ANTI-NEUROTENSIN 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 in the, 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 human neurotensin, 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.

2. The neutralising 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 the H-CDR1 region, SEQ ID NO:3 for the H-CDR2 region and SEQ ID NO:4 for the H-CDR3 region.

3. The neutralising 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. The neutralising 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 and 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.

5. The neutralising antibody of claim 1 wherein the heavy chain variable region comprises 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 wherein the light chain variable region comprises 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 neutralising antibody of claim 1 wherein the heavy chain variable region has at least 70% identity with SEQ ID NO:1 and/or the light chain variable region has at least 70% identity with SEQ ID NO:5.

7. The neutralising 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 neutralising antibody of claim 1 which is a chimeric antibody.

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

10. The neutralising 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 human neurotensin, 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 human neurotensin, 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 a nucleic acid sequence which encodes a heavy chain and/or a light chain of an antibody which binds to human neurotensin, 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:81; or a vector comprising the nucleic acid.

14. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an antibody which binds to human neurotensin, 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:81.

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; hemangio sarcoma; 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 human neurotensin, 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:81.

Description

FIGURES

[0100] FIG. 1: NTSp27-7.4 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.

[0101] FIG. 2: NTSp27-7.4 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 type 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.

[0102] FIG. 3. NTSp27-7.4 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 type 1 invasion assay of Hep 3B, HepR1a, and HepR2b cells in a presence of 3.75 μg/ml of purified NTSp27-7.4 or mouse IgG. Results represent the mean±SEM of 3 experiments. Results are expressed as a % on invasive cells of respective control cells.

[0103] FIG. 4. NTSp27-7.4 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 NTSp27-7.4. At day one, 10, and 8 mice were randomized on LNM-R tumors size reaching approximately 40 mm3 for control and NTSp27-7.4 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).

[0104] FIG. 5. NTSp27-7.4 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 NTSp26-8.2. At day one, 7, and 6 mice were randomized for the size of MDA-MB231 tumors size; reaching approximately 50 mm3. Mice were treated and measured every other day.

[0105] FIG. 6. NTSp27-7.4 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 NTSp27-7.4 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).

[0106] FIG. 7. NTSp27-7.4 induces apoptosis in ovarian cancer cell lines expressing NTS and NTSR1. A) NTS, NTSR2, and NTSR1 transcript analysis from 100 ng of A2780 and SKOV3 total RNA. B) The Annexin-V-FITC Apoptosis Detection Kit (Roche) was used to detect apoptosis by flow cytometry. (Left) Example of FACS analysis on cells treated or not with 50 μg/ml of NTS p27-7.4 for 24 h. (Right) Apoptosis levels of NTS p27-7.4 treated cells presented as the fold increase relative to the apoptosis of untreated cells n=3.

[0107] FIG. 8. NTSp27-7.4 restores cisplatin response to ovarian cancer cell lines expressing NTS and NTSR1. 3 million A2780 cells were injected into mice. When tumors generated by A2780 reached an average volume of 200 mm.sup.3, mice were randomized into 4 groups. Mice were treated with PBS, or 15 mg/kg NTSp27-7.4 every other day and/or with cisplatin 1 mg/kg day at 1, 3, 5, and 7 days. Results are the mean of 8 to 12 mice performed in two different independent experiments.

EXAMPLES

Example 1: Cloning and Sequencing of Antibody Variable Regions

[0108] Step 0: Peptide Synthesis and Conjugation

[0109] 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 (CQLYENKPRRPYIL-amide MW 1792.2). 1. Peptide synthesis was controlled by MS and HPLC. The peptide used was the lyophilised form as TFA-salt and conjugated with BSA.

[0110] Step 1 Immunisation

[0111] 5 mice were immunized with the antigen.

TABLE-US-00001 OD.sub.405 nm after 15 min incubation with the substrate. The ELISA plates were coated with 50 μl/well p12027-BSA-conjugate (concentration 4 μg/ml). dilution of normal antiserum mouse 1 mouse 2 mouse 3 mouse 4 mouse 5 serum 1:100 2.282 2.236 2.110 2.262 2.364 0.019 1:200 2.278 2.260 2.207 2.281 2.332 0.015 1:400 2.404 2.372 2.346 2.404 2.262 0.011 1:800 2.423 2.381 2.488 2.475 2.292 0.009 1:1600 2.306 2.236 2.400 2.391 2.016 0.015 1:3200 2.058 2.018 2.285 2.199 1.726 0.008 1:6400 1.668 1.620 1.927 1.866 1.286 0.000 1:12800 1.198 1.183 1.451 1.426 0.885 0.000

[0112] 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: Serum from mice #3 and 5 inhibited the morphology changes by 15% and 10% respectively, and the mouse #1 only by 5% compare to preimmune serum. The serum of the mice 2 and 4 did not inhibit the morphology changes.

[0113] Step 2 Fusion:

[0114] The mouse 3 and 5 were selected. Two clones were positive after the first cloning test. Clones 1-5, 7-4 were tested.

[0115] Two test were performed 1) the NTS induced CHO NTSR1 morphology changes test and 2) the invasion test on type I collagen matrices of MCF-7 cell overexpressing NTS. The experiment was repeated twice.

[0116] The clone 1.5 inhibited the NTS effect in both tests from 50 to 60%, and the clone 7.4 inhibited the NTS effect in both tests from 10 to 20% according to the control.

[0117] Unfortunately the clone 1.5 was very unstable and got lost.

[0118] Step 3: Final Selection

[0119] Finally, clone 7.4 (i.e. NTSp27-7.4) hybridoma was selected. Antibody was purified and tested in proliferation assay of CHO NTSR1, invasion assay of breast cancer cells expressing of not NTS, hepatocellular carcinoma expressing or not NTSR1. NTSp27-7.4 was also tested on tumor growth of breast and lung cancer cells, and on the response to cisplatin in lung cancer model. (See result section).

[0120] Step 4: Cloning and Sequencing:

[0121] 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)

[0122] 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.

[0123] A single V.sub.H 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 NTSp27-7.4 antibody Domain Sequences VH DVKLVESGGGLVKLGGSLKLSCAASGFTFSGYYMSWVRQTP EKRLELVAAINNYGDNTNYPDTVKGRFSVSRDNAKNTLYLE MNSLKSEDTALYYCARLANYANQRGAMDYWGQGTSVTVSS (SEQ ID NO: 1) H- GFTFSGYYMS (SEQ ID NO: 2) CDR1 H- AINNYGDNTNYPDTVKG (SEQ ID NO: 3) CDR2 H- LANYANQRGAMDY (SEQ ID NO: 4) CDR3 VL DIQMTHTTSSLSASLGDRVTISCRASQDIANYLNWYQQKPD GTVTLLIYYTSRLHSGVPSRFSGSGSGTDYSLTINNLDQED IATYFCQQGYTLPPTFGGGTKLEIK (SEQ ID NO: 5) L- RASQDIANYLN (SEQ ID NO: 6) CDR1 L- YTSRLHS (SEQ ID NO: 7) CDR2 L- QQGYTLPPT (SEQ ID NO: 8) CDR3

Example 2: Functional Assays

[0124] Material & Methods:

[0125] Cell Proliferation

[0126] 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 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 10 ml 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.

[0127] Invasion Assay

[0128] 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 NTSp27-7.4. 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.

[0129] Tumor Xenografts

[0130] 1×10.sup.6 LMN-R cells and 1×10.sup.6R-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 NTSpP27-7.4 (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.

[0131] 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 NTSpP27-7.4 (i.p. every other day, 15 mg/kg) for total 12 times. PBS was used as the vehicle control.

[0132] 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 NTSp27-7.4 (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 NTSp27-7.4 (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.

[0133] FACS Analysis

[0134] The Annexin-V-FITC Apoptosis Detection Kit (Roche) was used to detect apoptosis by flow cytometry. Cells were exposed or not to 50 μg/ml of NTS p27-7.4 in 0.5% FCS for 24 h. Cells were harvested by accutase (including detached cells), and detected in a Flow Cytometer (BD LSR II).

[0135] A2780 cells (3×10.sup.6) cells were combined with 50 μl PBS and 50 μl matrigel, then injected subcutaneously into the right hind leg of 4 week old female NMRI-Nude Foxn1 mice. Treatment started when tumors reached approximately 200 mm3. Tumor volume was calculated every two days by using the following equation: tumor volume (mm3)=4/3×π× (length/2)×(width/2)×(height/2). After treatment, the mice were sacrificed and tumors were fixed in formalin.

[0136] Results:

[0137] Neutralization of NTS Induced Proliferation Inhibition of CHO Over Expressing NTSR1.

[0138] 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 NTSp27-7.4 is added to the media this cellular growth inhibition is reduced to 30% and 26%, for NTS or CM treated cells, respectively.

[0139] Neutralization of NTS Induced Invasion of Breast Cancer Cell Expressing NTS.

[0140] 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 NTSp27-7.4 only in the NTS-overexpressing clones. This confirms the neutralizing properties of the NTS monoclonal antibody NTSp27-7.4.

[0141] Neutralization of NTS Induced Invasion of Liver Cancer Cell Expressing NTSR1.

[0142] 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 NTSp27-7.4, 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.

[0143] NTSp27-7.4 Reduced Tumor Growth Specifically in Tumor Expressing NTS and NSTR1.

[0144] The efficiency of NTSp27-7.4 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 NTSp27-7.4 treated group, respectively. The tumor size of animals treated with the NTSp27-7.4 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 5.11±0.38 days for control and NTSp27-7.4 treated animals, respectively. The growth from D1 was 24.68±4.05 fold for PBS treated animals and only of 8.22±1.84 for NTSp27-7.4 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 NTSp27-7.4 (FIGS. 4C and 4E).

[0145] In the same vein NTSp27-7.4 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 50.7±6.3 mm.sup.3 for NTSp27-7.4 group. FIG. 5A shows a strong reduction of MDA-MB231 tumor growth from cells by NTSp27-7.4 as compared to PBS treated animals. The growth rate from D1 was 4.4±0.33 fold for PBS treated animals and only of 1.5±0.1 for NTSp27-7.4 treated animals. The doubling time after 24 days of treatment was 11.63±1 and 52.8±17.6 days for control and NTSp27-7.4 treated animals, respectively. The same parameters analyzed on the MDA Si2 tumors (NTSR1−) showed no difference between the tumor sizes (FIG. 5B), the growth rate and the doubling time.

[0146] NTSp27-7.4 Restores Cisplatin Response

[0147] The ability of NTSp27-7.4 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 express only NTS. Mice were grafted with both cell lines. LNM-R cells on the left flank and R-SI NTSR1 cells on the right flank.

[0148] Mice were randomized with LNM-R tumors 96.3±18.5, 96.4±14.2, 91.3±14.9, 92.9±10.6 and 91.4±10.1 mm.sup.3 for PBS, NTSp27-7.4, PBS and cisplatin, NTSp27-7.4 and cisplatin, or Mouse IgG treated group, respectively. Because the large size of the tumors the experiment was stopped after 21 for the control group, 23 days for IgG and PBS cisplatin group, 25 days for NTSp27-7.4 group and 27 days for NTSp27-7.4 and cisplatin group. For LNM-R tumors, the size or the tumor with time are shown in FIG. 6A. As R-SI NTSR1 tumors could not be randomized, the result are presented as the growth ratio to D1 (FIG. 6B).

[0149] LNM-R tumor growth rate 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 NTSp27-7.4 the LNM-R tumor size is smaller. The size tumor is stabilized when animals are treated with NTSp27-7.4 and cisplatin (FIG. 6A).

[0150] 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 NTSp27-7.4 do not change the tumor growth rate.

[0151] Neutralization of NTS Induced Apoptosis of Ovarian Cancer Cell Expressing NTSR1.

[0152] A 2780 and SKOV3 are human ovarian carcinoma cells which constitutively express NTS, NTSR1 and NTSR2 as shown in FIG. 7A. Apoptosis induced by NTS antibody was by Annexin V-FITC/PI staining after treating cells with NTS p27-7.4 for 24 hours, followed by fluorescence activated cell sorting analysis. Gate settings distinguish between living (bottom left), necrotic (top left), early apoptotic (bottom right), and late apoptotic (top right) cells. The two-dimensional dot plots show an increase of the fluorescent dots in early apoptotic, and late apoptotic cells in both cell lines. Levels of NTS p27-7.4 induced apoptosis presented as fold increase relative to control cells is 2.5% and 8.2% for A2780 and SKOV3 cells, respectively (FIG. 7B).

[0153] The ability of NTSp27-7.4 to restore cisplatin response was tested on ovarian cancer experimental tumors generated by A 2780 cells, expressing NTS and NTSR1. Mice were randomized as follows 201.3±33.8, 217.15±47.6, 203.5±32.6, and 205.9±46 5 mm.sup.3 for PBS, NTSp27-7.4, PBS and cisplatin, or NTSp27-7.4 and cisplatin, treated group, respectively. A2780 tumor growth rates were slightly but not significantly altered by cisplatin treatment as compared to PBS treated mice (FIG. 8). When animals are treated with NTSp27-7.4 the A2780 tumor size was smaller 2062±371.5 mm.sup.3, and 1280±262.0 mm.sup.3 for PBS and NTSp27-7.4 treated group, respectively, p=0.054 vs PBS group. The tumor growth rate is more reduced when animals are treated with NTS p27-7.4 and cisplatin to 870.75±92.8 mm.sup.3 p=0.0058 vs cisplatin group (FIG. 8).

[0154] In conclusion, the treatments with NTSp27-7.4 restore the sensitivity to cisplatin of cells expressing NTS and NTSR1, and can be proposed to patients with NSLCL and Ovarian cancer expressing high levels of NTSR1.

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