tumour-derived cell lines are among the target cells that may be stained with MAb anti-CD117, in such assay procedures.

[0186] Cell lines. The established human erythroleukemia cells HEL (available from DSMZ were grown in RPMI-1640 Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France). The established human erythroleukemia cells TF1 (available from ECACC) was grown in RPMI-1640 Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycin (Sigma, St Quentin Fallavier, France), 1% sodium pyruvate (Sigma, St Quentin Fallavier, France) and 5 ng/mL GM-CSF (R&D systems, Lille, France)

Example 4: MAb Competition by Flow Cytometry

[0187] Flow cytometry experiments for MAb competition binding. Briefly, 2.10.sup.5 cells HEL or TF1 per 96 wells are incubated with or without different concentration of unconjugated antibody and incubated at 4° C. for 30 min. Then different concentrations of biotinylated murine anti-CD117 antibody as a reference were added and incubated at 4° C. for 30 min. Only data obtained with 1 μg/mL of biotinylated antibody is shown. Unbound antibody is washed away with PBS (Invitrogen, Villebon sur Yvette, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). Subsequently, cells are centrifuged (5 min at 400 g) and bound antibody is detected with Phycoerythrin conjugated Streptavidin (Interchim, Montluçon, France) at 4° C. for 30 min. Detection reagent is washed away and cells are centrifuged (5 min at 400 g) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACScan or a FACScalibur (BD Biosciences, Rungis, France), (FL2 channel, at least 2000 events per acquisition.

[0188] Table 3 shows the results of the level ot MAb competition (%) resulting of the inhibition of biotinylaled MAb binding (1 μg/mL, FACS analysis) in the presence of other unconjugated antibody (5 g/mL) using the HEL cells (1.10.sup.6 cells/mL), (mean +/− SD, n=2).

TABLE-US-00004 Biotinylated Mab Codification R036-05 hIgG1 − ch36-05 +++ 36-NOR − 36-KOR − 36-IMR +++ Legend: % of inhibition of mean fluorescence intensity of cell labeling with biotinylated MAb in the presence of unconjugated Mab: −: −10%; +: 10-40%; ++: 50%; +++: 90%)

[0189] For example the unconjugated ch36-05 is not in competition with the biotinylated 36-NOR or 36-KOR. By contrast, unconjugated ch36-05 is in competition with the biotinylated 36-IMR. Therefore, the epitope ch36-05 is not common or adjacent with the epitopes 36-NOR or 36-KOR.

Example 5: Antibody Potency on SCF Binding

[0190] Flow cytometry experiments of MAb impact on human SCF binding. HEL cell lines were seeded at a density of 1.10.sup.5 per 96-wells. Cells were incubated for 30 min at 4° C. with or without MAb anti-CD117 tested at 5 μg/mL then diluted at 1/10. Unbound antibodies were washed away with PBS (Invitrogen, Villebon sur Yvette, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). Subsequently, cells are incubated with the human SCF (10 ng/mL), for 30 min at 4° C. Unbound antibodies were washed away with PBS (Invitrogen, Villebon sur Yvette, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). The bound human SCF is detected with biotinylated conjugated goat anti-human SCF (R&D Systems, Lille, France). After washings, Phycoerthrin conjugated Streptavidin (Interchim, Montluçon, France) was added at 4° C. for 30 min. Detection reagent is washed away and cells are centrifuged (5 min at 400 g) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL2 channel, 2 000 events per acquisition). During the experiment, the respective isotype controls are included to exclude any unspecific binding events.

[0191] Results of experiments to determine the antagonist activity on SCF binding of anti-CD117 antibodies are shown in FIG. 1. All of the anti-CD117 MAbs inhibit human SCF binding on HEL CD117 positive cells.

Example 6: Antibody Potency on SCF Induced Cell Proliferation

[0192] Biochemical reagents. Biochemical reagents used for the cell proliferation related studies were: recombinant human SCF (R&D Systems, Lille, France) and Cell Titer GLo-ATP (Promega, Charbonnières-les-bains, France).

[0193] Cell viability analysis following ATP level determination. The CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Charbonnières les Bains, France) was used to determine the number of viable cells in culture based on quantification of the ATP present, an indicator of metabolically active cells. Detection is based on using the luciferase reaction to measure the amount of ATP from viable cells. Within minutes after a loss of membrane integrity, cells lose the ability to synthesize ATP, and endogenous ATPases destroy any remaining ATP; thus the levels of ATP fall precipitously. Human erythroleukemia cell line TF1 show complete growth dependency on GM-CSF. Cell cultures are incubated for 24 hours before the test with culture medium without GM-CSF and only 1% FCS. Cell cultures (5.10.sup.4 cells/mL) are incubated for 72 at 37° C. hours with different concentration of interest MAbs and controls. The human SCF concentration was used at 10 ng/mL. The CellTiter-Glo® reagent was added directly to cells in culture at a ratio of 504 of reagent to 2004 of culture medium. The assay plates are incubated at room temperature for 10 min and the bioluminescent signal is recorded using a standard multiwell fluorometer Mithras LB940, (Berthold, Thoiry, France).

[0194] Results of experiments to determine the antagonist activity of anti-CD117 antibodies on SCF induced myeloid cell proliferation are shown in FIG. 2. All of the anti-CD117 MAbs inhibit human SCF induced TF1 cell proliferation.

Example 7: Antibody Potency on c-Kit Phosphorylation

[0195] Activation of CD117 occurs when an SCF dimer binds to its extracellular domain. CD117 phosphorylation triggers several signal transduction pathways, including JAK/STAT, RAS/MAP kinase pathway, PI3 kinase, PLCγ pathway, and SRC pathway. TF1 cells were serum-starved 24 hours at a density of 0.5.10.sup.6 cells/ml and the following day treated in presence of anti-CD117 MAbs, benchmark MAbs or control IgG at 10 μg/ml for 30 minutes at 37° C. Human SCF (R&D Systems, Lille, France) stimulation at 10 ng/mL was performed for 10 min at 37° C. Cells were rinsed in PBS1× (Life Technologies, Courtaboeuf, France) and lysates prepared in cell lysis buffer (Life Technologies, Courtaboeuf, France) containing Protease 1× (Sigma, St Quentin Fallavier, France) and Phosphatase Inhibitor cocktails 1× (Life Technologies, Courtaboeuf, France). Samples were sonicated on ice 10 times (amplitude value 70%) for 10 seconds on/50 seconds off. Lysate analysis was performed on NuPage 4-12% Bis-Tris Midigels (Life Technologies, Courtaboeuf, France) and protein transferred onto Immuno-Blot PVDF membrane (Biorad, Oxfordshire, United Kingdom). Blots were incubated with primary antibody dilutions: 1:500 Phospho-CD117/c-Kit (Y719) antibody (Life Technologies, Courtaboeuf, France), 1:500 Phospho-CD117/c-Kit (Y730) antibody (Life Technologies, Courtaboeuf, France), 1:2 000 CD117/c-Kit antibody (R&D Systems, Lille, France) and 1:10 000 Histone H3 antibody (Abcam, Cambridge, United Kingdom). Secondary antibody-HRP conjugates were used at 1/10000 (Biorad, Oxfordshire, United Kingdom) and at 1/500 (R&D Systems, Lille, France). Enhanced chemiluminescence (ECL) reagents (GE Healthcare, Saint Cyr au Mont d'or, France) were used to detect protein band signal. Enhanced chemiluminescence (ECL) reagents (Amersham; RPN2235) were used to detect protein band signal. All blot images were captured with an PXi4 imaging system (Syngene). Band density analysis were performed using Gene Tools software from Syngene. Band density value was attributed (#100) to Ctrl Human IgG1 band. Band density values were calculated for each band according to this reference value, for all PAbs tested: Anti-Histone H3 (Loading control—PAb0), Anti-c-kit (PAb1) and Anti-Phospho-c-kit (PY719)/PAb4 or PY730/PAb6). Band density Ratios [Anti-(Phospho)-c-kit/Anti-Histone H3] were calculated for each condition tested. For a ratio ≈1, its means no variation for (Phospho)-c-kit detection level. For a ratio >1, its means a up-regulation (Phospho)-c-kit detection and with a ratio <1, its means a down-modulation of (Phospho)-c-kit detection level by Western Blot compared with Histone H3.

[0196] Results of experiments to determine the antagonist activity of anti-CD117 antibodies on CD117 activation on TF1 cells are shown in FIG. 3. Western-blot analysis showed that all tested anti-CD117 antibodies tested down-modulate stem-cell factor induced PY719 phosphorylated CD117 (FIG. 3). By contrast all of anti-CD117 antibodies excepted antibody R036-05 significantly up-modulate stem-cell factor induced PY730 phosphorylated CD117 (FIG. 4). Thus the R036-05 antibody according to the invention selectively induces a restricted transduction pathway on transphosphorylation of tyrosine c-kit residues. The major substrates for activated KIT are specific tyrosine residues contained within the cytosolic domain of KIT. Therefore restricted Pi CD117 will generate subsequent events differing compared to the other tested anti-CD117 antibodies. (gene transcription, cell adhesion, cell proliferation, Phospholipase Cγ (PLCγ), phosphoinositol-3-kinase (PI3K), protein tyrosine phosphatase 1C (PTP1C), mitogen-activate kinase kinase (MAPKK).

Example 8: Peptide Mapping by ELISA

[0197] ELISA experiments were performed by using a panel of 52 peptides covering the extracellular domain of CD117 antigen. Each BSA conjugated peptide vas coated on 96 wells ELISA plates, (from 50 to 100 ng/mL) The MAb panel was tested from 10 to 0.01 μg/mL and revealed by using a goat polyclonal anti specie (mouse or human) IgG1 Horse Radish Peroxydase (HRP) conjugated (AbD Serotec, Colmar, France).

[0198] Among the panel of coated BSA peptides (52 peptides) related to the extracellular region of CD117, some of them are detected with the 36-NOR, 36-KOR and 36-ImR. However, R036-05 did not detect them, revealing that the R036-05 antibody recognizes a conformational CD117 epitope (data not shown).

Example 9: Preparation of Chimeric Monoclonal Antibody Directed Against CD117

[0199] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g. by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Conversion of murine MAb to native chimeric MAb:

[0200] The cDNA corresponding to the variable region of the hybridoma was obtained using two approaches. The first approach consisted in the use in PCR of the degenerate N-term amino acid related primer set generated from the N-Terminal sequencing. The second approach consisted in the use in PCR of degenerate primer set generated by IMGT® primer database and specific primers previously described (Essono et al., J Immunol Methods. 2003; 203: 279:25-66, Wang et al., Mol Immunol. 1991; 28:1387-97). The sequence of N-terminal variable region was determined by Edman degradation. Total RNA extraction was carried out using the Tri Reagent kit according to the protocol described by the supplier Sigma. The amplified VL and VH fragments were cloned into the TOPO-TA cloning vector (Invitrogen) for sequence analyses by the dideoxytermination method (Sanger et al., Nature. 1977; 265:687-95). Then antibody variant constructs were amplified by PCR and cloned into the expression vector.

[0201] Positions were numbered according to IMGT and to Kabat index (Identical V region amino acid sequences and segments of sequences in antibodies of different specificities). Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites were analyzed (Kabat et al., NIH Publ. 1991; No. 91-3242, Vol. 1, 647-669).

Example 10: Preparation of Humanized Monoclonal Antibodies

[0202] Antibody CDR and FR regions have been determined according to various numbering approaches such as IMGT (ImMunoGeneTics Information SystemR http://imgt.cines.fr), Kabat or Common Numbering System. However, IMGT determined CDRs for a given antibody are not necessarily identical to the CDRs defined by the other numbering systems. The CDRs and framework regions (FR) have been identified by the inventor thanks to IMGT numbering systems. (ImMunoGeneTics Information SystemR http://imgt.cines.fr). For Chotia, reference can be done to Antibodies—www.bioinf.org.uk.

[0203] Conversion of Chimeric MAb to Humanized MAb:

[0204] Humanized CD117 antibody Heavy (H) and Light (L) chain were generated using CDR-grafting by the PCR method. In order to generate a humanized antibody in which the CDRs of a mouse monoclonal antibody are grafted onto a human antibody, there is preferably a high homology between the variable region of a mouse monoclonal antibody and the variable region of a human antibody. Thus, the H chain and L chain V regions of a mouse anti-human CD117 monoclonal antibody are compared to the V region of all known human antibodies using the software IMGT/DomainGapAlign. When a mouse antibody is humanized by a conventional technology, the amino acid sequence of some of the V region FRs of a mouse antibody supporting the CDR may be grafted onto the FR of a human V region, as desired. Then the sequence of the Humanized variable region of is determined. The variables regions of H and L were amplified by PCR and cloned into the expression vector p3U containing the human IgG1 constant region. Three humanized antibodies were selected sharing a common light chain, but with three distinct heavy chains as the antibody 36-5 Hz114 related to Heavy chain SEQ ID NO: 25 and Light chain SEQ ID NO: 27, the antibody 36-5 Hz121 related to Heavy chain SEQ ID NO: 29 and Light chain SEQ ID NO: 27; and the antibody 36-5 Hz128 Heavy chain SEQ ID NO: 31 and Light chain SEQ ID NO: 27.

[0205] By flow cytometry, the humanized mAb staining was analyzed on HEL CD117 positive cell line. As shown in table 4, similar mAb cell staining (percentage of labelled cells/mean fluorescence Intensity) was observed whatever the mAb design (chimeric versus humanized mAb). No staining was observed on the cell line CEM (CD117 negative, (data not shown).

TABLE-US-00005 TABLE 4 Comparative mAb analysis by flow cytometry of HEL cellular staining according the mA design. μl/2.10.sup.5 cells 100 10 1 0.1 mAb design ref % labelled HEL cells/MFI chimeric mAb chR036-05 80/251 81/262 80/259 43/259 humanized mAb 36-5 Hz114 74/242 70/136 66/228 64/225 humanized mAb 36-5 Hz121 73/241 70/236 67/228 59/210 humanized mAb 36-5 Hz128 94/291 77/250 72/240 61/214