Compositions and methods for detecting and treating prostate cancer using progastrin binding molecule

Abstract

The present invention relates to compositions and methods for the prevention or the treatment of prostate cancer, wherein said compositions comprise an antibody binding to progastrin and said methods comprise the use of an antibody binding to progastrin.

Claims

1. A method for inhibiting prostate adenocarcinoma cell proliferation in a patient in need thereof, said method comprising administering a progastrin-binding antibody, or an antigen-binding fragment thereof, to said patient, wherein said antibody is a monoclonal antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of amino acid sequences SEQ ID NOs 28, 29, and 30, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of amino acid sequences SEQ ID NOs 31, 32, and 33, respectively.

2. The method of claim 1, wherein said antibody is a monoclonal antibody comprising a heavy chain variable region of amino acid sequence SEQ ID NO:49 and a light chain variable region of amino acid sequence SEQ ID NO:50.

3. The method of claim 1, wherein said antibody is a humanized antibody.

4. The method of claim 3, wherein said antibody is a humanized antibody comprising a heavy chain variable region of amino acid sequence selected from SEQ ID NO:69 and SEQ ID NO:71, and a light chain variable region of amino acid sequence selected from SEQ ID NO:70 and SEQ ID NO:72; wherein said antibody also comprises constant regions of the light-chain and the heavy-chain derived from a human antibody.

5. The method of claim 3, wherein said antibody comprises a heavy chain variable region of amino acid sequence SEQ ID NO:71 and a light chain variable region of amino acid sequence SEQ ID NO:72, said antibody also comprising constant regions of the light-chain and the heavy-chain derived from a human antibody.

6. The method of claim 3, wherein said antibody comprises a heavy chain of amino acid sequence SEQ ID NO:73 and a light chain of amino acid sequence SEQ ID NO:74.

7. A method for inhibiting prostate adenocarcinoma cell proliferation in a patient in need thereof, said method comprising administering a pharmaceutical composition comprising the progastrin-binding antibody, or an antigen-binding fragment thereof, of claim 1 and a pharmaceutically acceptable carrier and/or an excipient to said patient.

8. The method of claim 7, further comprising a second therapeutic agent.

9. The method of claim 8, wherein said agent is a biological agent or a chemotherapeutic agent.

10. The method of claim 9, wherein said biological agent is an anti-EGFR monoclonal antibody or an anti-VEGF monoclonal antibody.

11. The method of claim 9, wherein said chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, anti-tumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenesis agents, anti-estrogens, anti-androgens, and immunomodulators.

Description

FIGURE LEGENDS

(1) FIG. 1

(2) DU145cells proliferation assay: cells were treated either with a control antibody or with anti-hPG Hz 8CV2 (PG Hz), a C-terminal anti-hPG humanized antibody.

(3) FIG. 2

(4) Effect of treatment with anti-hPG Hz 8CV2 (PG Hz), a C-terminal anti-hPG humanized antibody, or with an N-terminal anti-hPG humanized antibody, on sphere formation of LNCaP cells.

EXAMPLES

Example 1: Neutralizing Activity of Anti-hPG Antibodies on Cancer Cell Lines

(5) 1.1. Neutralizing Activity of Anti-hPG Monoclonal Antibodies

(6) Monoclonal antibodies to PG are tested for their ability to inhibit proliferation of several cell lines commonly used to study prostate cancer, which produce and secrete progastrin. Survival of cells from each of these cell lines is tested using different anti-hPG monoclonal antibodies.

(7) For each experiment, 50,000 cells are seeded into 6-well plates in medium containing fetal calf serum and incubated for 8 hours. Cells are serum-starved overnight, and starting at 24 hours after seeding (time “T0”), cells are treated in sextuplicates every 12 h for 48 hours, in the absence of fetal calf serum, with 1 to 20 μg/ml of monoclonal control antibodies (monoclonal antibody anti-puromycin)(CT mAb), or with 1 to 20 μg/ml anti-hPG mAb, wherein said mAb is a C-terminal anti-hPG monoclonal antibody or a N-terminal anti-hPG monoclonal antibody.

(8) Said mAb is a C-terminal anti-hPG antibody, selected among: An antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NOs 28, 29 and 30, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID NOs 31, 32 and 33, An antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NOs 34, 35 and 36, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID NOs 37, 38 and 39.

(9) or a N-terminal anti-hPG antibody selected among: An monoclonal antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NOs 4, 5 and 6, respectively, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID NOs 7, 8 and 9, An antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NOs 10, 11 and 12, respectively, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID NOs 13, 14 and 15, respectively, An antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NOs 16, 17 and 18, respectively, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID NOs 19, 20 and 21, respectively, An antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NOs 22, 23 and 24, respectively, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID NOs 25, 26 and 27, respectively,

(10) The number of cells at T0 is counted in a control well, for each experiment.

(11) Specifically, the number of live cells in both control and anti-hPG mAb treated wells is counted at 48 hours, then the difference between each cell count and the cell count determined at T0, is calculated. The resulting number of anti-hPG mAb-treated cells is then expressed as a percentage of the number of control mAb-treated cells.

(12) Treatment with anti-hPG monoclonal antibodies reduces cell number as compared to treatment with control antibody. Statistical significance is determined using a one-way ANOVA with a Tukey post-hoc test: *=p<0.05, **=p<0.01, and ***=p<0.001. In each cell line, anti-hPG antibodies reduce cell survival.

(13) 1.2. Neutralizing Activity of Anti-hPG Humanized Antibodies on Cell Survival

(14) Humanized antibodies to PG are tested for their ability to inhibit proliferation of several cell lines commonly used to study prostate cancer, which produce and secrete progastrin. Survival of cells from each of these cell lines is tested using different anti-hPG monoclonal antibodies.

(15) For each experiment, 50,000 cells are seeded into 6-well plates in medium containing fetal calf serum and incubated for 8 hours. Cells are serum-starved overnight, and starting at 24 hours after seeding (time “T0”), cells are treated in sextuplicates every 12 h for 48 hours, in the absence of fetal calf serum, with 1 to 20 μg/ml of humanized control antibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 1 to 20 μg/ml anti-hPG Hz, wherein said Hz is a C-terminal anti-hPG humanized antibody or a N-terminal anti-hPG humanized antibody. The number of cells at T0 is counted in a control well, for each experiment.

(16) Specifically, the number of live cells in both control and anti-hPG Hz treated wells is counted at 48 hours, then the difference between each cell count and the cell count determined at T0, is calculated. The resulting number of anti-hPG Hz-treated cells is then expressed as a percentage of the number of control mAb-treated cells.

(17) Treatment with anti-hPG Hz antibodies reduces cell number as compared to treatment with control antibody. Statistical significance is determined using a one-way ANOVA with a Tukey post-hoc test: *=p<0.05, **=p<0.01, and ***=p<0.001. In each cell line, anti-hPG antibodies reduce cell survival.

(18) 1.3. Neutralizing Activity of Anti-hPG Monoclonal Antibodies on Cancer Stem Cell Frequency

(19) Monoclonal antibodies to PG are tested for their ability to reduce cancer stem cell (CSC) frequency using Extreme Limiting Dilution Assay (ELDA), in several cell lines commonly used to study prostate cancer, which produce and secrete progastrin. CSC frequency from each of these cell lines is tested using different anti-hPG monoclonal antibodies.

(20) For each experiment, cells are seeded in ultra-low attachment (ULA) P96 (96-well plates) at fixed cellular concentrations per well using a FACS Aria flow cytometer, and a range of concentrations is used from one to 500 cells per well. The cells are cultivated for up to 11 days in ULA plates with M11 medium (Macari et al, Oncogene, 2015) and treated every 3 or 4 days with 1 to 20 μg/ml of monoclonal control antibodies (monoclonal antibody anti-puromycin)(CT mAb), or with 1 to 20 μg/ml anti-hPG mAb, wherein said mAb is a C-terminal anti-hPG monoclonal antibody or a N-terminal anti-hPG monoclonal antibody.

(21) Specifically, at the end of the incubation phase, the plates are observed with a phase-contrast microscope and the number of positive wells per cellular concentration is assessed. Finally, the ELDA webtool (http://www.bioinf.wehi.edu.au/software/elda/) is used to calculate the CSC frequencies of each treatment group and test for any statistical difference between groups (modified Chi-square test).

(22) Treatment with anti-hPG monoclonal antibodies reduces CSC frequency as compared to treatment with control antibody.

(23) 1.4. Neutralizing Activity of Anti-hPG Humanized Antibodies on Cancer Stem Cell Frequency Sphere Formation Assay

(24) Humanized antibodies to PG are tested for their ability to reduce cancer stem cell (CSC) frequency using sphere formation assay in several cell lines commonly used to study prostate cancer, which produce and secrete progastrin.

(25) For each experiment, 700 cells are seeded in 24-well ultra-low attachment (ULA). The cells are cultivated for up to 7 days in ULA plates with M11 medium (Macari et al, Oncogene, 2015) and treated every 3 or 4 days with 20 pg/ml of humanized control antibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 20 pg/ml anti-hPG Hz (PG Hz), wherein said Hz is a C-terminal anti-hPG humanized antibody or a N-terminal anti-hPG humanized antibody.

(26) Specifically, at the end of the incubation phase, the wells are photographed via brightfield microscopy, the pictures are analyzed and the spheres with a mean diameter above 25 μm are counted.

(27) Treatment with anti-hPG humanized antibodies reduces CSC frequency as compared to treatment with control antibody. Extreme Limiting Dilution Assay

(28) Humanized antibodies to PG are tested for their ability to reduce cancer stem cell (CSC) frequency using Extreme Limiting Dilution Assay (ELDA) in several cell lines commonly used to study prostate cancer, which produce and secrete progastrin. CSC frequency from each of these cell lines is tested using different anti-hPG humanized antibodies.

(29) For each experiment, cells are seeded in ultra-low attachment (ULA) P96 (96-well plates) at fixed cellular concentrations per well using a FACS Aria flow cytometer, and a range of concentrations is used from one to 500 cells per well. The cells are cultivated for up to 11 days in ULA plates with M11 medium (Macari et al, Oncogene, 2015) and treated every 3 or 4 days with 1 to 20 μg/ml of humanized control antibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 1 to 20 μg/ml anti-hPG Hz, wherein said Hz is a C-terminal anti-hPG humanized antibody or a N-terminal anti-hPG humanized antibody.

(30) Specifically, at the end of the incubation phase, the plates are observed with a phase-contrast microscope and the number of positive wells per cellular concentration is assessed. Finally, the ELDA webtool (http://www.bioinf.wehi.edu.au/software/elda/) is used to calculate the CSC frequencies of each treatment group and test for any statistical difference between groups (modified Chi-square test).

(31) Treatment with anti-hPG humanized antibodies reduces CSC frequency as compared to treatment with control antibody.

(32) 1.5. Neutralizing Activity of Anti-hPG Monoclonal Antibodies on the WNT/β-Catenin Pathway

(33) Monoclonal antibodies to PG are tested for their ability to inhibit the WNT/β-catenin pathway in several cell lines commonly used to study prostate cancer, which produce and secrete progastrin, using the expression of the protein survivin, a well-known WNT/β-catenin pathway targeted gene, as read-out. Survivin expression from each of these cell lines is tested using different anti-hPG monoclonal antibodies.

(34) For each experiment, 50,000 cells are seeded into 6-well plates in medium containing fetal calf serum and incubated for 8 hours. Cells are serum-starved overnight, and starting 24 hours after seeding cells are treated in quadruplicate every 12 h for 72 hours, in the absence of fetal calf serum, with 1 to 20 μg/ml of monoclonal control antibodies (monoclonal antibody anti-puromycin)(CT mAb), or with 1 to 20 μg/ml anti-hPG mAb, wherein said mAb is a C-terminal anti-hPG monoclonal antibody or a N-terminal anti-hPG monoclonal antibody.

(35) Specifically, after 72 hours of treatment, cells are harvested and total proteins are extracted using RIPA buffer. An equal amount of protein from CT mAb or anti-hPG mAb treated cells are then subjected to a western blot using anti-survivin antibody (monoclonal antibody, #2802 from Cell Signaling) and anti-actin antibody as loading control (monoclonal antibody, #A4700 from SIGMA). Quantification is performed using the GBOX chemi system from Syngene.

(36) Treatment with anti-hPG monoclonal antibodies reduces survivin expression as compared to treatment with control antibody. Statistical significance is determined using a unpaired Student's T-test: *=p<0.05, **=p<0.01, and ***=p<0.001.

(37) 1.6. Neutralizing Activity of Anti-hPG Humanized Antibodies on the WNT/13-Catenin Pathway

(38) Humanized antibodies to PG are tested for their ability to inhibit the WNT/β-catenin pathway in several cell lines commonly used to study prostate cancer, which produce and secrete progastrin, using the expression of the protein survivin, a well-known WNT/β-catenin pathway targeted gene, as read-out. Survivin expression from each of these cell lines is tested using different anti-hPG humanized antibodies.

(39) For each experiment, 50,000 cells are seeded into 6-well plates in medium containing fetal calf serum and incubated for 8 hours. Cells are serum-starved overnight, and starting 24 hours after seeding cells are treated in quadruplicate every 12 h for 72 hours, in the absence of fetal calf serum, with 1 to 20 μg/ml of humanized control antibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 1 to 20 μg/ml anti-hPG Hz, wherein said Hz is a C-terminal anti-hPG humanized antibody or a N-terminal anti-hPG humanized antibody.

(40) Specifically, after 72 hours of treatment, cells are harvested and total proteins are extracted using RIPA buffer. An equal amount of protein from CT Hz or anti-hPG Hz treated cells are then subjected to a western blot using anti-survivin antibody (monoclonal antibody, #2802 from Cell Signaling) and anti-actin antibody as loading control (monoclonal antibody, #A4700 from SIGMA). Quantification is performed using the GBOX chemi system from Syngene.

(41) Treatment with anti-hPG humanized antibodies reduces survivin expression as compared to treatment with control antibody. Statistical significance is determined using a unpaired Student's T-test: *=p<0.05, **=p<0.01, and ***=p<0.001.

Example 2: Neutralizing Activity of Anti-hPG Antibodies on Cancer Cell Lines

(42) 2.1 Neutralizing Activity of Anti-hPG Humanized Antibodies on Cell Survival

(43) Humanized anti-PG antibodies were tested for their ability to inhibit proliferation of several cell lines commonly used to study prostate cancer (i.e., PC3, LNCAP, DU145, etc.) which produce and secrete progastrin. Survival of cells of each of these lines was tested using different antihPG antibodies.

(44) 125,000 DU145 cells were seeded into 6-well plates in medium containing fetal calf serum and incubated for 8 hours. Cells were serum-starved overnight, and starting at 24 hours after seeding (time “T0”), cells were treated in every 12 h for 48 hours, in the absence of fetal calf serum, with 20 pg/ml of humanized control antibodies (anti-human FcG1, from BioXCell) (CT Hz), or with 20 pg/ml anti-hPG Hz 8CV2 (PG Hz), wherein said Hz is a C-terminal anti-hPG humanized antibody. The number of cells at T0 was counted in a control well, for each experiment.

(45) Specifically, the number of live cells in both control and anti-hPG Hz treated wells was counted at 48 hours. The difference between each cell count and the cell count determined at T0, was then calculated.

(46) Treatment with anti-hPG Hz antibodies reduced cell number as compared to treatment with control antibody. Statistical significance was determined using t-test: *=p<0.05.

(47) 2.2 Neutralizing Activity of Anti-hPG Humanized Antibodies on Sphere Formation

(48) 150 LNCaP cells were seeded in 24-well ultra-low attachment (ULA). The cells were cultivated for 11 days in ULA plates with M11 medium (Macari et al, Oncogene, 2015) and treated every 3 or 4 days with 10 pg/ml of humanized control antibodies (anti-human FcG1, from BioXCell) (CT Hz), or with 10 pg/ml anti-hPG Hz 8CV2 (PG Hz), wherein said Hz is a C-terminal anti-hPG humanized antibody or a N-terminal anti-hPG humanized antibody.

(49) Specifically, at the end of the incubation phase, the wells were photographed via brightfield microscopy, the pictures analyzed and the spheres with a mean diameter above 20 μm counted.

(50) The results displayed in FIG. 2 show clearly that treatment with anti-hPG monoclonal antibodies of prostate cancer cells substantially reduced the number of spheroids that formed during growth under low adherence culture conditions compared to control monoclonal antibody. Statistical significance was determined using t-test: *=p<0.05.