Abstract
The present disclosure relates to human monoclonal antibodies against orexin receptor type 1 (OX1R, hyprocretin 1) and uses thereof for the treatment of cancer. The antibodies are characterized by their CDRs: NYYMN, YISGSSRNIYYADFVKG, SNYDGMDV (Heavy chain) and AGTSSDVGGSNYVS, PGKAP, SSYTYYSTRV (Light Chain)) or the CDRS having at least 50% or 70% identity with the above listed sequences.
Claims
1. A human monoclonal antibody against OX1R comprising a heavy chain comprising i) a H-CDR1 having at least 50% identity with a H-CDR1 of C2, ii) a H-CDR2 having at least 50% identity with a H-CDR2 of C2 and iii) a H-CDR3 having at least 50% identity with a H-CDR3 of C2 of and/or a light chain comprising i) a L-CDR1 having at least 50% identity with a L-CDR1 of C2, ii) a L-CDR2 having at least 50% identity with a L-CDR2 of C2 and iii) a L-CDR3 having at least 50% identity with a L-CDR3 of C2 wherein the H-CDR1 of C2 is defined by a sequence ranging from an amino acid residue at position 31 to an amino acid residue at position 35 in SEQ ID NO:1, the H-CDR2 of C2 is defined by a sequence ranging from an amino acid residue at position 50 to an amino acid residue at position 66 in SEQ ID NO:1, the H-CDR3 of C2 is defined by a sequence ranging from an amino acid residue at position 99 to an amino acid residue at position 109 in SEQ ID NO:1, the L-CDR1 of C2 is defined by a sequence ranging from an amino acid residue at position 23 to an amino acid residue at position 36 in SEQ ID NO:2, the L-CDR2 of C2 is defined by a sequence ranging from an amino acid residue at position 52 to an amino acid residue at position 58 in SEQ ID NO:2, and, the L-CDR3 of C2 is defined by a sequence ranging from an amino acid residue at position 91 to an amino acid residue at position 100 in SEQ ID NO:2.
2. The human monoclonal antibody of claim 1 comprising a heavy chain comprising i) a H-CDR1 having at least 50% identity with the H-CDR1 of C2, ii) a H-CDR2 having at least 50% identity with the H-CDR2 of C2 and iii) a H-CDR3 having at least 50% identity with the H-CDR3 of C2 and a light chain comprising i) a L-CDR2 having at least 50% identity with the L-CDR1 of C2, ii) a L-CDR2 having at least 50% identity with the L-CDR2 of C2 and iii) a L-CDR3 having at least 50% identity with the L-CDR3 of C2.
3. The human monoclonal antibody of claim 1 which comprises a heavy chain having i) the H-CDR1 of C2, ii) the H-CDR2 of C2 and iii) the H-CDR3 of C2.
4. The human monoclonal antibody of claim 1 which comprises a light chain having i) the L-CDR1 of C2, ii) the L-CDR2 of C2 and iii) the L-CDR3 of C2.
5. The human monoclonal antibody of claim 1 which comprises a heavy chain having i) the H-CDR1 of C2, ii) the H-CDR2 of C2 and iii) the H-CDR3 of C2 and a light chain having i) the L-CDR1 of C2, ii) the L-CDR2 of C2 and iii) the L-CDR3 of C2.
6. The human monoclonal antibody of claim 1 which is an antibody comprising a heavy chain having at least 70% identity with SEQ ID NO:1.
7. The human monoclonal antibody of claim 1 which is an antibody comprising a light chain having at least 70% identity with SEQ ID NO:2.
8. The human monoclonal antibody of claim 1 which is an antibody comprising a heavy chain having at least 70% identity with SEQ ID NO:1 and a light chain having at least 70% identity with SEQ ID NO:2.
9. The human monoclonal antibody of claim 1 which is an antibody comprising a heavy chain which is identical to SEQ ID NO:1.
10. The human monoclonal antibody of claim 1 which is an antibody comprising a light chain identical to SEQ ID NO:2.
11. The human monoclonal antibody of claim 1 which is an antibody comprising a heavy chain identical to SEQ ID NO:1 and a light chain identical to SEQ ID NO:2.
12. A fragment of the human monoclonal 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.
13. A nucleic acid molecule which encodes a heavy chain and/or a light chain of the human monoclonal antibody of claim 1.
14. A vector comprising the nucleic acid molecule of claim 13.
15. A host cell comprising the nucleic acid molecule of claim 13.
16. (canceled)
17. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the human monoclonal antibody of claim 1.
18. A pharmaceutical composition which comprises the human monoclonal antibody of claim 1.
19. A host cell comprising the vector of claim 14.
Description
FIGURES
[0127] FIG. 1: Effect of OxB and anti-OX1R antibodies including B4, B10, C1, C2, D4, E7, H7 on the cell growth of HEK-OX1R cells expressing recombinant OX1R. Cells were treated for 48 h with 0.1 μM of each compound and then cells were counted. Results were expressed as the percentage of untreated cell number (Control).
[0128] FIG. 2: Effect of OxB and anti-OX1R antibodies including B4, B10, C1, C2, D4, E7, 117 on the cell growth of HEK cells which do not express OX1R. Cells were treated for 48 h with 0.1 μM of each compound and then cells were counted. Results were expressed as the percentage of untreated cell number (Control).
[0129] FIG. 3: Effect of OxB and anti-OX1R antibodies including B4, B10, C1, C2, D4, E7, H7 on the cell growth of HEK-OX1R in the presence of NSC87877. SHP-2 protein tyrosine phosphatase inhibitor, NSC-87877, blocks orexin-induced apoptosis. Cells were treated for 48 h with 0.104 of each compound and then cells were counted. Results were expressed as the percentage of untreated cell number (Control).
[0130] FIG. 4: Effect of OxB, OxA and anti-OX1R antibodies including C1, C2, B6, H7 on the cell growth of HEK-mouseOX1R (left) and CHO-ratOX1R. Cells were treated for 48 h with 0.1 μM of each compound and then cells were counted. Results were expressed as the percentage of untreated cell number (Control).
[0131] FIG. 5: Effect of OxB and anti-OX1R antibodies including B4, B10, C1, C2, D4, E7, H7 on the cell growth of colon cancer cells lines SW48 (top left) and LoVo (top right) which express OX1R; colon cancer cell line HCT-116 (bottom right) which does not expressed OX1R; and pancreas cancer cell line AsPC-1 (bottom left) which expressed OX1R. Cells were treated for 48 h with 0.1 μM of each compound and then cells were counted. Results were expressed as the percentage of untreated cell number (Control).
[0132] FIG. 6: Effect of OxB and anti-OX1R antibodies including B4, B6, B10, C1, C2, E7, and H7 on apoptosis in HEK-OX1R cells expressing recombinant OX1R. SHP-2 protein tyrosine phosphatase inhibitor, NSC-87877, blocks orexin-induced apoptosis. HEK-OX1R cells were challenged with 0.1 μM of each compound for 48 hr in the absence (black bars) or the presence (white bars) of NSC-87877 (50 μM). Apoptosis was measured by determination of annexin V-PE binding, and results are expressed as the percentage of apoptotic cells.
[0133] FIG. 7: Competitive inhibition of specific .sup.125I-OxA binding to HEK-OX1R cells by increasing concentrations of unlabeled OxB, Cetuximab (irrelevant antibody) and anti-OX1R antibodies including B6, C1 and C2. Cells were incubated with the indicated concentration of OxB (), Cetuximab (.box-tangle-solidup.), B6 (.square-solid.), C1 (◯) and C2 (Δ). Results were expressed as the percentage of radioactivity specifically bound in the absence of added unlabeled compound. Each point is the mean of three separate experiments. ND, not determined.
[0134] FIG. 8: Effect of inoculation of orexin-A and C2 antibody on the growth of tumors developed by xenografted human HT-29 cells in nude mice. Colon adenocarcinoma derived cells, HT-29, were inoculated in the flank of nude mice at day 0. Mice were injected at day 1 (2 injections/week) intraperitoneally with 100 μl of orexin-A solution (1.4 μmoles of orexin-A/Kg (white circles)) or with 100 μl of C2 antibody solution (0.065 μmoles/Kg (black triangles) or 0.02 μmoles/Kg (white triangles)) or with 100 μl of PBS (black circles) for control.
EXAMPLES
Example 1
[0135] The development of antibodies directed against OX1R were produced by a phage display strategy and the antibody selection was performed by using HEK and HEK stably expressing OX1R (HEK-OX1R) cell lines. As a first step, a batch of 7 different antibodies named B4, B10, C1, C2, D4, E7 and H7 was tested for their ability to inhibit the cell growth of HEK-OX1R. Cells were incubated with 0.1 μM of OxB or antibodies for 48 h in culture medium and then cells were counted in order to estimate the cellular growth. As shown in FIG. 1, C1 and C2 reduced the HEK-OX1R cell number of about 46%±3 and 37±3% respectively as compared to orexin-B (OxB, 0.1 μM) which reduced of 40±3% the cell number (FIG. 1). In contrast, B4, B10, D4, E7 and H7 have a weak effect on cellular growth (range from 14% to 20%) as compared to OxB (FIG. 1). To determine the specificity of the cellular growth inhibition induced by these antibodies, we test it on HEK cells which does not expressing OX1R. As shown in FIG. 2 no anti-OX1R antibodies induced a cellular growth inhibition demonstrating that the observed effects of C1, C2 and, to a lesser extent for B4, B10, D4, E7 and H7 on HEK-OX1R cellular growth were clearly associated to the presence of OX1R. As previously described orexins (A & B) induced a mitochondrial apoptosis mediated by an entirely novel mechanism, not related to Gq-mediated phospholipase C activation. In fact, orexins induced the tyrosine phosphorylation of two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in the OX1R sequence. The resulting phosphorylated receptor could then recruit and activate the phosphotyrosine phosphatase, SHP-2, which is responsible for mitochondrial apoptosis, involving cytochrome c release from mitochondria to cytosol and caspase-3 and caspase-7 activation. Here, we tested the effect of SHP inhibitor (NSC 87877) on the ability of anti-OX1R to inhibit the cellular growth of HEK-OX1R (FIG. 3). HEK-OX1R cells were incubated 48 h with 0.1 μM of OxB or antibodies in the presence of 50 μM NSC 87877. As shown in FIG. 3, the inhibition of cellular growth induced by OxB (FIG. 1) was totally reverted in the presence of SHP inhibitor as compared to untreated cells, indicating that the orexin effect was well related to the recruitment of SHP2. Similarly, the inhibition of cellular growth induced by C1 and C2 but also by other antibodies (B4, B10, D4, E7 and H7) was totally reversed by NSC 87877. These results demonstrate that the cellular growth inhibition mediated by C1 and C2 was associated to the SHP2 signaling pathway as previously described for orexins/OX1R. Finally, we show that the antibodies are able to cross react between interspecies (human, rat and mouse), since the antibodies C1 and C2 are able to promote apotosis of CHO cells lines transfected with OX1R of mouse or rat (FIG. 4).
[0136] We have tested the ability of antibodies to inhibit the cellular growth in cancer cell lines derived from colon cancer (SW48, LoVo and HCT-116 cells) and pancreas cancer (AsPC-1 cells). Data reveal that: 1) C1, C2 and also B4 inhibit the cell growth in SW48 cells similarly to OxB (FIG. 5). D4 and H7 have a weak effect. In contrast, B10 and E7 have no effect on cell growth (FIG. 5). Moreover, all observed effects induced by antibodies were totally reversed in the presence of NSC 87877 inhibitor (not shown); 2) C1 and C2 inhibit the cell growth in LoVo cells similarly to OxB (FIG. 5). Inversely, B4, B10, D4, E7 and H7 have no or weak effect on cell growth; 3) all antibodies except B10 and E7 inhibit the cell growth in AsPC-1 cells derived from pancreas cancer. It should be noted that C1, C2 and B4 display a cell growth inhibition similar to OxB effect (FIG. 5); 4) all antibodies have no effect on cell growth of HCT-116 cells (FIG. 5) which do not express OX1R confirming that in the absence of orexin receptor, antibodies have no effect on cell growth of cancer cell lines. As previously described (see above) orexins are able to induce a mitochondrial apoptosis in cancer cell lines. We test the ability of antibodies to induce apoptosis in HEK-OX1R cells and colonic cancer cell line LoVo. Cells were incubated for 48 h in the presence of 0.1 μM OxB or 0.1 μM of each antibody and then, apoptosis was determined using the Guava PCA system and the Guava nexin kit. FIG. 6 shows that C1 and C2 are able to induce apoptosis in HEK-OX1R cells, respectively, 12±1% and 16±2% of total cells as compared to OxB, 37±2%. This effect was dose-dependent. Indeed when HEK-OX1R cells were treated with 0.01 μM of C2 antibody, cell apoptosis was only of 6±0.7% of total cells. In contrast, B4, E7 and H7 have no effect on cell apoptosis. It should be noted that B10 antibody has a weak effect on the induction of apoptosis but independent of the doses suggesting a non-specific response. In the same way, C2 antibody (0.1 μM) was able to induce apoptosis in LoVo cancer cell line. This apoptotic effect was dose dependent since the treatment of LoVo with 0.01 μM of C2 strongly reduce the apoptotic response. Taken together these results demonstrate that C2 and C1 are able 1) to induce a strong inhibition of cell growth and; 2) to stimulate the apoptosis in HEK-OX1R and cancer cell lines. These properties are specific since in the absence of OX1R expression (HEK and HCT-116 cells) or in the presence of SHP inhibitor (NSC 87877) these effects are totally abolished.
[0137] The ability of C1 and C2 antibodies to interact with the OX1R binding site was determined by competitive inhibition of .sup.125I-OxA binding study. HEK-OX1R cells were incubated with .sup.125I-OxA in the presence of increasing concentration of native OxB, C1 or C2 antibody and the resulting .sup.125I-OxA specific binding was measured. As shown in FIG. 8, C1 and C2 antibodies were able to competitively inhibit the binding of .sup.125I-OxA to HEK-OX1R with an estimated IC.sub.50 of about 5 μM as compared to OxB (IC.sup.50=10 nM). These results indicate that C1 and C2 antibodies specifically displace the OxA binding to its OX1R receptor.
Example 2
[0138] The inventors have tested the ability of C2 antibody to inhibit the tumor development of xenografted nude mice with the human colon adenocarcinoma cell line, HT-29. After subcutaneous injection of 1.5×10.sup.6 HT-29 cells in mice, animals were treated by 2 intraperitoneal (ip) injection/week of Orexin-A (100 μg/injection corresponding to 1.4 μmoles of orexin-A/Kg) or two doses of C2 antibody (200 μg/injection or 50 μg/injection corresponding to 0.065 μmoles/Kg and 0.02 μmoles/Kg, respectively). Control was determined by ip injection of 100 μl of PBS. The experiment was conducted during 45 days and the tumor volume was estimated by measuring the short and long axes of developed tumors each ⅔ days. As shown in FIG. 8, the inventors observed that HT-29 cells induced a tumor volume of about 1600±120 mm.sup.3 (Day 43) in the absence of treatment (black circles). In contrast, injection of C2 antibody (black triangle) strongly reduced the tumor development of about 60% (tumor volume #650±98 mm.sup.3) as compared to orexin treatment which induced an inhibition of about 65% (tumor volume #570±100 mm.sup.3). It should be noted that the use of low dose of C2 antibody (white triangles) induced a lowest inhibition of tumor growth of 45% (tumor volume #885±130 mm.sup.3) revealing a dose response relationship.
REFERENCES
[0139] 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.
