COMPOSITIONS AND METHODS FOR TREATING LUNG CANCER

Abstract

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

Claims

1-15. (canceled)

16. A method of treating lung cancer, comprising administering an effective amount of a progastrin-binding antibody, or an antigen-binding fragment thereof, to a patient in need thereof.

17. The method of claim 16, wherein said progastrin-binding antibody, or antigen-binding fragment thereof, is selected in the group consisting of: single chain antibodies, camelized antibodies, IgA1 antibodies, IgA2 antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, and IgM antibodies.

18. The method of claim 16, wherein said progastrin-binding antibody, or an antigen-binding fragment thereof, is selected from N-terminal anti-progastrin antibodies and C-terminal anti-progastrin antibodies.

19. The method of claim 16, wherein said progastrin-binding antibody, or an antigen-binding fragment thereof, is a neutralizing antibody.

20. The method of claim 16, wherein said antibody is selected in the group consisting of: an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively, an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively, and an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively.

21. The method of claim 16, wherein said antibody is selected in the group consisting of: a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:41 and a light chain variable region of SEQ ID NO:42; a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:43 and a light chain variable region of SEQ ID NO:44; a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:45 and a light chain variable region of SEQ ID NO:46; a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:47 and a light chain variable region of SEQ ID NO:48; a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:49 and a light chain variable region of SEQ ID NO:50; and a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:51 and a light chain variable region of SEQ ID NO:52.

22. The method of claim 16, wherein said antibody is a humanized antibody.

23. The method of claim 16, wherein said antibody is selected in the group consisting of: a humanized antibody comprising a heavy chain variable region of SEQ ID NO:53, and a light chain variable region of SEQ ID NO:54; a humanized antibody comprising a heavy chain variable region of SEQ ID NO:55, and a light chain variable region of SEQ ID NO:56; a humanized antibody comprising a heavy chain variable region of SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:59, and a light chain variable region of SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:62; a humanized antibody comprising a heavy chain variable region of SEQ ID NO:63, SEQ ID NO:64, or SEQ ID NO:65, and a light chain variable region of SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68; a humanized antibody comprising a heavy chain variable region of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71, and a light chain variable region of SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72; and a humanized antibody comprising a heavy chain variable region of SEQ ID NO:75 or SEQ ID NO:76, and a light chain variable region of SEQ ID NO:77 or SEQ ID NO:78; wherein said antibody also comprises constant regions of the light-chain and the heavy-chain derived from a human antibody.

24. The method of claim 16, wherein said antibody comprises a heavy chain variable region of SEQ ID NO:71 and a light chain variable region of SEQ ID NO:72, said antibody also comprising constant regions of the light-chain and the heavy-chain derived from a human antibody.

25. The method of claim 16, wherein said antibody comprises a heavy chain of SEQ ID NO:73 and a light chain of SEQ ID NO:74.

26. A method of treating lung cancer, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a progastrin-binding antibody, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and/or an excipient.

27. The method of claim 26, wherein said progastrin-binding antibody, or antigen-binding fragment thereof, is selected in the group consisting of: single chain antibodies, camelized antibodies, IgA1 antibodies, IgA2 antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, and IgM antibodies.

28. The method of claim 26, wherein said progastrin-binding antibody, or an antigen-binding fragment thereof, is selected from N-terminal anti-progastrin antibodies and C-terminal anti-progastrin antibodies.

29. The method of claim 26, wherein said progastrin-binding antibody, or an antigen-binding fragment thereof, is a neutralizing antibody.

30. The method of claim 26, wherein said antibody is selected in the group consisting of: an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively, an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively, and an antibody comprising a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively, and a light chain comprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively.

31. The method of claim 26, wherein said antibody is selected in the group consisting of: a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:41 and a light chain variable region of SEQ ID NO:42; a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:43 and a light chain variable region of SEQ ID NO:44; a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:45 and a light chain variable region of SEQ ID NO:46; a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:47 and a light chain variable region of SEQ ID NO:48; a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:49 and a light chain variable region of SEQ ID NO:50; and a monoclonal antibody comprising a heavy chain variable region of SEQ ID NO:51 and a light chain variable region of SEQ ID NO:52.

32. The method of claim 26, wherein said antibody is a humanized antibody.

33. The method of claim 26, wherein said antibody is selected in the group consisting of: a humanized antibody comprising a heavy chain variable region of SEQ ID NO:53, and a light chain variable region of SEQ ID NO:54; a humanized antibody comprising a heavy chain variable region of SEQ ID NO:55, and a light chain variable region of SEQ ID NO:56; a humanized antibody comprising a heavy chain variable region of SEQ ID NO:57, SEQ ID NO:58, or SEQ ID NO:59, and a light chain variable region of SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:62; a humanized antibody comprising a heavy chain variable region of SEQ ID NO:63, SEQ ID NO:64, or SEQ ID NO:65, and a light chain variable region of SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68; a humanized antibody comprising a heavy chain variable region of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71, and a light chain variable region of SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72; and a humanized antibody comprising a heavy chain variable region of SEQ ID NO:75 or SEQ ID NO:76, and a light chain variable region of SEQ ID NO:77 or SEQ ID NO:78; wherein said antibody also comprises constant regions of the light-chain and the heavy-chain derived from a human antibody.

34. The method of claim 26, wherein said antibody comprises a heavy chain variable region of SEQ ID NO:71 and a light chain variable region of SEQ ID NO:72, said antibody also comprising constant regions of the light-chain and the heavy-chain derived from a human antibody.

35. The method of claim 26, wherein said antibody comprises a heavy chain of SEQ ID NO:73 and a light chain of SEQ ID NO:74.

36. The method of claim 26, said method further comprising administering a second therapeutic agent to said subject.

37. The method of claim 36, wherein said agent is a biological agent or a chemotherapeutic agent.

38. The method of claim 37, wherein said biological agent is an anti-EGFR monoclonal antibody or an anti-VEGF monoclonal antibody.

39. The method of claim 37, wherein said chemotherapeutic agent is selected from the group consisting of alkylating agents, anti-metabolites, anti-tumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenesis agents, anti-estrogens, anti-androgens, and immunomodulators.

Description

FIGURE LEGENDS

[0136] FIG. 1:

[0137] Cell proliferation assay: NCI-H358 cells were treated with either a control antibody or the anti-hPG Hz 8CV2 (PG Hz), a C-terminal anti-hPG humanized antibody.

EXAMPLES

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

[0138] 1.1. Neutralizing Activity of Anti-hPG Monoclonal Antibodies

[0139] Monoclonal antibodies to PG are tested for their ability to inhibit proliferation of several cell lines commonly used to study lung cancer, which produce and secrete progastrin. Survival of cells from each of these cell lines is tested using different anti-hPG monoclonal antibodies.

[0140] 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 TO), cells are treated in sextuplicates every 12 h for 48 hours, in the absence of fetal calf serum, with 1 to 20 pg/ml of monoclonal control antibodies (monoclonal antibody anti-puromycin)(CT mAb), or with 1 to 20 g/ml anti-hPG mAb, wherein said mAb is a C-terminal anti-hPG monoclonal antibody or a N-terminal anti-hPG monoclonal antibody.

[0141] Said mAb is a C-terminal anti-hPG antibody, selected among: [0142] An antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 28, 29 and 30, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 31, 32 and 33, [0143] An antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 34, 35 and 36, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 37, 38 and 39.

[0144] or a N-terminal anti-hPG antibody selected among: [0145] An monoclonal antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 4, 5 and 6, respectively, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 7, 8 and 9, [0146] An antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 10, 11 and 12, respectively, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 13, 14 and 15, respectively, [0147] An antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 16, 17 and 18, respectively, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 19, 20 and 21, respectively, [0148] An antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 22, 23 and 24, respectively, and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 25, 26 and 27, respectively,

[0149] The number of cells at TO is counted in a control well, for each experiment.

[0150] 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 TO, 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.

[0151] 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.

[0152] 1.2. Neutralizing Activity of Anti-hPG Humanized Antibodies on Cell Survival

[0153] Humanized antibodies to PG are tested for their ability to inhibit proliferation of several cell lines commonly used to study lung cancer, which produce and secrete progastrin. Survival of cells from each of these cell lines is tested using different anti-hPG monoclonal antibodies.

[0154] 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 TO), cells are treated in sextuplicates every 12 h for 48 hours, in the absence of fetal calf serum, with 1 to 20 g/ml of humanized control antibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 1 to 20 g/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 TO is counted in a control well, for each experiment.

[0155] 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 TO, 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.

[0156] 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.

[0157] 1.3. Neutralizing Activity of Anti-hPG Monoclonal Antibodies on Cancer Stem Cell Frequency

[0158] 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 lung cancer, which produce and secrete progastrin. CSC frequency from each of these cell lines is tested using different anti-hPG monoclonal antibodies.

[0159] 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 g/ml of monoclonal control antibodies (monoclonal antiboby anti-puromycin)(CT mAb), or with 1 to 20 g/ml anti-hPG mAb, wherein said mAb is a C-terminal anti-hPG monoclonal antibody or a N-terminal anti-hPG monoclonal antibody.

[0160] 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).

[0161] Treatment with anti-hPG monoclonal antibodies reduces CSC frequency as compared to treatment with control antibody.

[0162] 1.4. Neutralizing Activity of Anti-hPG Humanized Antibodies on Cancer Stem Cell Frequency [0163] Sphere formation assay

[0164] 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 lung cancer, which produce and secrete progastrin.

[0165] 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 g/ml of humanized control antibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 20 g/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.

[0166] 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 m are counted.

[0167] Treatment with anti-hPG humanized antibodies reduces CSC frequency as compared to treatment with control antibody. [0168] Extreme Limiting Dilution Assay

[0169] 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 lung cancer, which produce and secrete progastrin. CSC frequency from each of these cell lines is tested using different anti-hPG humanized antibodies.

[0170] 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 g/ml of humanized control antibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 1 to 20 g/ml anti-hPG Hz, wherein said Hz is a C-terminal anti-hPG humanized antibody or a N-terminal anti-hPG humanized antibody.

[0171] 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).

[0172] Treatment with anti-hPG humanized antibodies reduces CSC frequency as compared to treatment with control antibody.

[0173] 1.5. Neutralizing Activity of Anti-hPG Monoclonal Antibodies on the WNT/-Catenin Pathway

[0174] Monoclonal antibodies to PG are tested for their ability to inhibit the WNT/-catenin pathway in several cell lines commonly used to study lung cancer, which produce and secrete progastrin, using the expression of the protein survivin, a well-known WNT/-catenin pathway targeted gene, as read-out. Survivin expression from each of these cell lines is tested using different anti-hPG monoclonal antibodies.

[0175] 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 g/ml of monoclonal control antibodies (monoclonal antiboby anti-puromycin)(CT mAb), or with 1 to 20 g/ml anti-hPG mAb, wherein said mAb is a C-terminal anti-hPG monoclonal antibody or a N-terminal anti-hPG monoclonal antibody.

[0176] 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.

[0177] 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.

[0178] 1.6. Neutralizing Activity of Anti-hPG Humanized Antibodies on the WNT/-Catenin Pathway

[0179] Humanized antibodies to PG are tested for their ability to inhibit the WNT/-catenin pathway in several cell lines commonly used to study lung cancer, which produce and secrete progastrin, using the expression of the protein survivin, a well-known WNT/-catenin pathway targeted gene, as read-out. Survivin expression from each of these cell lines is tested using different anti-hPG humanized antibodies.

[0180] 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 g/ml of humanized control antibodies (anti-human FcG1, from BioXCell)(CT Hz), or with 1 to g/ml anti-hPG Hz, wherein said Hz is a C-terminal anti-hPG humanized antibody or a N-terminal anti-hPG humanized antibody.

[0181] 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.

[0182] 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

[0183] Neutralizing Activity of Anti-hPG Humanized Antibodies on Cell Survival

[0184] Humanized antibodies to PG were tested for their ability to inhibit proliferation of several cell lines commonly used to study lung cancer (i.e., NCI-H358, A549 or NCI-H460), which produce and secrete progastrin. Survival of cells from each of these cell lines was assayed using different anti-hPG monoclonal antibodies.

[0185] 200,000 NCI-H358 cells were seeded into 6-well plates in medium containing foetal calf serum and incubated for 8 hours. Cells were serum-starved overnight.

[0186] From 24 hours after seeding (time TO), cells were treated in every 12 h for 48 hours, in the absence of foetal calf serum, with 20 g/ml of humanized control antibodies (anti-human FcG1, from BioXCell) (CT Hz), or with 20 g/ml anti-hPG Hz 8CV2 (PG Hz), wherein said Hz is a C-terminal anti-hPG humanized antibody. The number of cells at TO was counted in a control well, for each experiment.

[0187] 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 TO, was then calculated.

[0188] Treatment with anti-hPG Hz antibodies reduced cell number as compared to treatment with control antibody. Statistical significance was determined using a t-test: **=p<0.01, In each cell line, anti-hPG antibodies reduced cell survival (see FIG. 1).

BIBLIOGRAPHIC REFERENCES

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