IMMUNE MODULATORS FOR REDUCING IMMUNE-RESISTANCE IN MELANOMA AND OTHER PROLIFERATIVE DISEASES

Abstract

The present invention pertains to novel modulators of resistance against T-cell mediated cytotoxic immune responses. The invention provides antagonists of immune escape mechanisms and therefore offers a novel approach for treating, or aiding a treatment, of various proliferative diseases such as cancerous diseases, in particular melanoma, pancreatic cancer and colorectal cancer. The invention specifically discloses the receptor Olfactory Receptor, Family 10, Subfamily H, Member 1 (OR10H1)as a checkpoint molecule in tumor resistance against cytotoxic T-cells. Provided is the inhibition of OR10H1 expression and/or function as a strategy for enhancing tumor susceptibility to a patients T-cell mediated immune response. Provided are antigen binding constructs for the detection of the OR10H1 protein, as well as inhibitory compounds, such as siRNA/shRNA molecules targeting OR10H1 and anti-OR10H1 antibodies, forimpairing the immune escape mediated by OR10H1. The invention furthermore provides screening methods for the identification of novel cancer therapeutics based on the modulation OR10H1 expression/function, diagnostic methods for the detection of immune resistance of a tumor to cytotoxic T-cell responses, as well as pharmaceutical compositions and diagnostic kits for performing these methods.

Claims

1. A compound for use in the treatment of a disease of a subject, wherein the compound is a modulator of the expression, function and/or stability of Olfactory Receptor, Family 10, Subfamily H, Member 1 (OR10H1), or of a variant of OR10H1.

2. The compound for use according to claim 1, wherein the disease is characterized by a pathological immune response.

3. The compound for use according to claim 1 or 2, wherein the disease is characterized by expression of said OR10H1, or the variant of OR10H1.

4. The compound for use according to any one of claims 1 to 3, wherein the variant of OR10H1 is a protein comprising an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 25 (OR10H1 amino acid sequence).

5. The compound for use according to any one of claims 1 to 4, wherein the compound is an inhibitor or antagonist of expression, function and/or stability of OR10H1, or of the variant of OR10H1.

6. The compound for use according to any one of claims 1 to 5, wherein the compound is selected from a polypeptide, peptide, glycoprotein, a peptidomimetic, an antigen binding construct (for example, an antibody, antibody-like molecule or other antigen binding derivative, or an or antigen binding fragment thereof), a nucleic acid such as a DNA or RNA, for example an antisense or inhibitory DNA or RNA, a ribozyme, an RNA or DNA aptamer, RNAi, siRNA, shRNA and the like, including variants or derivatives thereof such as a peptide nucleic acid (PNA), a genetic construct for targeted gene editing, such as a CRISPR/Cas9 construct and/or a guide nucleic acid (gRNA or gDNA) and/or tracrRNA.

7. The compound for use according to any one of claims 1 to 6, wherein the compound is an antigen binding construct (for example, an antibody, antibody-like molecule or other antigen binding derivative, or an antigen binding fragment thereof), that binds said OR10H1, or the variant of OR10H1.

8. The compound for use according to any one of claims 1 to 7, wherein the compound is an OR10H1 inhibitory antibody, or an inhibitory antigen binding fragment thereof, or an inhibitory antibody of a variant of OR10H1, or an inhibitory antigen binding fragment thereof.

9. The compound for use according to any one of claims 1 to 6, wherein the compound is a nucleic acid (for example an anti-sense nucleotide molecule such as a siRNA or shRNA molecule) that binds to a nucleic acid that encodes or regulates the expression of OR10H1, or of a variant of OR10H1.

10. The compound for use according to any one of claims 1 to 6, wherein the compound is a nucleic acid (for example an anti-sense nucleotide molecule such as a siRNA or shRNA molecule) that binds to a nucleic acid that encodes or regulates the expression of a gene that controls the expression, function and/or stability of OR10H1, or of a variant of OR10H1.

11. The compound for use according to any one of claim 1 to 6, 9 or 10, wherein the compound is a nucleic acid inhibitor of expression of OR10H1, or of a variant of OR10H1.

12. The compound for use according to any one of claims 1 to 11, wherein the disease is selected from a proliferative disease.

13. The compound for use according to any one of claims 1 to 12, wherein the disease is cancer, preferably a cancer disease selected from lung cancer, bladder cancer, ovarian cancer, uterine cancer, endometrial cancer, breast cancer, liver cancer, pancreatic cancer, stomach cancer, cervical cancer, lymphoma, leukemia, acute myeloid leukemia, acute lymphocytic leukemia, salivary gland cancer, bone cancer, brain cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer, skin cancer, melanoma, squamous cell carcinoma, pleomorphic adenoma, hepatocellular carcinoma, and/or adenocarcinoma.

14. The compound for use according to claim 13, wherein the cancer is an OR10H1 positive cancer, or a cancer positive for the variant of OR10H1.

15. The compound for use according to any one of claims 1 to 14, wherein the compound is for use in enhancing an immune response in the subject, preferably for use in aiding a cell-mediated immune response in the subject such as the subject's T-cell mediated immune response, for example for treating a proliferative disease such as a cancer disease.

16. The compound for use according to any one of claims 1 to 15, wherein the treatment comprises a transfer of cells to the subject, preferably a transfer of immune cells to the subject, more preferably an adoptive T-cell transfer.

17. The compound for use according to claim 16, wherein the cells are autologous cells of the subject, for example autologous immune cells, such as T-cells or Natural Killer (NK)-cells, of the subject.

18. The compound for use according to any one of claims 1 to 17, wherein the compound is an inhibitor or antagonist of expression, function and/or stability of said OR10H1, or the variant of OR10H1, and wherein the inhibition of the expression, function and/or stability of said OR10H1, or the variant of OR10H1, enhances an immune response, preferably enhances a cell-mediated immune response in the subject such as a T-cell mediated immune response in the subject, for example for treating a proliferative disease such as a cancer disease.

19. The compound for use according to claim 18, wherein the immune response is enhanced by an increase in T-cell activity and/or survival.

20. The compound for use according to claim 19, wherein the increase in T-cell activity and/or survival is associated with the inhibition of OR10H1-mediated or OR10H1-variant-mediated cAMP signaling.

21. The compound for use according to any one of claims 1 to 20, wherein the subject is a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or preferably a human, for example a human patient.

22. The compound for use according to claim 21, wherein the subject is a human, for example a human patient.

23. The compound for use according to any one of claims 1 to 22, wherein the treatment comprises a step of administering a therapeutically effective amount of the compound to the subject.

24. An isolated antigen binding construct, capable of specifically binding to OR10H1, or of a variant of OR10H1, optionally wherein the antigen binding construct inhibits the expression, function and/or stability of OR10H1, or the variant of OR10H1.

25. The isolated antigen binding construct according to claim 24, which comprises a sequence of an antibody variable heavy and/or light chain of an antibody obtainable from hybridoma Di-8A11-H12-E6 (DSMZ Deposition Number: DSM ACC3310, deposited 26 Oct. 2016).

26. The isolated antigen binding construct according to claim 25, which is an antibody obtainable from hybridoma Di-8A11-H12-E6 (DSMZ Deposition Number: DSM ACC3310, deposited 26 Oct. 2016), or an antigen binding fragment obtainable from such antibody.

27. An isolated antigen binding construct comprising at least one Complementary Determining Region (CDR) 3 having an amino acid sequence with at least 80% sequence identity to an amino acid sequence selected from SEQ ID NOs. 3, 7, 11, 15, 19, and 23.

28. The isolated antigen binding construct according to claim 27, comprising an antibody variable chain sequence having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 4, 8, 12, 16, 20, and 24.

29. The isolated antigen binding construct according to claim 27 or 28, comprising an antigen binding fragment of an antibody, wherein said antigen binding fragment comprises CDR1, CDR2 and CDR3, optionally selected from the CDR1, CDR2 and CDR3 sequences having the respective amino acid sequences of SEQ ID Nos. 1, 2, 3; or 5, 6, 7; or 9, 10, 11; or 13, 14, 15; or 17, 18, 19; or 21, 22, 23; in each case independently, optionally with not more than three or two, preferably one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.

30. The isolated antigen binding construct according to claim 29, wherein said CDR1 has an amino acid sequence of SEQ ID No 1, 5, 9, 13, 17 or 21, and CDR2 has an amino acid sequence of SEQ ID No 2, 6, 10, 14, 18, or 22, and CDR3 has an amino acid sequence of SEQ ID No 3, 7, 11, 15, 19, and 23; in each case independently, optionally with not more than three or two, preferably one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.

31. The isolated antigen binding construct according to any one of claims 27 to 30, wherein the antigen binding construct is an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of said antibody heavy chain sequences comprise CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 1 to 3, and at least one, preferably both, of said antibody light chain sequences comprise CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 5 to 7; or wherein at least one, preferably both, of said antibody heavy chain sequences comprise CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 9 to 11, and at least one, preferably both, of said antibody light chain sequences comprise CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 13 to 15; or wherein at least one, preferably both, of said antibody heavy chain sequences comprise CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 17 to 19, and at least one, preferably both, of said antibody light chain sequences comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NO: 21 to 23; in each case of a CDR independently, optionally with not more than three or two, preferably one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.

32. The isolated antigen binding construct according to any one of claims 27 to 31, wherein the antigen binding construct is an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequence, and at least one, preferably two, antibody light chain sequence, wherein said antibody heavy chain sequence comprises a variable region having the amino acid sequence of SEQ ID NO: 4, and wherein said antibody light chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 8; or wherein said antibody heavy chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 12, and wherein said antibody light chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 16; or wherein said antibody heavy chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 20, and wherein said antibody light chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 24; in each case of a variable region sequence independently, optionally with not more than ten, nine, eight, seven, six, five, four, three, two or one, preferably not more than three, amino acid substitutions, insertions or deletions compared to these sequences.

33. An isolated antigen binding construct that competes for antigen binding to an isolated antigen binding construct according to any one of claims 24 to 32.

34. The compound for use according to any one of claims 1 to 23 or the isolated antigen binding construct according to any one of claims 24 to 33 which decreases resistance and/or enhances sensitivity of cells expressing OR10H1, or a variant of OR10H1, to an immune response.

35. The compound for use or the isolated antigen binding construct according to claim 34, wherein said an immune response is a cytotoxic immune response.

36. The compound for use or the isolated antigen binding construct according to claim 34 or 35, wherein the immune response is a cell-mediated immune response, in particular a T-cell-mediated immune response.

37. The compound for use or the isolated antigen binding construct according to any one of claims 34 to 36, wherein said immune response is lysis and/or killing of said cells mediated by cytotoxic T-cells.

38. The compound for use or the isolated antigen binding construct according to any one of claims 34 to 37, wherein said cells are cancer cells or have originated from a tumor cell.

39. The compound for use according to any one of claims 1 to 23 or the isolated antigen binding construct according to any one of claims 24 to 38 which increases T-cell activity and/or survival.

40. The compound for use according to any one of claims 1 to 23 or the isolated antigen binding construct according to any one of claims 24 to 39, wherein the compound enhances killing and/or lysis of cells expressing said OR10H1, or the variant of OR10H1.

41. The compound for use according to any one of claims 1 to 23, and 34 to 40, or the isolated antigen binding construct according to any one of claims 24 to 40, which is an anti-body or comprises an antibody variable heavy and/or light chain sequence, or is an antigen binding fragment thereof, wherein the construct is chimerized, optionally is humanized or murinized

42. The compound for use according to any one of claims 1 to 23 and 34 to 41, or the isolated antigen binding construct of any one of claims 24 to 41, which is a monoclonal antibody.

43. The compound for use according to any one of claims 1 to 23 and 34 to 42, or the isolated antigen binding construct of any one of claims 24 to 42, wherein said compound or antigen binding construct is an IgG antibody.

44. An isolated nucleic acid encoding for a compound according to any one of claims 41 to 43 or an antigen binding construct according to any one of claims 23 to 43, or encoding for a part or a monomer of an antigen binding construct.

45. A vector comprising a nucleic acid according to claim 44.

46. A recombinant host cell comprising a compound according to any one of claims 1 to 23 or 41 to 43, an antigen binding construct according to any one of claims 24 to 43, or a nucleic acid according to claim 44, or a vector according to claim 45.

47. The recombinant host cell according to claim 46, wherein the cell is a human cell, preferably an autologous human cell.

48. The recombinant host cell according to claim 46, wherein the cell is a Chinese hamster ovary (CHO) cell.

49. A pharmaceutical composition comprising the compound according to any one of claims 1 to 23 or 41 to 43, the isolated antigen binding construct according to any one of claims 24 to 43, or the isolated nucleic acid according to claim 44, or the vector according to claim 45, or the recombinant host cell according to claim 46 or 47; and a pharmaceutically acceptable carrier, stabilizer and/or excipient.

50. The compound according to any one of claims 1 to 23 or 41 to 43, or the isolated antigen binding construct according to any one of claims 24 to 43, or a isolated nucleic acid according to claim 44, or a vector according to claim 45, or a recombinant host cell according to claim 46 or 47, or the pharmaceutical composition according to claim 49, for use in medicine, preferably for use in the diagnosis, prevention, and/or treatment of a proliferative disease, for example a disease comprising a malignant or benign tumor disease, such as a cancer.

51. A method of producing a recombinant cell line capable of expressing an antigen binding construct specific for OR10H1, or for a OR10H1 variant, comprising (a) providing a suitable host cell, (b) providing at least one genetic construct comprising coding sequence(s) encoding the antigen binding construct according to any one of claims 24 to 43 or the compound according to any one of claims 41 to 43, (c) introducing into said suitable host cell said genetic construct(s), (d) optionally, expressing said genetic construct(s) by said suitable host cell under conditions that allow for the expression of the antigen binding construct.

52. A method of producing an antigen binding construct specific for OR10H1, or for a OR10H1 variant, comprising (c) providing a hybridoma or host cell capable of expressing a compound of any one of claims 41 to 43 or an antigen binding construct according to any one of claims 24 to 43, for example a recombinant cell line comprising at least one genetic construct comprising coding sequence(s) encoding said compound or antigen binding construct, (d) culturing said hybridoma or host cell under conditions that allow for the expression of the antigen binding construct.

53. A hybridoma or host cell capable of expressing a compound of any one of claims 41 to 43 or an antigen binding construct according to any one of claims claim 24 to 43.

54. A method for detecting an OR10H1 protein, or a variant of OR10H1, in a sample comprising contacting the sample with an antigen binding construct specifically binding to said OR10H1 protein, or to the variant of OR10H1, and detecting the binding between said antigen binding construct and said OR10H1 protein, or the variant of OR10H1.

55. The method according to claim 54, wherein the antigen binding construct is an antibody or an antigen binding fragment thereof.

56. The method according to claim 54 or 55, wherein the sample is a sample of a tumor or a cancer, preferably a tumor sample of a patient suffering from a refractory/recurrent tumor disease, a metastatic tumor disease, or a multidrug resistant tumor disease.

57. A method for diagnosing a resistance phenotype of a cancer disease against an immune response such as a cell-mediated immune response in a subject, the method comprising the steps of (a) Providing a sample of tumor or cancer cells of the subject, (b) Determining the presence or absence of protein or mRNA of OR10H1, or of a variant of OR10H1, in the tumor or cancer cells, and (c) Diagnosing a resistance phenotype of the cancer disease against an immune response in the subject when protein or mRNA of said OR10H1, or of the variant of OR10H1, is present in the tumor or cancer cells.

58. A kit, comprising means for the detection of protein or mRNA of OR10H1, or of a variant of OR10H1, wherein said means is preferably selected from: (i) an antigen binding construct specifically binding said protein of OR10H1, or the variant of OR10H1, and/or (ii) a nucleic acid for detecting said mRNA of OR10H1, or of the variant of OR10H1; optionally together with instructions for use and/or with one or more other components useful for said detection.

59. The method according to claim 57 or the kit according to claim 58, wherein said means are coupled to a detectable label.

60. The kit according to claim 58 or 59, for use in the diagnosis of a resistance of a proliferative disease, for example a cancer disease, against an immune response such as a cell-mediated immune response; preferably the kit is for use in a method according to claim 57 or 59.

61. A method for identifying a compound suitable for the treatment of a disease characterized by expression of OR10H1, or a variant thereof, the method comprising the steps of (a) Providing a first cell expressing a protein or mRNA of OR10H1, or of a variant of OR10H1, and (b) Providing a candidate compound, and (c) Optionally, providing a second cell which is a cytotoxic immune cell, for example a cytotoxic T-lymphocyte (CTL), capable of immunologically recognizing said first cell, and (d) Bringing into contact the first cell and the candidate compound, and optionally the second cell, and (e) Determining subsequent to step (d), either or both of: (i) expression, function and/or stability of said protein or mRNA of OR10H1, or of a variant of OR10H1, in said first cell, wherein a reduced expression, function and/or stability of said protein or mRNA of OR10H1, or of a variant of OR10H1, in said first cell contacted with the candidate compound compared to said first cell not contacted with said candidate compound indicates that the candidate compound is a compound suitable for the treatment of a disease characterized by expression of said protein or mRNA of OR10H1, or of a variant of OR10H1; and/or (ii) cytotoxicity of said second cell against said first cell, wherein an enhanced cytotoxicity of said second cell against said first cell contacted with the candidate compound compared to the cytotoxicity of said second cell against said first cell not contacted with the candidate compound indicates that the candidate compound is a compound suitable for the treatment of a disease characterized by expression of said OR10H1, or the variant of OR10H1.

62. The method according to claim 61, wherein said first cell is a tumor cell or a cell derived from a tumor cell.

Description

IN THE FIGURES

[0285] FIG. 1AA: OR10H1 is expressed by various cancer cell-lines. RT-PCR shows expression of OR10H1 by the cell lines: M579-A2 (melanoma), PANC-1 (PDAC) and SW480 (colorectal), but not by the myeloma cell line KMM-1.

[0286] FIG. 1 cont.: OR10H1 prevents lysis of solid tumors by TILs. AB M579-A2 cells were transfected with the siRNA sequences for OR10H1 and mRNA was measured by RT-PCR after 72 h. Beta-actin served as a control. B, C Killing assays of M579-A2 with two different TIL-cultures. B M579-A2-luc cells, which were transfected with individual (s1-s4), pooled OR10H1 siRNA or control siRNA (PD-L1 as positive and non-specific as negative control) and TIL-mediated lysis was measured by Luc-CTL cytotoxicity assay (cumulative data). C Chromium-release assay showing specific lysis of M579-A2 by TIL209 at different E:T ratios after transfection with OR10H1 siRNA 1 (), positive control PD-L1 siRNA () or control siRNA (.square-solid.). D Luc-CTL assay with an autologous pair of melanoma (M615-luc) and TIL (TIL615) conducted in parallel to C (cumulative data,). E, F Chromium-release assay showing, respectively lysis of SW480 colorectal cancer and PANC-1 pancreatic adenocarcinoma cells by patient-derived HLA-matched TILs at different E:T ratios upon OR10H1 (), PD-L1 positive control () or control (.square-solid.) knockdown. All experiments were performed in triplicates and are representative (if not indicated otherwise) of at least three independent experiments. Error bars denoteSEM, and statistical significance was calculated using unpaired, two-tailed Student's t-test.

[0287] FIG. 2: OR10H1 inhibits TIL type I cytokine secretion and induces apoptosis. A Cumulative data of type I vs. type II cytokine secretion of TIL412 after 20 h co-culture with OR10H1 knock-down (siRNA1 or s1) or OR10H1 expressing (control siRNA) M579-A2 measured by Luminex assay. B ELISpot assay showing the number of IFN- secreting TILs (spot numbers) after co-culture with OR10H1 knockdown M579-A2 (or control siRNA). TILs without stimulation were used as a negative control. C cumulative data of apoptosis induction (measured by FACS staining for Annexin V.sup.+) in CD8.sup. TILs after co-culture (6 h) with OR10H1 knock-down (siRNA1 or siRNA pool) or OR10H1 expressing (control siRNA) M579-A2. TILs stimulated with PMA/ionomycin and unstimulated TILs (no melanoma encounter) were used as positive and negative controls, respectively. A and C show cumulative data from three independent experiments. B shows representative data of two independent experiments (performed in triplicates). Error bars denoteSEM, and statistical significance was calculated using unpaired, two-tailed Student's t-test.

[0288] FIG. 3: OR10H1 functions as an immune checkpoint in vivo. A Stable OR10H1 knock-down M579-A2 (transduced with OR10H1-targeting shRNA) or control transduced M579-A2 (non-targeting sequence shRNA; NTS) were injected subcutaneously (mixed with matrigel) into the left and right flank of NSG mice. On day 2 and 9, mice (n=6) received adoptive cell transfer of TIL412 (i.v. injection) and the tumor growth was measured. Mice (n=4) with tumors but without ACT serve as a control group for tumor growth. B, C Tumor growth curves showing meanSEM tumor volume of OR10H1-negative (ie OR10H1 knockdown; kd) or OR10H1 -positive (ie NTS shRNA control) M579-A2 tumors in TIL412-treated mice, ie, with ACT (B), or in the growth control group without ACT (C). Statistical difference was calculated using the Mann-Whitney U test.

[0289] FIG. 4: OR10H1 reduces Lck and increases CREB activity by cAMP signaling via PKA in TILs. A, B TIL412 were co-cultured with OR10H1-positive (control siRNA) or OR10H1-negative (OR10H1 siRNA) M579-A2 cells for 10 h and mRNAs were extracted from the respective TILs for transcriptome analysis by RNA sequencing. A Smear plot showing differential gene expression (log 2 of fold change) between OR10H-negative (kd) and OR10H1-postive, control siRNA)-tumor cell-treated TILs versus the average expression (log of count per million). LogFC cutoff at 0.5 is represented by horizontal lines. Genes with a false discovery rate (FDR) above 0.05 (depicted in gray) were excluded from subsequent analysis. B Functional enrichment analysis (Ingenuity pathway analysis; IPA) based on top upregulated (LogFC>0.5) and downregulated (LogFC<0.5) genes with FDR 0.05. Differential gene expression-associated functions were selected based on enrichment p-value (log of p-value; threshold=1.3) and activation Z-score (threshold 2). RNA-Sequencing was performed in duplicates. C, D, E, F Phospho pathway analysis for key signaling proteins in TILs after co-culture (up to 2 h) with OR10H1-positive (ie control siRNA) and OR10H1-negative (ie OR10H1 siRNA) M579-A2. Phosphoplex analysis showing phosphorylation state changes in TILs upon encountering OR10H1-positive- or OR10H1-negative M579-A2 tumor cells (log 2 ratio) of classical (C) or T cell receptor (TCR)-associated (D, E) signaling proteins (CREB and Lck) compared to unstimulated TILs. In each case, TILs stimulated with PMA and ionomycin serve as positive control. F Immunoblot analysis showing phospho-CREB (Ser133), phospho-ATF1, phospho-PKA (Thr197) and phospho-Lck (Tyr505) levels in OR10H1-positive tumor cell-treated, OR10H1-negative tumor cell-treated, PMA and iono-mycin-treated or unstimulated TILs using the according phospho-specific antibodies. Beta-actin was used as a loading control. C, D and E are cumulative data of three independent experiments. F is representative of three independent experiments. MeanSEM are shown, unless stated otherwise, and statistical significance was calculated using unpaired, two-tailed Student's t-test. G Lck inhibition by a small molecule abrogates OR10H1 knockdown effect on T-cell mediated cytotoxicity (as reflected the ratio of cytotoxicity/viability) of melanoma cells. H Western blot showing that olfactory receptor signalling activates a unique G protein (G-alpha-Olf) and subsequently adenylate cyclase type III. I In the presence of TILs the cAMP response in M579-A2 cells is reduced if OR10H1 is knocked-down on the melanoma cells.

[0290] FIG. 5: Generation of monoclonal blocking antibodies against OR10H1. Mother clones of OR10H1-blocking antibodies were generated by genetic immunization of three rats with an OR10H1-IgG construct and screening for binding and blocking activity. The four most promising mother clones were enriched and tested for their impact on TIL-mediated killing by Luc-CTL cytotoxicity assay (A and B) and for two of such clones in an IncuCyte cytotoxicity assay (C and D), based on caspase activation. A, B Blocking efficacy was tested using different antibody concentration (0-500 g/ml) for the co-culture of TIL412 with OR10H1.sup.- or OR10H1.sup.M579-A2-luc, compared to pre-immunization rat serum. IncuCyte cytotoxicity assays depicting the impact of mother clones 8A1 l (C) and 1B11 (D) on TIL-mediated induction of apoptosis in M579-A2 over 48 hours (measurement every 5 min). The Y axis represents the number of green object counts (event of caspase-3 activation) per well. E, F, G Summary of screening for monoclonal antibodies derived from aforementioned mother clones. Luc-CTL cytotoxicity assays showing the impact of monoclonal supernatants (unknown concentration) on the TIL-mediated killing of wild-type (E), control knockdown (F) or OR10H1 knockdown (G) M579-A2-luc. A and B are representative of two independent experiments. C and D are exemplary experiments. E, F and G are results from one screening. A-E were performed in triplicates, F and G were performed in duplicates. Error bars denoteSEM, and statistical significance was calculated using unpaired, two-tailed Student's t-test with *p0.05; **p0.01; ***p0.001; ****p0.0001. Clone IDs: A=1C3, B=1B11, C=8A11, D=4B4, A-1=1C3-B2, A-2=1C3-A9, D-1=4B4-A10, C-1=8A11-G12, C-2=8A11-E7, B-1=1B11-B12 and B-2=1B11-B9.

[0291] FIG. 6: Detection of OR10H1 using an antibody of the invention. A Surface expression of OR10H1 on melanoma M579-A2 transfected with non-specific control siRNA or OR10H1 siRNA 1 detected with post-immunisation polysera against OR10H1. B Surface expression of OR10H1 detected by the antibody purified from clone Di-8A11-H12-E6 on M579-A2 melanoma cells that express OR10H1 (determined by RT-PCR), but not detected on KMM-1 myeloma cells that do not express OR10H1 (determined by RT-PCR).

[0292] FIG. 7: Amino acid sequences of certain antibodies of or for use in the present invention. A. Sequence of the amino acid sequence of the heavy and light chains of antibody 1C3-A1-A1; B. Sequence of the amino acid sequence of the heavy and light chains of antibody 1C3-Al-A2; C. Sequence of the amino acid sequence of the heavy and light chains of anti-body 8A11-B9-A1.

[0293] And in the Sequences:

TABLE-US-00001 TABLE1D AntibodySequences SEQIDNO: Antibody Chain Region AminoAcidSequence 1 1C3-A1-A1 heavy CDR1 FTFDNAW 2 1C3-A1-A1 heavy CDR2 IKAKSNNYAT 3 1C3-A1-A1 heavy CDR3 TRLYYD 4 1C3-A1-A1 heavy variabledomain EVQLVETGGNLVQPGKSL KLTCATSGFTFDNAWMH WVRQSPEKQLEWVAQIKA KSNNYATYYAESVKGRFTI SRDDSKSSVYLQMNRLKE EDTAIYYCTRLYYDWGQG VMVTVSS 5 1C3-A1-A1 light CDR1 QDIGNY 6 1C3-A1-A1 light CDR2 RAT 7 1C3-A1-A1 light CDR3 LQHKQYPL 8 1C3-A1-A1 light variabledomain DIQMTQSPSSISVSLGDRFT FTCRASQDIGNYLSWFQQ KPEKSPKLMIYRATNLEDG VPSRFSGSRSGSDYSLTINS LESEDTGIYFCLQHKQYPL TFGSGTKLEIKR 9 1C3-A1-A2 heavy CDR1 FTFSNYD 10 1C3-A1-A2 heavy CDR2 ISHSGDSTYF 11 1C3-A1-A2 heavy CDR3 HGVYTN 12 1C3-A1-A2 heavy variabledomain EVQLQESGGGLIQPGRSLK LSCAASGFTFSNYDMAWV RQAPTKGLEWVASISHSGD STYFRASVKGRFTVSRDNA KSSLYLQMDSLRSEDTATY YCARHGVYTNYGIWFAY WGQGTLVTVSS 13 1C3-A1-A2 light CDR1 QDIGNY 14 1C3-A1-A2 light CDR2 RAT 15 1C3-A1-A2 light CDR3 LQHKQYPL 16 1C3-A1-A2 light variabledomain DIQMTQSPSSISVSLGDRFT FTCRASQDIGNYLSWFQQ KPEKSPKLMIYRATNLEDG VPSRFSGSRSGSDYSLTINS LESEDTGIYFCLQHKQYPL TFGSGTKLEIKR 17 8A11-B9-A1 heavy CDR1 FTFSSAW 18 8A11-B9-A1 heavy CDR2 IKGKSNNYAT 19 8A11-B9-A1 heavy CDR3 TWFGPMDA 20 8A11-B9A1 heavy variabledomain EVQLQESGGRLVQPGKSL KLTCAASGFTFSSAWIHW VRQSPEKQLEWVAQIKGK SNNYATYYAESVKGRFTIS RDDSKSSVYLQMNSLKEE DTAIYYCTWFGPMDAWG QGASVTVSS 21 8A11-B9-A1 light CDR1 KSLLHNNGKTF 22 8A11-B9-A1 light CDR2 WMS 23 8A11-B9-A1 light CDR3 QQFLEYPL 24 8A11-B9-A1 light variabledomain DIVMTQGALPNPVPSGESA SITCQSSKSLLHNNGKTFL NWYLQRPGQSPQLLIYWM STRASGVSDRFSGSGSGAD FTLKISSVEAEDVGVYYCQ QFLEYPLTFGSGTKLEIKR SEQIDNO25:showstheOR10H1FullLengthProteinSequence: MQRANHSTVTQFILVGFSVFPHLQLMLFLLFLLMYLFTLLGNLLIMATVWSERSLHTP MYLFLCALSVSEILYTVAIIPRMLADLLSTQRSIAFLACASQMFFSFSFGFTHSFLLTVM GYDRYVAICHPLRYNVLMSPRGCACLVGCSWAGGLVMGMVVTSAIFHLAFCGHKEI HHFACHVPPLLKLACGDDVLVVAKGVGLVCITALLGCFLLILLSYAFIVAAILKIPSAE GRNKAFSTCASHLTVVVVHYGFASVIYLKPKSPQSLEGDTLMGITYTVLTPFLSPIIFSL RNKELKVAMKKTFFSKLYPEKNVMM SEQIDNO:26to34showOR10H1siRNA/shRNAsequences(seetableE1/E2below).

EXAMPLES

Example 1

OR10H1 Knock-Down Increases TIL-Mediated Killing of Solid Tumors (FIG. 1)

[0294] RT-PCR data and expression database searches suggest that the cell surface-expressed gene OR10H1 is expressed by melanoma, PDAC and colorectal cancer. Therefore, two OR10H1-specific PCR primers are tested by sequencing the respective RT-PCR amplicons according to standard procedures. The results show that OR10H1 is expressed by the following cells: M579-A2 (melanoma) (Machlenkin et al, 2008; Cancer Res 68:2006-13), PANC-1 (PDAC) and SW480 (colorectal) (both, ATCC), but not expressed by KMM-1 (myeloma) (Namba et al, 1989; In Vitro Cell Dev Biol 8:723-9 (FIG. 1AA).

[0295] SMART pool siRNA against OR10H1 (GE Dharmacon) were tested (by RT-PCR) for their activity in downregulating OR10H1 mRNA in M579 (melanoma) cells. Following transfection into M579-A2 cells, all siRNAs show a knock-down of OR10H1 (FIG. 1AB). siRNA 1 shows a complete absence of OR10H1 mRNA, siRNA 3 and pooled OR10H1 siRNAs show a strong reduction, siRNA 2 and 4 show a weaker but clear reduction in OR10H1 transcription. Details of the respective siRNAs are set out in Table E1.

TABLE-US-00002 TABLEE1 ExemplarysiRNAsagainstORH10H1 GEDharmacon SEQID Name Sequence order# No. OR10H1 GGAGACACCUUGAUGGGCA D-020479-01 26 siRNA1 OR10H1 AGUAAACUCUACCCAGAAA D-020479-02 27 siRNA2 OR10H1 GCAGAGAGCCAAUCACUCC D-020479-03 28 siRNA3 OR10H1 GGUCGUGCACUAUGGCUUU D-020479-04 29 siRNA4 OR10H1 M-020479-01 pool

[0296] The inventors were surprised to find that all siRNAs show an impact on TIL412-mediated killing of M579-A2-luc at 5:1 Effector to Target cell (E:T) ratio (FIG. 1 B). Transfection of M579-A2-luc cells with OR10H1 siRNA 1 increased HLA-matched patient-derived tumor-infiltrating lymphocyte (TIL, clone 412)-mediated killing to 70% (30% remaining melanoma cells compared to a scrambled negative control siRNA; p=<0.0001). This increase in TIL-mediated lysis is stronger than for PD-L1 positive control knock-down (39% remaining cells). Overall, the effect on the phenotype is comparable to the knock-down efficacy on OR10H1 mRNA. Only siRNA 3 shows an effect on M579-A2-luc viability (without the presence of TILs; data not shown).

[0297] TIL-mediated lysis of M579-A2 was validated in chromium release assay (4 h co-culture). Another patient-derived and HLA-matched TIL culture (TIL209) was used to show that the knock-down effect on TIL-mediated killing is not depending specifically on TIL412. Knock-down of OR10H1 (siRNA 1) strongly increased the lysis of M579-A2-luc in all effector to target (E:T) ratios compared to the negative control siRNA (57% to 27% specific lysis at 12.5 E:T ratio; FIG. 1C). Indeed, this effect of OR10H1 knock-down was stronger than the PD-L1 positive control knock-down (41% specific lysis at 12.5 E:T ratio; FIG. 1C).

[0298] OR10H1 knock-down increased TIL-mediated killing in an autologous melanoma setting. For this, M615 tumor cells and TIL615 were derived from the same patient, and M615 was stably transfected with luciferase to produce M615-luc. The basic killing of M615-luc by TIL615 was very low. However, knock-down of OR10H1 increased TIL-mediated killing compared to the negative control siRNA (FIG. 1D).

[0299] Furthermore, it was tested whether OR10H1 knock-down also has an effect in other cancers. Surprisingly, OR10H1 knock-down increased the TIL-mediated tumor cell killing in colorectal cancer (CRC) and pancreatic cancer cells. SW480 (ATCC) and PANC-1 cells were analyzed for their expression of OR10H1 (see above) and the knock-down efficacy of the siRNAs were validated. The CRC cell line SW480 and the PANC-1 cell line were each co-cultured with corresponding HLA-matched patient-derived TILs for chromium release (4 h co-culture). OR10H1 siRNA 1 almost tripled the lysis of SW480 by TILs in all effector to target ratios compared to the negative control siRNA (33% to 12.8% specific lysis at 50:1 E:T ratio; FIG. 1E). Indeed, knock-down of OR10H1 was almost as effective as knock-down of PD-L1 positive control on TIL-mediated lysis (FIG. 1E). Such effect was also surprising to see in pancreatic cancer cells: knock-down of OR10H1 (siRNA 1) doubled the lysis of PANC-1 by TILs in all effector to target ratios compared to the negative control siRNA (53% to 26% specific lysis at 50:1 E:T ratio; FIG. 1F)

[0300] The effects of siRNA knock-down on TIL-mediated killing and lysis in a chromium release assay were investigated as described in Khandelwal et al, 2015 (EMBO Mol Med 7:450-63). Tumor-infiltrating lymphocytes 412 and 209 microcultures were expanded from an inguinal lymph node of a melanoma patient as described in Dudley et al, 2010 (Clin Cancer Res 16:6122-31). M615, TIL615 and other patient-derived TIL cells were obtained analogously as described in Dudley et al, 2010. M579-A2-luc cells and M615-luc cells were produced from M579-A2 and M615, respectively (Machlenkin et al, 2008; Cancer Res 68:2006-13).

Example 2

OR10H1 Knock-Down Increases TIL Activity and Survival (FIG. 2)

[0301] A co-culture of M579-A2 and TIL412 leads to a switch in cytokines secreted by TILs (from immune-suppressive type II to immune-activating type I), showing an increase in anti-tumor response. Cells were co-cultured for 20 hours and the cytokine concentrations in the supernatant were measured by Luminex as described in Khandelwal et al, 2015. As controls unstimulated TILs (no tumor cells) and over-stimulated TILs (PMA and ionomycin) were used (not shown).

[0302] As expected unstimulated TILs did not secrete considerable amounts of cytokines, whereas over-stimulated TILs secreted dramatically increased amounts. However, the inventors were surprised to observe that knock-down of OR10H1 on melanoma increased type I cytokine secretion by TIL412 (FIG. 2A). IFN- secretion increased significantly from 2825 to 3037 pg/ml (increase of 8%; p=0.0236) compared to the negative control. IL-2 secretion significantly increased from 1079 to 1701 pg/ml (increase of 58%; p=0.045). Furthermore, knock-down of OR10H1 was found to surprisingly decrease type II cytokine secretion by TIL412 (FIG. 2A). MCP-1 secretion significantly decreased from 6658 to 4035 pg/ml (decrease of 39%; p=0.016). IL-6 secretion significantly decreased from 49.4 to 31 pg/ml (decrease of 37%; p=0.028). IL-4 secretion decreased from 14.75 to 11.8 pg/ml (20%; trend).

[0303] Knock-down of OR10H1 increased the number of IFN- producing TILs. Melanoma cells and TILs were co-cultured for 20 h and the number of IFN- producing cells was measured using ELISpot as described in Khandelwal et al, 2015. Although TILs did not produce IFN-g without interaction with melanoma cells, knock-down of OR10H1 (pooled siRNA) significantly increased the number of IFN-g spots/104 TILs from 107.75 to 187.75 (increase of 73%; p=0.02; FIG. 2B).

[0304] Without being bound by theory, the effect of OR10H1 knock-down (in melanoma cells) on TIL activity appeared to depend on the HLA-TCR interaction (not shown). The inventors then compared the amounts of IFN- after the co-culture (6 h) of M579-A2 melanoma cells with TILs. If melanoma cells did not express HLA-A2 (M579), IFN- secretion was abrogated regardless of the knock-down of OR10H1. However, knock-down of OR10H1 on melanoma cells surprisingly reduced the induction of apoptosis in TILs after co-culture suggesting that OR10H1 knock-down prolonged survival and/or increased proliferation of TILs. Melanoma cells and TILs were co-cultured for 6 h and the percentage of Annexin V-positive CD8+ TILs (FACS staining) were calculated, in each case according to standard procedures. TILs which were not co-cultured or activated showed 35% of Annexin V-positive CD8+ T cells. Over-activation with PMA and ionomycin resulted in 60% apoptotic CD8+ TCs. Interestingly, co-culture with OR10H1-positive M579-A2 (scrambled siRNA control) increased the percentage of apoptotic CD8+ TCs to 62%. Surprisingly, knock-down of OR10H1 prior to co-culture reduced the number of apoptotic CD8+ TILs to 47% (FIG. 2C). The induction of apoptosis (compared to unstimulated TILs) is half as large if OR10H1 is not expressed in the melanoma cells (p=0.032). Knock-down of PD-L1 decreased the percentage of apoptotic CD8+ TCs to 53% (non-significant compared to scrambled siRNA control)

Example 3

OR10H1 Functions as an Immune Checkpoint In Vivo (FIG. 3)

[0305] In order to assess OR10H1 function in vivo, a stable OR10H1 knock-down M579-A2 line was generated using lentiviral shRNA particles (MISSION shRNA for OR10H1; Sigma Aldrich), analogously as described in Khandelwal et al, 2015 for the stable CCR9 knockdown in M579-A2. The stable knock-down of OR10H1 increased the specific lysis of M579-A2 by TIL412 and TIL209 in vitro (chromium release) compared to negative target sequence (NTS) control-transduced M579-A2 (not shown). ShRNA 4 showed the strongest impact on OR10H1 expression and functionality, whereas stable knock-down of OR10H1 did not affect viability or proliferation of M579-A2 in vitro (not shown). Details of the respective shRNAs are set out in Table E2.

TABLE-US-00003 TABLEE2 ExemplaryshRNAsagainstORH10H1 SEQ SigmaAldrich ID Name Sequence order# No. OR10H1 GTTCCTGCTGATGTACCTGTT TRCN0000011786 30 shRNA1 OR10H1 TGCGCTACAACGTGCTCATGA TRCN0000357706 31 shRNA2 OR10H1 TGGCTTTGCCTCCGTCATTTA TRCN0000357707 32 shRNA3 OR10H1 TCTGCTGAAGGTCGGAACAAG TRCN0000357708 33 shRNA4 OR10H15 ACACAAGGAGATCCACCATTT TRCN0000357775 34 shRNA

[0306] OR10H1 knock-down and NTS control-transduced M579-A2 cells (in matrigel) were injected subcutaneously into the flanks of immunodeficient (NOD scid gamma; NSG) mice (FIG. 3A) and on day 2 and 9 after tumor inoculation, TIL412 cells were injected intravenously (adoptive cell transfer; ACT). Subsequently the tumor size was measured for 24 days. A control group of mice was injected with tumor cells but did not receive ACT, so as to validate the effect of OR10H1 knock-down on tumor growth without the presence of TILs.

[0307] Tumor growth is strongly reduced in OR10H1 knock-down (kd) tumors (ie, OR10H1-negative tumors) after ACT with TIL412 but not without the ACT (FIG. 3B and C). On day 7 (273 mm.sup.3) the tumors start to grow regardless of the presence or absence of OR10H1. There is no significant difference in tumor growth of OR10H1 knock-down M579-A2 compared to that of the NTS shRNA control group (FIG. 3C). In contrast, and to the surprise of the inventors, if the mice were i.v. injected with TIL412 as ACT, M579-A2 tumor growth of OR10H1-negative (kd), but not growth of shRNA control M579-A2 tumors (ie, OR10H1-negative), was significantly reduced after day 11 (FIG. 3B). OR10H1 knock down tumors significantly reduced in volume from 260 to 207 mm.sup.3 between day 11 and 14 after inoculation. In contrast, and over the same period, the NTS control tumors grew substantially from 296 to 389 mm.sup.3 (p=0.013). On day 18 the average OR10H1-negative tumor (kd) had a volume of only 203 mm.sup.3 compared to 401 mm.sup.3 for the average size of NTS control (ie OR10H1-positive) tumors (p=0.004). Although after day 18, the OR10H1-negative tumors do start to grow again, their tumor mass remains significantly reduced compared to NTS control (ie OR10H1-positive) tumors.

Example 4

OR10H1 Inhibits TIL Functionality Utilizing cAMP (FIG. 4)

[0308] TIL412 were co-cultured with (OR10H1 knock-down or negative siRNA control) M579-A2 for 10 h cells to activate signaling pathways in the TILs, and then the melanoma cells were then removed from the co-culture using magnetic melanoma beads specific for MCSP (Miltenyi Biotech). The purity of the remaining TILs was around 99.5% (not shown). Differential gene expression between the TILs isolated from the OR10H1 knock-down and the negative siRNA control was measured by RNA-Sequencing according to standard procedures. Several genes were significantly up or down regulated (fold change above 0.5 or below -0.5 respectively) in the setting with OR10H1 knock-down M579-A2 (siRNA 1) compared to the negative siRNA control (FIG. 4A). Down-regulated in the TILs were the following genes: Early growth response gene 3 (Egr3) is a key negative transcriptional regulator of T cell activation and induces anergy [1-3]. Interferon-gamma receptor chain 2 (IFNGR2) expressed in high levels inhibits T cell proliferation and may induce apoptosis [4, 5]. Nuclear Receptor Subfamily 4, Group A, Member 2 (NR4A2) is associated with T cell exhaustion in chronic viral infection [6] and might be involved in apoptosis [7]. Up-regulated in the TILs were the following genes: Chemokine (C-X-C motif) ligand 13 (CXCL13) promotes T cell recruitment [8] and facilitates the inflammatory response of antigen-experienced T helper cells [9]. Cytotoxic And Regulatory T Cell Molecule (CRTAM) is expressed (upon TCR activation) on activated T cells and its interaction with Nec1-2 (on tumors) promotes IFN-g secretion by CD8+ T cells [10]. Vav guanine nucleotide exchange factor 3 (VAV3) is involved in TCR signaling via NFAT and SRF activation [11]. FBJ Murine Osteosarcoma Viral Oncogene Homolog (FOS) together with c-Jun builds up activating protein 1 (AP-1), one of three major transcription factors downstream of TCR signaling [12]. V-Myc Avian Myelocytomatosis Viral Oncogene Homolog (c-Myc) is a transcription factor which is involved in proliferation and metabolic reprogramming upon T lymphocyte activation [13, 14]. It modulates the generation of CD8+ immunological memory in tumors and viral infection [15, 16]. Interferon regulatory factor 4 (IRF4) is a transcription factor necessary for the expansion and effector differentiation of CD8+ T cells and represses genes involved in cell cycle arrest and apoptosis. [17, 18]

[0309] Upon Ingenuity pathway analysis (Qiagen; FIG. 4B) to investigate functions in which the differential expressed genes are enriched (positive or negative activation) in TILs and which signaling networks play a role, the inventors surprisingly found that of the eight pathways analyzed, only CREB is differentially phosphorylated in the setting with OR10H1-negative.sup. melanoma. The log 2 ratio (compared to unstimulated TILs) for CREB is significantly reduced after the knock-down of OR10H1 (p=0.006; FIG. 4C).

[0310] Western blot analysis for phospho-CREB and ATFL (same phosphorylation site) in TIL412 confirmed the reduced activation depending on OR10H1 knock-down on melanoma cells (FIG. 4F). cAMP response element-binding protein (CREB) downstream of TCR activation is widely known to be important for proliferation and survival of T cells [19] but has been associated with T cell anergy as well [20]. Therefore, key factors of TCR signaling (Lck, LAT, CREB, ZAP-70, Syk, CD3e and ERK1/2) were analyzed.

[0311] Knock-down of OR10H1 on melanoma cells leads to decreased phosphorylation of CREB and Lck (inhibitory phospho-Tyrosin 505) in TILs (FIGS. 4D and 4E). Importantly however, OR10H1 does not play a role in modulating CD3e activation (phosphylation): CD3e does not show increased phosphorylation levels after co-culture with melanoma cells/TILs (regardless of OR10H1 knock-down) compared to unstimulated TILs, and the same is observed for and ERK1/2. Yet, over-stimulation with PMA and ionomycin does dramatically increased phosphorylation of ERK1/2 but does not affect CD3e.

[0312] Phosphorylation of Linker of activated T cells (LAT) slightly increases in TILs in the first 30 minutes of co-culture and stays stable up to two hours (for OR10H1-positive and OR10H1-negative melanoma cell/TIL co-cultures and over-activation PMA and ionomycin). Spleen tyrosine kinase (Syk) gets activated in 5 minutes and later on decreases below the level of unstimulated TILs in all three settings. Finally, phosphorylation of Zeta-chain-associated protein kinase 70 (ZAP70) increases in the first 5 minutes and stays stable for 2 h if co-cultured with melanoma cells/TILs (regardless of OR10H1 knock-down). Over-activation with PMA and ionomycin leads to an increased phosphorylation of ZAP70.

[0313] Co-culture of TIL412 with OR10H1-positive M579-A2 melanoma cells leads to a dramatically increased phosphorylation of CREB after 2 h in TILs (FIG. 4D). Interestingly, the first 30 min after activation the phosphorylation levels are similar to the OR10H1 knock-down setting. Over activation with PMA and ionomycin leads to a faster onset of CREB phosphorylation but the levels are similar to the control siRNA (OR10H1-positive) setup. In contrast, Lymphocyte-specific protein tyrosine kinase (Lck) becomes strongly dephosphorylated after 30 min in TILs if co-cultured with OR10H-negative M579-A2 melanoma cells or overactivated with PMA and ionomycin but not in the control siRNA (OR10H1-positive) setup (FIG. 4E).

[0314] As described above, TCRs of TILs which were co-cultured with melanoma cells were activated in the same way, but showed different signaling at two important hubs, namely CREB and Lck, depending on whether OR10H1 was present or absent on the target melanoma cells. Therefore, OR10H1-mediated inhibition of T cell activity might converge here. Lck has two phosphorylation sites but phosphoplex analysis of Lck measures total Lck phosphorylation. Phosphorylation of Lck-Tyr384 stabilizes the active conformation whereas phosphorylation of Lck-Tyr505 promotes the auto-inhibited conformation of Lck [21-23]. Western blots for phospho-Lck confirmed that phosphorylation of the inhibitory Tyr505 residue was strongly reduced after the knock-down of OR10H1 suggesting an increased activation of Lck in TILs. Indeed, inhibition of Lck activity by a small molecule inhibitor is shown to abrogate OR10H1 knock-down effect on T-cell mediated cytotoxicity (as reflected by the ratio of cytotoxicity/viability) of melanoma cells (FIG. 4G). Protein kinase A (PKA) is activated by cAMP and in turn activates c-Src Tyrosine Kinase (Csk). Csk abrogates Lck activity by phosphorylation of Lck-Tyr505 [24, 25]. Indeed, the inventors were able to confirm by western blot analysis, an enhancement of PKA phosphorylation in TILs after the OR10H1-positive (ie control siRNA) M579-A2 melanoma cell/TIL co-culture. (FIG. 4F).

[0315] Olfactory receptor signaling activates a unique G protein (G-alpha-Olf) and subsequently adenylate cyclase type III (FIG. 4H). RNA-sequencing expression data suggest that both genes are expressed in M579-A2. M579-A2 melanoma cells (OR10H1-positive and OR10H1-negative) were transiently transfected with a cAMP reporter luciferase construct and co-cultured with TIL412. The production of cAMP in melanoma is altered by OR10H1 knock-down only if there is an interaction with TILs, while co-culture of M579-A2 cells with TILs alone was not sufficient to trigger a cAMP response (not shown). Indeed, after co-culture of M579-A2 (OR10H1-positive or OR10H1-negative) with TILs for 2 h, 10 uM forskolin was added to raise cAMP levels: without a preceding co-culture with TILs there is no difference in cAMP response in M579-A2 cells, whereas in the presence of TILs the cAMP response in M579-A2 cells is reduced if OR10H1 is knocked-down on the melanoma cells (FIG. 41).

[0316] The inventors were surprised to observe data suggesting that that TIL-driven cytotoxicity of the melanoma cells may be inhibited by a mechanism involving connexin 32 (Cx32) and the transport of cAMP from the tumor cell to the T cell. We validated by RNA-sequencing that M579-A2 cells express connexin 32, a protein involved in the formation of gap-junctions. Such gap-junctions are selective for the transport of cAMP and cGMP [28], and hence the potential for cAMP transport from M579-A2 to the TIL412. Indeed, preliminary results suggest that blockade of Cx32 leads to an increased killing of melanoma cells by TILs only if OR10H1 is present (not shown). This is consistent with recent studies have shown that regulatory T cells (Tregs) and tumor cells can transport cAMP via gap-junctions into T cells and lead to inhibition through phosphorylation of Lck and CREB [26, 27].

Example 5

Development of Antibodies Against OR10H1 and their Functional Activity (FIG. 5)

[0317] For generating antibodies against OR10H1, genomic immunization of rats was conducted at Aldevron (Freiburg), described briefly as follows.

[0318] Firstly, an OR10H1 construct was cloned into an Aldrevon immunization vector (pB8-OR10H1) and an Aldrevon screening vector (pB1-OR10H1). Transient transfection of these vectors into mammalian cells confirmed (weak) cell surface expression. Cell surface expression of such proteinscontaining a vector-derived N-terminal tag-sequencewas analyzed by flow cytometry on non-fixed living cells using an anti-tag murine antibody and a goat anti-mouse IgG R-phycoerythrin conjugate (Southern Biotech) as a secondary antibody. Secondly, three rats were immunized with the immunization vector (pB8-OR10H1) for 46 days and 5 genetic applications (IS46d-5) followed by an additional 2 genetic immunizations over a subsequent 35 days; in total immunization over for 81 days with 7 genetic applications (IS81d-7)). Thirdly, sera of immunized rats (diluted in PBS 3% FBS) were then tested (by flow cytometry) for reactivity against mammalian cells transiently transfected with the screening vector (pB1-OR10H1) using a goat anti-rat IgG R-phycoerythrin conjugate (Southern Biotech) as a secondary antibody. Compared to pre-immunization sera, a significant (but weak reactivity) was observed (data not shown). Fourthly, splenocytes were isolated from the rats' spleen, the resulting B cells fused with immortalized myeloma cells by standard technologies, and the resulting hybridoma clones screened for the reactivity of their supernatant against the recombinant mammalian cells transiently transfected with the screening vector (as described above). Screening and enrichment of such hybridoma clones resulted in 4 mother clones being selected (1C3, 1B11, 8A11 and 4B4).

[0319] Each of these antibodies produced by such clones showed a significant impact on TIL-mediated melanoma killing at a concentration of 100 g/ml in the OR10H1-positive M579-A1-Luc/TIL412 assay (FIGS. 5A and B) as well as for those tested (8A11 and 1B11) in the IncuCyte cytotoxicity assay based on caspase activation (FIGS. 5C and D), in each case as described above.

[0320] Next, the mother clones were single-cell sorted to generate individual hybridoma clones that produce monoclonal antibodies, which were tested for their functional activity in the OR10H1-positive M579-A1-Luc/TIL412 assay as described above (FIGS. 5E, F and G), and individual hybridoma clones were selected for sequencing of the antibody they produce.

[0321] Such results demonstrate the ability of antibodies of the present invention to have therapeutic and research utility as and/or in the development of compounds for the treatment of proliferative disease such as a cancer disease, in particular of OR10H1-positive cancers.

Example 6

Detection of OR10H1 using an Antibody of the Present Invention (FIG. 6)

[0322] The post-immunisation poly sera was used to demonstrate that it can specifically detect OR10H1 expressed on the surface of tumor cells. FACS detection of melanoma M579-A2 cells transiently transfected with negative control siRNA or OR10H1 knock-down siRNA1 was conducted (FIG. 6A), using a chicken anti-rat IgG-AlexaFluor 647 conjugate (ThermoFisher A-21472) as a secondary antibody. Cells knocked-down for OR10H1 show reduced fluorescence compared to wild-type (ie, OR10H1-positive) cells.

[0323] In particular, the antibody purified from clone Di-8A11-H12-E6 shows specificity (compared to control IgG2a isotype antibody) by binding to those melanoma cells (M579-A2) that express OR10H1 but not binding to myeloma cells (KMM-1) that do not express OR10H1 (FIG. 6B). OR10H1 expression is determined by RT-PCR.

[0324] Such results demonstrate the ability of antibodies of the present invention to detected the expression of OR10H1 protein and their utility to determine increased resistance of a cell against an immune response, such as of a cancer cell.

Example 7

Sequences of Antibodies of the Invention (FIG. 7)

[0325] The sequences of variable domains of the antibodies produced by hybridoma clones of Example 5 were determined by standard procedures at Antibody Designs Laboratories (San Diego). Briefly, the following general procedure was followed: (1) total RNA extraction and cDNA

[0326] Synthesis; (2) 5'RACE extension; (3) amplification of VH and VL domains including leader sequence and partial constant regions CH1 and CL; (4) cloning of PCR positive reactions; (5) colony PCR and sequencing of clones with proper insert size; and (6) sequencing analysis up to 5 coverage or 10 clones per chain. From the nucleic acid sequence the amino acid sequence of the variable domains of the chains of each antibody was determined. The amino acid sequence of the variable domains of heavy and light chain of exemplary antibodies of the invention is shown in FIGS. 7A, B and C. Hybridoma clone Di-8A11-H12-E6 is deposited at the DSMZ (Leibniz-Institut DSMZDeutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstr. 7B, 38124 Braunschweig, Germany) in accordance with the Budapest Treaty. (DSMZ Deposition Number: DSM ACC3310, deposited 26 Oct. 2016, Depositor: Deutsches Krebsforschungszentrum Stiftung des offentlichen Rechts, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany). Hybridoma clone Di-8A11-H12-E6 is a hybridoma comprising Rat B lymphocytes fused with murine Ag8 myeloma cells, and can be cultured at 37 C. in a 5% CO.sub.2 atmosphere in RPMI-1640 medium supplemented with 10% Fetal calf serum, 10 U/ml human recombinant IL-6 and 1% penicillin-streptomycin (10,000 U/mL). The optimal split ratio is 1:1. The viability of these hybridoma cells may be relatively low when first thawed, however this recovers over a few days in culture. Each antibody chain sequence comprises c-terminally a constant region (Table E3).

TABLE-US-00004 TABLE E3 Rat Constant Domain Regions: Rat IgG Uniprot ID for constant-region Constant region subclass (accessed 20 Oct. 2016) length (aa) IgG1 P20759 (Sequence version 1 of 1 Feb. 1991; 326 Entry version 113; 5 Oct. 2016) IgG2a P20760 (Sequence version 1 of 1 Feb. 1991; 322 Entry version 116; 5 Oct. 2016) IgG2b P20761 (Sequence version 1 of 1 Feb. 1991; 333 Entry version 105; 5 Oct. 2016) IgG2c P20762 (Sequence version 1 of 1 Feb. 1991; 329 Entry version 96; 5 Oct. 2016) IgG kappa P01836 (Sequence version 1 of 21 Jul. 1986; 106 (A allele) Entry version 80; 5 Oct. 2016) IgG kappa P01835 (Sequence version 1 of 21 Jul. 1986; 106 (B allele) Entry version 89; 5 Oct. 2016)

Example 8

Peptide Fingerprint Mapping of Antibodies of the Invention

[0327] Using standard proceduresbriefly, trypsin digestion of antibody protein followed by peptide mass fingerprinting using MALDI-TOF mass spectrometry(Toplab GmbH, Martinsried, Germany), the IgG subclass of the constant region of the antibody chain(s) is determined, by comparison of the peptide mass-peaks measured by MALDI-TOF mass spectrometry to the mass of peptide fragments predicted to be obtained from trypsin digestion of the respective rat IgG subclasses (eg Table E3).

REFERENCES FOR EXAMPLES

[0328] 1. Safford, M., et al., Egr-2 and Egr-3 are negative regulators of T cell activation. Nat Immunol, 2005. 6(5): p. 472-480.

[0329] 2. Collins, S., et al., Opposing regulation of T cell function by Egr-1/NAB2 and Egr-2/Egr-3. European Journal of Immunology, 2008. 38(2): p. 528-536.

[0330] 3. Zheng, Y., Y. Zha, and T. F. Gajewski, Molecular regulation of T-cell anergy. EMBO Rep., 2008. 9: p. 50-55.

[0331] 4. Bernabei, P., et al., Interferon- receptor 2 expression as the deciding factor in human T, B, and myeloid cell proliferation or death. Journal of Leukocyte Biology, 2001. 70(6): p. 950-960.

[0332] 5. Schroder, K., et al., Interferon-: an overview of signals, mechanisms and functions. Journal of Leukocyte Biology, 2004. 75(2): p. 163-189.

[0333] 6. Wherry, E. J., et al., Molecular Signature of CD8+ T Cell Exhaustion during Chronic Viral Infection. Immunity, 2007. 27(4): p. 670-684.

[0334] 7. Zhou, T., et al , Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self-reactive T cells. J Exp Med, 1996. 183(4): p. 1879-92.

[0335] 8. Hui, W., C. Zhao, and S. G. Bourgoin, LPA Promotes T Cell Recruitment through Synthesis of CXCL13. 2015. 2015: p. 1-10.

[0336] 9. Manzo, A., et al., Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid joint. Arthritis & Rheumatism, 2008. 58(11): p. 3377-3387.

[0337] 10. Boles, K. S., et al., The tumor suppressor TSLC1/NECL-2 triggers NK-cell and CD8+ T-cell responses through the cell-surface receptor CRTAM. Blood, 2005. 106(3): p. 779-786.

[0338] 11. Charvet, C., et al., Membrane localization and function of Vav3 in T cells depend on its association with the adapter SLP-76. Journal of Biological Chemistry, 2005. 280(15): p. 15289-15299.

[0339] 12. Huang, Y. and R. L. Wange, T Cell Receptor Signaling: Beyond Complex Complexes. Journal of Biological Chemistry, 2004. 279(28): p. 28827-28830.

[0340] 13. Dose, M., et al., c-Myc mediates pre-TCR-induced proliferation but not developmental progression. Blood, 2006. 108(8): p. 2669-2677.

[0341] 14. Wang, R., et al., The Transcription Factor Myc Controls Metabolic Reprogramming upon T Lymphocyte Activation. Immunity, 2011. 35(6): p. 871-882.

[0342] 15. Hague, M., et al., C-Myc regulation by costimulatory signals modulates the generation of CD8+ memory T cells during viral infection. Open Biology, 2016. 6(1).

[0343] 16. Chandran, S. S., et al., Tumor-Specific Effector CD8+ T Cells That Can Establish Immunological Memory in Humans after Adoptive Transfer Are Marked by Expression of IL7 Receptor and c-myc. Cancer Research, 2015. 75(16): p. 3216-3226.

[0344] 17. Yao, S., et al., Interferon Regulatory Factor 4 Sustains CD8+ T Cell Expansion and Effector Differentiation. Immunity, 2013. 39(5): p. 833-845.

[0345] 18. Huber, M. and M. Lohoff, IRF4 at the crossroads of effector T-cell fate decision. European Journal of Immunology, 2014. 44(7): p. 1886-1895.

[0346] 19. Wen, A. Y., K. M. Sakamoto, and L. S. Miller, The role of the transcription factor CREB in immune function. Journal of immunology (Baltimore, Md.: 1950), 2010. 185(11): p. 6413-6419.

[0347] 20. Powell, J. D., et al., The-180 Site of the IL-2 Promoter Is the Target of CREB/CREM Binding in T Cell Anergy. The Journal of Immunology, 1999. 163(12): p. 6631-6639.

[0348] 21. Abraham, N., et al., Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p561ck. Nature, 1991. 350(6313): p. 62-66.

[0349] 22. Gervais, F. G., et al., The SH2 domain is required for stable phosphorylation of p561ck at tyrosine 505, the negative regulatory site. Molecular and Cellular Biology, 1993. 13(11): p. 7112-7121.

[0350] 23. Yamaguchi, H. and W. A. Hendrickson, Structural basis for activation of human lymphocyte kinase Lck upon tyrosine phosphorylation. Nature, 1996. 384(6608): p. 484-489.

[0351] 24. Vang, T., et al., Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor. The Journal of experimental medicine, 2001. 193(4): p. 497-507.

[0352] 25. Taskn, K. and a. J. Stokka, The molecular machinery for cAMP-dependent immuno-modulation in T-cells. Biochemical Society transactions, 2006. 34(Pt 4): p. 476-479.

[0353] 26. Bopp, T., et al., Cyclic adenosine monophosphate is a key component of regulatory T cell-mediated suppression. The Journal of Experimental Medicine, 2007. 204(6): p. 1303-1310.

[0354] 27. Ye, J., et al., TLR8 signaling enhances tumor immunity by preventing tumor-induced T-cell senescence. EMBO Molecular Medicine, 2014. 6(10): p. 1294-1311.

[0355] 28. Bevans, C. G., Isoform Composition of Connexin Channels Determines Selectivity among Second Messengers and Uncharged Molecules. Journal of Biological Chemistry, 1998. 273(5): p. 2808-2816.