ANTI-NEUROTENSIN LONG FRAGMENT ANTIBODIES AND USES THEREOF

Abstract

The present invention relates to a neutralising antibody which is capable of binding to neurotensin with high affinity. The antibody of the present invention neutralises the activity of neurotensin, in particular the oncogenic activities of neurotensin. In particular, the present invention relates to a neutralising antibody which binds to the human neurotensin long fragment, and having a heavy chain variable region which comprises a H-CDR1 region having at least 90% of identity with SEQ ID NO:2, a H-CDR2 region having at least 90% of identify with SEQ ID NO:3 and a H-CDR3 region having at least 90% of identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% of identity with SEQ ID NO:6, a L-CDR2 having at least 90% of identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% of identity with SEQ ID NO:8. The present invention also provides the use of such antibodies in the treatment of cancer.

Claims

1. A neutralising antibody which binds to the a human neurotensin long fragment, and has a heavy chain variable region which comprises a H-CDR1 region having at least 90% of identity with SEQ ID NO:2, a H-CDR2 region having at least 90% identify with SEQ ID NO:3 and a H-CDR3 region having at least 90% identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% identity with SEQ ID NO:6, a L-CDR2 having at least 90% identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% identity with SEQ ID NO:8.

2. The antibody of claim 1 which comprises a heavy chain wherein a variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:2 for H-CDR1, SEQ ID NO:3 for H-CDR2 and SEQ ID NO:4 for H-CDR3.

3. The antibody of claim 1 which comprises a light chain wherein a variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:6 for L-CDR1, SEQ ID NO:7 for L-CDR2 and SEQ ID NO:8 for L-CDR3.

4. (canceled)

5. The antibody of claim 1 which comprises a heavy chain variable region comprising SEQ ID NO:2 in the H-CDR1 region, SEQ ID NO:3 in the H-CDR2 region and SEQ ID NO:4 in the H-CDR3 region; and a light chain variable region comprising SEQ ID NO:6 in the L-CDR1 region, SEQ ID NO:7 in the L-CDR2 region and SEQ ID NO:8 in the L-CDR3 region.

6. The antibody of claim 1 which comprises a heavy chain variable region having at least 70% identity with SEQ ID NO:1 and/or a light chain variable region having at least 70% identity with SEQ ID NO:5.

7. The antibody of claim 1 wherein the heavy chain variable region has an amino acid sequence set forth as SEQ ID NO:1 and/or the light chain variable region has an amino acid sequence set forth as SEQ ID NO:5.

8. The antibody of claim 1 which is a chimeric antibody

9. The antibody of claim 1 which is a humanized antibody.

10. The antibody of claim 1 which is selected from the group consisting of Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2 and diabodies.

11. A nucleic acid sequence which encodes a heavy chain and/or a light chain of an antibody which binds to a human neurotensin long fragment, and has a heavy chain variable region which comprises a H-CDR1 region having at least 90% identity with SEQ ID NO:2, a H-CDR2 region having at least 90% identify with SEQ ID NO:3 and a H-CDR3 region having at least 90% identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% identity with SEQ ID NO:6, a L-CDR2 having at least 90% identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% identity with SEQ ID NO:8.

12. A vector which comprises a nucleic acid sequence which encodes a heavy chain and/or a light chain of an antibody which binds to a human neurotensin long fragment, and has a heavy chain variable region which comprises a H-CDR1 region having at least 90% identity with SEQ ID NO:2, a H-CDR2 region having at least 90% identify with SEQ ID NO:3 and a H-CDR3 region having at least 90% identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% identity with SEQ ID NO:6, a L-CDR2 having at least 90% identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% identity with SEQ ID NO:8.

13. A host cell which has been transfected, infected or transformed by i) a nucleic acid sequence which encodes a heavy chain and/or a light chain of an antibody which binds to a human neurotensin long fragment, and has a heavy chain variable region which comprises a H-CDR1 region having at least 90% identity with SEQ ID NO:2, a H-CDR2 region having at least 90% identify with SEQ ID NO:3 and a H-CDR3 region having at least 90% identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% identity with SEQ ID NO:6, a L-CDR2 having at least 90% identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% identity with SEQ ID NO:8; or ii) a vector encoding the nucleic acid.

14. A method of treating cancer in a subject in need thereof comprising administering to the subject with a therapeutically effective amount of an antibody which binds to a human neurotensin long fragment, and has a heavy chain variable region which comprises a H-CDR1 region having at least 90% identity with SEQ ID NO:2, a H-CDR2 region having at least 90% identify with SEQ ID NO:3 and a H-CDR3 region having at least 90% identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% identity with SEQ ID NO:6, a L-CDR2 having at least 90% identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% identity with SEQ ID NO:8 or a heavy chain variable region having at least 70% identity with SEQ ID NO:1 and/or a light chain variable region having at least 70% identity with SEQ ID NO:5.

15. The method of claim 14 wherein the cancer is selected from the group consisting of neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

16. The method of claim 14 wherein the antibody is used in combination with a chemotherapeutic agent.

17. The method of claim 16 wherein the chemotherapeutic agent is cisplatin.

18. The method of claim 14 wherein the antibody is used in combination with a HER inhibitor.

19. The method of claim 18 wherein the HER inhibitor is a HER antibody selected from the group consisting of EGFR antibodies, HER2 antibodies, HER3 antibodies, and HER4 antibodies.

20. The method of claim 18 wherein the HER inhibitor is selected from the group consisting of small organic molecule HER antagonists; HER tyrosine kinase inhibitors; HER2 and EGFR dual tyrosine kinase inhibitors, and HER dimerization inhibitors.

21. The method of claim 18 wherein the HER inhibitor is selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab, erlotinib, gefitinib, lapatinib, neratinib, canertinib, vandetanib, afatinib, TAK-285, ARRY334543, Dacomitinib, OSI-420, AZD8931, AEE788, Pelitinib, CUDC-101, XL647, BMS-599626, PKC412, BIBX1382 and AP261 13.

22. The method of claim 18 wherein the HER inhibitor is a pan-HER inhibitor.

23. A pharmaceutical composition which comprises an antibody which binds to a human neurotensin long fragment, and has a heavy chain variable region which comprises a H-CDR1 region having at least 90% identity with SEQ ID NO:2, a H-CDR2 region having at least 90% identify with SEQ ID NO:3 and a H-CDR3 region having at least 90% identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% identity with SEQ ID NO:6, a L-CDR2 having at least 90% identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% identity with SEQ ID NO:8.

24. The antibody of claim 8, wherein the chimeric antibody is a chimeric mouse/human antibody.

Description

FIGURES

[0099] FIG. 1: FLp26-8.2 inhibits the proliferation inhibition of CHO over expressing NTSR1 induced by NTS or conditioned media from lung cancer cells overexpressing NTS. Results represent the mean±SEM of 3 independent experiments.

[0100] FIG. 2: FLp26-8.2 inhibits the cellular invasion induced by EGF and NTS in breast cancer cells. Synergism between NTS and EGF on invasion in a collagen 1 invasion assay of MCF-7 and NTS-1 cells. Cells were seeded on the top of a collagen 1 gel and treated with EGF (100 ng/mL). Results represent the mean±SEM of 3 experiments. Inset, NTS and NTSR1 transcript analysis from 200 ng of MCF-7, and NTS-h, total RNA.

[0101] FIG. 3. FLp26-8.2 inhibits the cellular invasion induced by NTS in Hepatocellular carcinoma. A) Migration in a collagen 1 invasion assay of Hep 3B, HepR1a, and HepR2b cells. Results represent the mean±SEM of 3 experiments. Results are expressed as a % on invasive cells of control cells, Hep3B. Inset, NTS and NTSR1 transcript analysis from 200 ng of Hep 3B, HepR1a, and HepR2b total RNA. B) Migration in a collagen 1 invasion assay of Hep 3B, HepR1a, and HepR2b cells in a presence of 3.75 μg/ml of purified FLp26-8.2 or mouse IgG. Results represent the mean±SEM of 3 experiments. Results are expressed as a % on invasive cells of respective control cells.

[0102] FIG. 4. FLp26-8.2 inhibits experimental tumor growth generated by lung cancer cell lines. A) LNM-R or R-SI NTSR1 cells (LNM-R expressing sh-RNA for NTSR1) were injected into the left and the right flank of the mice, respectively. Here is shown an example of a mouse from each group after 15 days of treatment. B) Tumor growth generated by LNM-R cells (left flank) xenografted into nude mice and treated for 15 days with PBS, or 15 mg/kg FLp26-8.2. At day one, 10, and 8 mice were randomized on LNM-R tumors size reaching approximately 40 mm3 for control and FLp26-8.2 group, respectively. Mice were treated every other day and measured every day. C) Tumor growth generated by R-SI NTSR1 cells (right flank) xenografted into the same mice. D and E) Tumor growth rate from day one generated by LNM-R cells (left flank) and R-SI NTSR1 cells (right flank).

[0103] FIG. 5. FLp26-8.2 inhibits experimental tumor growth generated by breast cancer cell lines. Tumor growth generated by MDA-MB 231 cells (left flank) (A), and MDA Si2 cells (right flank) (B), and treated for 24 days with PBS, or 15 mg/kg FLp26-8.2. At day one, 7, and 6 mice were randomized for the size of MDA-MB231 tumors; reaching approximately 50 mm3. Mice were treated and measured every other day.

[0104] FIG. 6. FLp26-8.2 restores cisplatin response to lung cancer cell lines expressing NTS and NTSR1. A) LNM-R or R-SI NTSR1 cells (LNM-R expressing sh-RNA for NTSR1) were injected into the left and the right flanks of the mice, respectively. Mice were treated with PBS, or 15 mg/kg FLp26-8.2 every other day and/or with cisplatin 1 mg/kg day 1,3, 5, 7, 15, 17, and 19. At day one, 9 and 8 mice were randomized for the size of the LNM-R tumors; reaching approximately 95 mm3 on average. Mice were treated every other day and measured every day. B) Tumor growth rate from day one generated by R-SI NTSR1 cells (right flank).

EXAMPLES

Example 1

Cloning and Sequencing of Antibody Variable Regions

[0105] Step 0: Peptide Synthesis and Conjugation

[0106] In order to inhibit NTS oncogenic action, we produced NTS monoclonal antibody directed against NTS long fragment (SEQ ID NO:9: mmagmkiqlv cmlllafssw slcsdseeem kaleadfltn mhtskiskah vpswkmtlln vcslvnnlns paeetgevhe eelvarrklp taldgfslea mltiyqlhki chsrafqhwe liqedildtg ndkngkeevi krkipyilkr qlyenkprrp yilkrdsyyy). The antigen peptide sequence chosen was SEQ ID NO:10 (CQEDILDTGNDKNGKE-amide MW 1777.9). 1.

[0107] Peptide synthesis was controlled by MS and HPLC. The peptide used was the lyophilised form as TFA-salt and conjugated with BSA.

[0108] Step 1 Immunisation

[0109] 5 mice were immunized with the antigen.

TABLE-US-00001 OD.sub.405nm after 15 min incubation with the substrate. The ELISA plates were coated with 50 μl/well p12026-BSA-conjugate (concentration 4 μg/ml). dilution of normal antiserum mouse 1 mouse 2 mouse 3 mouse 4 mouse 5 serum 1:100 2.523 2.918 2.796 3.236 2.183 0.032 1:200 2.167 2.428 2.216 2.899 1.764 0.016 1:400 1.442 1.629 1.221 2.336 1.291 0.016 1:800 0.759 0.879 0.435 1.398 0.881 0.012 1:1600 0.342 0.446 0.131 0.687 0.497 0.008 1:3200 0.145 0.215 0.042 0.289 0.273 0.010 1:6400 0.056 0.092 0.011 0.094 0.132 0.009 1:12800 0.027 0.037 0.002 0.032 0.063 0.004

[0110] The functional test was inhibition of morphological changes of CHO stably overexpressing NTSR1 and induced by 10.sup.−8 or 10.sup.−7 M JMV449 a weekly degradable NTS agonist, or the culture medium of LNM35 cells expressing NTS and NTSR1. The results were the following ones:

[0111] Serum from mouse #4 inhibited the morphology changes by 10%. The mice #3 and #5 were only inhibited by 5% as compared to pre-immune serum. The serum from mice 1 and mice 2 did not inhibit the morphology changes. Mice having a low antibody titer were reboosted in order to be able to perform the fusion.

[0112] Step 2 Fusion:

[0113] The mouse #4 was selected. 5 clones were obtained. Clones 1-6, 7-12, 8-2, 13-1, and 16-12 were tested. Two tests were performed for NTS induced CHO NTSR1 morphology changes and invasion test on type I collagen matrices of MCF-7 cells with overexpressing NTS. The experiments were repeated twice. Only the clone 8.2 inhibited the NTS effect in both tests from 40 to 70% according to the control. No effects were observed with the other clones.

[0114] Step 3: Final Selection

[0115] The clone 8-2 (i.e. FLp26-8.2) hybridoma was selected. Antibody was purified and tested in proliferation assays for CHO NTSR1, invasion assays of breast cancer cells expressing, or not NTS, and hepatocellular carcinomas expressing, or not, NTSR1. FLp26-8.2 was also tested on tumor growths of breast and lung cancer cells, and on their response to cisplatin in lung cancer model.

[0116] Step 4: Cloning and Sequencing:

[0117] Total RNA was prepared from 2×10.sup.7 of the cells from the first tube provided for each hybridoma using the Qiagen RNeasy mini kit (Cat No: 74104). RNA was eluted in 60 μL water and checked on a 1.2% agarose gel alongside Qarta Bio 1 Kb Markers (cat: M-DNA-1 Kb). V.sub.H and V.sub.K cDNAs were prepared using reverse transcriptase with IgG and kappa constant region primers. The first strand cDNAs were amplified by PCR using a large set of signal sequence primers. The amplified DNAs were gel-purified and cloned into the vector pGem T Easy (Promega). The V.sub.H and V.sub.K clones obtained were screened for inserts of the expected size. The DNA sequence of selected clones was determined in both directions by automated DNA sequencing. The locations of the CDRs in the sequences were determined with reference to other antibody sequences (Kabat E A et al., 1991)

[0118] A single productive V.sub.K sequence was identified in ten clones (eight independent). The sequences were identical apart from a single base change in one clone at position 33 and a single base change in another clone at position 315. A non-productive aberrant V.sub.K with an error in V-J joining and the aberrant V.sub.K sequence that arises from the fusion partner were also found. The deduced protein sequence with CDRs annotated is shown in Table A.

[0119] A single V.sub.K sequence was identified. Identical sequence was found in six independent clones apart from two single base changes at residues 22 and 243 in one clone, a single base change in one clone at position 103 and a single base change in another clone at position 261. The deduced protein sequence with CDRs annotated is shown in Table A.

TABLE-US-00002 TABLE A Sequences of FLp26-8.2 antibody Domain Sequences VH QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAP GKGLKWMGWITTNTGEPTYAEEFKGRFAFSLETSASTAYLQ INNLKNEDTATYFCARRAFAMDYWGQGTSVTVSS  (SEQ ID NO: 1) H-CDR1 GYTFTNYGMN (SEQ ID NO: 2) H-CDR2 WITTNTGEPTYAEEFKG (SEQ ID NO: 3) H-CDR3 RAFAMDY (SEQ ID NO: 4) VL DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLY WFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLR ISRVEAEDVGVYYCMQHLEYPYTEGGGTKLEIK  (SEQ ID NO: 5) L-CDR1 RSSKSLLHSNGNTYLY (SEQ ID NO: 6) L-CDR2 RMSNLAS (SEQ ID NO: 7) L-CDR3 MQHLEYPYT (SEQ ID NO: 8)

Example 2

Functional Assays

[0120] Material & Methods:

[0121] Cell Proliferation 20 000 CHO-NTSR1 cells were seeded in 48 well dishes, in 200 ul media 10% with FCS. The next day the cells were treated with 10.sup.−7 M Neurotensin or ½ LNM-R conditioned media which were pre-incubated for 2 h at room temperature in the presence or not of 2.8 μg antibody FLp26-8.2 or P27-7.4. The conditioned media was prepared as follows. 3 million LNM-R cells were seeded in 75 cm.sup.2 flask in media with 10% FCS. Cells were grown for 24 h, media were removed, and 10m1 serum free media were added. The media were collected and centrifuged 5 mins at 500 g after 48 h. The supernatant was aliquoted and freeze at −20° C. until used. Cells were treated for 40 h in 200 μl media with 2.5% FCS. Cellular proliferation was evaluated by counting using Beckman Coulter's cell counting.

[0122] Invasion Assay

[0123] The cell culture insert (8 μm, Beckton Dickinson®) was coated with type I collagen (100 μl/well, 4×10.sup.2 μg/ml, Sigma®) at 37° C. 24 h before the assay. 1 million MCF, NTS-h, Hep 3B, HepR1a, and HepR2b cells were seeded in the insert with 250 μl of serum free medium in presence or absence of 7 μg antibodies FLp26-8.2. Outside the insert, 750 μl medium with 10% FCS was added in the well as chemoattractant. After 48 h of incubation, the non-invading cells and collagen are removed from the upper surface of the membrane by scrubbing with a cotton swab. Invading cells (those adhering to the bottom surface of the membrane) were fixed and stained with the Kwif diff stain kit(Thermo®) and the number of stained cells were counted with an inverted microscope at 200× magnification.

[0124] Tumor Xenografts

[0125] 1×10.sup.6 LMN-R cells and 1×10.sup.6 R-SI NTSR1 were subcutaneously inoculated in NMRI nu/nu mice, LMN-R in the left flank and R-SI NTSR1 in the right flank. When tumors generated by LMN-R reached an average volume of 40 mm.sup.3, mice were randomized in 2 groups, then administered antibody FLp26-8.2 (i.p. every other day, 15 mg/kg) for total 8 times. PBS was used as the vehicle control. The volume of tumor was measured daily.

[0126] 3×10.sup.6 MDA-MB 231 cells and 3×10.sup.6 MDA si2 were subcutaneously inoculated in 100 μl of matrigel the left flank and the right flank of the NMRI nu/nu mice, respectively. When tumors generated by MDA reached an average volume of 50 mm.sup.3, mice were randomized in 2 groups, then administered antibody FLp26-8.2 (i.p. every other day, 15 mg/kg) for total 12 times. PBS was used as the vehicle control.

[0127] Cisplatin test, 1×10.sup.6 LMN-R cells and 1×10.sup.6 R-SI NTSR1 were subcutaneously inoculated in NMRI nu/nu mice, LMN-R in the left flank and R-SI NTSR1 in the right flank. When tumors generated by LMN-R reached an average volume of 95 mm.sup.3, mice were randomized in 5 groups of 8 to 10 mice. Group 1 was administered with PBS, group 2 with antibody FLp26-8.2 (i.p. every other day, 15 mg/kg) for total of 13 times and group 3 with mouse IgG (i.p. every other day, 15 mg/kg). Group 4 was administered with PBS and cisplatin (1 mg/kg at day 1,3 5 7, 15, 17, and 19) and groups 5 with antibody FLp26-8.2 (i.p. every other day, 15 mg/kg) and cisplatin (1 mg/kg at day 1,3 5 7, 15, 17, and 19). The volume of tumor was measured every other day.

[0128] Results:

[0129] Neutralization of NTS Induced Proliferation Inhibition of CHO Over Expressing NTSR1.

[0130] LNM-R cells expressed NTS and NTSR1. NTS is released in the media and was assay by radioimmunoassay. Culture medium (CM) of LNM-R cells contained 76.4±10.3, 153.2±25.3, and 624.3±81.8 fmol/mL of NTS corresponding to 14, 48, and 72 hours of culture, respectively. When CHO NTSR1 overexpressing cells are exposed to NTS, cells change shape which induced a decrease in proliferation rate. In FIG. 1, cell growth is reduced by 60% when cells are treated by NTS or CM, when the purified monoclonal antibody FLp26-8.2 is added to the media this cellular growth inhibition is reduced to 48% and 15%, for NTS or CM treated cells, respectively.

[0131] Neutralization of NTS Induced Invasion of Breast Cancer Cell Expressing NTS.

[0132] The breast cancer cell line, MCF-7, constitutively expressing NTSR1 was stably transfected with the neurotensin full length coding sequence. An overexpressing NTS clone, NTS-h, was selected (FIG. 2 inset). The invasiveness properties of NTS-h, was studied using a 3 dimensional collagen invasion assay. The ectopic NTS expression of MCF-7 cells induced a small increase in invasiveness properties. EGF-induced invasion was tripled in NTS-overexpressing cells as compared to MCF-7 (FIG. 2). The induction of invasiveness induced or not by EGF was inhibited by FLp26-8.2 only in the NTS-overexpressing clones. This confirms the neutralizing properties of the NTS monoclonal antibody FLp26-8.2.

[0133] Neutralization of NTS Induced Invasion of Liver Cancer Cell Expressing NTSR1.

[0134] Hep3B is a Hepatocellular carcinoma which constitutively expresses NTS, but not NTSR1. The Hep3B cells were stably transfected with NTSR1 coding sequence and two clones were selected, Hep-R1a and Hep-R1b (FIG. 3A inset). The invasiveness properties of cells were studied using a 3 dimensional collagen invasion assay. The ectopic expression of NTSR1 largely increased the invasive properties of the cells as shown in FIG. 3A. When cells were exposed to FLp26-8.2, the invasive rate of the cells expressing NTS and NTSR1 was reduced, and more drastic for the cells for which the increase of invasive rate was moderate, Hep-R1a. Both results confirm the neutralizing properties of this NTS antibody on cell invasiveness.

[0135] FLp26-8.2 Reduced Tumor Growth Specifically in Tumor Expressing NTS and NSTR1.

[0136] The efficiency of FLp26-8.2 to decrease tumor growth was tested on lung cancer experimental tumors generated by cells expressing LNM-R cells, expressing NTS and NTSR1 or R-SI NTSR1 cells which express only NTS. The R-SI NTSR1 cells are a clone obtained from LNM-R and stably transfected with NTSR1 Sh RNA. Mice were grafted with both cell lines. LNM-R cells on the left flank and R-SI NTSR1 cells on the right flank (FIG. 4A). Mice were randomized with LNM-R tumors 42±11 mm.sup.3 and 43±8 mm.sup.3 for PBS and FLp26-8.2 treated group, respectively. The tumor size of animals treated with the FLp26-8.2 was 2.6 times smaller as compared to controls (FIG. 4B). The doubling time after 16 days of treatment was 3.43±0.34 and 5.96±0.55 days for control and FLp26-8.2 treated animals, respectively. The growth from D1 was 24.68±4.05 fold for PBS treated animals and only of 5.92±0.99 for FLp26-8.2 treated animals (FIG. 4D). The specificity and the efficiency of the antibody was confirmed when the tumors carrying the non-expressing NTSR1, in the same mice, were analyzed. For R-I NTSR1 tumors, the size of the tumor and the growth rate were not different whether the mice were treated with PBS or FLp26-8.2 (FIGS. 4C and 4E).

[0137] In the same vein FLp26-8.2 was also shown to efficiently reduce tumor growth generated by breast cancer cells. MDA-MD 231 expressing NTS and NTSR1 and its subclone MDA Si2 stably transfected with sh NSTR1, were xenografted on the flank on mice. MDA-MD 231 cells on the left flank and MDA Si2 cells on the right flank. MDA Si2 cells were injected a few days before MDA MD 231. Mice were randomized with MDA-MB231 tumors, as follows 52±10 mm.sup.3 for control group and 51.8±6 mm.sup.3 for FLp26-8.2 group. FIG. 5A shows a strong reduction of MDA-MB231 tumor growth from cells by FLp26-8.2 as compared to PBS treated animals. The growth rate from D1 was 4.4±0.33 fold for PBS treated animals and only of 2.1±0.18 for FLp26-8.2 treated animals. The doubling time after 24 days of treatment was 11.63±1 and 55.5±32 days for control and FLp26-8.2 treated animals, respectively. The same parameters analyzed on the MDA Si2 tumors (NTSR1-) showed no difference between the tumor size (FIG. 5B), the growth rate and the doubling time.

[0138] FLp26-8.2 Restores Cisplatin Response

[0139] The ability of FLp26-8.2 to restore cisplatin response was tested on lung cancer experimental tumors generated by cells expressing LNM-R cells, expressing NTS and NTSR1, or R-SI NTSR1 cells which only express NTS. Mice were grafted with both cell lines: LNM-R cells on the left flank and R-SI NTSR1 cells on the right flank.

[0140] Mice were randomized with LNM-R tumors 96.3±18.5, 87.1±8.4, 91.3±14.9, 96.9±11.2 and 91.4±10.1 mm.sup.3 for PBS, FLp26-8.2, PBS and cisplatin, FLp26-8.2 and cisplatin, or Mouse IgG treated group, respectively. Due to the large size of the generated tumors the experiments were stopped after 21 days for the control group, 23 days for IgG and PBS cisplatin group, 25 days for FLp26-8.2 group and 27 days for FLp26-8.2 and cisplatin group. For LNM-R tumors, the size of the tumors is expressed as a function of time in FIG. 6A. As R-SI NTSR1 tumors could not be randomized, the result are presented as the growth ratio to D1 (FIG. 6B).

[0141] LNM-R tumor growth rates were not altered by cisplatin treatment or purified IgG treatment, as compared to PBS treated mice (FIG. 6A). As previously shown, when animals are treated with FLp26-8.2, the LNM-R tumor size is smaller. The size tumor is stabilized when animals are treated with FLp26-8.2 and cisplatin (FIG. 6A).

[0142] The R-SI NTSR1 tumor size is decreased when animals are treated with cisplatin, indicating that NTS/NTSR1 complex is implicated in the cellular resistance to cisplatin (FIG. 6B). As expected, combined treatment with FLp26-8.2 did not change the tumor growth rate.

[0143] In conclusion, treatment with FLp26-8.2 restores cisplatin sensitivity in cells expressing NTS and NTSR1, and can be proposed to patients with NSLCL expressing high levels of NTSR1.

REFERENCES

[0144] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0145] 1. Carraway, R. & Leeman, S. E. (1973) J. Biol. Chem. 248, 6854-6861.

[0146] 2. Rosell, S. (1980) Soc. Gen. Physiol Ser. 35, 147-162.

[0147] 3. Rosell, S., Al-Saffar, A., & Thor, K. (1984) Scand. J. Gastroenterol. Suppl 96, 69-75

[0148] 4. Vincent, J. P., Mazella, J., & Kitabgi, P. (1999) Trends Pharmacol. Sci. 20, 302-309.

[0149] 5. Thomas, R. P., Hellmich, M. R., Townsend, C. M., Jr., & Evers, B. M. (2003) Endocr. Rev. 24, 571-599.

[0150] 6. Wu, Z., Martinez-Fong, D., Tredaniel, J., & Forgez, P. (2012) Front Endocrinol. (Lausanne) 3, 184.

[0151] 7. Dupouy, S., Mourra, N., Doan, V. K., Gompel, A., Alifano, M., & Forgez, P. (2011) Biochimie 93, 1369-1378.

[0152] 8. Alifano, M., Souaze, F., Dupouy, S., Camilleri-Broet, S., Younes, M., hmed-Zaid, S. M., Takahashi, T., Cancellieri, A., Damiani, S., Boaron, M. et al. (2010) Clin. Cancer Res. 16, 4401-4410.

[0153] 9. Dupouy, S., Viardot-Foucault, V., Alifano, M., Souaze, F., P1u-Bureau, Chaouat, M., Lavaur, A., Hugol, D., Gespach, C., Gompel, A. et al. (2009) PLoS. One. 4, e4223.

[0154] 10. Shimizu, S., Tsukada, J., Sugimoto, T., Kikkawa, N., Sasaki, K., Chazono, H., Hanazawa, T., Okamoto, Y., & Seki, N. (2008) Int. J. Cancer 123, 1816-1823.

[0155] 11. Guha, S., Rey, O., & Rozengurt, E. (2002) Cancer Res. 62, 1632-1640.

[0156] 12. Heakal, Y., Woll, M. P., Fox, T., Seaton, K., Levenson, R., & Kester, M. (2011) Cancer Biol. Ther. 12, 427-435.

[0157] 13. Ishizuka, J., Townsend, C. M., Jr., & Thompson, J. C. (1993) Ann. Surg. 217, 439-445.

[0158] 14. Somai, S., Gompel, A., Rostene, W., & Forgez, P. (2002) Biochem. Biophys. Res. Commun. 295, 482-488.

[0159] 15. Souaze, F., Dupouy, S., Viardot-Foucault, V., Bruyneel, E., Attoub, S., Gespach, C., Gompel, A., & Forgez, P. (2006) Cancer Res. 66, 6243-6249.

[0160] 16. Iwase, K., Evers, B. M., Hellmich, M. R., Kim, H. J., Higashide, S., Gully, D., & Townsend, C. M., Jr. (1996) Surg. Oncol. 5, 245-251.

[0161] 17. Yoshinaga, K., Evers, B. M., Izukura, M., Parekh, D., Uchida, T., Townsend, C. M., Jr., & Thompson, J. C. (1992) Surg. Oncol. 1, 127-134.

[0162] 18. Nakaizumi, A., Uehara, H., Baba, M., Iishi, H., & Tatsuta, M. (1996) Cancer Lett. 110, 57-61.

[0163] 19. Tatsuta, M., Iishi, H., Baba, M., & Nakaizumi, A. (1991) Int. J. Cancer 47, 408-412.

[0164] 20. Servotte, S., Camby, I., Debeir, 0., Deroanne, C., Lambert, C. A., Lapiere, C. M., Kiss, R., Nusgens, B., & Decaestecker, C. (2006) Neuropathol. Appl. Neurobiol. 32, 575-584.

[0165] 21. Wu, Z., Martinez-Fong, D., Tredaniel, J., & Forgez, P. (2012) Front Endocrinol. (Lausanne) 3, 184.

[0166] 22. Ehlers, R. A., Zhang, Y., Hellmich, M. R., & Evers, B. M. (2000) Biochem. Biophys. Res. Commun. 269, 704-708.

[0167] 23. Gully, D., Labeeuw, B., Boigegrain, R., Oury-Donat, F., Bachy, A., Poncelet, M., Steinberg, R., Suaud-Chagny, M. F., Santucci, V., Vita, N. et al. (1997) J. Pharmacol. Exp. Ther. 280, 802-812.

[0168] 24. Hassan, S. & Carraway, R. E. (2006) Regul. Pept. 133, 105-114.

[0169] 25. Hassan, S., Dobner, P. R., & Carraway, R. E. (2004) Regul. Pept. 120, 155-166.

[0170] 26. Kisfalvi, K., Guha, S., & Rozengurt, E. (2005) J. Cell Physiol 202, 880-890.

[0171] 27. Servotte, S., Camby, I., Debeir, O., Deroanne, C., Lambert, C. A., Lapiere, C. M., Kiss, R., Nusgens, B., & Decaestecker, C. (2006) Neuropathol. Appl. Neurobiol. 32, 575-584.

[0172] 28. Zhao, D., Kuhnt-Moore, S., Zeng, H., Wu, J. S., Moyer, M. P., & Pothoulakis, C. (2003) Am. J. Physiol Cell Physiol 284, C1397-C1404.

[0173] 29. Leyton, J., Garcia-Marin, L., Jensen, R. T., & Moody, T. W. (2002) Eur. J. Pharmacol. 442, 179-186.

[0174] 30. Lee, L. F., Guan, J., Qiu, Y., & Kung, H. J. (2001) Mol. Cell Biol. 21, 8385-8397.