MODULATORS OF TUMOR IMMUNE RESISTANCE FOR THE TREATMENT OF CANCER

Abstract

The present invention pertains to novel modulators of tumor resistance against T-cell mediated cytotoxic immune responses. The invention provides antagonists of tumor immune escape mechanisms and methods and other aspects related thereto, and therefore provides novel approaches for treating or aiding a treatment of various cancerous diseases and/or the diagnosis thereof. The invention pertains to both negative and positive regulators of tumor cell resistance and suggests the use inhibitors or activators of these genes for therapeutic purposes. In particular aspects, the invention provides combination therapeutics and/or therapies involving such inhibitors or activators. The invention furthermore provides screening methods for novel cancer therapeutics modulating the action of the identified genes, diagnostic approaches to detect cancer resistance to cytotoxic T-cells as well as pharmaceutical compositions and diagnostic kits, for use with or related to for performing these methods.

Claims

1. A combination comprising (a) and (b); or (a) and (c); or (a), (b) and (c); wherein (a) Is an inhibitor or antagonist of: FZD3, TRHDE, IL8, OR3A2, P2RY11, GHRHR, FLJ31393, CXCL3, GNRHR2, HTR1D, CCL23, CCL2, CCR7, IL8RA, GPR34, TACR2, GPR43, GCG, GRK6, TRAR5, TRAR3 or CCR9, (b) Is an inhibitor or antagonist of PI3K-Akt signaling and/or an inhibitor or antagonist of p70S6 kinase signaling, and (c) Is an activator or agonist of ERK1/2 signaling and/or an activator or agonist of JNK signaling.

2. The combination according to claim 1, wherein the combination is a pharmaceutical composition, or is a plurality of pharmaceutical compositions, comprising (a) and (b), or (a) and (c), or (a) and (b) and (c).

3. The combination according to claim 1 or 2, wherein said inhibitor or antagonist of (a) is an inhibitor or antagonist of FZD3.

4. The combination according to any one of claims 1 to 3, comprising (a) and (b), wherein (b) is, an inhibitor or antagonist of PI3K-Akt signaling.

5. The combination according to any one of claims 1 to 5, comprising (a) and (b), wherein (b) is, an inhibitor or antagonist of p70S6 kinases signaling, preferably an inhibitor of S6K.

6. The combination according to any one of claims 1 to 3, comprising (a) and (b), wherein (b) is an mTOR/PI3K dual inhibitor or a dual S6K and Akt inhibitor.

7. The combination according to any one of claims 1 to 3, comprising (a) and (b), wherein (b) comprises MK-2206 (CAS NO: 1032350-13-2).

8. The combination according to any one of claims 1 to 3, comprising (a) and (b), wherein (b) comprises MSC-2363318A (CAS NO: 1379545-95-5).

9. The combination according to any one of claims 1 to 8, wherein (a) is an antibody that binds to the protein that is the target of the inhibitor or antagonist of (a) and inhibits expression of said protein and/or said protein-T-cell interaction and/or said protein cell signaling.

10. The combination according to any one of claims 1 to 8, wherein (a) is a nucleic acid, preferably an siRNA, that inhibits expression of the protein that is the target of the inhibitor or antagonist of (a) and/or said protein-T-cell interaction and/or said protein cell signaling.

11. A method for treating a tumor disease of a patient, wherein the tumor disease is characterized by a resistance of a tumor cell to a T cell mediated immune response of the patient, the method comprising a step of administering to the patient a therapeutically effective amount of the combination according to any one of claims 1 to 12, preferably by administering to the patient a therapeutically effective amount of the components (a) and (b), or (a) and (c), or (a) and (b) and (c) of such combination.

12. The method according to claim 11, wherein the inhibitor or antagonist of (a) is selected from an inhibitor or antagonist of expression of the protein that is the target of the inhibitor or antagonist of (a), an inhibitor or antagonist of said protein signaling, or an inhibitor or antagonist of said protein-T-cell interaction.

13. The method according to claim 11 or 12, wherein said tumor cell is characterized by a detectable cell surface expression of the protein that is the target of the inhibitor or antagonist of (a).

14. The method according to claim 12 or 13, wherein said inhibitor of said protein-T-cell interaction is an inhibitor of said protein mediated STAT1 impairment in T-cells.

15. The combination according to any one of claims 1 to 10, or the method according to any one of claims 11 to 14, wherein said inhibitor or antagonist of (a), said inhibitor or antagonist of PI3K-Akt signaling and/or said inhibitor or antagonist of p70S6 kinase signaling, and/or said activator or agonist of ERK1/2 signaling and/or said activator or agonist of JNK signaling, is a compound selected from a polypeptide, peptide, glycoprotein, a peptidomimetic, an antibody or antibody-like molecule; a nucleic acid such as a DNA or RNA, for example an antisense DNA or RNA, a ribozyme, an RNA or DNA aptamer, siRNA, shRNA and the like, including variants or derivatives thereof such as a peptide nucleic acid (PNA); a targeted gene editing construct, such as a CRISPR/Cas9 construct, a carbohydrate such as a polysaccharide or oligosaccharide and the like, including variants or derivatives thereof; a lipid such as a fatty acid and the like, including variants or derivatives thereof; or a small organic molecules including but not limited to small molecule ligands, small cell-permeable molecules, and peptidomimetic compounds.

16. The method according to any one of claims 11 to 15, wherein said tumor cell, tumor or tumor disease is characterized by a resistance against T-cell mediated cytotoxicity.

17. The method according to any one of claims 11 to 16, wherein said tumor cell, tumor or tumor disease is selected from a liquid or solid tumor, and preferably is breast cancer, ovarian cancer, cancer of the colon and generally the gastro-intestinal tract, lung cancer, e.g., small-cell lung cancer and non-small-cell lung cancer, renal cancer, bladder cancer, prostate cancer, skin cancer like melanoma, head and neck cancer or a tumor disease of the central nervous system, e.g., cervix cancer and, in particular, a brain tumor, more especially astrocytoma, e.g., glioma, or blood cancer such as leukemia (or a tumor cell derived therefrom).

18. The method according to any one of claims 11 to 17, wherein said inhibitor or antagonist of (a) is an inhibitor or antagonist of an interaction between a T-cell and the protein that is the target of the inhibitor or antagonist of (a), and said protein-T-cell interaction is a said protein mediated binding of said tumor cell to said T-cell, for example by intermolecular interaction between cell surface expressed said protein on said tumor cell and at least one T-cell component expressed on the cellular surface of said T-cell.

19. The method according to any one of claims 11 to 18, wherein components (a) and (b), or (a) and (c), or (a) and (b) and (c) of said combination are combined by sequential or concomitant administration to a subject suffering from the tumor disease during said treatment, preferably wherein (a) and (b), or (a) and (c), or (a) and (b) and (c) are concomitantly administered during said treatment.

20. A combination comprising (a) and (b), or (a) and (c), or (a) and (b) and (c), wherein (a) Is an activator or agonist of: CXCL9, CXCR3, GRM4, GRK5, CCR2, ENPP2, GRM6, OR1G1, ADMR, MASS1, or OR1D4, (b) Is an inhibitor or antagonist of PI3K-Akt signaling and/or an inhibitor or antagonist of p70S6 kinase signaling, and (c) Is an activator or agonist of ERK1/2 signaling and/or an activator or agonist of JNK signaling.

21. The combination according to claim 20, wherein said activator or agonist of (a) is an activator or agonist of one or other member of the receptor/ligand pair CXCL9 or CXCR3.

22. The combination according to claim 20 or 21 comprising (a) and (b), wherein (b) comprises MK-2206 (CAS NO: 1032350-13-2).

23. The combination according to any one of claims 20 to 22, wherein (a) is an antibody that binds to the protein that is the target of the activator or agonist of (a) and activates expression of said protein and/or said protein-T-cell interaction and/or said protein cell signaling.

24. A method for treating a tumor disease of a patient, wherein the tumor disease is characterized by a resistance of a tumor cell to a T cell mediated immune response of the patient, the method comprising a step of administering to the patient a therapeutically effective amount of the combination according to any one of claims 1 to 9, preferably by administering to the patient a therapeutically effective amount of the components (a) and (b), or (a) and (c), or (a) and (b) and (c) of such combination.

25. The combination according to any one of claims 20 to 23, or the method according to claim 24, wherein said activator or agonist of (a), said inhibitor or antagonist of PI3K-Akt signaling and/or said inhibitor or antagonist of p70S6 kinase signaling, and/or said activator or agonist of ERK1/2 signaling and/or said activator or agonist of JNK signaling, is a compound selected from a polypeptide, peptide, glycoprotein, a peptidomimetic, an antibody or antibody-like molecule; a nucleic acid such as a DNA or RNA, for example an antisense DNA or RNA, a ribozyme, an RNA or DNA aptamer, siRNA, shRNA and the like, including variants or derivatives thereof such as a peptide nucleic acid (PNA); a targeted gene editing construct, such as a CRISPR/Cas9 construct, a carbohydrate such as a polysaccharide or oligosaccharide and the like, including variants or derivatives thereof; a lipid such as a fatty acid and the like, including variants or derivatives thereof; or a small organic molecules including but not limited to small molecule ligands, small cell-permeable molecules, and peptidomimetic compounds.

26. A method for reducing resistance of a tumor cell to an immune response, the method comprising a step of contacting the tumor cell with a modulator of tumor resistance selected from an (a) inhibitor or antagonist of: FZD3, TRHDE, IL8, OR3A2, P2RY11, GHRHR, FLJ31393, CXCL3, GNRHR2, HTR1D, CCL23, CCL2, CCR7, IL8RA, GPR34, TACR2, GPR43, GCG, GRK6, TRAR5, TRAR3 or CCR9, or (b) an activator or agonist of: ENPP2, GRM6, GRK5, OR1G1, CCR2, ADMR, GRM4, MASS1, CXCL9, CXCR3, or OR1D4.

27. The method according to claim 26, wherein the resistance of a tumor cell to an immune response is a resistance of the tumor cell to a T cell mediated immune response.

28. The method according to claim 26 or 27, wherein said modulator of tumor resistance is (a) An inhibitor or antagonist of expression, protein function, or signaling of a protein selected from: FZD3, TRHDE, IL8, OR3A2, P2RY11, GHRHR, FLJ31393, CXCL3, GNRHR2, HTR1D, CCL23, CCL2, CCR7, IL8RA, GPR34, TACR2, GPR43, GCG, GRK6, TRAR5, TRAR3 or CCR9, as applicable, or (b) an activator or agonist of the expression, protein function, or signaling of a protein selected from: CXCL9, CXCR3, GRM4, GRK5, CCR2, ENPP2, GRM6, OR1G1, ADMR, MASS1, or OR1D4, as applicable.

29. The method according to claim 27 or 28, comprising a step of contacting the tumor cell with at least one additional compound effective in the treatment of cancer, preferably wherein the at least one additional compound effective in the treatment of cancer is one or more modulators of tumor resistance selected from (a) An inhibitor or antagonist of expression, protein function, or signaling of a protein selected from CCR9, GHRHR, FLJ31393, FZD3, OR3A2, CXCL3, GNRHR2, IL8, HTR1D, CCL23, CCL2, P2RY11, TRHDE, CCR7, IL8RA, GPR34, TACR2, GPR43, GCG, GRK6, TRAR5 or TRAR3, and/or (b) an activator or agonist of the expression, protein function, or signaling of a protein selected from ENPP2, GRM6, GRK5, OR1G1, CCR2, ADMR, GRM4, MASS1, CXCL9, CXCR3, or OR1D4.

30. The method according to claim 27 or 28, comprising a step of contacting the tumor cell with at least one additional compound effective in the treatment of cancer, preferably wherein the at least one additional compound effective in the treatment of cancer is one or more modulators of tumor resistance selected from an inhibitor or antagonist of expression, protein function, or signaling of a protein selected from OR2J2, VN1R4 or OR1F1.

31. The method according to claim 27 or 28, comprising a step of contacting the tumor cell with at least one additional compound effective in the treatment of cancer, preferably wherein the at least one additional compound effective in the treatment of cancer is one or more modulators of tumor resistance selected from an inhibitor or antagonist of expression, protein function, or signaling of a protein selected from CEACAM-6 or CD274.

32. The method according to any one of claims 27 to 31, wherein said tumor cell is characterized by a detectable cell surface expression of: FZD3, TRHDE, IL8, OR3A2, P2RY11, GHRHR, FLJ31393, CXCL3, GNRHR2, HTR1D, CCL23, CCL2, CCR7, IL8RA, GPR34, TACR2, GPR43, GCG, GRK6, TRAR5, TRAR3 or CCR9, as applicable, before contacting the tumor cell with the corresponding modulator of tumor resistance.

33. The method according to claim 28, wherein said inhibitor of said protein-T-cell interaction is an inhibitor of said protein mediated STAT1 impairment in T-cells.

34. A method for treating a tumor disease in a patient, wherein said tumor disease is characterized by a resistance of said tumor against immune responses, the method comprising a step of (a) Inhibiting or antagonizing in said patient: FZD3, TRHDE, IL8, OR3A2, P2RY11, GHRHR, FLJ31393, CXCL3, GNRHR2, HTR1D, CCL23, CCL2, CCR7, IL8RA, GPR34, TACR2, GPR43, GCG, GRK6, TRAR5, TRAR3 or CCR9, or (b) Activating or agonizing: CXCL9, CXCR3, GRM4, GRK5, CCR2, ENPP2, GRM6, OR1G1, ADMR, MASS1, or OR1D4.

35. A method for aiding a patient's immune response against a tumor disease comprising a step of (a) Inhibiting or antagonizing in said patient: FZD3, TRHDE, IL8, OR3A2, P2RY11, GHRHR, FLJ31393, CXCL3, GNRHR2, HTR1D, CCL23, CCL2, CCR7, IL8RA, GPR34, TACR2, GPR43, GCG, GRK6, TRAR5, TRAR3 or CCR9, or (b) Activating or agonizing: CXCL9, CXCR3, GRM4, GRK5, CCR2, ENPP2, GRM6, OR1G1, ADMR, MASS1, or OR1D4.

36. The method according to claim 34 or 35, comprising a step of administering to said patient a therapeutically effective amount of a modulator of tumor resistance selected from an (a) inhibitor or antagonist of: FZD3, TRHDE, IL8, OR3A2, P2RY11, GHRHR, FLJ31393, CXCL3, GNRHR2, HTR1D, CCL23, CCL2, CCR7, IL8RA, GPR34, TACR2, GPR43, GCG, GRK6, TRAR5, TRAR3 or CCR9, as applicable, or (b) an activator or agonist of CXCL9, CXCR3, GRM4, GRK5, CCR2, ENPP2, GRM6, OR1G1, ADMR, MASS1, or OR1D4, as applicable.

37. The method according to any one of claims 26 to 36, wherein said inhibitor or antagonist of (a), or said activator or agonist of (b), is a compound is selected from a polypeptide, peptide, glycoprotein, a peptidomimetic, an antibody or antibody-like molecule; a nucleic acid such as a DNA or RNA, for example an antisense DNA or RNA, a ribozyme, an RNA or DNA aptamer, siRNA, shRNA and the like, including variants or derivatives thereof such as a peptide nucleic acid (PNA); a targeted gene editing construct, such as a CRISPR/Cas9 construct, a carbohydrate such as a polysaccharide or oligosaccharide and the like, including variants or derivatives thereof; a lipid such as a fatty acid and the like, including variants or derivatives thereof; or a small organic molecules including but not limited to small molecule ligands, small cell-permeable molecules, and peptidomimetic compounds.

38. The method according to any one of claims 26 to 37, wherein said tumor cell, tumor or tumor disease is selected from a liquid or solid tumor, and preferably is breast cancer, ovarian cancer, cancer of the colon and generally the gastro-intestinal tract, lung cancer, e.g., small-cell lung cancer and non-small-cell lung cancer, renal cancer, bladder cancer, prostate cancer, skin cancer like melanoma, head and neck cancer or a tumor disease of the central nervous system, e.g., cervix cancer and, in particular, a brain tumor, more especially astrocytoma, e.g., glioma, or blood cancer such as leukemia.

39. A method for identifying a compound suitable for the treatment of a tumor disease, the method comprising the steps of (a) Providing a first cell expressing a protein on the cellular surface, wherein the protein is selected from: (x) FZD3, TRHDE, IL8, OR3A2, P2RY11, GHRHR, FLJ31393, CXCL3, GNRHR2, HTR1D, CCL23, CCL2, CCR7, IL8RA, GPR34, TACR2, GPR43, GCG, GRK6, TRAR5, TRAR3 or CCR9, or is selected from: (y) CXCL9, CXCR3, GRM4, GRK5, CCR2, ENPP2, GRM6, OR1G1, ADMR, MASS1, or OR1D4, (b) Providing a candidate compound, (c) Optionally, providing a second cell which is a cytotoxic T-lymphocyte (CTL), preferably that is 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 of said protein in said first cell, wherein a differential protein expression 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 tumor disease; and/or ii. cytotoxicity of said CTL against said first cell, wherein an enhanced cytotoxicity of said CTL against said first cell contacted with the candidate compound compared to the cytotoxicity of said CTL against said first cell not contacted with the candidate compound indicates that the candidate compound is a compound suitable for the treatment of a tumor disease.

40. The method according to claim 39, wherein a reduced protein expression/function of said protein of (x) 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 tumor disease.

41. The method according to claim 39, wherein an increased protein expression/function of said protein of (y) 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 tumor disease.

42. The method according to any one of claims 39 to 41, wherein, said tumor disease or tumor derived cell is selected from a liquid or solid tumor, and preferably is breast cancer, ovarian cancer, cancer of the colon and generally the gastro-intestinal tract, lung cancer, e.g., small-cell lung cancer and non-small-cell lung cancer, renal cancer, bladder cancer, prostate cancer, skin cancer like melanoma, head and neck cancer or a tumor disease of the central nervous system, e.g., cervix cancer and, in particular, a brain tumor, more especially astrocytoma, e.g., glioma, or blood cancer such as leukemia (or a tumor cell derived therefrom).

43. The method according to any one of claims 39 to 42, wherein said first cell is a cell resistant to cytotoxicity mediated by T-lymphocytes, preferably a tumor derived cell.

44. The method according to any one of claims 39 to 43, wherein said candidate compound is selected from a polypeptide, peptide, glycoprotein, a peptidomimetic, an antibody or antibody-like molecule; a nucleic acid such as a DNA or RNA, for example an antisense DNA or RNA, a ribozyme, an RNA or DNA aptamer, siRNA, shRNA and the like, including variants or derivatives thereof such as a peptide nucleic acid (PNA); a targeted gene editing construct, such as a CRISPR/Cas9 construct, a carbohydrate such as a polysaccharide or oligosaccharide and the like, including variants or derivatives thereof; a lipid such as a fatty acid and the like, including variants or derivatives thereof; or a small organic molecules including but not limited to small molecule ligands, small cell-permeable molecules, and peptidomimetic compounds.

45. A method for diagnosing in a patient a resistance of a tumor disease against T cell mediated immune responses, the method comprising a step of (a) determining expression of: FZD3, TRHDE, IL8, OR3A2, P2RY11, GHRHR, FLJ31393, CXCL3, GNRHR2, HTR1D, CCL23, CCL2, CCR7, IL8RA, GPR34, TACR2, GPR43, GCG, GRK6, TRAR5, TRAR3 or CCR9 in a tumor cell from the tumor of the patient, wherein a detectable or increased expression of any one of the proteins in the tumor cell compared to a negative control is indicative for a resistance of the tumor disease against T cell mediated immune responses; or (b) determining expression of: CXCL9, CXCR3, GRM4, GRK5, CCR2, ENPP2, GRM6, OR1G1, ADMR, MASS1, or OR1D4 in a tumor cell from the tumor of the patient, wherein a reduced expression of any one of the proteins in the tumor cell compared to a negative control is indicative for a resistance of the tumor disease against T cell mediated immune responses.

46. The method according to claim 45, comprising a preceding step of obtaining a tumor cell from the patient.

47. The method according to claim 45 or 46, wherein said expression is a cell surface expression of said protein of (a) or (b) on the tumor cell.

48. The method according to any one of claims 45 to 47, wherein, said tumor disease is selected from a liquid or solid tumor, and preferably is breast cancer, ovarian cancer, cancer of the colon and generally the gastro-intestinal tract, lung cancer, e.g., small-cell lung cancer and non-small-cell lung cancer, renal cancer, bladder cancer, prostate cancer, skin cancer like melanoma, head and neck cancer or a tumor disease of the central nervous system, e.g., cervix cancer and, in particular, a brain tumor, more especially astrocytoma, e.g., glioma, or blood cancer such as leukemia.

Description

IN THE FIGURES

[0345] FIG. 1: Heat map representation of potential positive and negative immune modulators identified from the RNAi screens Differential scores were used to identify positive immune modulators (yellow) the knockdown of which enhance CTL-mediated cell killing and negative immune modulators (blue) the knockdown of which reduce CTL-mediated cell killing. Differential scores prior to filtering are shown for all genes tested in the 3 different screens (see Materials and Methods). Selected representative clusters of high-confidence hits are displayed herein.

[0346] FIG. 2: CCR9 knockdown sensitizes tumor cells to immune attack A MCF7 cells were transfected with the described siRNA sequences and harvested after 72 h for mRNA and protein estimation using RT-PCR (upper) and immunoblot (lower) analysis, respectively. GAPDH and beta-actin were used as controls for RNA and protein normalization, respectively. B Luc-CTL cytotoxicity assay with PBMC-derived CTLs and bi-specific Ab as effector population and MCF7 as target cells, which were transfected with individual (s1-s4) or pooled CCR9 siRNA sequences. PD-L1 and non-specific control siRNAs were used as positive and negative controls, respectively, for CTL-mediated cytotoxicity. C, D Cr-release assay showing % specific lysis of MCF7 cells by survivin-specific T cells at different ratios upon CCR9 knockdown (C) or overexpression (D). MCF7 cells were transfected with either CCR9 siRNA s1 (), pooled siRNA sequences (), positive control PD-L1 (), and non-specific control siRNA (.square-solid.) (C) or with pCMV6-AC-His control vector (.square-solid.) and pCMV6-AC-His-CCR9 expression construct () (D) 72 h prior to the assay. E Cr-release assay showing % specific lysis of MDA-MB-231 breast tumor cell line by survivin-specific T cells at different ratios upon CCR9 knockdown () in comparison to the control knockdown (.square-solid.). F, G Cr-release assay showing lysis of patient-derived melanoma cells (M579-A2) by tumor-infiltrating lymphocytes (TIL 412) (F) or lysis of PANC-1 pancreatic adenocarcinoma cells by patient-derived pancreatic TIL 53 (G) at different E:T ratios upon CCR9 () or control (.square-solid.) knockdown. Data information: All experiments were performed in triplicates and are representative of at least three independent experiments. Error bars denote SEM, and statistical significance was calculated using the unpaired, two-tailed Student's t-test.

[0347] FIG. 3: Tumor-specific CCR9 impedes Th1-type immune response A, B ELISpot assay showing IFN- (A) and granzyme B (B) secretion by survivin-specific T cells, as spot numbers, upon CCR9 knockdown (black bars) in MCF7 cells compared to the control knockdown (white bars). T cells (TC) alone (grey bars) were used as control for background spot numbers. C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin-specific TC and either CCR9.sup.hi MCF7 (transfected with CCR9-specific siRNA) or CCR9.sup.lo MCF7 (transfected with control siRNA) cells. D Phosphoplex analysis showing the phospho-STAT levels in survivin-specific TC upon encountering CCR9.sup.hi or CCR9.sup.lo MCF7 cells. Log 2 ratio of mean fluorescent intensity (MFI) of the respective analytes to the unstimulated TC is plotted herein. E Immunoblot analysis showing the phospho-STAT1 levels in the CCR9.sup.hi-treated, CCR9.sup.lo-treated or unstimulated TC using the phospho-specific STAT1 (pTyr701) antibody. Beta-actin was used as the loading control. Data information: In all the cases, experiments were performed in triplicate with at least two independent repeats. MeanSEM are shown herein, unless stated otherwise, with statistical significance assessed using unpaired, two-tailed Student's t-test. Source data are available online for this figure.

[0348] FIG. 4: Tumor-specific CCR9 interacts directly with T cells inducing prominent changes in the gene expression signature A ELISA showing CCL25 levels in cell lysates from indicated tumor cell lines. CCR9 knockdown (k.d.) in MCF7 cells was achieved using specific shRNA (see Materials and Methods). B Cr-release assay showing % specific lysis of MCF7 cells by survivin TC upon CCL25 () or CCR9 () inhibition using specific siRNAs in comparison to the control siRNA (.square-solid.). MeanSEM are depicted herein. C MCF7 cells were transfected with control or CCR9-specific siRNAs, and 48 h later, the supernatants (CCR91.sup.lo or CCR9.sup.hi SSN, respectively) were used to culture survivin TCs overnight. Supernatant-treated TCs were then used as effector cells against CCR9.sup.lo or CCR9.sup.hi MCF7 tumor cells in the Cr-release assay along with wild-type MCF7 cells. MeanSEM are depicted herein. D Cr-release assay showing % specific lysis of MCF7 cells that were pre-treated with or without pertussis toxin (PTX), or knocked down for CCR9 using specific siRNA. MeanSEM are depicted herein. E, F MCF7 cells transfected with control siRNA (CCR9.sup.hi) or CCR9 siRNA (CCR9.sup.lo) were cocultured with survivin TCs for 12 h. Gene microarray was performed with the total RNA extracted from purified T cells after the coculture. Volcano plot (E) illustrating fold change (FC; log 2) in gene expression intensities compared with P-value (log 2) between CCR9.sup.hi- and CCR9.sup.lo-treated TCs. Horizontal bar at y=4.32 represents a statistical significance of P=0.05 (genes in gray below this line did not reach significance). Log FC cutoff at +0.5 is represented by the vertical lines. Heatmap representation of the top upregulated (Log FC>0.5) and downregulated (Log FC<0.85) genes (F) with P0.05. Individual replicates per sample group are shown herein.

[0349] FIG. 5: In vivo inhibition of CCR9 significantly reduces tumor outgrowth in response to adoptive TIL therapy A Cr-release assay showing TIL 209-mediated lysis of CCR9.sup.+ M579-A2 (transduced with control shRNA) or CCR9.sup. M579-A2 cells (transduced with CCR9-specific shRNA). Curves represent meanSEM. B Scheme for the in vivo mouse experiment involving the s.c. injection of CCR9+(shControl) or CCR9 (shCCR9) M579-A2 tumor cells in the left and right flank, respectively, of the NSG mice. Following this, at d2 and d9, mice received i.v. injection of TIL 209 in PBS (n=7) or PBS alone (control group for tumor growth; n=3) and measured for tumor growth. C, D Tumor growth curves showing meanSEM tumor volume of CCR9.sup.+ or CCR9.sup. M579-A2 tumors in TIL-treated mice (C) or the PBS alone group (D). Statistical difference was calculated using the unpaired one-sided Mann-Whitney U-test.

[0350] FIG. 6: Altered signaling cascades in MCF7 tumor cells upon CCR9 knockdown. MCF7 cells were reverse transfected with control or CCR9-specific siRNA and after 72 h protein lysates were used for phospho-plex analysis of the major transcription factors indicated on x-axis (studied phopho-sites are indicated in brackets). Statistical differences between the two groups were analyzed using student's two-sided t-test, n=3. Error bars represent SEM.

IN THE SEQUENCES: [INCLUDED TO BE CONSISTENT IN CASE WE PROSECUTE A CCR9 ASPECT OUT OF HERE]

[0351]

TABLE-US-00001 SEQIDNO:1showsHomosapiensIsoform1ofC-Cchemokine receptortype9CCR9: MTPTDFTSPIPNMADDYGSESTSSMEDYVNFNFTDFYCEKNNVRQFASHFLPPLYWLVFIVG ALGNSLVILVYWYCTRVKTMTDMFLLNLAIADLLFLVTLPFWAIAAADQWKFQTFMCKVVNS MYKMNFYSCVLLIMCISVDRYIAIAQAMRAHTWREKRLLYSKMVCFTIWVLAAALCIPEILY SQIKEESGIAICTMVYPSDESTKLKSAVLTLKVILGFFLPFVVMACCYTIIIHTLIQAKKSS KHKALKVTITVLTVFVLSQFPYNCILLVQTIDAYAMFISNCAVSTNIDICFQVTQTIAFFHS CLNPVLYVFVGERFRRDLVKTLKNLGCISQAQWVSFTRREGSLKLSSMLLETTSGALSL SEQIDNO:2showsHomosapiensIsoform2ofC-Cchemokine receptortype9CCR9: MADDYGSESTSSMEDYVNFNFTDFYCEKNNVRQFASHFLPPLYWLVFIVGALGNSLVILVYW YCTRVKTMTDMFLLNLAIADLLFLVTLPFWAIAAADQWKFQTFMCKVVNSMYKMNFYSCVLL IMCISVDRYIAIAQAMRAHTWREKRLLYSKMVCFTIWVLAAALCIPEILYSQIKEESGIAIC TMVYPSDESTKLKSAVLTLKVILGFFLPFVVMACCYTIIIHTLIQAKKSSKHKALKVTITVL TVFVLSQFPYNCILLVQTIDAYAMFISNCAVSTNIDICFQVTQTIAFFHSCLNPVLYVFVGE RFRRDLVKTLKNLGCISQAQWVSFTRREGSLKLSSMLLETTSGALSL SEQIDNO:3showsHomosapiensC-Cmotifchemokinereceptor9 (CCR9),isoform1,mRNA: GCTTCCTTTCTCGTGTTGTTATCGGGTAGCTGCCTGCTCAGAACCCACAAAGCCTGCCCCTC ATCCCAGGCAGAGAGCAACCCAGCTCTTTCCCCAGACACTGAGAGCTGGTGGTGCCTGCTGT CCCAGGGAGAGTTGCATCGCCCTCCACAGAGCAGGCTTGCATCTGACTGACCCACCATGACA CCCACAGACTTCACAAGCCCTATTCCTAACATGGCTGATGACTATGGCTCTGAATCCACATC TTCCATGGAAGACTACGTTAACTTCAACTTCACTGACTTCTACTGTGAGAAAAACAATGTCA GGCAGTTTGCGAGCCATTTCCTCCCACCCTTGTACTGGCTCGTGTTCATCGTGGGTGCCTTG GGCAACAGTCTTGTTATCCTTGTCTACTGGTACTGCACAAGAGTGAAGACCATGACCGACAT GTTCCTTTTGAATTTGGCAATTGCTGACCTCCTCTTTCTTGTCACTCTTCCCTTCTGGGCCA TTGCTGCTGCTGACCAGTGGAAGTTCCAGACCTTCATGTGCAAGGTGGTCAACAGCATGTAC AAGATGAACTTCTACAGCTGTGTGTTGCTGATCATGTGCATCAGCGTGGACAGGTACATTGC CATTGCCCAGGCCATGAGAGCACATACTTGGAGGGAGAAAAGGCTTTTGTACAGCAAAATGG TTTGCTTTACCATCTGGGTATTGGCAGCTGCTCTCTGCATCCCAGAAATCTTATACAGCCAA ATCAAGGAGGAATCCGGCATTGCTATCTGCACCATGGTTTACCCTAGCGATGAGAGCACCAA ACTGAAGTCAGCTGTCTTGACCCTGAAGGTCATTCTGGGGTTCTTCCTTCCCTTCGTGGTCA TGGCTTGCTGCTATACCATCATCATTCACACCCTGATACAAGCCAAGAAGTCTTCCAAGCAC AAAGCCCTAAAAGTGACCATCACTGTCCTGACCGTCTTTGTCTTGTCTCAGTTTCCCTACAA CTGCATTTTGTTGGTGCAGACCATTGACGCCTATGCCATGTTCATCTCCAACTGTGCCGTTT CCACCAACATTGACATCTGCTTCCAGGTCACCCAGACCATCGCCTTCTTCCACAGTTGCCTG AACCCTGTTCTCTATGTTTTTGTGGGTGAGAGATTCCGCCGGGATCTCGTGAAAACCCTGAA GAACTTGGGTTGCATCAGCCAGGCCCAGTGGGTTTCATTTACAAGGAGAGAGGGAAGCTTGA AGCTGTCGTCTATGTTGCTGGAGACAACCTCAGGAGCACTCTCCCTCTGAGGGGTCTTCTCT GAGGTGCATGGTTCTTTTGGAAGAAATGAGAAATACAGAAACAGTTTCCCCACTGATGGGAC CAGAGAGAGTGAAAGAGAAAAGAAAACTCAGAAAGGGATGAATCTGAACTATATGATTACTT GTAGTCAGAATTTGCCAAAGCAAATATTTCAAAATCAACTGACTAGTGCAGGAGGCTGTTGA TTGGCTCTTGACTGTGATGCCCGCAATTCTCAAAGGAGGACTAAGGACCGGCACTGTGGAGC ACCCTGGCTTTGCCACTCGCCGGAGCATCAATGCCGCTGCCTCTGGAGGAGCCCTTGGATTT TCTCCATGCACTGTGAACTTCTGTGGCTTCAGTTCTCATGCTGCCTCTTCCAAAAGGGGACA CAGAAGCACTGGCTGCTGCTACAGACCGCAAAAGCAGAAAGTTTCGTGAAAATGTCCATCTT TGGGAAATTTTCTACCCTGCTCTTGAGCCTGATAACCCATGCCAGGTCTTATAGATTCCTGA TCTAGAACCTTTCCAGGCAATCTCAGACCTAATTTCCTTCTGTTCTCCTTGTTCTGTTCTGG GCCAGTGAAGGTCCTTGTTCTGATTTTGAAACGATCTGCAGGTCTTGCCAGTGAACCCCTGG ACAACTGACCACACCCACAAGGCATCCAAAGTCTGTTGGCTTCCAATCCATTTCTGTGTCCT GCTGGAGGTTTTAACCTAGACAAGGATTCCGCTTATTCCTTGGTATGGTGACAGTGTCTCTC CATGGCCTGAGCAGGGAGATTATAACAGCTGGGTTCGCAGGAGCCAGCCTTGGCCCTGTTGT AGGCTTGTTCTGTTGAGTGGCACTTGCTTTGGGTCCACCGTCTGTCTGCTCCCTAGAAAATG GGCTGGTTCTTTTGGCCCTCTTCTTTCTGAGGCCCACTTTATTCTGAGGAATACAGTGAGCA GATATGGGCAGCAGCCAGGTAGGGCAAAGGGGTGAAGCGCAGGCCTTGCTGGAAGGCTATTT ACTTCCATGCTTCTCCTTTTCTTACTCTATAGTGGCAACATTTTAAAAGCTTTTAACTTAGA GATTAGGCTGAAAAAAATAAGTAATGGAATTCACCTTTGCATCTTTTGTGTCTTTCTTATCA TGATTTGGCAAAATGCATCACCTTTGAAAATATTTCACATATTGGAAAAGTGCTTTTTAATG TGTATATGAAGCATTAATTACTTGTCACTTTCTTTACCCTGTCTCAATATTTTAAGTGTGTG CAATTAAAGATCAAATAGATACATT SEQIDNO:4showsHomosapiensC-Cmotifchemokinereceptor9 (CCR9),isoform2mRNA: GCTTCCTTTCTCGTGTTGTTATCGGGTAGCTGCCTGCTCAGAACCCACAAAGCCTGCCCCTC ATCCCAGGCAGAGAGCAACCCAGCTCTTTCCCCAGACACTGAGAGCTGGTGGTGCCTGCTGT CCCAGGGAGAGTTGCATCGCCCTCCACAAGCCCTATTCCTAACATGGCTGATGACTATGGCT CTGAATCCACATCTTCCATGGAAGACTACGTTAACTTCAACTTCACTGACTTCTACTGTGAG AAAAACAATGTCAGGCAGTTTGCGAGCCATTTCCTCCCACCCTTGTACTGGCTCGTGTTCAT CGTGGGTGCCTTGGGCAACAGTCTTGTTATCCTTGTCTACTGGTACTGCACAAGAGTGAAGA CCATGACCGACATGTTCCTTTTGAATTTGGCAATTGCTGACCTCCTCTTTCTTGTCACTCTT CCCTTCTGGGCCATTGCTGCTGCTGACCAGTGGAAGTTCCAGACCTTCATGTGCAAGGTGGT CAACAGCATGTACAAGATGAACTTCTACAGCTGTGTGTTGCTGATCATGTGCATCAGCGTGG ACAGGTACATTGCCATTGCCCAGGCCATGAGAGCACATACTTGGAGGGAGAAAAGGCTTTTG TACAGCAAAATGGTTTGCTTTACCATCTGGGTATTGGCAGCTGCTCTCTGCATCCCAGAAAT CTTATACAGCCAAATCAAGGAGGAATCCGGCATTGCTATCTGCACCATGGTTTACCCTAGCG ATGAGAGCACCAAACTGAAGTCAGCTGTCTTGACCCTGAAGGTCATTCTGGGGTTCTTCCTT CCCTTCGTGGTCATGGCTTGCTGCTATACCATCATCATTCACACCCTGATACAAGCCAAGAA GTCTTCCAAGCACAAAGCCCTAAAAGTGACCATCACTGTCCTGACCGTCTTTGTCTTGTCTC AGTTTCCCTACAACTGCATTTTGTTGGTGCAGACCATTGACGCCTATGCCATGTTCATCTCC AACTGTGCCGTTTCCACCAACATTGACATCTGCTTCCAGGTCACCCAGACCATCGCCTTCTT CCACAGTTGCCTGAACCCTGTTCTCTATGTTTTTGTGGGTGAGAGATTCCGCCGGGATCTCG TGAAAACCCTGAAGAACTTGGGTTGCATCAGCCAGGCCCAGTGGGTTTCATTTACAAGGAGA GAGGGAAGCTTGAAGCTGTCGTCTATGTTGCTGGAGACAACCTCAGGAGCACTCTCCCTCTG AGGGGTCTTCTCTGAGGTGCATGGTTCTTTTGGAAGAAATGAGAAATACAGAAACAGTTTCC CCACTGATGGGACCAGAGAGAGTGAAAGAGAAAAGAAAACTCAGAAAGGGATGAATCTGAAC TATATGATTACTTGTAGTCAGAATTTGCCAAAGCAAATATTTCAAAATCAACTGACTAGTGC AGGAGGCTGTTGATTGGCTCTTGACTGTGATGCCCGCAATTCTCAAAGGAGGACTAAGGACC GGCACTGTGGAGCACCCTGGCTTTGCCACTCGCCGGAGCATCAATGCCGCTGCCTCTGGAGG AGCCCTTGGATTTTCTCCATGCACTGTGAACTTCTGTGGCTTCAGTTCTCATGCTGCCTCTT CCAAAAGGGGACACAGAAGCACTGGCTGCTGCTACAGACCGCAAAAGCAGAAAGTTTCGTGA AAATGTCCATCTTTGGGAAATTTTCTACCCTGCTCTTGAGCCTGATAACCCATGCCAGGTCT TATAGATTCCTGATCTAGAACCTTTCCAGGCAATCTCAGACCTAATTTCCTTCTGTTCTCCT TGTTCTGTTCTGGGCCAGTGAAGGTCCTTGTTCTGATTTTGAAACGATCTGCAGGTCTTGCC AGTGAACCCCTGGACAACTGACCACACCCACAAGGCATCCAAAGTCTGTTGGCTTCCAATCC ATTTCTGTGTCCTGCTGGAGGTTTTAACCTAGACAAGGATTCCGCTTATTCCTTGGTATGGT GACAGTGTCTCTCCATGGCCTGAGCAGGGAGATTATAACAGCTGGGTTCGCAGGAGCCAGCC TTGGCCCTGTTGTAGGCTTGTTCTGTTGAGTGGCACTTGCTTTGGGTCCACCGTCTGTCTGC TCCCTAGAAAATGGGCTGGTTCTTTTGGCCCTCTTCTTTCTGAGGCCCACTTTATTCTGAGG AATACAGTGAGCAGATATGGGCAGCAGCCAGGTAGGGCAAAGGGGTGAAGCGCAGGCCTTGC TGGAAGGCTATTTACTTCCATGCTTCTCCTTTTCTTACTCTATAGTGGCAACATTTTAAAAG CTTTTAACTTAGAGATTAGGCTGAAAAAAATAAGTAATGGAATTCACCTTTGCATCTTTTGT GTCTTTCTTATCATGATTTGGCAAAATGCATCACCTTTGAAAATATTTCACATATTGGAAAA GTGCTTTTTAATGTGTATATGAAGCATTAATTACTTGTCACTTTCTTTACCCTGTCTCAATA TTTTAAGTGTGTGCAATTAAAGATCAAATAGATACATT SEQIDNO:5showsaCCR9-specificshRNAhairpin: ACCGGGCCAGTGGAGGTCTTTGTTCTGTTAATATTCATAGCAGAACAAGGACCTTCACTGGC TTTT

IN THE EXAMPLES

Example 1: Validation of Immune-Modulatory Function of CCR9

[0352] An siRNA screen for immunomodulatory factors was performed as in Khandelwal N et al 2015. FIG. 1 shows the screening results and selected new candidates. For exemplary functional validation of the screening approach, the C-C chemokine receptor type 9 (CCR9) was chosen as it was found to be highly immunosuppressive in all the three screens despite the divergent biological background, inhibiting T cell function in an antigen-dependent as well as antigen-independent manner (FIG. 1). CCR9 is a chemokine receptor involved in immune cell trafficking (Kunkel et al, 2000; Uehara et al, 2002) and is expressed on tolerogenic plasmacytoid dendritic cells (Hadeiba et al, 2008). So far, an implication of CCR9 in T cell function or tumor-immune resistance has not been reported.

[0353] The mRNA and protein knockdown efficiency of single siRNAs within the CCR9 siRNA pool correlated well with the functional effect on T cell cytotoxicity (FIGS. 2A and B), while none of the CCR9 siRNAs influenced cell viability. Surface expression of CCR9 on MCF7 cells was also found to be reduced by 50% in flow cytometry staining using CCR9 s1 siRNA. Knockdown of CCR9 using siRNA markedly increased MCF7 lysis by survivin-specific CTL (FIG. 2C) in the classical chromium-release assay.

[0354] Conversely, overexpression of CCR9 inhibited tumor lysis, demonstrating that CCR9 expression enables immune escape of cancer cells (FIG. 2D). CCR9 inhibition in MDA-MB-231 metastatic breast cancer cell line also resulted in marked increase in immune-mediated tumor lysis (FIG. 2E). To explore the broad applicability of CCR9-mediated immune suppression in different tumor entities under clinical setting, the inventors next silenced CCR9 in patient-derived primary melanoma cells (M579-A2 cells) and co-cultured them with HLA-matched tumor-infiltrating lymphocytes (TIL; clone 412) derived from melanoma patient and found a remarkable increase in melanoma cell lysis upon CCR9 knockdown in comparison to the control knockdown (FIG. 2F). Similarly, HLA-matched TIL cultures (TIL 53) from pancreatic adenocarcinoma patients recognized and lysed PANC-1 pancreatic cancer cells more effectively upon CCR9 knockdown as shown in FIG. 2G, stressing that CCR9-mediated immune suppression may be a clinically relevant phenomenon in multiple tumor entities.

Example 2: CCR9 Influence on CTL Function

[0355] The influence of CCR9 expression on CTL functions was explored. CCR9 knockdown in MCF7 cells significantly increased the secretion of IFN- and granzyme B by survivinspecific CTL in response to MCF7 cells (FIGS. 3A and 3B), supporting the increased cytotoxicity observed in the kill assays. To assess whether this correlated with increased TCR activation and signaling, TCR phospho-plex analysis in survivin-specific CTLs was performed after contact with CCR9.sup.hi or CCR9.sup.lo MCF7 cells. With the exception of some degree of reduced Lck phosphorylation (which was detectable only 5 min after exposure to CCR9.sup.lo tumor cells), not any CCR9-dependent changes in TCR signaling was observed. Nevertheless, TCR engagement was found to be necessary for CCR9-mediated immunosuppression as polyclonal T cells failed to secrete higher levels of IFN- in response to CCR910 MCF7 cells in the absence of anti-EpCAMCD3 bi-specific antibody.

[0356] One alternative route of T cell activation is the STAT (signal transducer and activator of transcription) family of transcription factors that regulate cytokine expression in T cells (Yu et al, 2009). CCR9 expressed on MCF7 cells significantly inhibited the secretion of the T-helper-1 (Th1) cytokines including tumor necrosis factor-alpha (TNF-), interleukin-2 (IL-2), and (to a minor extent) of IFN- as well as IL-17, while the secretion of IL-10 was slightly but consistently increased (FIG. 3C). Accordingly, a significant increase in STAT1 and STAT2 signaling in survivin-specific T cells upon coculture with CCR910 MCF7 cells was observed, suggesting that anti-tumor type-1 immune response is impeded by tumor-specific CCR9 (FIGS. 3D and 3E).

Example 3: CCR9 Modulates T-Cell Responses Directly and Independent from Intracellular CCR9 Signalling

[0357] Next, the inventors assessed whether CCR9 expression in breast tumor cells affected T cell recognition directly or indirectly, for example, through CCR9 signaling-mediated increase in secretion of immune-suppressive factors. Since, the C-C chemokine ligand 25 (CCL25) is the only known interacting partner and ligand for CCR9, it was first assessed whether CCL25 was involved in defining CCR9's tolerogenic phenotype. CCL25 was found to be produced by all the studied tumor cell lines, although at varied levels, as determined by ELISA (FIG. 4A). Interestingly, shRNA-mediated stable knockdown of CCR9 did not affect CCL25 production by MCF7 breast cancer cells (FIG. 4A). Next, inhibition of CCL25 using siRNAs (FIG. 4B) or blocking antibody showed no effect on antigen-specific lysis of MCF7 cells, in contrast to the CCR9 knockdown. However, it might still be possible that CCR9 mediates its immunesuppressive effect via other unknown soluble ligands or mediators.

[0358] To examine this possibility, survivin-specific T cells were treated with the cell culture supernatants from either the CCR9 siRNA knockdown (CCR9.sup.lo) or control (CCR9.sup.hi) MCF7 tumor cells overnight and then challenged against CCR9.sup.hi or CCR9.sup.lo MCF7 cells in the cytotoxicity assay. Against the same tumor target, neither of the supernatant-treated T cells showed any difference in their recognition and lytic capacity. The difference in lysis between the different groups depended upon CCR9's expression on the tumor targets rather than on the T cell treatment (FIG. 4C), hinting to the possibility that T cells can interact directly with CCR9 on tumor cells.

[0359] To further assess whether intracellular signaling in tumor cells mediated by the surface-bound CCR9 plays any role in immunosuppression, pertussis toxin (PTX), a G.sub.i inhibitor, was used. Although, pertussis toxin inhibited the migration of CCR9.sup.+ tumor cells toward CCL25 in a transwell migration assay, proving its effectiveness in blocking CCR9's downstream signaling that is responsible for the chemotaxis, it, however, did not elicit elevated tumor lysis by antigen-specific T cells when compared to the CCR9 gene knockdown (FIG. 4D). This further supported the notion that CCR9-mediated immune suppression on T cells might be independent of its intracellular signaling in the tumor cells and rather affects the T cells directly. Additionally, the inventors evaluated whether CCR9 knockdown influences MHC-I expression on the tumor targets that could possibly explain their impact on T cell recognition and lysis. However, flow cytometric analysis revealed no major alterations in the surface expression of HLA-A2 on the target tumor cell lines upon CCR9 knockdown.

Example 4: Influence of CCR9 on the Transcriptome of T Cells

[0360] To better understand the mode of CCR9-mediated immune suppression on T cells, a broadscale transcriptomics study was performed to compare the changes in the transcriptome of T cells that encounter CCR9.sup.hi versus CCR9.sup.lo MCF7 tumor cells. Microarray analysis comparing these two T cell populations revealed a list of differentially up- and downregulated genes in CCR9.sup.lo-treated T cells, which are represented in the volcano plot of FIG. 4E and in the associated heat map of FIG. 4F. Immune response-related genes such as integrin alpha-2 (ITGA2; Yan et al, 2008), lymphotoxin alpha LTA; (Dobrzanski et al, 2004), interleukin 2 receptor alpha (IL2RA; Pipkin et al, 2010), and cytokine-inducible SH2-containing protein (CISH; Li et al, 2000) were upregulated, whereas genes that inhibit T cell maturation and effector function such as ephrin-A1 (EFNA1; Abouzahr et al, 2006), Kruppel-like factor 4 (KLF4; Wen et al, 2011), inhibitor of DNA binding-1 (ID1; Qi & Sun, 2004), transducer of ERBB2, 1 (TOB1; Tzachanis et al, 2001) were downregulated in T cells encountering CCR9.sup.lo tumor cells, which was found to be in accordance with the observed increase in cytotoxicity as shown before. Gene annotation/ontology (GO) analysis of the top upregulated genes revealed an enrichment of genes involved in positive regulation of immune response, while genes involved in lymphocyte maturation and apoptosis were enriched in the list of downregulated genes. The question arose whether these gene signatures observed in T cells upon tumor-specific CCR9 knockdown overlap with gene signatures generally associated with an activated T cell population. Using a publically available gene expression study comparing unstimulated CD8.sup.+ T cells to CD3/CD28 antibody and IL-2-activated T cells (Wang et al, 2008), we indeed identified overlapping gene signatures in both these studies, suggesting that CCR9 knockdown on tumor cells favors better survival, proliferation, and activation of the encountering T cells.

Example 5: In Vivo Relevance of CCR9 in Human Cancer

[0361] To evaluate the in vivo relevance of CCR9 as a tumor-associated immunosuppressive entity, CCR9 was stably knocked down in the melanoma patient-derived M579-A2 tumor cell culture using CCR9-specific shRNA (shCCR9) or the control non-targeting shRNA (shControl). As expected, stable CCR9 knockdown tumor cell variants were more susceptible to immune lysis by melanoma patient-derived tumor-infiltrating lymphocytes (TIL 209) than their counterparts in the chromium-release cytotoxicity assay (FIG. 5A), with no significant difference noted on the surface HLA-A2 expression upon CCR9 knockdown. For the in vivo analysis, 510.sup.5 cells each of the CCR9.sup.+ M579-A2 (shControl) and CCR9.sup. M579-A2 (shCCR9) tumor cell lines were subcutaneously implanted in the left and the right flank, respectively, of the NSG immune-deficient mice (scheme in FIG. 5B). These mice then received intravenous injection of 110.sup.7 tumor-infiltrating lymphocytes (TIL 209) at Day 2 and Day 9. As shown in FIG. 5C, CCR9.sup. M579-A2 tumors grew significantly slower than the CCR9.sup.+ tumors in response to the adoptive T cell transfer, indicating that CCR9 suppresses the anti-tumor activity of the transferred T cells in vivo as well. No difference in the tumor growth kinetic between the CCR9.sup.+ and the CCR9 tumor cells was observed in mice that received no T cell transfer (FIG. 5D). Taken together, these results suggest an important role for tumor-associated CCR9 as an immune-checkpoint node for application in cancer immunotherapy.

Example 6: Combination Therapies for Reducing Tumor Resistance

[0362] For a rational design of efficient combinatorial therapies for cancer treatment, it is essential to identify whether redundant or divergent signaling pathways underlying the potential immune modulatory function of CCR9 and other immune-checkpoint entities exist, which in a combination therapy are targeted synergistically. In order to identify the signaling pathways involved in CCR9 mediated modulation of tumor cell immune resistance, (intracellular) signaling pathways modulated by (eg, downstream of) CCR9 were characterized using the phosphoprotein analysis of major transcription factors in WT versus CCR9 knockdown MCF7 cells. Knockdown of CCR9 resulted in a significantly reduced signaling via Akt and S6-kinase, whereas a compensatory upregulation in the ERK kinase pathway and in the JNK pathway was noted, indicating their involvement with (eg in the downstream) CCR9 signaling (FIG. 6).

Example 7: Demonstrating Combination Therapies for the Reduction of Tumor Resistance to Immune Response

[0363] To demonstrate the synergy between CCR9-mediated immune suppression and the other relevant signal transduction pathways set forth in the present invention, luciferase-tagged tumor cell lines (based on MCF-7, MDA-MB-231, PANC-1 etc cell lines) are generated analogously to the approach described in the Materials and Methods. Each such luciferase-tagged tumor cell line is then reverse transfected with either control siRNA or CCR9-specific siRNA (Dharmacon, GE healthcare) as described in Khandelwal et al, 2015. Following culture for 72 hours, the cells are incubated with either DMSO alone as control or various concentrations (ranging from 0 nM, 0.1 nM, 10 nM, 100 nM, 1 M, 10 M, 100 M or 1000 M) of: (i) an inhibitor or antagonist of PI3K-Akt signaling (for example, MK-2206 or MSC-2363318A); (ii) an inhibitor or antagonist of p70S6 kinase signaling (for example, LY-2584702 or LY2780301 or MSC-2363318A); (iii) an activator or agonist of ERK1/2 signaling; or (iv) an activator or agonist of JNK signaling. 1-hour after treatment with the inhibitor/antagonist (or activator/agonists, as applicable), tumor cells are co-cultured with HLA-matched (to the tumor cell line used) T cells (CTLs)at T-cell to tumor cell ratios of between about 10:1 to 1:1for an additional 8-10 hours, followed by the Luc-CTL assay readout for assessment of tumor lysis (Khandelwal et al, 2015). For comparison, a sample of the corresponding luciferase-tagged tumor cells is treated solely with CCR9 inhibitor or with the respective modulator of the mentioned pathway. Control experimentswithout co-culture with CTLsare also conducted. The corresponding IC50 values are calculated for each treatment, and the IC50 value of CCR9 inhibitor alone, as well as IC50 of the respective modulator of the aforementioned pathways when used alone, are higher than the IC50 value for treatment of CCR9 inhibitor in combination together with the respective modulator of the aforementioned pathways; thus demonstrating the principle of such CCR9 inhibitor-based combinations as therapies for reducing the resistance of a tumor to an immune response.

[0364] Conducting the above experiment in a similar fashion, CCR9 activity in the tumor cells can instead be inhibited by using varying concentrations of an inhibitory anti-CCR9 antibody (or a small-molecule CCR9 inhibitor), and the synergy of such CCR9 inhibition with modulation of the other relevant signal transduction pathways set forth in the present invention can also be demonstrated. Tumor cell lysis can be measured for: (1) the CCR9 inhibitor and for the respective pathway modulator alone; (2) the CCR9 inhibitor in a series of concentrations plus the respective pathway modulator at a set concentration; and (3) the respective modulator in a series of concentrations plus the CCR9 inhibitor at a set concentration. Using such data, a Combination Index can be calculated from the algorithm of Chou & Talala, 1984 (Adv Enzyme Regul; 22:27) using XLfit software (IDBS, Guilford, UK); where Combination Index values of <1, 1 and >1 indicate synergisms, additive effect and antagonism, respectively. These data can also be represented using an isobologram. Synergy can also be evaluated by calculation of Bliss independence (Bliss, 1939; Ann Appl Biol 26:585).

[0365] Analogous experiments can be conducted to demonstrate the synergy between eg FZD3-mediated immune suppression (or any other negative modulator gene) and the other relevant signal transduction pathways, by using eg FZD3 inhibitors or antagonists, such as eg anti-FZD3 antibodies or eg anti-FZD3 siRNA.

[0366] Analogous experiments can be conducted to demonstrate the synergy between eg CXCL9- (or CXCR3-) mediated immune suppression (or any other positive modulator gene) and the other relevant signal transduction pathways, by using eg CXCL9 or CXCR3 activators or agonists.

[0367] Materials and Methods

[0368] Cell Culture and Reagents

[0369] MCF7, MDA-MB-231 (breast cancer), and PANC-1 pancreatic cancer cells were acquired from American Type Cell Culture (Wesel, Germany). MCF7luc cells were generated by electroporation with pEGFP-Luc plasmid and expansion of sorted GFP+ clones in selection medium containing 550 jtg/ml G418 (Gibco, UK). M579-A2 melanoma culture was established from a patient and stably transfected with HLA-A2 expression construct as described before (Machlenkin et al, 2008). For stable CCR9 knockdown, lentiviral particles were produced using the pRSI9-U6-TagRFP-2APuro lentiviral expression vector (Cellecta) that contained either the CCR9-specific shRNA hairpin (ACCGGGCCAGTGGAGGTCTTTGTTCTGTTAATAT TCATAGCAGAACAAGGACCTTCACTGGCTTTT: SEQ ID NO. 5) or control nontargeting shRNA. Viruses were packaged using the psPAX2 and pMD2.G packaging plasmids (Addgene), and tumor cell lines were transduced with the viral particles as per the manufacturer's protocol.

[0370] For RNAi screens, CD8+ T cells were isolated from PBMC of healthy donors using CD8 Flow Comp kit (Invitrogen; Karlsruhe, Germany) and activated for 3 days in X-vivo medium (Lonza, Belgium) containing anti-CD3/CD28 activation beads (Dynal, Invitrogen) and 100 U/ml interleukin 2 (IL-2). HLA-A0201-restricted survivin95-104 (clone SK-1)specific CTL clones were generated from PBMC of healthy donors as described (Brackertz et al, 2011). Tumor-infiltrating lymphocytes 412 and 209 microcultures were expanded from an inguinal lymph node of a melanoma patient as described (Dudley et al, 2010). TIL 53 microculture was established from a male patient with poorly differentiated pancreatic adenocarcinoma (PDAC) (Poschke & Offringa, unpublished data) and expanded using the rapid expansion protocol (REP) as described elsewhere (Dudley et al, 2003).

[0371] RNAi Screen and Data Analysis

[0372] The GPCR-targeting sub-library of the genome-wide siRNA library siGENOME (Dharmacon, GE Healthcare) contained 520 siRNA pools, consisting of four synthetic siRNA duplexes each and was prepared as described (Gilbert et al, 2011). Four RNAi screens were performed in duplicate wells. Positive and negative siRNA controls were distributed into empty wells prior to screening. Reverse siRNA transfection was performed by delivering 0.05 l of RNAiMAX in 15 l RPMI (Invitrogen). After 30 min, 3,000 MCF7 cells (screens 1 and 3: MCF7luc, screens 2 and 4: MCF7) in 30 l DMEM medium (Invitrogen) supplemented with 10% FBS (Invitrogen) were added. Plates were incubated at 37 C. for 24 h, and for screen 2, cells were transiently transfected with a luciferase expression plasmid (pEGFP-Luc) using TransIT-LT1 transfection reagent (Mirius Bio LLC, Madison, USA). 72 h post siRNA transfection, cancer cells were either challenged with CTLs and anti-EpCAMCD3 bi-specific antibody (0.2 g/well; screens 1 and 2) or survivin-specific CTLs (screen 3) or left untreated (condition without addition of CTLs and screen 4). Tumor lysis was quantified by analysis of residual luciferase expression in tumor cells (Brown et al, 2005). Screen 1 contained CTLs from one single donor and screen 2 contained CTLs from 2 different donors; one for each technical replicate within the screen. 18 h later, supernatant was removed, cells were lysed, and luciferase measurements (screens 1, 2, and 3) or viability measurements using CellTiterGlo (Promega) (screen 4) were performed as previously described (Muller et al, 2005; Gilbert et al, 2011). Plate reader data from RNAi screens were analyzed using the cellHTS2 package in R/Bioconductor (Boutros et al, 2006). Scores from both conditions, that is, addition of CTLs and without addition of CTLs, were quantile normalized against each other using the aroma.light package in R. Differential scores were calculated using a loess regression fitting.

[0373] To reveal high-confidence hits, unsupervised hierarchical clustering of differential score of all genes from all screens was performed using the loess score. In order to robustly identify genes that positively modulate CTL-mediated cytotoxicity and to avoid biases potentially introduced by employing CTLs from different donors and employing genetically engineered as well as unmodified MCF7 cells, we filtered out genes that had a score >2, and <2 in the condition without addition of CTLs and had a score >0.5, and <0.5 in the condition with addition of CTLs. Finally, genes scoring in a CTG-based viability screen were filtered out from the candidate list (score <1.5 and >1.5). Thereby, siRNAs generally affecting cell viability, as determined by intracellular ATP levels, were excluded.

[0374] Chromium-Release Cytotoxicity Assay

[0375] Tumor cells were transfected with the described siRNAs using RNAiMAX or with pCMV6-AC-His-CCR9 encoding vector and empty control vector (OriGene, Rockville, USA) using TransIT-LT1. 72 h later, transfected cells were harvested for chromium-release cytotoxicity assay as detailed in Supplementary Methods. For CCR9 blockade using pertussis toxin (PTX), 106 tumor cells were incubated with 250 ng/ml of PTX (Sigma Aldrich) for 1 h at 37 C. before labeling with radioactive chromium.

[0376] ELISpot Assay

[0377] IFN- and granzyme B secretion from T cells was determined using ELISpot assay as described by the manufacturer (Mabtech, Nacka Strand, Sweden) and detailed in the Supplementary Methods.

[0378] Cytokine and Phospho-Plex Analysis

[0379] Cytokines in T cell stimulation cultures were determined with Bio-Plex Pro Assay kit (Biorad, Germany). For phospho-TCR and phospho-STAT analysis, 2106 survivin-specific TCs were cocultured with the respective target tumor cells at 20:1 ratio for defined time points, then isolated and lysed. Protein lysates were used for 7-plex TCR phosphoprotein kit and phospho-STAT 5-plex kit (Millipore, Billerica, USA) as detailed in the manufacturer's protocol. Measurements were performed using Luminex100 Bio-Plex System (Luminex, Austin, US; see also Supplementary Methods).

[0380] Global Gene Expression Analysis

[0381] For transcriptomic analysis, 2.5105 MCF7 cells per group were reverse transfected with control or CCR9 s1 siRNA in 6-well plates and cocultured with 5106 survivin T cells after 72 h for an additional 12 h. Following co-incubation, TCs were purified using the anti-EpCAM antibody-coated mouse IgG beads (detailed in Supplementary Methods) and total RNA was isolated using the RNeasy Mini kit (Qiagen) as instructed by the manufacturer. Gene expression analysis was performed using the GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). Gene expression intensity was quantile normalized, and significant differences in the log fold change of gene expression between the CCR9hi- versus the CCR9.sup.lo treated TCs were evaluated using the Welch's t-test. Top differentially up- and downregulated genes were plotted as heat maps using heatmap.2 function in R. Expression data can be accessed using the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3244. CCR9-induced gene expression signature was compared with a publically available gene expression dataset from a previous study (Wang et al, 2008), which compared CD8+ T cells from the peripheral blood of healthy donors before and after 24 h of activation with anti-CD3/CD28 antibody plus IL-2. The published dataset was retrieved from the Gene Expression Omnibus using the accession code GSE7572 and analyzed using standard methods in R.

[0382] In Vivo Experiments

[0383] Appropriate approval for animal work was obtained from the regulatory authorities (Regierungsprasidium, Karlsruhe) before the start of the experiment. Four- to six-week-old female NSG mice were ordered from the Animal Core Facility at DKFZ, Heidelberg. Mice were subcutaneously injected with 5105 cells (in 100 l of matrigel per injection) of each CCR9-M579-A2 (transduced with CCR9-specific shRNA) and CCR9+M579-A2 (transduced with non-targeting control shRNA) cell lines in the left and the right flank, respectively. Following this, at Day 2 and Day 9, 7 out of the 10 tumor-bearing mice received adoptive transfer of expanded TIL 209 cells intravenously into the tail vein (1107 cells/100 l PBS/mouse). The remaining three mice were injected with PBS alone to assess tumor growth in the absence of adoptive TIL transfer. Tumor measurements were performed using a digital caliper (Carl Roth) at the indicated time points, and tumor volume was measured using the formula: volume=height*width*width*(n/3).

[0384] Statistical Evaluation

[0385] Differences between test and control groups were analyzed by two-sided Student's t-test. In all statistical tests, a P-value <0.05 was considered significant. Statistical difference between the tumor growth curves in vivo was assessed using the unpaired one-sided Mann-Whitney U-test.

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TABLE-US-00002 TABLE 1 Protein/Gene Annotation: Gene symbol Gene Name Entrez Gene ID GHRHR Growth Hormone Releasing Hormone Receptor 2692 FLJ31393 (alias for Olfactory Receptor, Family 7, Subfamily E, 219445 OR7E5P) Member 5 Pseudogene FZD3 Frizzled class receptor 3 7976 OR3A2 Olfactory Receptor, Family 3, Subfamily A, 4995 Member 2 CXCL3 Chemokine (C-X-C Motif) Ligand 3 2921 GNRHR2 Gonadotropin-Releasing Hormone (Type 2) 114814 Receptor 2 IL8 Interleukin 8 3576 HTR1D 5-Hydroxytryptamine (Serotonin) Receptor 3352 1D CCL23 Chemokine (C-C Motif) Ligand 23 3352 CCL2 Chemokine (C-C Motif) Ligand 2 6347 P2RY11 Purinergic Receptor P2Y, G-Protein Coupled, 11 6347 TRHDE Thyrotropin-Releasing Hormone Degrading 29953 Enzyme CCR7 Chemokine (C-C Motif) Receptor 7 1236 IL8RA Interleukin 8 Receptor Alpha 3577 GPR34 G Protein-Coupled Receptor 34 2857 TACR2 Tachykinin Receptor 2 6865 GPR43 G protein-coupled receptors 43 (alias FFAR2) 2867 GCG Glucagon 2641 GRK6 G Protein-Coupled Receptor Kinase 6 2870 TAAR8, also known as Trace Amine Associated Receptor 8 83551 TRAR5 TAAR9, also known as Trace Amine Associated Receptor 9 134860 TRAR3 ENPP2 Ectonucleotide Pyrophospha- 5168 tase/Phosphodiesterase 2 GRM6 Glutamate Receptor, Metabotropic 6 2916 GRK5 G Protein-Coupled Receptor Kinase 5 2869 OR1G1 Olfactory Receptor, Family 1, Subfamily G, Member 1 8390 CCR2 Chemokine (C-C Motif) Receptor 2 729230 ADMR, also known as Adrenomedullin receptor 11318 GPR182 GRM4 Glutamate Receptor, Metabotropic 4 2914 ADGRV1, also known as Adhesion G Protein-Coupled Receptor V1 84059 MASS1 CXCL9 Chemokine (C-X-C Motif) Ligand 9 4283 CXCR3 Chemokine (C-X-C Motif) Receptor 3 2833 Olfactory Receptor, Family 1, Subfamily D, OR1D4 Member 4 653166 Olfactory receptor family 2 subfamily J 26707 OR2J2 member 2 VN1R4 Vomeronasal 1 receptor 4 317703 OR1F1 Olfactory receptor family 1subfamily F 4992 member 1 CEACAM-6 Carcinoembryonic antigen related cell adhesion 4680 molecule 6 CD272 B and T lymphocyte associated 151888 Gene Symbol ENSEMBLE information GHRHR Location: Chromosome 7: 30,938,669-30,993,254 forward strand. GRCh38: CM000669.2 Human CCDS set: CCDS5432.1 UniProtKB identifiers: Q02643 Ensembl version: ENSG00000106128.18 FLJ31393 Location: Chromosome 11: 55,979,398-55,980,103 reverse (alias for OR7E5P) strand. GRCh38: CM000673.2 Ensembl version: ENSG00000214880.3 FZD3 Location: Chromosome 8: 28,494,205-28,574,268 forward strand. GRCh38: CM000670.2 Human CCDS set: CCDS6069.1 UniProtKB identifiers: Q9NPG1 Ensembl version: ENSG00000104290.10 OR3A2 Location: Chromosome 17: 3,277,899-3,278,974 reverse strand. GRCh38: CM000679.2 Human CCDS set: CCDS42233.1 UniProtKB identifiers: P47893 Ensembl version: ENSG00000221882.2 CXCL3 Location: Chromosome 4: 74,036,589-74,038,807 reverse strand. GRCh38: CM000666.2 Human CCDS set: CCDS34007.1 UniProtKB identifiers: P19876 Ensembl version: ENSG00000163734.4 GNRHR2 Location: Chromosome 1: 145,919,013 -145,925,341 forward strand. GRCh38: CM000663.2 Ensembl version: ENSG00000211451.11 IL8 Location: Chromosome 1: 145,919,013-145,925,341 forward strand. GRCh38: CM000663.2 Ensembl version: ENSG00000211451.11 HTR1D Location: Chromosome 1: 23,191,895-23,194,729 reverse strand. GRCh38: CM000663.2 human CCDS set: CCDS231.1 UniProtKB identifiers: P28221 Ensembl version: ENSG00000179546.4 CCL23 Location: Chromosome 17: 36,013,056-36,017,968 reverse strand. GRCh38: CM000679.2 Human CCDS set: CCDS11305.1, CCDS59282.1 UniProtKB identifiers: P55773 Ensembl version: ENSG00000274736.4 CCL2 Location: Chromosome 17: 34,255,218-34,257,203 forward strand. GRCh38: CM000679.2 Human CCDS set: CCDS11277.1 UniProtKB identifiers: P13500 Ensembl version: ENSG00000108691.9 P2RY11 Location: Chromosome 19: 10,111,538-10,115,372 forward strand. GRCh38: CM000681.2 Human CCDS set: CCDS12226.1 UniProtKB identifiers: Q96G91 Ensembl version: ENSG00000244165. TRHDE Location: Chromosome 12: 72,087,266-72,670,757 forward strand. GRCh38: CM000674.2 Human CCDS set: CCDS9004.1 UniProtKB identifiers: Q9UKU6 Ensembl version: ENSG00000072657.8 CCR7 Location: Chromosome 17: 40,553,769-40,565,472 reverse strand. GRCh38: CM000679.2 Human CCDS set: CCDS11369.1, CCDS77026.1 UniProtKB identifiers: P32248 Ensembl version: ENSG00000126353.3 IL8RA Location: Chromosome 2: 218,162,845-218,166,995 reverse strand. GRCh38: CM000664.2 Human CCDS set: CCDS2409.1 UniProtKB identifiers: P25024 Ensembl version: ENSG00000163464.7 GPR34 Location: Chromosome X: 41,688,973-41,697,277 forward strand. GRCh38: CM000685.2 Human CCDS set: CCDS14258.1 UniProtKB identifiers: Q9UPC5 Ensembl version: ENSG00000171659.13 TACR2 Location: Chromosome 10: 69,403,903-69,416,867 reverse strand. GRCh38: CM000672.2 Human CCDS set: CCDS7293.1 UniProtKB identifiers: P21452 Ensembl version: ENSG00000075073.14 GPR43 Location: Chromosome 19: 35,443,907-35,451,767 forward strand. GRCh38: CM000681.2 Human CCDS set: CCDS12461.1 UniProtKB identifiers: O15552 Ensembl version: ENSG00000126262.4 GCG Location: Chromosome 2: 162,142,873-162,152,404 reverse strand. GRCh38: CM000664.2 Human CCDS set: CCDS46439.1 UniProtK identifiers: P01275 Ensembl version: ENSG00000115263.14 GRK6 Location: Chromosome 5: 177,403,204-177,442,901 forward strand. GRCh38: CM000667.2 Human CCDS set: CCDS34303.1, CCDS43406.1, CCDS47348.1 UniProtKB identifiers: P43250 Ensembl version: ENSG00000198055.10 TAAR8, also known as TRAR5 Location: Chromosome 6: 132,552,693-132,553,721 forward strand. GRCh38: CM000668.2 Human CCDS set: CCDS5154.1 UniProtKB identifiers: Q969N4 Ensembl version: ENSG00000146385.1 TAAR9 also known as TRAR3 Location: Chromosome 6: 132,538, 290-132,539,336 forward strand. GRCh38: CM000668.2 Human CCDS set: CCDS75520.1 UniProtKB identifiers: Q96RI9 Ensembl version: ENSG00000237110.2 ENPP2 Location: Chromosome 8: 119 557 086-119 67 453 reverse strand. GRCh38: CM000670.2 Human CCDS set: CCDS34936.1, CCDS47914.1, CCDS6329.1 UniProtKB identifiers: Q13822 Ensembl version: ENSG00000136960.12 GRM6 Location: Chromosome 5: 178,978,327-178,996,206 reverse strand. GRCh38: CM000667.2 Human CCDS set: CCDS4442.1 UniProtKB identifiers: O15303 Ensembl version: ENSG00000113262.14 GRK5 Location: Chromosome 10: 119,207,589-119,459,742 forward strand. GRCh38: CM000672.2 Human CCDS set: CCDS7612.1 UniProtkt identifiers: P34947 Ensembl version: ENSG00000198873.11 OR1G1 Location: Chromosome 17: 3,126,584-3,127,581 reverse strand. GRCh38: CM000679.2 Human CCDS set: CCDS11020.1 UniProtKB identifiers: P47890 Ensembl version: ENSG00000183024.3 CCR2 Location: Chromosome 3: 46,353,734-46,360,928 forward strand. GRCh38: CM000665.2 Human CCDS set: CCDS43078.1, CCDS46813.1 UniProtKB identifiers: P41597 Ensembl version: ENSG00000121807.5 ADMR, also known as GPR182 Location: Chromosome 12: 56,994,446-56,998,441 forward strand. GRCh38: CM000674.2 Human CCDS set: CCDS8927.1 UniProtKB identifiers: O15218 Ensembl version: ENSG00000166856.2 GRM4 Location: Chromosome 6: 34,018,645-34,155,622 reverse strand. GRCh38: CM000668.2 Human CCDS set: CCDS4787.1, CCDS59010.1, CCDS59011.1, UniProtKB identifiers: Q14833 Ensembl version: ENSG00000124493.13 ADGRV1, also known as MASS1 Location: Chromosome 5: 90,529,344-91,164,221 forward strand. GRCh38: CM000667.2 Human CCDS set: CCDS47246.1 UniProtKB identifiers: Q8WXG9 Ensembl version: ENSG00000164199.15 CXCL9 Location: Chromosome 4: 76,001,275-76,007,488 reverse strand. GRCh38: CM000666.2 Human CCDS set: CCDS34014.1 UniProtKB identifiers: Q07325 Ensembl version: ENSG00000138755.5 CXCR3 Location: Chromosome X: 71,615,916-71,618,517 reverse strand. GRCh38: CM000685.2 Human CCDS set: CCDS14416.1, CCDS48135.1 UniProtKB identifiers: P49682 Ensembl version: ENSG00000186810.7 OR1D4 Location: Chromosome 17: 3,240,676-3,241,614 forward strand. GRCh38: CM000679.2 Ensembl version: EN5G00000255095.1 OR2J2 Location: Chromosome 6: 29,173,303-29,174,574 forward strand. GRCh38: CM000668.2 Human CCDS set: CCDS43434.1 UniProtKB identifiers: O76002 Ensembl version: ENSG00000204700.4 VN1R4 Location: Chromosome 19: 53,266,676-53,267,723 reverse strand. GRCh38: CM000681.2 Human CCDS set: CCDS33099.1 UniProtKB identifiers: Q7Z5H5 Ensembl version: ENSG00000228567.3 OR1F1 Location: Chromosome 16: 3,204,247-3,205,188 forward strand. GRCh38: CM000678.2 Human CCDS set: CCDS10496.1 UniProtKB identifiers: O43749 Ensembl version: ENSG00000168124.2 CEACAM-6 Location: Chromosome 19: 41,750,977-41,772,208 forward strand. GRCh38: CM000681.2 Human CCDS set: CCDS12585.1 UniProtKB identifiers: P40199 Ensembl version: ENSG00000086548.8 CD272 Location: Chromosome 3: 112,463,968-112,499,561 reverse strand. GRCh38: CM000665.2 Human CCDS set: CCDS33819.1, CCDS43130.1 UniProtKB identifiers: Q7Z6A9 Ensembl version: ENSG00000186265.9