MR1 LIGANDS AND PHARMACEUTICAL COMPOSITIONS FOR IMMUNOMODULATION - Patent application

Abstract

The present invention relates to a method for modulating the interaction between an MR1 polypeptide and an MR1-specific T cell receptor molecule, according to which the MR1 polypeptide is contacted with an MR1 ligand compound that is a nucleobase adduct product that reflects a metabolically disordered state of the cell. The present invention further relates to the use of compounds identified as MR1 ​​ligands in vaccination or modulation of an MR1-restricted immune response.

Description

Technical Field

[0001] This application claims the benefit of European Patent Application No. 20152326.3 filed on January 16, 2020, European Patent Application No. 20166918.1 filed on March 30, 2020, and European Patent Application No. 20166919.9 filed on March 30, 2020, all of which are incorporated herein by reference.

[0002] The present invention relates to ligands that are specifically presented by the MR1 molecule to MR1-specific T cells. These ligands are nucleic acid-forming bases produced in eukaryotic cells under specific conditions, in particular derivatives or analogs of ribonucleoside adducts and deoxyribonucleoside adducts. The present invention further relates to pharmaceutical formulations and methods for the use of such ligands in therapy and research. The present invention further relates to pharmaceutical formulations provided for the purpose of increasing the presence of MR1 ligands in clinical situations where an increase in the presence of MR1 ligands is clinically beneficial.

Background Art

[0003] MR1 (Uniprot ID 95460) is a non-polymorphic MHC class I-like protein that is expressed at low levels on the surface of most cell types. MR1 is highly conserved across multiple species, and human and mouse MR1 have more than 90% sequence homology at the protein level.

[0004] In recent years, the inventors have published research results confirming the existence of human T cells that recognize tumor-associated antigens (TAAs) presented by MR1 (Lepore et al., ELIFE 6, DOI: 10.7554 / eLife.24476). These novel T cells are involved in tumor immune surveillance and represent a new tool for cancer immunotherapy. However, the antigens recognized by these MR1-specific T cells remain unknown.

[0005] Adoptive therapy with donor- or patient-derived T cells engineered to express T cell receptors (TCRs) specific for selected TAAs represents a promising and safe strategy to induce clinically relevant antitumor immune responses in cancer patients. Targeting TAAs bound to MR1 nonpolymorphic antigen-presenting molecules overcomes this constraint and could in principle be applicable to all patients with MR1-expressing tumors. The use of tumor-reactive TCRs recognizing MR1-presented antigens could also have the advantage of complementing antitumor responses mediated by MHC-presented peptide antigens, except for cross-competition of TAAs for binding to the same type of presenting molecule. Moreover, this strategy may offer the possibility to target antigens of different nature on the same tumor cells, thereby minimizing the possible occurrence of tumor escape variants under selective immune pressure. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lepore et al., ELIFE 6, DOI:10.7554 / eLife.24476 Summary of the Invention [Problem to be solved by the invention]

[0007] The lack of information regarding the nature of this presented antigen, as well as the lack of tools capable of probing, analyzing, and modulating MR1 presentation, its interaction with antigen, and its interaction with the cognate TCR, represents an obstacle in the development of improved MR1-centered immuno-oncological therapies.

[0008] Therefore, the identification of MR1-presenting TAAs, and the identification and isolation of MR1-restricted TCRs that recognize these antigens, may have important implications for cancer immunotherapy. [Means for solving the problem]

[0009] Based on the above-mentioned state of the art, the object of the present invention is to provide means and methods for modulating MR1-TCR interaction in order to enable and improve the clinical application of MR1-based immunotherapy. This object is achieved by the subject matter of the independent claims herein. [Brief description of the drawings]

[0010] [Figure 1] Figure 1 shows an example of upregulation of cell surface MR1 on APCs by two compounds: a) 2-methyladenosine and b) N6,N6-dimethyladenosine, used at the three indicated concentrations. Expression of MR1 on THP1-MR1 cells was assessed by flow cytometry and median fluorescence intensity (MFI) is shown compared to baseline expression on cells incubated with vehicle alone. [Diagram 2] Figure 2 shows an example of a competition assay stimulating three MR1 T cell clones. 2-methyladenosine was used at the three indicated concentrations in THP-1 cells prior to the addition of optimal amounts of antigen for each of the indicated T cell clones (DGB129, MCA2E7, TC5A87). The responses of the T cell clones are shown as the mean ± sd (standard deviation) of IFN-γ release. Also shown are T cell responses in the presence of antigen alone, compound alone (○), and THP-1 cells alone (△). [Diagram 3] Figure 3 shows an example of compound-specific MR1 T cell activation. The responses of three MR1 T cell clones DGB129 (a), MCA2E7 (b) and TC5A87 (c) to THP-1 cells incubated with the compound N6,N6-dimethyladenosine (M6,2A) are shown as the mean ± sd of IFN-γ released after overnight stimulation. As a control, the T cell responses to THP-1 cells and compound vehicle are shown. [Figure 4]Figure 4 shows the generation of M3ADE-loaded MR1 monomer: a) Gel filtration chromatography purification of MR1 protein refolded in the presence of M3ADE. The y-axis shows absorbance at 280 nm, and the x-axis shows retention time (min). The indicated peaks 1, 2 and 3 were collected and b) used to activate DGB129 cells in a plate binding assay. IL-13 released by T cells is shown as the mean ± sd (standard deviation) of duplicate determinations. [Diagram 5] Figure 5 shows validation of MR1-M3ADE tetramer staining. MR1 T cell clone AVA34 was generated by two rounds of FACS sorting of CD3+,MR1-M3ADE tetramer+ cells from PBMCs, followed by PHA stimulation and cloning by limiting dilution of M3ADE-reactive T cells. a) Histogram overlay showing staining of AVA34 cells with MR1-M3ADE and MR1-5-OP-RU tetramers, and after incubation with anti-TCR mAb specific for Vβ8 (JR2, 1 μg / ml) to block M3ADE tetramer staining. b) Bar graph showing IL-13 release by AVA34 cells towards THP-1 cells in the presence of M3ADE. Low levels of IL-13 are released in the presence of MGdA but not the other indicated compounds. A375-MR1 cells were used as a positive control and T cells alone as a negative control. [Figure 6] Figure 6 shows the ex vivo frequency of MR1-M3ADE tetramer+ MR1 T cells in the blood of healthy donors. Dot plots of PBMCs from nine healthy donors co-stained with MR1-M3ADE tetramer and anti-CD3 (UCHT1). Each plot represents an individual donor. Cells were gated as CD3+,CD14-,CD19- live single cells and numbers indicate the percentage of MR1-M3ADE tetramer+ MR1 T cells within the oval gate. [Figure 7-1]FIG. 7 shows that treatment of APCs with drugs that induce accumulation of carbonyl-containing molecules stimulates MR1 ​​T cells. a) "Daidzin" and b) "Disulfiram" enhance the response of MCA2B9 MR1T clones. c) Disulfiram induces stimulation of MCA2B1 MR1T clones when co-administered with exogenous deoxycytidine. d) "Oleic acid" induces stimulation of TC5A87 MR1T clones, e) "Ellagic acid" induces stimulation of QY1A16 MR1T clones, f) EHNA induces stimulation of TC5A87 MR1T clones, and g) "Mycophenolic acid" induces stimulation of TC5A87 MR1T clones. APCs (THP-1 cells) were incubated with each drug for 18 hours before addition of MR1T cells and deoxycytidine (panel c only). Drugs did not induce stimulation of MR1 T cells in the absence of APC. Responses of T cell clones are expressed as IFN-γ release (mean±sd) of triplicates. [Figure 7-2] FIG. 7 shows that treatment of APCs with drugs that induce accumulation of carbonyl-containing molecules stimulates MR1 ​​T cells. a) "Daidzin" and b) "Disulfiram" enhance the response of MCA2B9 MR1T clones. c) Disulfiram induces stimulation of MCA2B1 MR1T clones when co-administered with exogenous deoxycytidine. d) "Oleic acid" induces stimulation of TC5A87 MR1T clones, e) "Ellagic acid" induces stimulation of QY1A16 MR1T clones, f) EHNA induces stimulation of TC5A87 MR1T clones, and g) "Mycophenolic acid" induces stimulation of TC5A87 MR1T clones. APCs (THP-1 cells) were incubated with each drug for 18 hours before addition of MR1T cells and deoxycytidine (panel c only). Drugs did not induce stimulation of MR1 T cells in the absence of APC. Responses of T cell clones are expressed as IFN-γ release (mean±sd) of triplicates. [Figure 8]Figure 8 shows purine metabolism involved in MR1T antigen accumulation. (A-F) Reactivity of MR1T clones TC5A87 (A-C) and DGB129 (D-F) to A375-MR1 cells transduced with sgRNAs targeting ADA (▲, A and D), LACC1 (▼, B and E), ADSSL1 (■, C and F) or scrambled sgRNA control (○, A-F). (G) Activation of MR1T clones TC5A87 (left), DGB129 (middle), MCA3C3 (right) by THP-1 cells preincubated with 250 μM of the indicated molecules or A375-MR1 or vehicle. Released IFN-γ is presented as mean ± SD of triplicate cultures. Experiments shown are representative of at least three independent runs. Each panel shows a representative experiment of at least three independent repeats. *p<0.05, **p≦0.01, and ***p≦0.001 compared to matched controls (A–F, multiple t test) or vehicle (G, one-way analysis with Dunnett's multiple comparisons). [Figure 9-1] Figure 9 shows that glycolysis and methylglyoxal lead to accumulation of MR1 T antigen. Schematic of methylglyoxal production. Dihydroxyacetone phosphate (DHAP), glyceraldehyde 3-phosphate (G3P). (A and B) Stimulation of MR1 T cell clones TC5A87 (A) and DGB129 (B) with A375-MR1 cells transduced with sgRNA targeting TPI1 (●) or scrambled control (○). (C and D) Stimulation of MR1 T cell clones TC5A87 (C) and DGB129 (D) in response to fixed A375-MR1 cells incubated with different concentrations of D-(+)-glucose (○) or 2-deoxy-D-glucose (■) for 6 hours and then fixed. [Figure 9-2](E and F) Stimulation of MR1T cell clones TC5A87 (E) and DGB129 (F) with A375-MR1 cells transduced with sgRNA against GLO1 (■), scrambled sgRNA control (○), or vector overexpressing GLO1 (▼). (G and H) Stimulation of MR1T cell clones TC5A87 (G) and DGB129 (H) with THP-1 cells pretreated with 25 μM erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA), 10 μM mycophenolic acid (MPA), and 20 μM S-bromobenzyl glutathione (BBG), either alone or in combination. (I-L) Activation of the MR1T clone DGB129 in response to methylglyoxal (I, J) or deoxyadenosine (K, L) in THP-1 cells (○), GLO1 overexpressing (▼) and GLO1 knockout (ko) (■) THP-1 cells. IFN-γ released is shown as mean ± SD of triplicate cultures. Data shown are representative of at least three independent experiments. (A, L) Mean ± SD, n=3, *p<0.05, **p≦0.01 and ***p≦0.001. (A, B and IL) Multiple t-test, (C and D) One-way ANOVA with Dunnett's multiple comparisons, (E and F) Two-way ANOVA with Dunnett's multiple comparisons, (G and H) One-way ANOVA with Tukey's multiple comparisons. [Figure 10-1] Figure 10 shows aldehydes (A-C) contributing to accumulation and scavenging of MR1T antigen. Stimulation of MR1T cell clones TC5A87 (A), DGB129 (B) and MCA3C3 (C) with THP-1 cells pretreated with "doxorubicin" (75 nM) or "paclitaxel" (5 μM) in the absence or presence of nucleosides (d-adenosine and guanosine, both at 150 μM). (D–F) Stimulation of MR1T cell clones TC5A87 (D), MCA2B1 (E), and MCA3C3 (F) with immobilized A375-MR1 cells treated with buthionine sulfoximine (400 μM, BSO), glutathione (4 mM, GSH), N-acetylcysteine ​​(4 mM, NAC), and apocynin (100 μM, APO). [Figure 10-2] (G-I) Stimulation of MR1 T cell clones TC5A87 (G), DGB129 (H), and MCA2B1 (I) on immobilized A375-MR1 cells treated with ML-210 (6 μM), RSL-3 (1 μM), and mercaptosuccinic acid (3.3 μM, MSA). (J-L) Stimulation of MR1 T cell clones TC5A87 (J), DGB129 (K), and MCA2B1 (L) on immobilized A375-MR1 cells treated with "hydralazine" (100 μM) or aminoguanidine (5 mM). Released IFN-γ is shown as the mean ± SD of triplicate cultures. Data shown are representative of at least three independent experiments. (A-L) Mean ± SD, n = 3, *p < 0.05, **p ≤ 0.01, and ***p ≤ 0.001. (A-C) Two-way ANOVA with Tukey's multiple comparisons, (DL) One-way ANOVA with Dunnett's multiple comparisons. See also Figures 15 and 16. [Figure 11] FIG. 11 shows that synthetic MDA or MG nucleoside adducts induce upregulation of MR1 and stimulate MR1T T cells. (A-E) Five synthetic adducts, M3ADE (A), OPdA (B), M1G (C), MGG (D), and OPdC (E), were purified and their structures are shown on the left. (Middle) Upregulation of MR1 surface expression in THP-1 MR1 cells after 6 h incubation with the adducts shown in each panel. The fold change in MFI of staining with anti-MR1 mAb ± SD is graphed. (Right) IFN-γ release response of several MR1 T cell clones cocultured overnight with THP-1 cells in the presence (○) or absence (●) of adduct. Blocking of T cell reactivity by anti-MR1 mAb is also shown for the highest dose of antigen (Ag) (▲). Data are shown as mean ± SD of triplicate cultures. Experiments were performed at least three times independently, and one representative experiment is shown. [Figure 12]FIG. 12 shows that MR1 T cell clones recognize unmodified nucleoside adducts and different tumor cells. (A) Recognition of plate-bound soluble MR1 (■) loaded with M3ADE, OPdA, M1G, and OPdC (○) by MR1 T clones AVA34, QY1A16, AC1A4, and TC5A87, respectively. (B) Activation assay of 14 MR1 T cell clones in the presence of THP-1 cells treated with each indicated antigen or vehicle. Stimulation of 14 MR1 T cell clones in the presence of M3ADE (100 μM~), OPdA (100 μM), M1G (300 μM~), or OPdC (100 μM) or THP-1 cells treated with vehicle. Heatmap depicts the cube root of the mean IFN-γ concentration. (C) MR1 T clones recognize different tumor cells. Black bars indicate coculture with the indicated tumor cell lines in the presence of anti-HLA-A, B, C mAb (monoclonal) (clone W6 / 32), anti-HLA-DR mAb (clone L243), and white bars indicate coculture with both anti-HLA and anti-MR1 mAbs. Graphs show IFN-γ release (mean ± SD of triplicate cultures). Data are mean ± SD of triplicate cultures (A, C) and representative of at least two independent experiments (A–C). (C) **p ≤ 0.01 and ***p ≤ 0.001 compared to anti-MR1 blocking control (multiple t-test). [Figure 13-1] Figure 13 shows that M3ADE-specific cells are detected in the periphery of healthy donors. (A) Histogram of MR1-M3ADE tetramer staining of MR1 T cell clone AC1A4 compared to MAIT cell clone MRC25. (B) Histogram of staining of AVA34 cells (representative clone obtained from MR1-M3ADE tetramer sorting) with MR1 tetramer loaded with 5-OP-RU (light grey), 6-FP (dark grey) and M3ADE (black). (C) Activation of clone AVA34 with five synthetic DNA adducts (including associated blocking with anti-MR1 mAb). Columns show IFN-γ release (mean ± SD of triplicate cultures). [Figure 13-2](D) MR1-M3ADE tetramer staining of PBMCs from two representative donors plotted against CD3 expression. Cells were pre-gated on live single cells and frequencies are expressed as percentage of CD3+ T cells. (E) Summary of ex vivo frequencies of MR1-M3ADE tetramer+ T cells from peripheral blood of nine healthy donors. Horizontal bars represent median values. (F) Dot plots show the ratios of CD4+ / CD8-, CD4- / CD8+ and CD4- / CD8- (DN (double negative)) T cells within MR1-M3ADE tetramer+ T cells. Horizontal bars represent median values. (G) Dot plots show the percentages of naive, central memory, effector memory and terminally differentiated effector memory (TEMRA) T cells within the MR1-M3ADE tetramer+ T cell fraction. Horizontal bars represent median values. [Figure 13-3] (H) Histograms of surface expression of CD8, CD4, CD45RA, and CCR7 markers in T cells pre-gated as M3ADE-MR1 tetramer+,CD3+ from each donor (D1-D9). Staining results for T cell clones are representative of at least two independent experiments. [Figure 14-1] FIG. 14 shows the presence of M3ADE tetramer+ T cells in human squamous cell lung carcinoma. (A and B) Dot plots show MR1-M3ADE tetramer+,CD3+ T cells in TILs obtained from two patients (donors 840 and 895) after in vitro expansion. The percentage of MR1-M3ADE tetramer+,CD3+ T cells among total CD3+ cells is shown (left). Dot plots show the ratios of CD4+ / CD8-, CD4- / CD8+, and CD4- / CD8- T cells among MR1-M3ADE tetramer+ T cells. Numbers in each quadrant indicate the percentage of each population calculated among the total M3ADE-MR1 tetramer+,CD3+ T cells (right). [Figure 14-2](C) Dot plots show FACS-sorted M3ADE-tetramer enriched T cell lines derived from TILs of each donor activated in the presence of A375-MR1-B2M KO cells or the same cells overexpressing MR1 treated with M3ADE or treated with M3ADE and blocked with anti-MR1 mAb. T cell activation was measured by TCR downregulation and shown as the percentage of tetramer+ cells and MFI indicated in each plot. (D) Activation of TIL lines in each condition corresponding to C. IFN-γ release (mean ± SD) was measured in duplicate for each condition. Data shown are representative of at least two independent experiments. [Figure 14-3] (E) MR1-dependent activation of tetramer-positive T cells as measured by IFN-γ release. [Figure 15-1] FIG. 15 shows characterization of knockout cell lines. (A, D, G, J, M) Activation assay of A375-MR1 cells and MRC25, a MAIT clone, in response to 5-OP-RU. (P and S) Activation assay of THP-1 cells and MRC25 with 5-OP-RU. Cells were either wild type (○), knockout (A▲, D■, G▼, J●, M▼, S▼) or overexpressing (M■ (gray), P■). IFN-γ is represented as mean ± SD of independent triplicate cultures. (B, E, H, K, N, Q, T) Surface MR1 expression of engineered cell lines. MR1 staining with anti-MR1 mAb26.5 of wild type cells (dark grey shading), ko (knockout) line (black line) and GLO1 overexpressing (GLO1++) A375-MR1 (N, grey thick dashed line). Isotype-matched control staining in wild-type cells (light grey shading and grey dotted line), ko cells (black dashed line), and GLO1++A375-MR1 (N, black dotted line) are shown. (C,F,I,L,O,R,U) Western blot analysis of target protein expression in the indicated cell lines. Tubulin or actin were used as loading controls. Experiments were repeated at least twice, and one representative experiment is shown. [Figure 15-2]FIG. 15 shows characterization of knockout cell lines. (A, D, G, J, M) Activation assay of A375-MR1 cells and MRC25, a MAIT clone, in response to 5-OP-RU. (P and S) Activation assay of THP-1 cells and MRC25 with 5-OP-RU. Cells were either wild type (○), knockout (A▲, D■, G▼, J●, M▼, S▼) or overexpressing (M■ (gray), P■). IFN-γ is represented as mean ± SD of independent triplicate cultures. (B, E, H, K, N, Q, T) Surface MR1 expression of engineered cell lines. MR1 staining with anti-MR1 mAb26.5 of wild type cells (dark grey shading), ko (knockout) line (black line) and GLO1 overexpressing (GLO1++) A375-MR1 (N, grey thick dashed line). Isotype-matched control staining in wild-type cells (light grey shading and grey dotted line), ko cells (black dashed line), and GLO1++A375-MR1 (N, black dotted line) are shown. (C,F,I,L,O,R,U) Western blot analysis of target protein expression in the indicated cell lines. Tubulin or actin were used as loading controls. Experiments were repeated at least twice, and one representative experiment is shown. [Figure 15-3]FIG. 15 shows characterization of knockout cell lines. (A, D, G, J, M) Activation assay of A375-MR1 cells and MRC25, a MAIT clone, in response to 5-OP-RU. (P and S) Activation assay of THP-1 cells and MRC25 with 5-OP-RU. Cells were either wild type (○), knockout (A▲, D■, G▼, J●, M▼, S▼) or overexpressing (M■ (gray), P■). IFN-γ is represented as mean ± SD of independent triplicate cultures. (B, E, H, K, N, Q, T) Surface MR1 expression of engineered cell lines. MR1 staining with anti-MR1 mAb26.5 of wild type cells (dark grey shading), ko (knockout) line (black line) and GLO1 overexpressing (GLO1++) A375-MR1 (N, grey thick dashed line). Isotype-matched control staining in wild-type cells (light grey shading and grey dotted line), ko cells (black dashed line), and GLO1++A375-MR1 (N, black dotted line) are shown. (C,F,I,L,O,R,U) Western blot analysis of target protein expression in the indicated cell lines. Tubulin or actin were used as loading controls. Experiments were repeated at least twice, and one representative experiment is shown. [Figure 16]FIG. 16 shows stimulation of MAIT clone MRC25 with nucleobases, inhibitors, and synthetic antigens. (A) MAIT clone MRC25 was stimulated with THP-1 cells in the presence of various nucleobases (250 μM), methylglyoxal (250 μM), or 5-OP-RU (30 nM). (B and D) MRC25 cells were stimulated with THP-1 cells treated with the indicated drugs. (C) MRC25 cells were stimulated with A375-MR1 cells treated with inhibitors of GSH, NAC, APO, BSO, or GPX and fixed, or with THP-1 cells pulsed with 5-OP-RU (10 nM). (E) MRC25 cells were stimulated with A375-MR1 cells treated with the indicated concentrations of carbonyl scavengers and fixed before addition of T cells (empty bars). As a control, the same experiment was performed using the same carbonyl scavengers in the presence of 6,7-dimethyl-8-ribityrumazine (20 M, black bars). (F) MRC25 cells were stimulated with THP-1 cells in the presence of M3ADE, OPdA, OPdC (all 100 μM), M1G (13 μM) or 5-OP-RU (10 nM). nd (not determined) = not determined. ***p<0.001 compared to vehicle-treated cells by one-way ANOVA (A, B, C, F) or two-way ANOVA (D and E) with Dunnett's multiple comparisons. IFN-γ is expressed as the mean ± SD of independent triplicate cultures. Experiments were repeated at least twice, and one representative experiment is shown. [Figure 17-1] FIG. 17 shows (A) quantification of ROS produced in THP-1 cells treated with doxorubicin, paclitaxel, or phorbol 12-myristate 13-acetate (PMA). Results are expressed as the median fluorescence intensity (MFI) of live cells ± SD of three independent cultures. (B) Surface expression of MR1 (black line) or HLA A, B, C (gray dotted line) in the indicated tumor cell lines. Isotype-matched staining controls are shaded gray. (C) Table describing the tissue source and diagnosed disease for each cell line. Each experiment was repeated at least twice, and one representative experiment is shown. **p 0.01 and ***p 0.001 using one-way ANOVA with Dunnett's multiple comparisons. [Figure 17-2] FIG. 17 shows (A) quantification of ROS produced in THP-1 cells treated with doxorubicin, paclitaxel, or phorbol 12-myristate 13-acetate (PMA). Results are expressed as the median fluorescence intensity (MFI) of live cells ± SD of three independent cultures. (B) Surface expression of MR1 (black line) or HLA A, B, C (gray dotted line) in the indicated tumor cell lines. Isotype-matched staining controls are shaded gray. (C) Table describing the tissue source and diagnosed disease for each cell line. Each experiment was repeated at least twice, and one representative experiment is shown. **p 0.01 and ***p 0.001 using one-way ANOVA with Dunnett's multiple comparisons. [Figure 18A] Figure 18 shows upregulation of MR1 on the cell surface of APCs by selected compounds. Expression of MR1 on tumor cells was assessed by flow cytometry and is shown as median fluorescence intensity (MFI) compared to baseline expression in cells incubated with vehicle alone. [Figure 18B] Figure 18 shows upregulation of MR1 on the cell surface of APCs by selected compounds. Expression of MR1 on tumor cells was assessed by flow cytometry and is shown as median fluorescence intensity (MFI) compared to baseline expression in cells incubated with vehicle alone. [Figure 19] Figure 19 shows compound-induced activation of MR1 T cells. The response of MR1 T cell clones to THP-1 cells incubated with different doses of each compound or a fixed dose of 50 μM (black bars) is shown as the mean ± sd of IFN-γ released after overnight stimulation. As a control, the response of T cells to THP-1 cells and compound vehicle (white bars) is shown. [Figure 20] Figure 20 shows the competitive ability of different compounds to stimulate MR1 T cells. Each compound was used at three indicated concentrations with THP-1 cells followed by addition of an optimal amount of antigen to specific MR1 T cells. T cell responses are shown as the mean ± sd of IFN-γ release. T cell responses in the presence of competitor and antigen (squares), compound only (triangles) or THP-1 cells only (circles) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Summary of the Invention To the best of the inventors' knowledge, the present invention is the first to identify MR1-specific eukaryotic antigen compounds that are presented to MR1 T cells in the context of an MR1-restricted immune response against intracellular antigens arising from the metabolic state of eukaryotic cells, thus facilitating the use of these compounds for therapeutic / prophylactic and diagnostic or research purposes.

[0012] As a result, the present invention provides a method that exploits the specificity of the MR1-TCR interaction (with the MR1 molecule presenting a specific MR1 ligand compound to T cells): i) A method for stimulating MR1 ligand compound-specific T cells and inducing a prophylactic or therapeutic immune response; ii) methods for identifying and isolating T cells or antibodies that react with compounds as presented by MR1; iii) A method for classifying metabolically altered cells, including but not limited to tumor cells, depending on the presence of said compound. Nucleobase-added MR1 ligand compounds, and in many embodiments nucleoside adducts or analogs, for use in

[0013] When applied to patient samples, the method of classifying metabolically altered cells can be employed as a diagnostic method to stratify patients according to their degree of response to MR1-specific T cell therapy, and can lead to the application of additional combination therapy, such as MR1 ​​ligand compounds as provided herein, or the administration of pharmaceutical agents known to interfere with intracellular metabolism in a manner that favors the production of MR1 ligands as provided herein, or both.

[0014] MR1 ligand compounds for use in the methods according to the invention are provided in claims 1-9 and Table 1.

[0015] The present invention further relates to a method for modulating (increasing or decreasing) the amount of MR1 ligand compound in or presented by a cell by pharmacological intervention leading to the accumulation or disposal of the MR1 ligand compound in the cell.

[0016] In another aspect, the present invention relates to a pharmaceutical composition comprising at least one of the MR1 ligand compounds of the present invention or a pharma- ceutically acceptable salt thereof and at least one of a pharma- ceutically acceptable carrier, diluent or excipient.

[0017] The present invention further relates to TCR molecules capable of specifically recognizing the MR1 ligand as disclosed herein that associate with (are presented by) the MR1 molecule, as well as polynucleotide sequences encoding such MR1 ligand-specific TCR molecules.

[0018] The present invention further provides a novel MR1 ligand compound, 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarboxaldehyde, identified and synthesized herein for the first time.

[0019] Detailed Description of the Invention Terms and Definitions The following definitions shall apply to the interpretation of this specification, and where appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0020] As used herein, the terms "comprising," "having," "containing," "including," and other similar forms and their grammatical equivalents are intended to be equivalent and open-ended in the sense that the item or items following any of these words are not an exhaustive list of such item or items or are not limited to only the listed item or items. For example, an item "comprising" components A, B, and C can consist of components A, B, and C (i.e., contain only components A, B, and C), or can contain not only components A, B, and C, but also one or more other components. Thus, "comprising" and similar forms and their grammatical equivalents are intended and understood to include the disclosure of embodiments that "consist essentially of" or "consist of."

[0021] Where a range of values ​​is stated, unless the context clearly indicates otherwise, it is understood that each intermediate value, to the tenth of the unit of the lower limit between the upper and lower limits of that range, and any other stated value or intermediate value within that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0022] As used herein, reference to "about" a value or parameter includes (and describes) variations that are directed to the value or parameter itself. For example, a statement referring to "about X" includes the statement of "X."

[0023] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural references unless the context clearly dictates otherwise.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic, and biochemical techniques (generally see Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th ed., John Wiley & Sons, Inc.) and chemical techniques.

[0025] The term "MR1" in the context of this specification refers to either the MR1 gene (Entrez 3140) or the MR1 gene product (Uniprot Q95460), also referred to herein as "MR1 polypeptide" or "MR1 molecule." As a non-limiting example, the MR1 polypeptide may be present in aspects or embodiments disclosed herein as an isolated MR1 polypeptide, e.g., in the form of an MR1 polypeptide tetramer (Gherardin, Immunol Cell Biol. 2018 May;96(5):507-525), or may be expressed on the patient's cells, either naturally or in response to introduction of an MR1-encoding gene construct.

[0026] The term "MR1 T cell" in the present context refers to a T cell expressing a T cell receptor capable of specifically binding to an MR1 molecule presenting an antigen molecule as defined herein.

[0027] "MR1 T cell receptor" in the context of this specification refers to a T cell receptor that is capable of specifically binding to an antigen presented by a cancer cell, for example, in association with an MR1 molecule.

[0028] Expression of markers such as MR1 ​​can be assayed by techniques such as fluorescence microscopy, flow cytometry, ELISPOT, ELISA, or multiplex analysis.

[0029] The TCR sequences or molecules described herein include fully functional TCR alpha and TCR beta polypeptide chains, or TCR gamma and TCR delta polypeptide chains. When referring to a TCR alpha or beta polypeptide having a particular sequence, it is understood that the presence of a complementary (beta or alpha, respectively) polypeptide chain is required for this to be fully functional in the methods and cells described herein. The same applies to the gamma and delta pair, mutatis mutandis. Reference to a specific TCR alpha, beta, gamma or delta sequence implies the possibility of pairing with the paired TCR sequence in the original clone, or with a certain identity to the original paired sequence, as described herein. Reference to a specific TCR alpha, beta, gamma, delta sequence also implies the possibility of pairing with another paired TCR sequence.

[0030] Recognition of MR1-presented cancer antigens is primarily via the CDR3 sequence. Reference is made herein to TCR sequences characterized solely by a specific CDR3 sequence, which may be a complete α, β, γ or δ TCR sequence as provided herein, and the resulting TCR molecule is paired with an appropriate second sequence.

[0031] In the present context, a "compound in association with MR1" refers to a compound that is non-covalently bound by the MR1 molecule. Binding can occur, for example, via electrostatic interactions, including van der Waals forces and hydrogen bonds.

[0032] The compound and the MR1 molecule form a complex that can be recognized by a specific T cell receptor. Recognition by a specific T cell receptor means that the T cell receptor can distinguish between an MR1 molecule that is not associated with the compound and an MR1-ligand compound-MR1 complex.

[0033] In the present context, the terms "sequence identity" and "percentage of sequence identity" refer to a single quantitative parameter that indicates the result of sequence comparison, which is determined by comparing two aligned sequences position by position. Methods for aligning sequences for comparison are well known in the art. Aligning sequences for comparison can be performed by the Smith and Waterman local homology algorithm, Adv. Appl. Math. 2: 482 (1981), the Needleman and Wunsch global alignment algorithm, J. Mol. Biol. 48: 443 (1970), the Pearson and Lipman similarity search method, Proc. Nat. Acad. Sci. 85: 2444 (1988), or computerized implementations of these algorithms, including but not limited to CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / ) and other sources.

[0034] An example of the comparison of amino acid sequences is the BLASTP algorithm using default settings: expectation threshold: 10; word size: 3; maximum match within query range: 0; matrix: BLOSUM62; gap cost: existence 11, extension 1; compositional adjustment: conditional compositional score matrix adjustment. An example of the comparison of nucleic acid sequences is the BLASTN algorithm using default settings: expectation threshold: 10; word size: 28; maximum match within query range: 0; match / mismatch score: 1.-2; gap cost: linear. Unless otherwise stated, the sequence identity values ​​provided herein refer to the values ​​obtained using the BLAST series of programs using the default parameters specified above for protein and nucleic acid comparisons, respectively (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)).

[0035] A description of identical sequences without specifying a percentage value implies 100% identical sequences (i.e., identical base sequences).

[0036] As used herein in the context of marker expression, the term "positive" refers to expression of an antigen as assayed by a fluorescently labeled antibody in which the median fluorescence intensity is at least 5% higher (≧5%) compared to staining with an isotype-matched antibody that does not specifically bind to the same target. Expression of such a marker can be, for example, MR1 + The marker name is followed by a superscript "plus" ( + ) as shown.

[0037] As used herein in the context of marker expression, the term "negative" refers to expression of an antigen as assayed by a fluorescently labeled antibody whose median fluorescence intensity is less than 5% higher than the median fluorescence intensity of an isotype-matched antibody that does not specifically bind to the same target. Such marker expression can be, for example, MR1- The marker name is followed by a superscript "minus" ( - ) as shown.

[0038] In the context of this specification, the term "checkpoint modulator agent" includes checkpoint inhibitors (particularly checkpoint inhibitor antibodies) and checkpoint agonist agents (particularly checkpoint agonist antibodies).

[0039] The term "having substantially the same biological activity" in the context of this specification, when used to define a TCR molecule capable of recognizing an MR1 ligand bound to an MR1 molecule, refers to the ability to recognize (or contribute to the recognition of) its cognate ligand (MR1 ligand associated with MR1). Assays and methods for determining such interactions are described herein.

[0040] The term "nucleic acid expression vector" in the context of this specification relates to a polynucleotide, such as a plasmid, a viral genome or a synthetic RNA molecule, that is used to transfect (in the case of a plasmid or RNA) or transduce (in the case of a viral genome) a target cell with a specific gene of interest. In the case of a DNA expression construct, the gene of interest is under the control of a promoter sequence that operates in the target cell so that the gene of interest is transcribed either constitutively, in response to a stimulus, or depending on the state of the cell. In the case of an RNA expression construct, the term relates to the translation of the RNA, and it is understood that the construct can be used by the target cell as m-RNA. In certain embodiments, the viral genome is encapsidated into a viral vector, which can transduce the target cell.

[0041] The term "transgenic MR1-reactive T cells" in the context of the present invention relates to autologous or allogeneic T cells expressing a T cell receptor (TCR) that specifically recognizes the MR1 molecule expressed on the patient's cells. In a particular embodiment, the TCR recognizes MR1-expressing tumor cells in the absence of added foreign antigen and in an MR1-dependent manner. MR-1-restricted TCR sequences are disclosed in PCT / EP2019 / 074284 and US20190389926(A1), both of which are incorporated herein by reference.

[0042] In the context of this specification, the term "checkpoint inhibitor" or "checkpoint blocking antibody" is meant to encompass agents, in particular antibodies (or antibody-like molecules), capable of disrupting the signaling cascade that leads to T cell inhibition following T cell activation, as part of what is known in the art as immune checkpoint mechanisms. Non-limiting examples of "checkpoint inhibitors" or "checkpoint inhibitor antibodies" include CTLA-4 (Uniprot P16410), PD-1 (Uniprot Q15116), TMIGD2 (Uniprot Q96BF3), BTLA (Uniprot Q7Z6A9), CD160 (Uniprot O95971), Lag-3 (Uniprot P18627), TIGIT (Uniprot Q495A1), CD96 (Uniprot P40200), TIM-3 (Uniprot Q8TDQ0), CEACAM1 (Uniprot P13688), SIRP alpha (Uniprot P78324), CD200R (Uniprot Q8TD46), KIR family (Uniprot protein Q99706, P43628, P43626, Q8NHK3, P43627, Q8N109, B0L652, Q86U48, B0L653, A0A191URI1, Q6H2H3, etc.), ILT family, or antibodies against PD-L1 (Uniprot Q9NZQ7), PD-L2 (Uniprot Q9BQ51), VISTA (Uniprot Q9H7M9), B7H3 (CD276; Uniprot Q5ZPR3), CD80 (Uniprot P33681), CD86 (Uniprot P42081), B7-H4 (Uniprot P42081), TNFRSF14 (HVEM, CD270, Uniprot Q92956), CD155 (Uniprot Examples of antibodies include those against CD200 (Uniprot Q8TD46), galectin 9 (Uniprot O00182), and CD200 (Uniprot P15151).

[0043] In the context of this specification, the term "checkpoint agonist agent" or "checkpoint agonist antibody" is meant to encompass agents, particularly but not limited to antibodies (or antibody-like molecules), that can participate in the signal cascade that leads to T cell activation as part of what is known in the art as an immune checkpoint mechanism. Non-limiting examples of receptors known to be upregulated upon T cell activation include CD25 (Uniprot P01589), CD122 (Uniprot P14784) and CD137 (4-1BB; Uniprot Q07011). The term "checkpoint agonist agent" or "checkpoint agonist antibody" encompasses antibodies against CD28 (P10747), ICOS (Q9Y6W8), CD137 (4-1BB; Uniprot Q07011), Light (HVEM, Uniprot O43557), OX40 (P23510), GITR (Q9Y5U5), DNAM-1 (CD226, Uniprot Q15762), 2B4 (CD244, Uniprot Q9BZW8), DR3 (Q93038), NKp80 (KLRF1, Uniprot Q9NZS2).

[0044] In the context of this specification, "C 1 ~C 4 The term "alkyl" refers to a saturated straight or branched chain hydrocarbon having 1, 2, 3 or 4 carbon atoms, in certain embodiments, one carbon-carbon bond is unsaturated and one CH 2 The moiety may be substituted at oxygen (ether bridges) or nitrogen (NH, or NR, where R is methyl, ethyl or propyl; amino bridges). 1 ~C 4 Non-limiting examples of alkyl include methyl, ethyl, propyl, prop-2-enyl, n-butyl, 2-methylpropyl, tert-butyl, but-3-enyl, prop-2-ynyl, and but-3-ynyl. 1 ~C 4 Alkyl is a methyl, ethyl, propyl or butyl moiety.

[0045] As used herein in its narrowest sense, "unsubstituted C n The term "alkyl" when used as a bridge between parts of a molecule is the moiety -C n H 2n -C when used in a terminal or in a related context n H 2n+1 This is regarding.

[0046] "Unsubstituted C n Alkyl and Substituted C n The term "alkyl" includes linear alkyls having linear alkyl substitution and including or linked to unsubstituted or substituted cyclic structures, such as cyclopropane, cyclobutane, cyclopentane, or cyclohexane moieties, depending on the context of the annotation or description. The total number of carbons and, where appropriate, N, O, or other heteroatoms in the linear or cyclic structure add up to n.

[0047] The term "substituted alkyl" in the broad sense refers to an alkyl as defined above, covalently bonded to an atom that is not carbon or hydrogen, in particular to an atom selected from N, O, F, B, Si, P, S, Cl, Br and I, which itself may - optionally - be bonded to one or more other atoms of this group, or to hydrogen, or to an unsaturated or saturated hydrocarbon (alkyl or aryl in the broad sense). In the narrow sense, "substituted alkyl" refers to an amine NH 2 , alkylamine NHR, imide NH, alkylimide NR, amino(carboxyalkyl) NHCOR or NRCOR, hydroxyl OH, oxyalkyl OR, oxy(carboxyalkyl) OCOR, carbonyl O and its ketal or acetal (OR) 2 , nitrile CN, isonitrile NC, cyanate CNO, isocyanate NCO, thiocyanate CNS, isothiocyanate NCS, fluoride F, chloride Cl, bromide Br, iodide I, phosphonate PO 3 H 2 , P.O. 3 R 2 , phosphate OPO 3 H2 and OPO 3 R 2 , sulfhydryl SH, sulfalkyl SR, sulfoxide SOR, sulfonyl SO 2 R, sulfanilamide SO 2 NHR, sulfate SO 3 H and sulfate esters SO 3 R refers to an alkyl group as defined above in the broad sense, in which one or more carbon atoms are substituted by a group selected from R, where the R substituent as used in this section is distinct from other uses assigned to R in the body of this specification, and as such, in the broad sense, refers to an unsubstituted or substituted C 1 ~C 12 In the narrow sense, unless otherwise specified, R is methyl, ethyl, or propyl.

[0048] The terms "amino-substituted alkyl" or "hydroxyl-substituted alkyl" refer to one or more amine groups or hydroxyl groups, NH 2 , N.H.R., N.R. 2 or OH, and the R substituent as used in this section is distinct from other uses assigned to R in the body of this specification and, in the broader sense, refers to an alkyl group as defined above modified by C 1 ~C 12 In the narrow sense, R is alkyl, and in the narrow sense, R is methyl, ethyl, or propyl, unless otherwise specified. An alkyl having more than one carbon may contain more than one amine or hydroxyl. Unless otherwise specified, the term "substituted alkyl" refers to an alkyl in which each C is substituted only with at most one amine or hydroxyl group, in addition to the bond to the alkyl chain, the terminal methyl, or hydrogen.

[0049] The term "carboxyl-substituted alkyl" refers to one or more carboxyl groups COOH, or derivatives thereof, in particular carboxyamides CONH 2 , CONHR and CONR 2or an alkyl as defined above modified by a carboxyl ester COOR, where R has the meaning given in the preceding paragraph, which is different from any other meaning assigned to R in the body of this specification.

[0050] The term "halogen substituted alkyl" refers to an alkyl, as defined above, modified by one or more halogen atoms selected (independently) from F, Cl, Br, I.

[0051] "Carboxylic acid ester" is a group -CO 2 R, where R is further defined herein. A carboxylic acid amide is the group -CONHR, where R is further defined herein.

[0052] As used herein, the term "pharmaceutical composition" refers to a compound of the present invention, or a pharma- ceutically acceptable salt thereof, together with at least one pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the present invention is provided in a form suitable for topical, parenteral, or injectable administration.

[0053] As used herein, the term "pharmaceutically acceptable carrier" includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, as known to those of skill in the art (see, e.g., Remington: the Science and Practice of Pharmacy, ISBN0857110624).

[0054] As used herein, the term "treating" or "treatment" of any disease or disorder (e.g., cancer) refers, in one embodiment, to ameliorating the disease or disorder (e.g., delaying or preventing or reducing the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and / or prevention of a disease are generally known in the art unless otherwise specified below.

[0055] In the present specification, the following abbreviations are used: APC (antigen-presenting cell), β2m, β2 microglobulin; DC (dendritic cell), dendritic cell; HLA (human leukocyte antigen), human leukocyte antigen; HPLC (high-pressure liquid chromatography), high-pressure liquid chromatography; IFN-γ (interferon-γ), interferon-γ; IL-13 (interleukin 13), interleukin 13; mAb (monoclonal antibody), monoclonal antibody; MAIT cell (mucosal associated invariant T cell), mucosal associated invariant T cell; MFI (median fluorescence intensity), fluorescence intensity; MHC (major histocompatibility complex), major histocompatibility complex; MR1 (MHC class I-related molecule 1), MHC class I-related molecule 1; MR1 T cell, MR1-restricted T cell, MR1-restricted T cell; MS (mass-spectrometry), mass spectrometry; PBMC (peripheral blood mononuclear cell), peripheral blood mononuclear cell; ROS (reactive oxygen species), reactive oxygen species; TAA (tumor-associated antigen), tumor-associated antigen; TCR (T cell receptor), T cell receptor; TIL (tumor-infiltrating lymphocyte), tumor-infiltrating lymphocyte.

[0056] In a particular embodiment, the invention relates to the use of MR1 (major histocompatibility complex class I-related gene protein 1) ligand compounds in methods involving modulation of the interaction of MR1 with MR1-specific T cells.

[0057] Certain MR1 ligand compounds according to the present invention can be described by adenine derivatives of the following general formula: [ka]

[0058] Other MR1 ligand compounds according to the invention can be described by the following general formula: guanine derivatives: [ka]

[0059] Other MR1 ligand compounds according to the invention can be described by pyrimidine nucleotide derivatives of the following general formula: [ka]

[0060] The substituents shown in the above general formula are understood to have the following meanings: - R 1A is H or methyl, R 1G is H or methyl; - R N3 is H or methyl: - R 2 is selected from H, methyl and -S-methyl; - R 3U and R 5U is selected from H and methyl; R 5C is selected from H and methyl; - R N1 and R N2 are all H or C 1 ~C 3 is alkyl, or R N1 is H or C 1 ~C 3 Alkyl (especially R N1 is H or methyl) and R N2 is C 1 ~C 6 Alkyl and C 2 ~C 6 Alkylene and C 1 ~C 6substituted carbamoyl of alkyl, where alkyl or alkylene is unsubstituted or substituted with carbonyl, carboxyl and / or hydroxyl; Especially R N1 is H and R N2 is selected from methyl, 2-hydroxy-ethyl, 1-carboxyethyl, 1,2-dicarboxyethyl, threonylcarbamoyl, isopent-2-enyl, cis-hydroxyisopent-2-enyl, 3-oxo-1-propenyl, and hexa-1,3,5-triene-1,1,3-tricarbaldehyde; or R N1 And, R N2 and the nitrogen together form the 2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarboxaldehyde bicyclic ring system; or - R N3 and R 1A and R N2 and R 3C and R N1 and R 1G and both are unsubstituted or C 4 ~C 16 -2-oxoalkyl- or ω-carboxy-2-oxo-alkyl-substituted imidazole ring; or R N3 and R 1A and R N2 and R 3C and R N1 and R 1G Both are -C(CH 3 )OH-CHOH- or C(R')OH-CH 2 -CHOH- or oxy-cyclopropylidene-malonaldehyde-substituted propane-2-ene, where R' is H, CH 3 , CH(OH)C 2 H 5 , C 2 H 5 and C 4 H 9 Selected from; or R N1 and R 1G and form a pyrimidine, or RN1 and R 1G or R N3 and R 1A and R form a 12-oxo-5,6,10,12-tetrahydro-3H-6,10-methano[1,3,5]oxadiazocine ring system, or N1 and R 1G and form the 2-oxa-6,8-diazabicyclo[3.3.1]non-3-ene-4-carbaldehyde bicyclic ring system; and - R O is H, unsubstituted or hydroxyl substituted C 1 ~C 5 Alkyl or C 2 ~C 5 alkylene; - R X , SH, C 1 ~C 5 Alkyl, C 2 ~C 5 Alkylene, and C 1 ~C 5 S-alkyl; - R R is selected from H, 1'-ribosyl, 2'-deoxy-1'-ribosyl, 5'-phospho-1'-ribosyl, 5'-methylthio-1'-ribosyl, 1'-(2'-O-ribosyl-5''-phosphate)ribosyl, 1'-(2'-O-ribosyl)-ribosyl 1'-(2'-O-methyl)ribosyl.

[0061] In our current understanding, the moiety R constitutes the structural difference between the adduct and the parent nucleoside. 1A , R 2、 R N1 , R N2 , R N3It is believed that "nucleoside modifications" such as those represented by nucleoside moieties, etc., facilitate the interaction with the MR1 pocket, resulting in a stable complex with MR1. Principle structural information on how the various adducts insert into the MR1 pocket is not yet available. It is possible that the various adducts adopt orthogonal positions. For example, the adenosine-containing adducts may have two rings oriented parallel to the alpha helix of MR1, whereas the guanosine-containing adducts may have two rings oriented perpendicular to it. This would be explained by the addition of residues at two different positions of the primary amines of adenosine and guanosine that are both compatible for binding. Data from the crystal structures of MR1-binding bacterial MR1 ligands support this idea.

[0062] Naturally occurring nucleobases and their ribosyl or deoxyribosyl derivatives, i.e., adenine, adenosine, deoxyadenosine, guanine, guanosine, deoxyguanosine, uracil, uridine, deoxyuridine, thymine, thymidine, deoxythymidine, cytosine and cytidine, and deoxycytidine, are not considered to be within the scope of the above contemplated general formulae I, II, III and IV and derivatives thereof and are not considered to be useful in the methods and uses disclosed herein.

[0063] The inventors also find that the compounds 3-methyladenine, 7-methyl-7-deaza-2'-deoxyguanosine, keuosine, wybutosine, hydroxywybutosine, pseudouridine, and (2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-(methylthio)-9H-purin-9-yl)tetrahydrofuran-3,4-diol can be used in the practice of the present invention, and therefore, modifications thereof made in the spirit of the invention as disclosed herein can also be used.

[0064] In certain embodiments of any of the nucleoside derivative compounds disclosed herein as MR1 ​​ligands, R R is represented by the general formula (V): [ka] In the formula, R B is the N of the formula (adenine nucleobase derivatives) of Group I specified above 9 to nitrogen or to N of the formulae II, III or IV above 1 This is the bond that connects this moiety to the nitrogen.

[0065] In certain embodiments, R of V 2’ is H and R 5’ is H. In certain embodiments, R of V 2’ is H and R 5’ PO 3 2- In certain embodiments, R of V is 2’ is H and R 5’ In certain embodiments, R of V is OH. 2’ is OH and R 5’ PO 3 2- In certain embodiments, R of V is 2’ OCH 3 and R 5’ is H. In certain embodiments, R of V 2’ OCH 3 and R 5’ PO 3 2- It is.

[0066] In certain embodiments, R of V 2’ is O-ribosyl or O-ribosyl-5″-phosphate, and R 5’ H and PO 3 2- is selected from.

[0067] In certain embodiments of any of the nucleoside derivative compounds disclosed herein as MR1 ​​ligands, R R is described by the general formula (Va): [ka] In the formula, R 2’is selected from H, OH, O-methyl, O-1-ribosyl and O-1-(5-phospho)-ribosyl. 2’ Let Va be Vb, where V is OH, and R 2’ Va in which is O-1-(5-phospho)-ribosyl is denoted as Vc.

[0068] In more particular embodiments of any of the nucleoside derivative compounds disclosed herein as MR1 ​​ligands, R R is represented by the general formula (Vb) or (Vc) [ka] It is described by:

[0069] The phosphate of the 5'' ribosyl oxygen of Vc is in the acid form (OPO 3 H 2 ) or may be a hydrogen phosphate or a phosphate with an appropriate anion.

[0070] In certain embodiments of any of the nucleoside derivative compounds disclosed herein as MR1 ​​ligands, the MR1 ligand compound is described by any one of the following general formulas: [ka] - In the formula, R N1 , R N2 , R 1A , R 1G , R 2’ and R 5’ may have the meaning given above, and - R 2’ is selected from H, OH, O-methyl, ribosyl and 5″ phosphoribosyl; R 5’ H, PO 3 2- and methyl; - Especially R 2’ is H or OH, and R 5’ is H.

[0071] In certain embodiments, the MR1 ligand compound is described by formula I, N1 And, R N2 and the nitrogen together form a 2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarboxaldehyde bicyclic ring system, where R is selected from H, ribosyl, and deoxyribosyl.

[0072] In certain embodiments of any of the nucleoside derivative compounds disclosed herein as MR1 ​​ligands, the MR1 ligand compound is described by: a. Formula (I), wherein R 2 is S-methyl and R N1 and R N2 and are both H; b. Formula (I), wherein R 2 is methyl and R N1 and R N2 and are both H; or c. Formula (I-1), wherein R 1A is methyl, R 2 is H and R N3 is H; or d. Formula (I-1), wherein R 1A is methyl, R 2 is methyl and R N3 is H; or e. Formula (I-1), R1A is methyl, and R 2 is S-methyl and R N3 is H; or f. Formula (I), wherein R 2 is H and R N1 and R N2 is selected from H and methyl, and R N1 and R N2 the other of which is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl, and threonylcarbamoyl; or g. Formula (I), wherein R 2 is S-methyl and R N1 and RN2 is selected from H and methyl, and R N1 and R N2 the other is selected from methyl, ethan-2-ol, 1,2-dicarboxy-ethyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl and threonylcarbamoyl; h. Formula (I), wherein R 2 is methyl and R N1 and R N2 is selected from H and methyl, and R N1 and R N2 the other of which is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl, and threonylcarbamoyl; i. Formula (II), wherein R 1G is methyl and R N1 and R N2 are both H; or j. Formula (II-plus), wherein R 1G is methyl and R N1 and R N2 are both H; or k. Formula (II-plus), wherein R 1G is H and R N1 and R N2 are both H; or l. Formula (II), wherein R 1G is methyl and R N1 and R N2 is selected from H and methyl, and R N1 and R N2 the other of which is selected from methyl, ethyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl, and threonylcarbamoyl; or m. Formula (II-plus), wherein R 1G is methyl and R N1 and R N2 is selected from H and methyl, and R N1 and R N2the other of which is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl, and threonylcarbamoyl; or n. Formula (II-plus), wherein R 1G is H and R N1 and R N2 is selected from H and methyl, and R N1 and R N2 the other of which is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl, and threonylcarbamoyl; or o. Formula (I-IM or II-IM), wherein R IM However, H, CH where n is 3 to 7 (especially n = 5) 2 COC n H (2n+1)、 and CH where m is 3 to 9 (especially m = 7). 2 CO(CH 2 ) m COO - Selected from; [ka] p. Formulas (II-c), (II-d), (II-e), (II-f) [ka] q. Formula (II-g) or (II-h), wherein R 2 is selected from H, methyl and S-methyl: [ka] r. Formula (II-i)(II-j), wherein R N1 is selected from H and methyl, and R 2 is selected from H, methyl and S-methyl: [ka] s. Formula (III), wherein R 3U is H and R 5Uis methyl, t. Formula (III), wherein R 3U is methyl and R 5U is H, u. Formula (III), wherein R 3U and R 5U are all methyl, v. Formula (IV), wherein R 5C is H and R N1 and R N2 is selected from H and methyl, and R N1 and R N2 the other of which is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl, and threonylcarbamoyl; w. Formula (IV-1), wherein R N2 is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl, and threonylcarbamoyl; or x. Formula (IV), wherein R 5C is methyl and R N1 and R N2 is selected from H and methyl, and R N1 and R N2 the other of which is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl, and threonylcarbamoyl; y. Formula (Ix), wherein R 2 is H and R X is selected from methyl and S-methyl; z. Formula (I), wherein R 2 is H and R N1 is H and R N2 is 3-oxo-1-propenyl; aa. Formula (Ia), [ka] bb. Formula (II-k), [ka] cc. Formula (I-2), wherein R 1A , R N1 and R 2 is H and R N2 is isopent-2-enyl, or cis-hydroxyisopent-2-enyl; dd. Formula (I), wherein R N1 and R 2 are both H, and R N2 is isopent-2-enyl, or cis-hydroxyisopent-2-enyl; ee. Formula (II-1), wherein R O is methyl or ethane-2-ol, and R N1 and R 2 are both H; ff. Formula (Ib), [ka] And in each formula, R R is selected from H, 1'-ribosyl, 2'-deoxy-1'-ribosyl, 5'-phospho-1'-ribosyl, 5'-methylthio-1'-ribosyl, 1'-(2'-O-ribosyl-5''-phosphate)ribosyl, 1'-(2'-O-ribosyl)-ribosyl 1'-(2'-O-methyl)ribosyl.

[0073] In certain particular embodiments of the above embodiments outlined as a., b., ...ff., gg., R R is H.

[0074] In certain particular embodiments of the above embodiments outlined as a., b., ...ff., gg., R R is (1)-ribosyl (Vb).

[0075] In certain particular embodiments of the above embodiments outlined as a., b., ...ff., gg., R R is (1)-deoxyribosyl (Vd).

[0076] MR1 ligand compounds defined by complete structure: In certain very specific embodiments of any of the nucleoside derivative compounds disclosed herein as MR1 ​​ligands, the MR1 ligand compound is selected from: a. 1-Methyladenosine (1) b. 2-Methyladenosine (2) c. 2'-O-methyladenosine (3) d. N6,N6-Dimethyladenosine (4) e. N6-Threonylcarbamoyl adenosine (5) f. N6-Isopent-2-enyladenosine (6) g. N6-(cis-hydroxyisopent-2-enyl)adenosine (7) h. 2-Methylthio-N6-(cis-hydroxyisopent-2-enyl)adenosine (8) i. 2-Methylthio-N6-isopent-2-enyladenosine (9) j. N6-Methyl-N6-threonylcarbamoyl adenosine (10) k. 2'-O-ribosyladenosine phosphate (11) l. N6-(3-oxo-1-propenyl)-2'-deoxyadenosine (12) m. 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarboxaldehyde (13) n. 1-(3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3H-imidazo[2,1-i]purin-7-yl)heptan-2-one (14) o. 1-Methylguanosine (15) p. N2-Methylguanosine (16) q. 7-Methylguanosine (17) r. 2'-O-methylguanosine (18) s. N2,N2-Dimethylguanosine (19) t. 2'-O-ribosylguanosine (20) u. 3-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,7-dihydroxy-6-methyl-6,7-dihydro-3H-imidazo[1,2-a]purin-9(5H)-one (21), 3-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,7-dihydroxy-7-methyl-6,7-dihydro-3H-imidazo[1,2-a]purin-9(5H)-one (22), or a mixture of the two; v. 2-((6-oxo-6,7-dihydro-1H-purin-2-yl)amino)propanoate (23) w. Pyrimido[1,2-α]purin-10(3H)-one (M 1 G)(24) x. 3-((2R,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7-(2-oxoheptyl)-3H-imidazo[1,2-a]purin-9(5H)-one (25) y. 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (26) z. N2-Oxopropenyl-deoxyguanosine (27) aa. 3-((2R,4S,5R)-4-Hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-12-oxo-5,6,10,12-tetrahydro-3H-6,10-methano[1,3,5]oxadiazocino[5,4-a]purine-9-carbaldehyde (28) bb. 2'-O-methylcytidine (29) cc. 3-Methyluridine (30) dd. 5-Methyluridine (31) ee. 3,2'-O-Dimethyluridine (32) ff. 6-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(2-oxoheptyl)-1,8a-dihydroimidazo[1,2-c]pyrimidin-5(6H)-one (37) gg. N4-(3-oxo-1-propenyl)-2'-deoxycytidine (38) hh. 8-(1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6 dicarbaldehyde (39) ii. 3-Methyladenine (41) jj. N6-Methyladenosine (42) kk. 6-Methylpurine (43) ll. 6-(Dimethylamino)purine (44) mm. N6-(Δ2-isopentenyl)adenine (45) nn. N1-Methyl-2'-deoxyguanosine (46) oo. 1-Methylguanine (47) pp. N2-Methyl-2'-deoxyguanosine (48) qq. 7-Methyl-7-deaza-2'-deoxyguanosine (49) rr. O6-Methyl-2'-deoxyguanosine (50) ss. N2-Ethyl-2'-deoxyguanosine (51) tt. 5'-deoxy-5'-(methylthio)adenosine (52) uu. N6-Methyl-2'-deoxyadenosine (53) vv. N6-(2-hydroxyethyl)-2'-deoxyadenosine (54) ww. O6-(2-hydroxyethyl)-2'-deoxyguanosine (55) xx. N6-Succinyladenosine (56) yy. 2-(2-((3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3,7-dihydropyrimido[2,1-i]purin-7-yl)oxy)cyclopropylidene)malonaldehyde (57).

[0077] The present invention further encompasses variations of the modifications shown herein that can be obtained by modifying a moiety that binds to the nucleoside core structure by addition of an amine functional group, a hydroxy functional group, a carboxylic acid moiety or a carboxylic acid ester moiety, or a halogen. Any of the compounds described above that exhibit an alkyl moiety may be modified to exhibit an aminoalkyl, hydroxyalkyl or haloalkyl as defined above. Mixed modifications are possible. The assays shown herein provide a reliable basis for testing whether such modifications can bind to MR1 and can induce or inhibit an MR1 T cell response.

[0078] In a general aspect, the present invention relates to a method for modulating the interaction between an MR1 polypeptide and an MR1-specific TCR molecule, particularly in vitro. Methods according to this aspect include contacting the MR1 polypeptide with an MR1 ligand compound as defined above.

[0079] Method for selecting a T cell, TCR, B cell or antibody that reacts with an MR1-presented MR1 ligand In a set of embodiments, the method for modulating the MR1-ligand interaction can be performed in vitro, for example, to search for and identify novel binders, particularly T cells, B cells or antibodies, that react with the MR1-ligand complex. Such binding molecules or polynucleotide sequences encoding them can then be developed as pharmaceuticals or diagnostic reagents, or particularly useful reagents can be selected from existing repertoires.

[0080] These sequences enable the use of various tools useful for pharmaceutical and diagnostic applications.

[0081] For example, MR-1 presenting cells are contacted with a ligand as disclosed herein in the presence of a T cell library or a B cell library (cells expressing various TCR or B cell receptor (BCR) sequences), and cells having TCR or BCR sequences that react with the MR1-ligand complex are identified and isolated by methods known to those of skill in the art.

[0082] Thus, one aspect of the invention relates to a method for identifying, isolating or selecting T cells reactive to an MR1 ligand compound as presented by MR1, as defined herein, in particular in any one of claims 1 to 9. The method comprises the steps of providing a preparation of T cells reactive to / capable of specifically recognizing MR1; contacting said preparation of T cells with isolated MR1 associated with said MR1 ligand compound, or a complex comprising MR1 of an MR1-presenting cell, followed by an isolation step to isolate T cells specifically reactive to said MR1 ligand compound in the context of MR1 presentation. Many methods are available to the skilled artisan for detecting TCR engagement and recognition of cognate antigen presented by MHC.

[0083] According to an alternative of this aspect of the invention, the method for modulating the interaction between an MR1 polypeptide and an MR1-specific TCR molecule may be employed as part of a method for identifying and isolating B cells or their receptors, antibodies specific for an MR1 polypeptide and / or reactive to an MR1 ligand compound presented on MR1.

[0084] An alternative of this aspect of the invention relates to a method for identifying, isolating or selecting B cells or antibodies reactive to an MR1 ligand compound as defined herein, in particular in any one of claims 1 to 9. The method comprises the steps of providing a preparation of B cells reactive to / capable of specifically recognizing MR1; contacting said preparation of B cells with a complex comprising isolated MR1 associated with said MR1 ligand compound, followed by an isolation step isolating B cells that specifically react to said MR1 ligand compound in the context of MR1 presentation. Many methods are available to the skilled artisan for detecting engagement of BCR with and recognition of cognate antigen presented by MHC.

[0085] According to another aspect of the invention, the method for modulating the interaction between an MR1 polypeptide and an MR1-specific TCR molecule may be employed as part of a diagnostic method for classifying metabolically altered cells, including but not limited to tumor cells, according to the presence of said compounds, in which samples obtained from patients are analyzed for the presence of the MR1 ligand compounds identified herein.

[0086] This embodiment is of particular use in selecting TCR molecules or transgenic T cells or vectors for obtaining transgenic T cells ("MR1-specific T cell reagents") from autologous T cell populations in patients with diseases, particularly tumors, characterized by a metabolic profile indicative of certain of the MR1 ligands provided herein. This embodiment is even better suited to selecting MR1-specific T cell reagents for patients whose tumors have been directly analyzed and found to be present with certain of the MR1 ligand compounds disclosed herein. Selecting the best / most specific MR1-specific T cell reagent from a panel of MR1-specific T cell reagents available to the clinician will provide the patient with the most optimal therapy.

[0087] Alternatively, this method allows the use of unbiased selection of T cells, which allows identification of T cells (from a much smaller population) to expand the MR1-specific T cell reagent repertoire.

[0088] Use of an MR1 ligand compound in the treatment or prevention of MR1-related diseases The present invention facilitates the use of T cells that specifically recognize and can react with MHC-presented ligands presented by invariant MR1 molecules, thereby providing treatment options that are not restricted by the patient's MHC genotype.

[0089] One aspect of the present invention is for use in the prevention or treatment of diseases associated with abnormal or absent MR1-specific T cell responses in the treatment of cancer, particularly cancers characterized by tumor cells expressing MR1, for the MR1 ligand compound defined in any one of the above or claims 1 to 9.

[0090] The inventors have for the first time found evidence to prove that the compounds disclosed herein are specifically presented and recognized by T cells in the context of cancer. Thus, the MR1 ligand compounds disclosed herein serve for use as "cancer vaccines" in the sense that their presence enhances the therapeutic approaches enabled by MR1-T cell interactions.

[0091] In certain embodiments, the MR1 ligand compound is co-administered with an anti-cancer agent. As disclosed herein, the inventors were able to demonstrate that the administration of the established anti-tumor drugs paclitaxel or doxorubicin likely increases the presentation of the MR1 ligand compounds disclosed herein by increasing metabolic stress within cancer cells. Such combinations can be expected to result in a synergistic effect as they enhance the loading of MR1 ligands in tissues.

[0092] In certain embodiments, the MR1 ligand compound is co-administered with a checkpoint modulator or checkpoint inhibitor. Such combinations are expected to produce synergistic effects, since the downstream immune effects of the engagement of the MR1 ligand with MR1-specific T cells physiologically present in the patient or optionally administered as part of an additional MR1-specific T cell therapy are expected to be enhanced by removing the inhibitory signal.

[0093] In a particular embodiment, the compound is administered in association with (before, concomitantly or after) a preparation comprising (transgenic) MR1-reactive T cells and / or (transgenic) MR1-reactive T cell receptor polynucleotide constructs (e.g., RNA constructs or DNA expression constructs encoding MR1-TCR, or viral vectors having the same function).

[0094] Optionally, a polynucleotide expression vector encoding MR1 can be provided. If affected disease tissue is found to downregulate MR1 expression, transgene expression of MR1 provides a means to increase MR1 expression in tissues.

[0095] The MR1 ligand and / or polynucleotide expression vector encoding the MR1 TCR or MR1 can be administered to the tumor, near the tumor, or to lymph nodes draining the tumor site. Local augmentation of either agent is believed to be more effective than systemic administration.

[0096] In particular, the present invention allows the administration of recombinant (allogeneic or autologous) T cells carrying a transgene TCR capable of specifically recognizing and reacting to a specific MR1 ligand compound of an MR1-presenting disease.

[0097] The invention further enables analysis of T cell samples obtained from patients for the presence of T cells capable of recognizing MR1-presented MR1 ligand compounds, and selectively stimulating and expanding, or de novo engineering, such MR1 patient T cells for subsequent therapeutic administration.

[0098] The present invention further allows for the targeted administration of MR1 ligand compounds as defined herein to patients in order to enhance or amplify MR1-specific T cell responses or MR1-targeted T cell therapy.

[0099] The compounds identified herein can be employed in methods relating to: - modulating the interaction between an MR1 (major histocompatibility complex class I-related gene protein 1) polypeptide and an MR1-specific T-cell receptor molecule, wherein the MR1 polypeptide is contacted with an MR1 ligand compound; - generating a panel of MR1 multimer reagents for use in identifying and selecting T cells that respond to MR1 ligand compounds presented on non-polymorphic MHC I-associated MR1 antigen-presenting molecules; - identifying TCR genes that specifically respond to said compound for use in personalized cellular immunotherapy, which can be achieved by providing a preparation of isolated tumor cells from a patient and subjecting them to mass spectrometry-based measurements to assess the presence and identity of the compound.

[0100] In certain embodiments, identifying TCR genes that specifically react with the compounds identified herein for use in personalized cellular immunotherapy can be performed by the following general sequence of processes: 1) detection of the compound in a tumor biopsy (tumors are classified based on the presence of a TAA), followed by 2) inducible TCR-mediated cellular immunotherapy based on the TCR that most reacts with the identified TAA in complex with MR1.

[0101] Combination of MR1 therapy and medicines MR1-specific T cell responses or MR1-targeted T cell therapy may be further enhanced by combination with drugs that increase the production of MR1 ligand compounds as defined herein.

[0102] The following pathways are targets for useful drugs in this context: glutathione-S-transferase (GST), aldehyde reductase and aldehyde-keto reductase (AKR), aldehyde dehydrogenase (ALDH), aldehyde oxidase (AOX), xanthine oxidase (XO), and short chain reductase / oxidase (SDR).

[0103] The following compound classes and specific drugs identified as part of the compound classes are possible combination partners for the MR1 TAA in a vaccine+drug therapy approach or a MR1-T cell transfer+drug therapy approach:

[0104] "Glutathione-S-transferase (GST) inhibitors", in particular any one of the glutathione-S-transferase (GST) inhibitors selected from the group consisting of ethacrynic acid, Terrapin 199, Terrapin 286, clofibrate, gossypol, indomethacin, piriprost, misonidazole and sulfasalazine.

[0105] "Inhibitors of aldo-keto reductase 1C3", in particular inhibitors of aldo-keto reductase 1C3 selected from the group comprising N-benzoyl anthranilates, 2,3-arylpropenoic acids, stylopne, 2'-hydroxyflavone, N-phenylsulfonylindole, N-(benzimidazolylcarbonyl)piperidine, N-(indolylcarbonyl)-piperidine, N-(pyridinepyrrolylcarbonyl)-piperidine, N-(benzimidazole or indole)benzoic acids, N-(phenylamino)-benzoates, N-(naphthylamino)-benzoates, isoquinolones, indomethacin analogues, nitrogen or sulfur substituted estrenes, β-naphthylacetic acids, baccarin and baccarin analogues.

[0106] "Aldehyde dehydrogenase inhibitors", in particular any one of the aldehyde dehydrogenase inhibitors selected from the group consisting of ampal, benomyl, citral, chloral hydrate, chlorpropamide analogues (NPI-1 and API-1), coprine, cyanamide, "daidzin", CVT-10216, 4-(diethylamino)benzaldehyde (DEAB), "disulfiram", gossypol, molinate, nitroglycerin, and pargyline.

[0107] "Aldehyde oxidase inhibitors", in particular any one of the aldehyde oxidase inhibitors selected from the group consisting of amodiaquine, chlorpromazine, domperidone, estradiol, felopidine, loratadine, maprotiline, metoclopramide, norclomipramine, nortriptyline, ondansetron, perphenazine, promazine, promethazine, raloxifene, salmeterol, tacrine, tamoxifen and thioridazine.

[0108] "Xanthine oxidase inhibitors", in particular any one of the xanthine oxidase inhibitors selected from the group consisting of allopurinol, febuxostat, oxypurinol, tisopurine, topiroxostat, and inositol (phytic acid, myo-inositol).

[0109] "Glyoxalase I inhibitors", particularly one of ethyl pyruvate, Sp-bromobenzyl glutathione cyclopentyl diester, S-ethyl glutathione, 2-(8-chloro-2-(4-chlorophenyl)-3-hydroxy-4-oxochroman-6-yl) acetic acid.

[0110] "GAPDH inhibitors", particularly one of gossypol, koningic acid (hepteric acid), arsenate, arsenic trioxide, 3-bromopyruvate, or iodoacetate.

[0111] Therefore, another aspect of the present invention is the use of compounds that can modulate intracellular metabolism, for example by inhibiting decarbonylation, to increase MR1-dependent immune responses.Exemplary agents tested by the present inventors include, but are not limited to, the group including paclitaxel, doxorubicin, disulfiram, daidzin, nitrobenzylthioinosine, ellagic acid, oleic acid, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), pentostatin, 1-deazaadenosine, and mycophenolic acid.These agents and related agents can be employed to prevent or treat diseases associated with abnormal or absent MR1 expression, particularly to treat or prevent recurrence of cancer diseases associated with tumor cells expressing MR1.

[0112] Particularly useful examples of agents that provide benefits according to this aspect of the invention are pharmaceutical agents that can increase the amount of reactive oxygen species (ROS) that lead to overproduction of MR1 ligand in cells. These include paclitaxel, docetaxel, epothilones, discodermolide, cabazitaxel, doxorubicin, daunorubicin, epirubicin, and idarubicin.

[0113] In one alternative of this aspect of the invention, "paclitaxel" is provided for treating cancer diseases associated with tumor cells expressing MR1 or preventing the recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Paclitaxel (CAS:33069-62-4) is a taxane-based small molecule chemotherapeutic agent with mitotic inhibitory properties. Paclitaxel interferes with the growth of microtubules by binding to the β-subunit of tubulin. As a result, the microtubule / paclitaxel complex is incapable of disassembly. Paclitaxel significantly increases reactive oxygen species (ROS) levels (Z. Yu et al., ACS Nano 2015, 9, 11064; M. Li, et al., J. Am. Chem. Soc. 2018, 140, 14851; C. Dai et al., ACS Nano 2017, 11, 9467; P. Zhu, et al., ACS Nano 2018, 12, 3780; Jiang, H. et al., Small 2019, 15, 1901787). Paclitaxel is generally administered as an intravenous injection solution of up to 600 mg per treatment unit, but is not limited to this.

[0114] In one alternative of this aspect of the invention, "docetaxel" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Docetaxel (CAS:114977-28-5) is another clinically established and approved taxane antitumor chemotherapeutic agent, which is primarily used to treat patients suffering from breast cancer, ovarian cancer, and non-small cell lung cancer. Docetaxel binds reversibly with high affinity to tubulin in a 1:1 stoichiometric ratio. It promotes the assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization.

[0115] Although docetaxel has a strong antitumor effect, it is known to cause excessive reactive oxygen species (ROS) generation, Ca 2+Its use is often limited due to systemic and adverse side effects due to influx and inflammatory markers such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1 beta (β), and IL-6 (Kutuk, SG et al., Biol Trace Elem Res 196, 184-194, 2020). Dosage forms include solutions for intravenous and parenteral injection, as well as powders for such preparation, in amounts up to 160 mg per therapeutic unit.

[0116] In one alternative of this aspect of the invention, "epothilones" are provided for treating cancer diseases associated with tumor cells expressing MR1 or preventing the recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Epothilones are a new class of compounds that can be isolated from myxobacteria or synthesized semi-synthetically and / or fully synthetically and exhibit anti-microtubule effects. Epothilones exert potent antiproliferative activity in various multidrug-resistant tumors, with a similar mechanism of action to taxanes. Epothilones stabilize microtubules and induce apoptosis. Currently, several epothilones and their derivatives / analogs are in clinical trials. Members of this group include epothilone A (CAS: 152044-53-6); epothilone B (CAS: 152044-54-7), also known as patupilone or BMS-310705 (a water-soluble derivative of patupilone), ixabepilone (a second-generation semisynthetic patupilone; approved by the FDA in 2007) or sagopilone (a fully synthetic third-generation analogue of patupilone), which are derivatives of epothilone A that bear a methyl group at the C12 atom. Recent studies have shown that epothilone B induces mitochondrial collapse and release of ROS, thereby promoting apoptosis. The above compounds are currently being investigated in clinical trials for their anticancer and proapoptotic effects; Epothilone D (CAS: 189453-10-9), also known as KOS-862 or desoxyepothilone B and KOS-1584 (second generation epothilone D), is a small molecule currently being investigated for the treatment of colorectal cancer, lung cancer, breast cancer, solid tumors, and prostate cancer. Epothilone D lacks the C12-13 epoxide and exhibits higher therapeutic efficacy than epothilone A.

[0117] In one alternative of this aspect of the invention, "discodermolide" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Discodermolide (CAS:127943-53-7) is a small molecule with microtubule stabilizing properties similar to other taxols. Discodermolide induces an increase in ROS following treatment.

[0118] In one alternative of this aspect of the invention, "cabazitaxel" is provided for treating cancer diseases associated with tumor cells expressing MR1 or preventing the recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Cabazitaxel (CAS:183133-96-2) is a small molecule anti-cancer agent. Cabazitaxel is administered to patients with advanced prostate cancer despite treatment with docetaxel. Cabazitaxel regulates microtubule inhibition by binding to tubulin and promoting its assembly into microtubules while simultaneously inhibiting its disassembly. This leads to the stabilization of microtubules, resulting in interference with mitotic and interphase cell functions, thus inhibiting further progression into the cell cycle and thereby inducing apoptosis. Recent studies have shown that cabazitaxel induces ROS production by inhibiting the expression of the antioxidant-sestrin3 (Kosaka T. et al., Oncotarget. 2017;8(50):87675-87683).

[0119] In one alternative of this aspect of the invention, "doxorubicin" is provided for treating cancer diseases associated with tumor cells expressing MR1 or preventing the recurrence of such tumor cells, especially in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Doxorubicin (CAS:23214-92-8) is a well-studied and widely applied small molecule cytotoxic anthracycline antibiotic. Doxorubicin binds to nucleic acids by specific intercalation of the planar anthracycline core with the DNA double helix. Doxorubicin is provided for causing regression of symptoms of disseminated tumors, including but not limited to acute lymphoblastic leukemia, acute myeloblastic leukemia, Wilms' tumor, neuroblastoma, sarcoma of soft tissue and bone, breast cancer, ovarian cancer, transitional cell bladder cancer, thyroid cancer, gastric cancer, Hodgkin's disease, malignant lymphoma, and bronchogenic carcinoma. Doxorubicin is also indicated for use as a component of adjuvant therapy in women with evidence of axillary lymph node metastasis following resection of primary breast cancer. Studies have shown that doxorubicin-induced ROS overproduction occurs within mitochondria and is regulated by mitochondrial NADPH oxidase (mitoNOX) activity (Asensio-Lopez MC et al., PLoS One. 2017;12(2):e0172803). Doxorubicin dosage forms include solutions for intravenous injection and powders for preparing such, with concentrations of approximately 2 mg / mL.

[0120] In one alternative of this aspect of the invention, "daunorubicin" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Daunorubicin (CAS:20830-81-3) is a small molecule, toxic anthracycline aminoglycoside anti-cancer drug approved for the treatment of patients suffering from leukemia and other neoplasms. Daunorubicin has mitotic inhibitory and cytotoxic activity through a number of proposed mechanisms of action, namely, inhibition of macromolecular synthesis by intercalation of daunorubicin into DNA strands, generation of ROS through interaction with molecular oxygen, which further generates DNA damage through double-strand breaks and topoisomerase II inhibition, and formation of DNA adducts (Al-Amri HM. et al., BMC Cancer. 2019;19(1):179).

[0121] In one alternative of this aspect of the invention, "epirubicin" is provided for treating cancer diseases associated with tumor cells expressing MR1 or preventing the recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Epirubicin (CAS:56420-45-2) is the 4'-epi isomer of doxorubicin and is a small molecule anthracycline that exerts its antitumor effect by its property of interfering with DNA synthesis and function. Epirubicin forms a complex with DNA by intercalation between base pairs, and epirubicin inhibits topoisomerase II activity by stabilizing the DNA-topoisomerase II complex, preventing the religation portion of the topoisomerase II catalyzed ligation-religation reaction. Furthermore, epirubicin interferes with DNA replication and transcription by inhibiting DNA helicase activity. Further studies have shown that epirubicin inhibits ROS and mitochondrial H 2 O 2It has been shown to significantly increase levels of mitochondrial cytokines, resulting in mitochondrial-mediated apoptosis caused by increased oxidative stress (Huang, T. C. et al., Apoptosis 23, 226-236, 2018).

[0122] In one alternative of this aspect of the present invention, "idarubicin" is provided for treating cancer diseases associated with tumor cells expressing MR1 or preventing recurrence of such tumor cells, especially in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Idarubicin (CAS:58957-92-9) is a small molecule anthracycline anti-cancer agent, and is provided for patients suffering from, but not limited to, breast cancer, lymphoma and leukemia. (Celik H,Arinc E. et al., J Pharm Pharm Sci.2008;11(4):68-82.PMID:19183515).

[0123] Studies have shown that DNA damage by idarubicin occurs through a mechanism involving redox cycling with P450 reductase, generating ROS. DNA damage by idarubicin has been shown to increase with increasing drug or enzyme concentrations and incubation times.

[0124] Disulfiram: In one alternative of this aspect of the invention, "disulfiram" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Disulfiram (CAS No. 97-77-8) is a well-characterized small molecule pharmaceutical agent that has been used to treat alcoholism in humans for over 50 years. Studies have shown that disulfiram has some efficacy against fungi (S. Khan et al., Jp. J. Med. Mycol (2007), 48, 109-113), protozoa (T. Nash and WG Rice, Antimicrob. Agents Chemother. (1998) 42, 1488-1492), and bacteria (MRSA, M. Phillips et al., Antimicrob. Agents Chemother. (1991), 35, 785-787). The results shown in FIG. 7 support the utility of the compound for administration as part of an MR1 T-targeted therapy (i.e., prevention or treatment of diseases associated with abnormal or absent MR1 expression, particularly treatment or prevention of recurrence of cancer diseases associated with tumor cells expressing MR1).

[0125] Mycophenolic acid: In another alternative of this aspect of the invention, "mycophenolic acid" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Mycophenolic acid (CAS number 24280-93-1) is a small molecule compound used as an immunosuppressant and antiproliferative agent.

[0126] Daidzin: In another alternative of this aspect of the invention, "daidzin" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Daidzin is an isoflavone phytochemical (CAS number 552-66-9). The results shown in Figure 7 support the utility of this compound administered as part of an MR1 T-targeted therapy.

[0127] Yet another alternative of this aspect of the invention relates to "Nitrobenzylthioinosine" (CAS No. 38048-32-7) for the treatment of cancer diseases associated with tumor cells expressing MR1 or for preventing the recurrence of such tumor cells.

[0128] In yet another alternative of this aspect of the invention, "ellagic acid" (CAS No. 476-66-4) and / or "oleanoic acid" (CAS No. 508-02-1) are provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. The results shown in Figure 7 support the utility of the compounds administered as part of an MR1 T-targeted therapy.

[0129] In another alternative of this aspect of the invention, "ethacrynic acid" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Ethacrynic acid (CAS number 58-54-8) is a commercially available drug (trade name: Edecrine) approved for the treatment of hypertension and / or edema due to diseases such as congestive heart failure, liver failure, and kidney failure. Ethacrynic acid has been found to inhibit components of the Wnt / β-catenin pathway, suppressing selective cytotoxicity against CLL cells, and to be a glutathione S-transferase inhibitor. Small molecules contained in pharmaceutical products are provided by various manufacturers in various dosage forms, such as tablets for oral intake and solutions for injection, in dosages ranging from 25 mg (tablets) to 50 mg (50 mL injection solution).

[0130] In another alternative of this aspect of the invention, "Terrapin 199" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Terrapin 199 (CAS No. 168682-53-9), also known as Ezatiostat, is a small molecule drug with promising activity for the treatment of patients suffering from myelodysplastic syndromes. Terrapin 199 is designed to inhibit glutathione S-transferase and increase the activity of c-Jun NH2-terminal kinase (JNK1) and ERK1 / ERK2 to promote cell proliferation under stress conditions, thereby inducing high levels of apoptosis. The drug is absorbed by oral ingestion (Hamilton et al., IDrugs 2005 Aug;8(8):662-9. PMID:16044376).

[0131] In another alternative of this aspect of the invention, "Terrapin 286" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Terrapin 286 (CAS No. 158382-37-7), also known as TLK-286 or Canfosamide, is a small molecule that is included in pharmaceuticals and shows promising activity as a drug against various forms of cancer (Rosen LS et al., Clin Cancer Res. 2004 Jun 1;10(11):3689-98. doi:10.1158 / 1078-0432. CCR-03-0687. PMID:15173075). Upon activation, TLK-286 splits into two fragments, one of which reacts with cellular components such as RNA and DNA, causing cell death, and the other is a glutathione analogue, thereby inhibiting glutathione S-transferase (Kavanagh JJ et al., Int J Gynecol Cancer. 2005 Jul-Aug;15(4):593-600. doi:10.1111 / j.1525-1438.2005.00114.x. PMID:16014111).

[0132] In another alternative of this aspect of the invention, "Clofibrate" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Clofibrate (CAS number 637-07-0) is a fibric acid derivative used in commercial drugs provided for the treatment of hyperlipoproteinemia type III and hypertriglyceridemia. It inhibits glutathione S-transferase activity and further agonizes PPAR-α receptors in soft tissues. This agonism ultimately leads to changes in gene expression resulting in increased β-oxidation. The compound is provided as a capsule (500-1000 mg) for oral intake.

[0133] In another alternative of this aspect of the invention, "Gossypol" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, especially in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Gossypol (CAS No. 303-45-7) is a small molecule drug with glutathione-S-transferase inhibitory properties and has high potential for the treatment of patients suffering from non-small cell lung cancer. Moreover, gossypol induces cell cycle arrest at the G0 / G1 phase and inhibits cell signaling enzymes, thereby inhibiting DNA replication, resulting in apoptosis as well as inhibition of cell proliferation.

[0134] In another alternative of this aspect of the invention, "indomethacin" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Indomethacin (CAS No. 53-86-1) is a well-studied small molecule drug with anti-inflammatory properties. Indomethacin is a drug that non-specifically and reversibly inhibits both isoforms of cyclooxygenase (COX) enzymes or prostaglandin G / H synthase. Chemical bulk synthesis is available from several manufacturers, and many of the commercially available products are administered orally, with dosages ranging from, but not limited to, 25 mg to 75 mg per treatment.

[0135] In another alternative of this aspect of the invention, "misonidazole" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, especially in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Misonidazole (CAS number 13551-87-6) is a pharmaceutical small molecule compound with glutathione-S-transferase inhibitory properties and shows high potential for the treatment of patients suffering from various tumor hypoxia. Misonidazole sensitizes hypoxic cells to the cytotoxic effects of ionizing radiation, thereby inhibiting DNA synthesis.

[0136] In another alternative of this aspect of the invention, "sulfasalazine" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Sulfasalazine (CAS No. 599-79-1) is a small molecule agent with glutathione-S-transferase inhibitory and anti-inflammatory properties. The agent is administered by tablet and rectal route at a dose of about 500 mg per tablet.

[0137] In another alternative of this aspect of the invention, N-benzoyl anthranilates are provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. N-benzoyl anthranilates include, but are not limited to, commercially available meclofenamic acid, a group of compounds that inhibit AKR1C3, known to catalyze reactions that stimulate tumor growth. The drug is provided as, but is not limited to, tablets for oral intake. Dosages range from 50 mg to 100 mg per treatment unit, particularly for meclofenamic acid.

[0138] In another alternative of this aspect of the invention, arylpropionic acids are provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Arylpropionic acids, including but not limited to commercially available ibuprofen, naproxen, flurbiprofen, are another group of compounds that exhibit inhibitory effects on AKR1C3. The drug is administered primarily by oral ingestion, with dosages ranging from but not limited to 50 mg to 1500 mg per treatment unit (Gazvoda M, et al., Eur J Med Chem. 2013 Apr; 62: 89-97. doi: 10.1016 / j.ejmech. 2012.12.045. Epub 2013 Jan 3. PMID: 23353746).

[0139] In another alternative of this aspect of the invention, "2'-hydroxyflavone" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. 2'-Hydroxyflavone (2-(2-hydroxyphenyl)chromen-4-one; CAS No. 35244-11-2) is a small molecule compound with AKR1C3 inhibitory properties and is associated with inhibiting tumor growth and / or recurrence.

[0140] In another alternative of this aspect of the invention, N-phenylsulfonylindoles, N-(benzimidazolylcarbonyl)-piperidines, N-(indolylcarbonyl)-piperidines, N-(pyridinepyrrolylcarbonyl)-piperidines, N-(pyridinepyrrolylcarbonyl)-piperidines are provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. N-Phenylsulfonylindole, N-(benzimidazolylcarbonyl)-piperidine, N-(indolylcarbonyl)-piperidine, N-(pyridinepyrrolylcarbonyl)-piperidine, N-(pyridinepyrrolylcarbonyl)-piperidine are compounds whose derivatives are non-steroidal AKR1C3 inhibitors (Trevor M. Penning Expert Opin Ther Pat. 2017 December;27(12):1329-1340.doi:10.1080 / 13543776.2017.1379503).

[0141] In another alternative of this aspect of the invention, β-naphthylacetic acid is provided for the treatment of cancer diseases associated with tumor cells expressing MR1, or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. β-naphthylacetic acid is a group of compounds that exhibit selective inhibitory activity against AKR1C3 (Adeniji et al., J Med Chem. 2016;59(16):7431-7444. doi:10.1021 / acs.jmedchem.6b00160).

[0142] In another alternative of this aspect of the invention, baccharin is provided for treating cancer diseases associated with tumor cells expressing MR1 or preventing the recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Baccharin derivatives have shown high potential for inhibiting AKR1C3 (Kshitij V. et al., J. Med. Chem. 2019, 62, 3590-3616).

[0143] In another alternative of this aspect of the invention, "AMPAL" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. 4-Amino-4-methyl-2-pentyne-1-al (AMPAL; CAS number 121188-32-7) is a small molecule compound known to inhibit aldehyde dehydrogenase activity and exhibit antitumor activity.

[0144] In another alternative of this aspect of the invention, "benomyl" and "carbendazim" are provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Benomyl (N-[1-[(butylamino)carbonyl]-1H-benzimidazol-2-yl]-, methyl-ester; CAS No. 17804-35-2) and its metabolite carbendazim (N-1H-benzimidazol-2-yl-, methyl-ester; CAS No. 10605-21-7) are small molecule compounds known to inhibit aldehyde dehydrogenase activity.

[0145] In another alternative of this aspect of the invention, "citral" is provided for treating cancer diseases associated with tumor cells expressing MR1 or preventing the recurrence of such tumor cells, especially in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Citral (3,7-dimethyl-2,6-octadienal; CAS number 5392-40-5) is a natural compound found in herbs and citrus fruits that has an inhibitory effect on aldehyde dehydrogenase. Citral is already used in dietary supplements.

[0146] In another alternative of this aspect of the invention, "chloral hydrate" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Chloral hydrate (2,2,2-trichloroethane-1,1-diol; CAS number 302-17-0) is a small molecule compound that inhibits aldehyde dehydrogenase and is provided for the treatment of insomnia. Various manufacturers are known; dosage forms include capsules and syrups for oral ingestion, with doses of around 500 mg per capsule and around 100 mg / mL.

[0147] In another alternative of this aspect of the invention, "chlorpropamide" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Chlorpropamide (1-(4-chlorobenzenesulfonyl)-3-propylurea; 94-20-2) and its analogs are small molecule compounds known to irreversibly inhibit aldehyde dehydrogenase and are provided for the treatment of non-insulin dependent diabetes mellitus.

[0148] In another alternative of this aspect of the invention, "cyanamide" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Cyanamide (Calcium (azanidylenemethylidene) azanide, CAS number 420-04-2) in the form of its citrated calcium salt is a small molecule found in pharmaceutical products and is commercially provided for the treatment of alcoholism. The dosage form is, but is not limited to, mainly a tablet for oral intake, with a dosage of around 50 mg per unit.

[0149] In another alternative of this aspect of the invention, "CVT-10216" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. CVT-10216 (3-[[[3-[4-[(methylsulfonyl)amino]phenyl]-4-oxo-4H-1-benzopyran-7yl]oxy]methyl]-benzoic acid; CAS number 1005334-57-5) is a small molecule and analogue of daidzin with aldehyde dehydrogenase inhibitory properties.

[0150] In another alternative of this aspect of the invention, "DEAB" is provided for treating cancer diseases associated with tumor cells expressing MR1 or preventing the recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. DEAB (4-(diethylamino)benzaldehyde; CAS number 120-21-8) is a commonly used selective inhibitor of aldehyde dehydrogenase in cancer stem cell biology.

[0151] In another alternative of this aspect of the invention, "nitroglycerin" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Nitroglycerin (1,3-bis(nitrooxy)propan-2-yl nitrate; CAS number 55-63-0) is a small molecule with aldehyde dehydrogenase inhibitory properties and is provided for the treatment of patients suffering from pain and hypertension, among others. Nitroglycerin is a well-characterized compound, first approved in 2000. Bulk chemical synthesis methods are known; extensive pharmacological and toxicological data exists for the compound (Ignarro LJ et al., Proc Natl Acad Sci USA. 2002 Jun 11;99(12):7816-7. doi:10.1073 / pnas.132271799, PMID:12060725; PMCID:PMC122975). Nitroglycerin is available in a variety of dosage forms, including but not limited to spray, sublingual tablet, intravenous, sustained release tablet, transdermal, and capsules. Dosage depends on the dosage form and can vary from 200 μg to 160 mg.

[0152] In another alternative of this aspect of the invention, "Pargyline" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Pargyline (benzyl(methyl)(prop-2-yn-1-yl)amine; CAS number 555-57-7) is a small molecule drug with aldehyde dehydrogenase inhibitory properties.

[0153] In another alternative of this aspect of the invention, "amodiaquine" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Amodiaquine (4-[(7-chloroquinolin-4-yl)amino]-2-[(diethylamino)methyl]phenol; CAS number 86-42-0) is a small molecule having aldehyde oxidase inhibitory properties. Amodiaquine is a well-characterized compound that has been known to be synthesized for over 70 years. Amodiaquine is used as an antimalarial drug and an anti-inflammatory drug and is administered via oral ingestion at a dose of about 100 mg per therapeutic unit.

[0154] In another alternative of this aspect of the invention, "chlorpromazine", "promazine", "perphenazine" are provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Chlorpromazine ([3-(2-chloro-10H-phenothiazin-10-yl)propyl]dimethylamine; CAS number 50-53-3), promazine (dimethyl[3-(10H-phenothiazin-10-yl)propyl]amine; CAS number 58-40-2) and perphenazine (2-{4-[3-(2-chloro-10H-phenothiazin-10-yl)propyl]piperazin-1-yl}ethan-1-ol; CAS number 58-39-9) are similarly small molecules included in drugs showing aldehyde oxidase inhibitory properties and are provided for the treatment of mental disorders. Chlorpromazine, promazine and perphenazine are manufactured and sold in different dosage forms, such as tablets for oral ingestion, solutions for intravenous administration, injections and syrups, by various known manufacturers, and the dosage ranges from 2 mg to 200 mg per therapeutic unit.

[0155] In another alternative of this aspect of the invention, "domperidone" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Domperidone (5-chloro-1-{1-[3-(2-oxo-2,3-dihydro-1H-1,3-benzodiazol-1-yl)propyl]piperidin-4-yl}-2,3-dihydro-1H-1,3-benzodiazol-2-one; CAS number 57808-66-9) is a small molecule contained in drugs that act as a specific blocker of dopamine receptors and have aldehyde oxidase inhibitory properties. The synthesis of domperidone is known to the expert (U.S. Patents: 4,066,772; 4,110,333; 4,126,687; 4,126,688; 4,160,836; and 4,175,129; Janssen Pharmaceutica NV). Dosage forms include tablets for oral ingestion, with a dosage of about 10 mg per therapeutic unit.

[0156] In another alternative of this aspect of the invention, "estradiol" is provided for treating cancer diseases associated with tumor cells expressing MR1 or preventing the recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Estradiol ((1S,3aS,3bR,9bS,11aS)-1-hydroxy-11a-methyl-1H,2H,3H,3aH,3bH,4H,5H,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-ylbenzoate; CAS number 50-28-2) is a naturally occurring small molecule that is also contained in some hormonal therapy-provided drugs for managing estrogen-depleted conditions. In addition, estradiol exhibits aldehyde oxidase inhibitory properties. Various dosage forms are provided: tablets, sprays, gels, and creams for oral ingestion, as well as injections, and vaginal rings or transdermal patches.

[0157] In another alternative of this aspect of the invention, "promethazine" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Promethazine (Dimethyl[1-(10H-phenothiazin-10-yl)propan-2-yl]amine; CAS No. 60-87-7) refers to a small molecule found in drugs for the treatment of various conditions, including but not limited to acute allergic reactions, upper respiratory tract symptoms, anaphylaxis, pain, nausea, and vomiting, as well as exhibiting an inhibitory effect on aldehyde oxidase. The compound was approved by the FDA about 70 years ago and is well known to experts. Bulk chemical synthesis is well documented with various manufacturers offering the compound in various dosage forms, such as tablets for oral intake, solutions for intravenous injection, or suppositories for rectal administration, with dosages ranging from 0.6 mg / mL to 50 mg per therapeutic unit.

[0158] In another alternative of this aspect of the invention, "salmeterol" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, especially in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Salmeterol (CAS number 89365-50-4) is a small molecule that exhibits an inhibitory effect of aldehyde oxidase. This compound is contained in drugs provided for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Patients suffering from said conditions generally inhale the drug. Dosage forms include tablets with doses of 20 μg to 50 μg per treatment unit.

[0159] In another alternative of this aspect of the invention, "raloxifene" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Raloxifene (CAS number 84449-90-1) is a well-documented small molecule with aldehyde oxidase inhibitory properties, mediating antiestrogenic effects on breast cancer and uterine tissues, as well as estrogenic effects on bone, i.e., maintaining bone density and reducing breast risk.

[0160] In another alternative of this aspect of the invention, "tacrine" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Tacrine (1,2,3,4-tetrahydroacridine-9-amine; CAS No. 321-64-2) is a small molecule drug provided as a respiratory stimulant and as a treatment for Alzheimer's disease, as well as other central nervous system disorders.

[0161] In another alternative of this aspect of the invention, "tamoxifen" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Tamoxifen (CAS number 10540-29-1) is a small molecule drug used in the treatment of various types of cancer diseases, including but not limited to breast cancer, ovarian cancer, and desmoid tumors. Tamoxifen is administered to patients via oral ingestion at doses ranging from 10 mg to 40 mg per treatment unit.

[0162] In another alternative of this aspect of the invention, "allopurinol" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Allopurinol (CAS No. 315-30-0) is a well-characterized small molecule drug that was first approved for the treatment of gout. Allopurinol is a structural analogue of hypoxanthine, which after oral ingestion is subsequently metabolized to the active metabolite oxypurinol (aloxanthine) (CAS No. 2465-59-0), which then acts as a xanthine oxidase inhibitor, resulting in an increase in nucleotide concentrations. Dosages vary depending on the manufacturer providing the drug and range from 100 mg to 300 mg per therapeutic unit.

[0163] In another alternative of this aspect of the invention, "Febuxostat" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Febuxostat (CAS No. 144060-53-7) is a small molecule drug and is provided to patients suffering from hyperuricemia. Febuxostat is a xanthine oxidase / dehydrogenase inhibitor and reduces serum uric acid after oral ingestion in patients (Stamp LK et al., Intern Med J. 2007 Apr;37(4):258-66). Dosage forms include tablets for oral ingestion, with doses ranging from 40 mg to 120 mg per treatment unit.

[0164] In another alternative of this aspect of the invention, "tisopurine" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Tisopurine (CAS No. 5334-23-6) is an alternative to allopurinol.

[0165] In another alternative of this aspect of the invention, "Topiroxostat" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Topiroxostat (CAS number 577778-58-6) was developed as a treatment for hyperuricemia and gout. This small molecule drug has been approved in Japan since 2013 and is sold under the names Topirolic and Uriadec. The drug is absorbed by oral ingestion. Topiroxostat exerts its effect by competitively inhibiting xanthine oxidase.

[0166] In another alternative of this aspect of the invention, "inositol" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Inositol, particularly phytic acid (CAS No. 83-86-3) and myo-inositol (cyclohexane-1,2,3,4,5,6-hexol; CAS No. 87-89-8), are small molecules contained in drugs that have high potential for the treatment of various diseases, such as depression, psychiatric disorders, and the prevention of cancer and cardiovascular calcification, as well as for the enhancement of fertility. Various drugs based on or consisting of myo-inositol, as shown in the basic structure above, have already been approved by the FDA. The dosage varies depending on the manufacturer, but is generally between 200 mg and 500 mg per treatment unit.

[0167] In another alternative of this aspect of the invention, "ethyl pyruvate" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Ethyl pyruvate (ethyl 2-oxopropanoate; CAS number 617-35-6) is a small molecule and a novel anti-inflammatory agent for the treatment of patients with severe inflammatory conditions. Ethyl pyruvate is an inhibitor of "glyoxalase I" and further inhibits the release of cytokines such as TNF-α and HMGB1. Studies have shown promising anti-inflammatory properties and tissue protective activity (Kou, QY et al., Chinese critical care medicine 20(1), 34-36).

[0168] In another alternative of this aspect of the invention, "Sp-bromobenzyl glutathione cyclopentyl diester" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Sp-Bromobenzyl glutathione cyclopentyl diester (N-[S-[(4-bromophenyl)methyl]-NL-γ-glutamyl-L-cysteinyl]-glycine dicyclopentyl ester; CAS number 166038-00-2) is a small molecule that has inhibitory effects on glyoxalase I and also induces apoptosis (Thornalley PJ et al., Biochem Pharmacol. 1996 May 17;51(10):1365-72. doi:10.1016 / 0006-2952(96)00059-7. PMID:8787553).

[0169] In another alternative of this aspect of the invention, "arsenic trioxide" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. Arsenic trioxide (diarsorhosoxidan: CAS number 1327-53-3), a small molecule chemotherapeutic agent, is administered to patients suffering from cancer, particularly APL, by intravenous injection at a concentration in a dose range of 1 mg / mL to 2 mg / mL. Arsenic trioxide targets and inhibits the thioredoxin and glutathione systems, causing DNA morphological changes and fragmentation that lead to apoptosis in cancer cells (Lu J. et al., Proc Natl Acad Sci USA. 2007 Jul 24;104(30):12288-93. doi:10.1073 / pnas.0701549104. Epub 2007 Jul 18. PMID:17640917; PMCID:PMC1940330).

[0170] In another alternative of this aspect of the invention, "3-bromopyruvate" is provided for the treatment of cancer diseases associated with tumor cells expressing MR1 or for the prevention of recurrence of such tumor cells, particularly in combination with MR1-reactive T cells, MR1-reactive T cell receptor polynucleotide constructs and / or polynucleotide expression vectors encoding MR1. 3-Bromopyruvate (3-bromo-2-oxopropanoic acid; CAS number 1113-59-3) is a selective uptake inhibitor of glycolysis in cancer cells, which further induces the release of apoptosis-inducing factors (Cal M. et al., Cells. 2020;9(5):1161. Published 2020 May 8. doi:10.3390 / cells9051161).

[0171] Another aspect of the present invention relates to the use of pharmaceutical agents that promote the accumulation of nucleobases, thereby increasing the availability of antigens. These drugs include, but are not limited to, inhibitors of adenosine deaminase 1 (ADA1), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), inhibitors of both ADA1 and ADA2, pentostatin (CAS No. 53910-25-1) and 1-deazaadenosine. Also included are inhibitors of dehydrogenase 1 (IMPDH1), such as, but not limited to, mycophenolic acid (CAS No. 24280-93-1). The results shown in Figure 7 support the utility of this compound administered as part of MR1 T targeted therapy.

[0172] A further aspect of the invention relates to an MR1 ligand compound identified herein according to the first aspect of the invention, in particular an MR1 ligand identified by the formula of claim 1 or any one of the subclaims and specific embodiments thereof given in Table 1, for use as a vaccine for eliciting or enhancing MR1 T cell responses, in particular in the treatment or prevention of recurrence of cancer. In a particular embodiment, the MR1 ligand compound identified herein may be employed as a combination drug for stimulating and enhancing the antitumor activity of MR1 T cells used in cell therapy and / or may be used in combination with immunostimulatory compounds and / or checkpoint modulators.

[0173] The MR1 ligand compounds identified herein can also be used as vaccines in subjects without overt disease but with a predisposition to developing such disease.Subjects, such as humans, can be treated with a prophylactic vaccine prior to each disease described herein.

[0174] Similarly, an alternative of this aspect of the invention relates to an MR1 ligand compound identified herein according to the first aspect of the invention, in particular an MR1 ligand identified by the chemical formula of claim 1 or any one of the specific embodiments thereof given in the dependent claims and Table 1, for use in a combination drug in combination with a cancer therapeutic agent.

[0175] One of the key advantages of the methods and compounds provided herein relates to the ability to test and provide TAAs to patients regardless of MHC haplotype. TAAs are targets of clinically relevant antitumor immune responses in cancer patients. However, the majority of TAAs identified to date are peptides presented by polymorphic MHC molecules. Because MHC genes are highly polymorphic, TAA targeting is limited to those patients expressing specific MHC alleles. Targeting TAAs bound to MR1 non-polymorphic antigen-presenting molecules overcomes this constraint and is applicable to all patients with MR1-expressing tumors. Furthermore, because tumor cells may express different non-peptide TAAs, this strategy can target multiple antigens displayed by the same tumor cells, thus minimizing the possibility of tumor escape mutants occurring under selective immune pressure. Thus, the identification of MR1-presenting TAAs matched with MR1-restricted TCRs that recognize these antigens has important implications for cancer immunotherapy.

[0176] In certain embodiments, the MR1 ligand compounds identified herein are provided for use in combination with immune checkpoint modulators, particularly in combination with immune checkpoint inhibitors.

[0177] In certain embodiments, the immune checkpoint inhibitor is ipilimumab (Yervoy; CAS number 477202-00-9).

[0178] In certain embodiments, the immune checkpoint inhibitor is an inhibitor of the interaction of programmed cell death protein 1 (PD-1) with its receptor PD-L1. In certain embodiments, the immune checkpoint inhibitor is selected from the clinically available antibody drugs nivolumab (Bristol-Myers Squibb; CAS No. 946414-94-4), pembrolizumab (Merck Inc.; CAS No. 1374853-91-4), pidilizumab (CAS No. 1036730-42-3), atezolizumab (Roche AG; CAS No. 1380723-44-3), and avelumab (Merck KGaA; CAS No. 1537032-82-8).

[0179] In certain embodiments, the MR1 ligand compounds identified herein are provided for use in combination, where any of the MR1 ligand compounds identified herein is a first combination partner, and a) modified T cells that respond to the MR1 molecule presenting the MR1 ligand compound and / or a nucleic acid expression vector encoding MR1 are the second combination partners; and b) An immune checkpoint modulator, in particular an immune checkpoint inhibitor, is the third combination partner.

[0180] Such combinations are likely to be in the form in which the combination partners are applied at different times during treatment and in different dosage forms.

[0181] Identification of the compounds identified herein that are presented by tumor cells in individual tumor patients represents a novel method of classifying tumors, which is relevant for selecting appropriate MR1T-derived TCRs for use in personalized TCR gene therapy.

[0182] Tumor patients can also be vaccinated with a selected compound that was previously detected in the tumor cells of the same patient, with the goal of eliciting and / or stimulating compound-specific MR1 T cells, which will then recognize and kill the tumor cells.

[0183] Some of the MR1 ligand compounds described herein are also present in tissues of patients with autoimmune and metabolic diseases, including rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, atherosclerosis, inflammatory bowel disease, and multiple sclerosis. In general, diseases involving abnormal generation of reactive oxygen species (ROS) are characterized by the accumulation of MR1-binding compounds, as described below. In such cases, the autoreactive MR1 T cells stimulated during these diseases represent a large T cell population that may be inhibited in a therapeutic setting.

[0184] We believe that what we have described for tumor immunotherapy can be used as a template to find suitable forms of therapeutic intervention, and thus potentially broaden the types of diseases that can be similarly treated due to the accumulation of the same types of MR1 ligands.

[0185] Yet another aspect of the present invention relates to the detection of the MR1 ligand compounds identified herein as part of a method for disease classification, in which the presence of at least one of the MR1 ligand compounds according to the present invention is identified in a patient sample. In the case of cancer patients, the identification of these compounds extracted from fresh tumor samples is considered as a diagnostic marker. In certain embodiments, the TAA MR1 ligand compounds identified herein can be used to guide cellular immunotherapy and can also be used for vaccination in combination with other therapeutic interventions; in other words, once MR1 ligand is identified to be present in tumors, patients can be treated with personalized immunotherapy interventions, including i) administration of a pharmaceutical agent capable of promoting the accumulation of the MR1 T cell stimulating compounds identified herein, ii) administration of selected TAA MR1 ligand compounds, iii) MR1 TCR mediated cell therapy alone or in combination with other tumor therapeutic agents.

[0186] The present invention also relates to a research method directed to identifying T cells that react with MR1 expressing cells, including a method for isolating MR1-restricted T cells selected from peripheral blood from normal donors or patients suffering from cancer, metabolic disease, autoimmune disease, etc. by using a compound carried on MR1 multimer molecules. Cell sources include, but are not limited to, T cells isolated from tissue biopsies.

[0187] The present invention also relates to the possibility to easily identify the TCR gene and protein sequences expressed by said T cells.

[0188] In certain embodiments, the present invention focuses on the identification of a novel class of compounds that bind to non-polymorphic MR1 molecules. Some of the compounds identified herein modulate the surface expression of MR1. Some of the compounds identified herein (not necessarily the same compounds found to modulate the surface expression of MR1) are antigens and stimulate specific human T cells that are restricted to MR1. Some of the compounds identified herein are isolated from tumor cells, where they are purified, identified, and synthetic analogs are produced. Compounds that exhibit antigen activity, when presented in association with the MR1 molecule, stimulate a population of human T cells that we have discovered, which we call MR1 T cells. Applications of the present invention include, but are not limited to, the following methods. i) a method for stimulating compound-specific T cells to induce a prophylactic immune response; ii) methods of stimulating compound-specific T cells to induce a therapeutic immune response; iii) methods for the identification and isolation of T cells that respond to said compounds; iv) a method for modulating (increasing or decreasing) the amount of a given compound within or presented by a cell; v) A method for classifying metabolically altered cells, including but not limited to tumor cells, according to the presence of said compounds.

[0189] Thus, in certain aspects and embodiments, the present invention relates to the use of the MR1-related compounds identified herein to guide personalized interventions in immunotherapy, cellular immunotherapy, vaccination strategies in at-risk humans, such as cancer, and for diagnostic testing of several diseases.

[0190] Novel T cell receptor Yet another aspect of the present invention relates to a novel isolated T cell receptor.

[0191] The isolated T cell receptor (TCR) according to the invention is composed of a T cell receptor (TCR) α chain and a TCR β chain, or a γ chain and a δ chain, which associates with an MR1 polypeptide and specifically binds and recognizes an MR1 ligand compound as specified herein, in particular in any one of claims 1 to 9.

[0192] The inventors have previously disclosed MR1-specific T cell receptor sequences (PCT / EP2019 / 074284, published as WO2020053312(A1) and incorporated herein by reference), which may be considered to be encompassed by the above definition to the extent that these previously published TCRs specifically recognize MR1-ligand complexes. Thus, the TCRs disclosed in WO2020053312A1 formed by the association of SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, 13 and 25, 14 and 26, 15 and 27, 16 and 28, 17 and 29, 18 and 30, 19 and 31, 20 and 32, 21 and 33, 22 and 34, 23 and 35, 24 and 36, and 61 and 62 are not claimed.

[0193] The inventors have found that a single TCR as disclosed herein can bind multiple MR1 ligand compounds in the context of MR1 presentation, and in this context, the term "specifically binds" includes MR1-specific TCRs that bind not only one ligand, but potentially multiple ligands as disclosed herein.

[0194] A determination of whether a TCR specifically binds and recognizes an MR1 ligand compound is made if, upon interaction of MR1 with a ligand compound, such TCR induces a higher activation of T cells than that measured in the absence of said MR1 ligand. Such a difference is statistically significant at a P value of 0.05 or less (P≦0.05). T cell activation can be assessed by measuring any of the following: cytokine release, chemokine release, proliferation, expression of activation markers, target cell killing, induction of transcription factors or reporter genes.

[0195] For the purposes of providing the definition of TCR given by the definition of binding to MR1-MR1 ligand complex claimed herein, the threshold for determining activation and therefore the specificity of the TCR for the MR1 ligand complex must be determined as described in the Methods section below (Activation Assay). The determination is positive if a statistically significant positive effect is observed for the MR1 ligand, particularly if the difference is at least 2-fold, more particularly if the difference is at least 10-fold.

[0196] In certain embodiments, the isolated TCR provided herein recognizes the following compounds that associate with MR1: a. 1-methyladenosine (1); b. 2-methyladenosine (2); c. 2'-O-methyladenosine (3), with the proviso that the TCR consisting of SEQ ID NOs: 22 and 34 is not claimed; d.N 6 ,N 6 - dimethyladenosine (4), with the proviso that TCRs consisting of SEQ ID NOs: 1 and 2, and 22 and 34 are not claimed; e.N 6 -threonylcarbamoyl adenosine (5), with the proviso that the TCR consisting of SEQ ID NOs: 22 and 34 is not claimed; f.N 6 -isopentenyl adenosine (6), with the proviso that TCRs consisting of SEQ ID NOs: 1 and 2, 16 and 28, and 22 and 34 are not claimed; g. N6-(cis-hydroxyisopentenyl)adenosine (7); h. 2-Methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine (8), with the proviso that TCRs consisting of SEQ ID NOs: 1 and 2, 16 and 28, and 22 and 34 are not claimed; i. 2-Methylthio-N6-isopentenyladenosine (9); j. N6-methyl-N 6 -threonylcarbamoyl adenosine (10), with the proviso that the TCR consisting of SEQ ID NOs: 22 and 34 is not claimed; k. 2'-O-ribosyladenosine(phosphate) (11); l.N 6 -(3-oxo-1-propenyl)-2'-deoxyadenosine (12), with the proviso that TCRs consisting of SEQ ID NOs: 1 and 2, 16 and 28, and 22 and 34 are not claimed; m. 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarboxaldehyde (13), except that the TCR consisting of SEQ ID NOs: 16 and 28 is not claimed; n. 1-(3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3H-imidazo[2,1-i]purin-7-yl)heptan-2-one (14); o. N2-methylguanosine (16); p. 7-methylguanosine (17), except that a TCR consisting of SEQ ID NOs: 22 and 34 is not claimed; q. 2'-O-methylguanosine (18); r. N2,N2-dimethylguanosine (19), except that a TCR consisting of SEQ ID NOs: 1 and 2 is not claimed; s. 2'-O-ribosylguanosine phosphate (20), except that the TCR consisting of SEQ ID NOs: 22 and 34 is not claimed; t. 3-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,7-dihydroxy-6-methyl-6,7-dihydro-3H-imidazo[1,2-a]purin-9(5H)-one (21), except that TCRs consisting of SEQ ID NOs: 16 and 28 are not claimed; u. 3-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,7-dihydroxy-7-methyl-6,7-dihydro-3H-imidazo[1,2-a]purin-9(5H)-one (22), except that TCRs consisting of SEQ ID NOs: 16 and 28 are not claimed; v. 1-methylguanosine (24), except that a TCR consisting of SEQ ID NOs: 22 and 34 is not claimed; w. 3-((2R,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7-(2-oxoheptyl)-3H-imidazo[1,2-a]purin-9(5H)-one (25); x. 2'-O-methylcytidine (29); y. 3-methyluridine (30), except that a TCR consisting of SEQ ID NOs: 22 and 34 is not claimed; z. 5-methyluridine (31); aa. 3,2'-O-dimethyluridine (32); bb. Kewosin(33); cc. Wybutosin (34); dd. Hydroxywvutocin (35); ee. Pseudouridine (36); ff. 6-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(2-oxoheptyl)-1,8a-dihydroimidazo[1,2-c]pyrimidin-5(6H)-one (37); gg. N4-(3-oxo-1-propenyl)-2'-deoxycytidine (38), except that the TCR consisting of SEQ ID NOs: 22 and 34 is not claimed; hh. 6-methylmercaptopurine (40), except that TCRs consisting of SEQ ID NOs: 16 and 28, 22 and 34, and 24 and 36 are not claimed; ii. N6-methyladenosine (42); jj. 6-methylpurine (43); kk. 6-(dimethylamino)purine (44); ll. N6-(Δ2-isopentenyl)adenine (45); mm. 1-methylguanine (47); nn. N2-methyl-2'-deoxyguanosine (48); oo. 5'-deoxy-5'-(methylthio)adenosine (52); pp. N 6 -methyl-2'-deoxyadenosine (53), with the proviso that a TCR consisting of SEQ ID NOs: 3 and 4 is not claimed; qq.N 6 -(2-hydroxyethyl)-2'-deoxyadenosine (54), with the exception that a TCR composed of SEQ ID NOs: 3 and 4 is not claimed.

[0197] In certain embodiments, the T cell receptor (TCR) provided by the present invention recognizes the following compound in association with MR1: a. N6-isopentenyladenosine (6), in particular the TCR comprises a CDR comprised of SEQ ID NOs: 99 and 100, in particular the TCR comprises a polypeptide chain characterized by SEQ ID NOs: 97 and 98; b. N 6 -(3-oxo-1-propenyl)-2'-deoxyadenosine (12), in particular the TCR comprises CDRs consisting of SEQ ID NOs: 99 and 100, or 103 and 104, or 108 and 108, or 111 and 112, or 123 and 124, in particular the TCR comprises a polypeptide chain characterized by SEQ ID NOs: 97 and 98, or 101 and 102, or 105 and 106, or 109 and 110, or 121 and 122; c. 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarboxaldehyde (13), in particular the TCR comprises CDRs consisting of SEQ ID NOs: 99 and 100, or 103 and 104, or 107 and 108, or 111 and 112, or 123 and 124, in particular the TCR comprises a polypeptide chain characterized by SEQ ID NOs: 97 and 98, or 101 and 102, or 105 and 106, or 109 and 110, or 121 and 122; d. pyrimido[1,2-a]purin-10(3H)-one (24), in particular the TCR comprises CDRs consisting of SEQ ID NOs: 99 and 100, or 103 and 104, or 111 and 112, in particular the TCR comprises a polypeptide chain characterized by SEQ ID NOs: 97 and 98, or 101 and 102, or 109 and 110; e.N 4 -(3-oxo-1-propenyl)-2'-deoxycytidine (38), in particular the TCR comprises a CDR consisting of SEQ ID NOs: 111 and 112, or 123 and 124, in particular the TCR comprises a polypeptide chain characterized by SEQ ID NOs: 109 and 110, or 121 and 122; f. 6-methylmercaptopurine (40), in particular wherein the TCR comprises CDRs consisting of SEQ ID NOs: 107 and 108, in particular wherein the TCR comprises a polypeptide chain characterized by SEQ ID NOs: 105 and 106.

[0198] The present inventors have succeeded in providing a novel TCR that specifically reacts with MR1 and MR1-MR1 ligand complex. The present invention provides an isolated T cell receptor (TCR) protein heterodimer comprising a TCR α chain and a TCR β chain, wherein the TCR α chain and the TCR β chain are each characterized by a CDR3 sequence, and the TCR protein heterodimer is characterized by a pair of α chain and β chain sequences selected from SEQ ID NOs: 99 and 100, 103 and 104, 107 and 108, 111 and 112, 115 and 116, 119 and 120, 123 and 124, 127 and 128, 131 and 132.

[0199] In a particular embodiment thereof, the TCR alpha chain and the TCR beta chain are selected from the pairs of alpha and beta chain amino acid sequences of SEQ ID NOs: 97 and 98, 101 and 102, 105 and 106, 109 and 110, 113 and 114, 117 and 118, 121 and 122, 125 and 126, and 129 and 130 (see Table 3B), or are sequences that are at least 85% (≧90%, ≧95%, ≧98%) identical to said pairs of alpha and beta chain amino acid sequences and have the same biological activity as the original TCR.

[0200] The present invention further provides the nucleotide sequence encoding the novel TCR as described hereinabove.In a particular embodiment, the polynucleotide is a DNA expression vector.In another particular embodiment, the polynucleotide encoding the TCR is an RNA molecule, in particular a stabilized messenger RNA molecule.In another particular embodiment, the polynucleotide encoding the TCR is a viral vector.

[0201] Another aspect of the invention relates to isolated T cells expressing a TCR defined by the above binding characteristics, or a sequence thereof, or both, or a polynucleotide encoding same, for use in the prevention or treatment of diseases associated with an abnormal or absent MR1-specific T cell response, in particular for use in the treatment of cancer.

[0202] A particular application of this embodiment is its use in cancers characterized by expression of MR1.

[0203] In a particular embodiment, the isolated T cells and / or polynucleotides for use according to the invention are co-administered with an MR1 ligand compound as defined herein, in particular as defined in any one of claims 1 to 9.

[0204] Alternatively, an MR1 ligand compound as defined herein, in particular in any one of claims 1 to 9, is provided for use in the treatment of cancer, comprising α and β as identified by the same row of Table 3. An isolated T cell expressing an MR1-specific TCR as defined in PCT / EP2019 / 074284 comprising a pair of CDR3 sequences, in particular SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, 13 and 25, 14 and 26, 15 and 27, 16 and 28, 17 and 29, 18 and 30, 19 and 31, 20 and 32, 21 and 33, 22 and 34, 23 and 35, 24 and 36, and 61 and 62, and / or a polynucleotide encoding said MR1-specific TCR, or a sequence that is at least 85% (≧90%, ≧95%, ≧98%) identical to the pair of amino acid sequences of the α chain and β chain and has the same biological activity as the original TCR (before, simultaneously, or after administration).

[0205] The invention further provides an isolated T cell and / or polynucleotide for use as defined in the preceding paragraph, wherein said isolated T cell and / or polynucleotide expressing a TCR is co-administered with a pharmaceutical compound selected from paclitaxel, doxorubicin, docetaxel, cabazitaxel, daunorubicin, epirubicin, idarubicin, disulfiram, ellagic acid, pentostatin and mycophenolic acid (MPA) amodiaquine, chlorpromazine, domperidone, estradiol, felopidine, loratadine, maprotiline, metoclopramide, nortriptyline, ondansetron, perphenazine, promazine, promethazine, raloxifene, salmeterol, tacrine, tamoxifen, and thioridazine, allopurinol, febuxostat, tisoprine, topiroxostat, inositol (phytic acid, and myo-inositol).

[0206] In certain embodiments, isolated T cells expressing a TCR and / or polynucleotides are co-administered with a pharmaceutical compound selected from paclitaxel, doxorubicin, disulfiram and MPA for the treatment or prevention of diseases associated with aberrant or absent MR1 expression, particularly for the treatment or prevention of recurrence of cancer diseases associated with tumor cells expressing MR1.

[0207] In a more particular embodiment, the isolated T cells expressing a TCR and / or polynucleotides are co-administered with a pharmaceutical compound selected from paclitaxel and doxorubicin for the treatment or prevention of a disease associated with aberrant or absent MR1 expression, in particular for the treatment or prevention of recurrence of a cancer disease associated with tumor cells expressing MR1.

[0208] The present invention further provides the compound 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarboxaldehyde, which was first synthesized by the present inventors and shown to be an MR1 ligand.

[0209] Medical Treatments, Dosage Forms, and Salts Also within the scope of the present invention is a method for treating a condition associated with a lack of an MR1-specific T cell response or an excess of an MR1-specific T cell response in a patient in need thereof, said method comprising administering to the patient a compound as defined in detail above.

[0210] Similarly, there is provided a dosage form for the prevention or treatment of a condition associated with a lack of an MR1-specific T cell response or an excess of an MR1-specific T cell response, said dosage form comprising a non-agonist ligand or an antisense molecule according to any of the above aspects or embodiments of the invention.

[0211] Those skilled in the art will understand that any specifically mentioned drug may exist as a pharma- ceutically acceptable salt of said drug. Pharmaceutically acceptable salts include ionized drug and oppositely charged counterions. Non-limiting examples of pharma- ceutically acceptable anionic salt forms include acetate, benzoate, besylate, tartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, naphsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharma-ceutically acceptable cationic salt forms include aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, and zinc.

[0212] The dosage form may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation dosage form or suppository. Alternatively, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection. Optionally, pharma-ceutically acceptable carriers and / or excipients may be present.

[0213] Topical administration is also within the scope of the advantageous application of the present invention.Those skilled in the art are aware of the wide range of possible formulations to provide topical formulations, as exemplified in Benson and Watkinson (eds.), Topical and Transdermal Drug Delivery: Principles and Practice (1st ed., Wiley 2011, ISBN-13:978-0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded Edition (2nd ed., CRC Press 2002, ISBN-13:978-0824708610); Osborne and Amann (eds.): Topical Drug Delivery Formulations (1st ed., CRC Press 1989; ISBN-13:978-0824781835).

[0214] Drug composition and administration Another aspect of the present invention relates to a pharmaceutical composition comprising a compound as defined herein in the context of the present invention, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.In a further embodiment, the composition comprises at least two pharma- ceutically acceptable carriers, such as those described herein.

[0215] In certain embodiments of the present invention, the compounds of the present invention are generally formulated into pharmaceutical dosage forms that provide easily controllable administration of the drug and provide the patient with an easy to administer, straightforward product.

[0216] In embodiments of the invention relating to topical use of the compounds of the invention, the pharmaceutical compositions are formulated in a manner suitable for topical administration, such as aqueous solutions, suspensions, ointments, creams, gels, or sprayable formulations (e.g., for delivery by aerosol, etc.), which contain the active ingredient together with one or more solubilizers, stabilizers, tonicity enhancing agents, buffers, and preservatives known to those of skill in the art.

[0217] The pharmaceutical compositions can be formulated for oral, parenteral, or rectal administration. Furthermore, the pharmaceutical compositions of the present invention can be in a solid form (including, without limitation, capsules, tablets, pills, granules, powders, or suppositories) or in a liquid form (including, without limitation, solutions, suspensions, or emulsions).

[0218] The dosing regimen of the compounds of the invention will vary depending on known factors such as the pharmacodynamic properties of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the subject; the nature and extent of the condition; type of concurrent treatment; frequency of treatment; route of administration, renal and hepatic function of the patient, and the desired effect. In certain embodiments, the compounds of the invention may be administered once daily, or the total daily dosage may be divided and administered two, three, or four times daily.

[0219] In certain embodiments, the pharmaceutical composition or combination of the present invention may be a unit dose of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg. The therapeutically effective dosage of the compound, pharmaceutical composition, or combination thereof depends on the subject's species, weight, age, and individual condition, the disorder or disease being treated, or its severity. A physician, clinician, or veterinarian of ordinary skill can readily determine the effective amount of each active ingredient required to prevent, treat, or inhibit the progression of a disorder or disease.

[0220] The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization, and / or can contain conventional inert diluents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers. They can be produced by standard processes, such as conventional mixing, granulation, dissolving, or lyophilization processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see, for example, L. Lachman et al., The Theory and Practice of Industrial Pharmacy, 4th ed., 2013 (ISBN 8123922892).

[0221] "Swiss-type" manufacturing method: Another aspect of the invention relates to the use of an MR1 ligand compound as described in table 1 in the manufacture of a medicament for the prevention or treatment of a disease associated with an abnormal or absent MR1-specific T cell response, especially in the treatment of cancers characterized by tumour cells expressing MR1, optionally in combination with a drug according to claim 27. Another embodiment relates to the use of an isolated T cell receptor according to claims 16-20 in the manufacture of a medicament for the prevention or treatment of a disease associated with an abnormal or absent MR1-specific T cell response, especially in the treatment of cancers characterized by tumour cells expressing MR1, optionally in combination with a drug according to claim 27. Another embodiment relates to the use of an isolated T cell according to claim 22 in the manufacture of a medicament for the prevention or treatment of a disease associated with an abnormal or absent MR1-specific T cell response, especially in the treatment of cancers characterized by tumour cells expressing MR1, optionally in combination with a drug according to claim 27. Another embodiment relates to the use of a polynucleotide encoding a TCR as claimed in claim 21 in the manufacture of a medicament for the prevention or treatment of a disease associated with an abnormal or absent MR1-specific T cell response, in particular in the treatment of cancers characterised by tumour cells expressing MR1, optionally in combination with an agent as claimed in claim 27.

[0222] Where single separable alternative features are described herein as "embodiments", it is to be understood that such alternatives can be freely combined to form separate embodiments of the invention disclosed herein.

[0223] The present invention is further illustrated by the following examples and figures from which further embodiments and advantages can be derived, these examples being intended to illustrate the invention without limiting its scope.

[0224] [Table 1] JPEG0007680769000016.jpg208153JPEG0007680769000017.jpg187152JPEG0007680769000018.jpg197153 JPEG0007680769000019.jpg224153JPEG0007680769000020.jpg185153JPEG0007680769000021.jpg175153 JPEG0007680769000022.jpg216153JPEG0007680769000023.jpg218153JPEG0007680769000024.jpg231152 JPEG0007680769000025.jpg220153JPEG0007680769000026.jpg221149JPEG0007680769000027.jpg195153

[0225] All exemplary compounds were shown to interact with MR1, elicit MR1-restricted T cell responses, and / or stabilize MR1 expression on cells, as evidenced by results obtained using assays such as those shown in the Examples. EXAMPLES

[0226] Materials and Methods Human blood samples Blood and tissue samples for T cell cloning, FACS analysis and antigen presentation assays were obtained from University Hospital Basel after informed consent according to protocol EKNZ2017-01888, which was ethically approved by the Swiss authorities (EKNZ, Ethics Committee North-West & Central Switzerland); all patients and healthy donors gave written consent to the analysis of their samples.

[0227] cell line The cell lines used as antigen-presenting cells (APCs) in this study were A375 (ATCC CRL-1619), THP-1 (ATCC TIB-202), A375-MR1 and THP1-MR1, which were generated and described previously (Lepore et al. 2017). HEL, Me67, Mel JUSO, H460, KMOE-2 and TF-1 tumor cell lines were cultured in RPMI-1640 supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1x MEM NEAA and 50 μg / ml kanamycin (all Bioconcept). The medium for TF-1 cells was further supplemented with 10 ng / ml recombinant human GM-CSF (Peprotech). All human T cell clones were maintained in culture as previously described. A representative MAIT clone (MRC25), generated from blood of a healthy donor, has been previously characterized (Schmaler et al. (2018). Mucosal Immunology 11:1060-1070). Cells were free of mycoplasma as assessed by PCR analysis of DNA samples. When possible, cells were confirmed by staining with mAbs against specific cell surface markers.

[0228] Lentiviral transduction was performed as previously described. Transduced cells were selected by FACS sorting based on expression of EGFP or mCherry reporters or by resistance to 2 μg / mL puromycin.

[0229] Screening of human knockout libraries A375-MR1-Cas9 cells, generated using the cell line and Lenti Cas9-Blast plasmid (Addgene) described previously, were transduced with both parts A and B of the pooled human GeCKO v2 CRISPR library (Addgene) at 0.3 MOI, followed by selection with 2 μg / mL puromycin (Calbiochem, Cat. No. 540411) for 96 h. Eight biological replicates of the resulting APCs, each with 64-fold representation of each guide in the library, were subjected to four successive rounds of killing with TC5A87 cells at a 2:1 E:T ratio, after which surviving cells were expanded for 24 h and DNA extracted with the NucleoSpin tissue kit (Macherey-Nagel, Cat. No. 740952). Eight further biological replicates were prepared similarly, but without killing with TC5A87 to serve as controls. Illumina libraries were prepared as described previously. Briefly, primers JScrispr1 and JScrispr3 were used to amplify genomic gRNA from extracted gDNA with a common Illumina primer handle for indexing the sequencing library. In addition, the former primer inserts 8 nt of degenerate sequence immediately downstream of the Illumina read 1 start site to mitigate issues with sequencing low complexity libraries. Each replicate was barcoded with a unique pair of Nextera indexes (Illumina, catalog no. 15055290) in a second step PCR performed as described in the Nextera DNA library preparation protocol (Illumina). Each PCR step involved in the preparation of the sequencing library used the High-fidelity Advantage HF2 PCR kit (Takara, catalog no. 639123).Libraries were quantified using the BioAnalyser high sensitivity DNA kit (Agilent, Cat. No. 5067-4626) and Qubit high-sensitivity dsDNA kit (ThermoFisher, Cat. No. Q32851), pooled to form equimolar sequencing libraries, denatured and diluted to 1.2 pM with 20% PhiX v3 control library (Illumina, Cat. No. FC-110-3001) as described in the Illumina denaturation and dilution protocol (Illumina), and sequenced on a NextSeq500 using the High-output 150-cycle v2 kit (Illumina, discontinued product). Both sets of sequencing libraries were sequenced at a depth of 25 million reads per replicate using a dual-index single-end protocol (131 cycles for read 1, 8 cycles for each barcode) to confirm that guide depletion could be detected after T cell-mediated killing.

[0230] Isolation and culture of primary cells from human blood and tissue samples MR1 T cells were isolated from peripheral blood of healthy donors. After separation of PBMCs by density gradient centrifugation, T cells were purified by negative selection using the EasySep Human T Cell Enrichment Kit and stimulated with irradiated (80Gray) A375-MR1 cells (2:1 ratio) and antigen once a week for 3 weeks. After each stimulation, human rIL-2 (5U / mL) was added on days +2 and +5. Twelve days after the final stimulation, cells were washed and co-cultured overnight with A375-MR1 cells (2:1 ratio) in the presence or absence of antigen.

[0231] Next is CD3 + CD69 + CD137 high Cells were FACS sorted and incubated with phytohemagglutinin (1 μg / mL, Remel, Cat. No. 30852801 HA16), human rIL-2 (100 U / mL) and irradiated PBMCs (5 × 10 5In some experiments, FACS-sorted CD3 T cells were cloned from T cell lines generated by expansion of purified T cells with synthetic Ag-pulsed A375-β2mKO-MR1 cells. + ,M 3 ADE-MR1-Tetramer + MR1 T cell clones were isolated by limiting dilution of the cells, and T cell clones were periodically restimulated according to the same protocol.

[0232] PBMCs were isolated from peripheral blood by density gradient centrifugation and frozen in liquid nitrogen until use.

[0233] T cells, B cells, monocytes, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs) were purified from PBMCs by immunomagnetic separation using kits shown in the KEY RESOURCES TABLE according to the manufacturer's protocols.

[0234] Tissue biopsies were obtained from small cell lung tumors and digested for 1 h at 37°C in medium containing Accutase (Innovative Cell; Catalog No. AT-104), Collagenase IV 200 U / mL (Worthington; Catalog No. LS004189), DNAse I 0.5 mg / mL (Sigma-Aldrich; Catalog No. D5025), and Hyaluronidase 50 mg / mL (Sigma; Catalog No. H6254). The digested material was passed through a 70 μM cell strainer to lyse red blood cells, and then cryopreserved in liquid nitrogen. After thawing, TILs were rested for 2 days and then transfected with A375-β2mKO-MR1 cells in a 1:1 ratio with 50 μM M 3 On day 5, human rIL-2 (5 U / mL) was added to the cultures for an additional 5 days, and expansion in this manner was repeated three times. The cells were then cultured in the presence of ADE. 3 After staining with ADE tetramers, anti-CD3, anti-CD4, and anti-CD8 mAbs, tetramer-positive cells were sorted into bulk lineages and then subjected to functional experiments.

[0235] CRISPR-Cas9-mediated gene disruption The results obtained in the screen were confirmed by knocking out the selected genes in A375-MR1-Cas9 cells transduced with gRNAs different from those present in the library (Table 4). After lentiviral transduction and selection, A375-MR1-Cas9 cells were maintained for a limited number of passages and used as a bulk population for activation assays. THP-1 cells were cloned by limiting dilution and screened for GLO1 expression. Target protein expression was assessed by Western blotting (Figure 15). MR1 surface expression was assessed by flow cytometry using APC-labeled mouse anti-MR1 mAb 26.5 (Biolegend) and APC-labeled mouse IgG2a (clone MOPC-173) as an isotype control (Figure 15).

[0236] The antigen-presenting capacity of the different cell lines was tested by pulsing APCs with the indicated concentrations of freshly prepared 5-OP-RU for 2 hours at 37° C. followed by stimulation with the MAIT clone MRC25 (FIG. 15).

[0237] TCR gene transfer Functional cDNAs for TCR α and β from the MR1T clone were cloned into a modified form of a lenti-expression vector (Addgene, Cat. No. 52962). SKW-3 or J76 cells lacking endogenous TCR were transduced with viral particle-containing supernatants produced as previously described (Lepore et al., 2017). When required, transduced cells were selected by FACS sorting based on CD3 expression.

[0238] Preparation and purification of synthetic antigens Compound M 3 ADE, OPdA, M 1 G, OPdC, M1dC, MGdA, MGG, m6,6A, io6A, m6t6A, Ar(p), M1dA, M3dA, ONEdA, m2, 2G, Gr, CEdG, ONEdG, M1dG, N 2OPdG, M2dG, m3U, m3Um, yW, OHyW, Psi, ONEdC, and M3dC were synthesized and subsequently purified before use in cells. All other compounds were purchased from various commercial sources, as shown in Table 1.

[0239] 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarboxaldehyde (M 3 Synthesis of ADE) (13) M 3 ADE was synthesized as previously described (Stone et al., Chemical Research in Toxicology 3, 33-38 (1990)) with minor modifications. 1,1,3,3-tetraethoxypropane (1.1 g, 5 mmol, 4.0 equiv) in aqueous HCl (25 mL, 1 M) was stirred at 40° C. for 1 h. A solution of adenine (168.9 mg, 1.25 mmol, 1.0 equiv) in water (25 mL) was then added. The mixture was adjusted to pH 4.0 with aqueous NaOH (1 M) and stirred at 37° C. for 5 days. M 3 The ADE was purified by solid-phase extraction on a Sep-Pak C18 2 g cartridge (Waters Corp., Milford, MA). The cartridge was preconditioned with 10 mL of water and 10 mL of acetonitrile. 3 The ADE was washed with 20 mL of water, 20 mL of 10% acetonitrile, and then eluted with 20 mL of 20% acetonitrile.

[0240] M 3 HPLC purification of ADE was performed on a JASCO RHPLC system equipped with an MD-4010 photodiode array detector. Semi-preparative HPLC purification was performed at 23 °C using a 250 × 10 mm 5 μM NUCLEODUR C18 Pyramid HPLC column, where mobile phases A and B were water and 95% aqueous methanol, respectively. Separation was performed at a flow rate of 6 mL / min with a linear gradient of 0–50% B from 0–15 min, 50–100% B from 15–38 min, 100% B from 38–43 min, 100–0% B from 43–44 min, and 0% B until 50 min. 3The yield of ADE was 12.5 mg (42 mol, 3.4%). The biologically active HPLC peak was collected and subjected to mass spectrometry and NMR analysis.

[0241] 1 H-NMR (600MHz, D 2 O, δ / ppm): 9.24 (s, 1H, H 21 ), 9.11(s,1H,H 19 ), 9.09(s,1H,H 15 ),8.61(s,1H,H 2 ),8.38(s,1H,H 8 ),7.64(s,1H,H 17 ), 7.33-7.29(m,1H,H 11 ), 4.09-4.07(m,1H,H 13 ),2.21(ddd, 2 J H12a-H12b =13.7Hz, 3 J H12a-H11 =2.8Hz, 3 J H12a-H13 =2.8Hz,1H,H 12a ),2.03(ddd, 2 J H12b-H12a =13.7Hz, 3 J H12b-H11 =2.2Hz, 3 J H12b-H13 =2.2Hz,1H,H 12b )

[0242] 13 C-NMR (151MHz, D 2 O, extracted from HSQC and HMBC, δ / ppm): 193.2 (C 19 ),192.2(C 21 ), 166.5(C 17 ), 154.3(C 4 ), 152.7(C 2 ), 150.3(C 6 ), 149.3(C 15 ), 144.8(C 8 ),126.2(C 14 ),125.7(C 16 ), 121.8(C 5 ),79.9(C 11), 25.0(C 12 ), 17.4(C 13 )

[0243] HR-ESI-MS: Calculated value [M+Na] + C 14 H 11 N 5 NaO 3 m / z=320.0754, detected value 320.0758

[0244] Pyrimido[1,2-α]purin-10(3H)-one (24) (M 1 Synthesis of G, CAS 103408-45-3) M 1 G was synthesized as previously reported (Seto et al., Bulletin of Chemical Society of Japan 58, 3431-3435 (1985); Hadley and Draper, Lipids 25, 82 (1990)) with minor modifications. 1,1,3,3-tetraethoxypropane (1.4 g, 6.25 mmol, 5.0 equiv.) in aqueous HCl (25 mL, 1 M) was stirred at 40° C. for 1 h. Then, a solution of guanine (188.9 mg, 1.25 mmol, 1.0 equiv.) in aqueous HCl (25 mL, 1 M) was slowly added. The mixture was stirred at 40° C. for 1 h and then kept at 4° C. for 16 h. The precipitate was washed three times with absolute ethanol at 2000×g for 10 min. Crude M 1 G was extracted from the precipitate three times with water at 65°C. The combined extracts were filtered through a 0.22 μm filter. The mixture was adjusted to pH 7.0 with aqueous NaOH (1 M).

[0245] M 1HPLC analysis of G was performed on a JASCO RHPLC system. Semi-preparative HPLC purification was performed using a 250 / 10 NUCLEODUR C18 Pyramid HPLC column at a column temperature of 23 °C. Solvent A consisted of Milli-Q water and solvent B consisted of 95% methanol and 5% Milli-Q water. The total run time was 55 min with a flow rate of 6 mL / min. The initial mobile phase was 100% solvent A for 10 min. Solvent B was increased linearly until the gradient reached 80% solvent A and 20% solvent B at 40 min. Solvent B was increased linearly again until it briefly reached 100% at 41 min. Isocratic flow at 100% B for 5 min, linear gradient to 100% solvent A for 1 min, and continued for 8 min. The biologically active HPLC peak was collected for mass spectrometry and NMR analysis (12.5 mg, 66.8 μmol, 5.3%).

[0246] 1 H-NMR (600MHz, D 2 O, δ / ppm): 9.31(d, 3 J H13-H12 =7.2Hz,1H,H 13 ),8.97(dd, 3 J H11-H12 =4.1Hz, 4 J H11-H13 =2.0Hz,1H,H 11 ),8.22(s,1H,H 8 ),7.30(dd, 3 J H12-H13 =7.2Hz, 3 J H12-H11 =4.2Hz,1H,H 12 )

[0247] 13 C-NMR (151MHz, D 2 O, extracted from HSQC and HMBC, δ / ppm): 162.9 (C 11 ), 154.4(C 4 ), 154.1(C 6 ),149.9(C 2 ), 146.0(C 8 ), 138.4(C 13 ), 116.7(C 5 ), 112.0(C12 )

[0248] HR-ESI-MS: Calculated value [M+H] + C 8 H 6 N 5 O m / z=188.0567, detected value 188.0571

[0249] N 6 Synthesis of -(3-oxo-1-propenyl)-2'-deoxyadenosine (12) (OPdA, CAS 178427-43-5) OPdA was synthesized as previously reported (Szekely et al., Nucleosides, Nucleotides and Nucleic Acids 27, 103-109 (2008)) with minor modifications. 2'-Deoxyadenosine monohydrate (219 mg, 0.813 mmol, 1 equiv.) was dissolved in 2 mL of anhydrous dimethylsulfoxide under argon atmosphere. To this stirred solution was added propargylaldehyde (12 μl, 11.0 mg, 0.203 mmol, 0.25 equiv.), followed by additional propargylaldehyde (1.25 equiv.) over 72 h. The reaction mixture was filtered and purified by preparative HPLC on a Shimadzu LC system (LC-20AT Prominence Liquid Chromatograph equipped with an SPD-20A Prominence UV / VIS detector (λ=254, 280 nm)). Preparative HPLC purification was performed using a Reprosil-Pur 120 ODS 3.5 μM, 150 × 20 mm column with mobile phases A and B of water and 90% acetonitrile in water, respectively. Separation was performed at a flow rate of 9 mL / min with a linear gradient of 1–30% B from 5 to 15 min, 30–100% B from 15 to 17 min, 100% B from 17 to 21 min, 100–0% B from 21 to 22 min, and 1% B until 25 min. Analytical HPLC was performed on a LC-20AD Prominence liquid chromatograph coupled with a Shimadzu LCMS-2020 liquid chromatograph mass spectrometer. Biologically active HPLC peaks were collected and subjected to mass and NMR analysis. OPdA yield was 13.5 mg (44.0 μmol, 5.4%).

[0250] 1 H-NMR(500MHz,D 2 O,δ / ppm):9.21(d, 3 J H13-H12 =8.7Hz,1H,H 13 ),8.49(d, 3 J H11-H12 =13.5Hz,1H,H 11 ),8.43(s,1H,H 8 ),8.38(s,1H,H 2 ),6.45(dd, 3 J H1’-H2’a= 6.8Hz, 3 J H1’-H2’b= 6.8Hz,1H,H 1’ ),5.89(dd, 3 J H12-H11 =13.5, 3 J H12-H13= 8.7Hz,1H,H 12 ),4.65(ddd, 3 J H3’-H2’a= 6.1Hz, 3 J H3’-H2’b= 3.5Hz, 3 J H3’-H4’= 3.5Hz,1H,H 3’ ),4.19(ddd, 3 J H4’-H5’b =3.8Hz, 3 J H4’-H5’a =3.5Hz, 3 J H4’-H3’ =3.5Hz,1H,H 4’ ),3.86(dd, 2 J H5’a-H5’b =12.5Hz, 3 J H5’a-H4’ =3.4Hz,1H,H 5’a ),3.80(dd, 2 J H5’b-H5’a =12.6Hz, 3 J H5’b-H4’ =4.3Hz,1H,H 5’b ),2.80(ddd, 2 J H2’a-H2’b =13.7Hz, 3 J H2’a-H1’ =7.1Hz, 3 J H2’a-H3’ =6.4Hz,1H,H 2’a ),2.58(ddd,2 J H2’b-H2’a =14.0Hz, 3 J H2’b-H1’ =6.3Hz, 3 J H2’b-H3’ =3.5Hz,1H,H 2’b )

[0251] 13 C-NMR (126MHz, D 2 O, extracted from HSQC and HMBC, δ / ppm): 195.7 (C 13 ), 151.9(C 2 ), 151.1(C 11 ), 150.6(C 4 ),148.9(C 6 ), 142.6(C 8 ), 120.8(C 5 ), 111.1(C 12 ),87.5(C 4’ ),84.7(C 1’ ), 71.1(C 3’ ),61.6(C 5’ ), 39.1(C 2’ )

[0252] HR-ESI-MS: Calculated value [M+Na] + C 13 H 15 N 5 NaO 4 m / z=328.1016, detected value 328.1020

[0253] N 4 Synthesis of -(3-oxo-1-propenyl)-2'-deoxycytidine (38) (OPdA, CAS 129124-79-4) OPdC was synthesized as previously described (Szekely et al., supra) with minor modifications. 2'-Deoxycytidine (185 mg, 0.813 mmol, 1 equiv) was dissolved in 2 mL of anhydrous dimethylsulfoxide under argon. To this stirred solution was added propargylaldehyde (12.0 μl, 11.0 mg, 0.203 mmol, 0.25 equiv), followed by additional propargylaldehyde (1.25 equiv) over 72 h. The reaction mixture was filtered and preparative HPLC purification was carried out as described for OPdA. The yield of OPdC was 7.00 mg (25.0 μmol, 3.1%). The biologically active HPLC peak was collected and subjected to mass and NMR analysis.

[0254] 1 H-NMR (500MHz, D 2 O, δ / ppm): 9.31(d, 3 J H10-H9 =8.6Hz,1H,H 10 ),8.29(d, 3 J H8-H9 =13.7Hz,1H,H 8 ),8.19(d, 3 J H6-H5 =7.4Hz,1H,H 6 ),6.28(d, 3 J H5-H6 =7.4Hz,1H,H 5 ),6.24(dd, 3 J H1’-H2’a= 6.1Hz, 3 J H1’-H2’b= 6.1Hz,1H,H 1’ ),5.91(dd, 3 J H9-H8 =13.7Hz, 3 J H9-H10 =8.6Hz,1H,H 9 ),4.43(ddd, 3 J H3’-H2’a =6.4Hz, 3 J H3’-H2’b =4.3Hz, 3 J H3’-H4’ =4.3Hz,1H,H 3’ ),4.12(ddd, 3 J H4’-H5’b =4.8Hz,3 J H4’-H5’a =4.1Hz, 3 J H4’-H3’ =4.1Hz,1H,H 4’ ),3.87(dd, 2 J H5’a-H5’b =12.5Hz, 3 J H5’a-H4’ =3.5Hz,1H,H 5’a ),3.77(dd, 2 J H5’b-H5’a =12.5Hz, 3 J H5’b-H4’ =5.3Hz,1H,H 5’b ),2.55(ddd, 2 J H2’b-H2’a =14.1Hz, 3 J H2’b-H1’ =6.3Hz, 3 J H2’b-H3’ =4.3Hz,1H,H 2’b ),2.32(ddd, 2 J H2’a-H2’b =14.2Hz, 3 J H2’a-H1’ =6.5Hz, 3 J H2’a-H3’ =6.5Hz,1H,H 2’a )

[0255] 13 C-NMR (126 MHz, D 2 O, HSQC and HMBC extraction, δ / ppm): 196.2 (C 10 ),161.5(C 4 ),156.8(C 2 ),149.6(C 8 ),144.3(C 6 ),112.1(C 9 ),96.8(C 5 ),87.1(C 1’ ),87.1(C 4’ ),70.3(C 3’ ),61.1(C 5’ ),39.8(C 2’ )

[0256] HR-ESI-MS: Calculated value [M+Na] + C 12 H 15 N3 NaO 5 m / z=304.0904, detected value 304.0902

[0257] M1dC was prepared by mixing 2'-deoxycytidine (25 mM, Sigma, Cat. No. D3897) and malondialdehyde tetrabutylammonium salt (100 mM). The mixture was incubated at 70°C for 18 hours with shaking at 400 rpm. The M1dC crude compound preparation was subjected to solid phase extraction as described above. M1dC was eluted with 20% acetonitrile.

[0258] Further HPLC purification was performed by reversed-phase HPLC using a C18 Pyramid column (Macherey-Nagel, Cat. No. 762204.40) as follows: Mobile phase A: deionized water, Mobile phase B: 95% methanol in deionized water. Flow rate 1.25 mL / min. Elution gradient: time 0 min, 100% A; time 1 min, 98% A; time 43 min, 50% A; time 46 min, 50% A; time 47 min, 100% A; time 56 min, 100% A. Biologically active HPLC separated peaks were collected for mass spectrometry and NMR analysis.

[0259] MGG was prepared by mixing guanosine (100 mM, Sigma, Cat. No. G6752) and methylglyoxal solution (100 mM) in DMSO (33.3% in water, v / v, Sigma Cat. No. D4540). The mixture was incubated at 70 °C for 2 h under shaking at 400 rpm. Further HPLC purification was performed by reversed-phase HPLC using a C18 Pyramid column (Macherey-Nagel, Cat. No. 762272.100) as follows: Mobile phase A: deionized water, Mobile phase B: 95% methanol in deionized water. Flow rate 5 mL / min. Elution gradient: time 0 min, A 97%; time 2.5 min, A 97%; time 30 min, A 87%; time 32.5 min, A 0%; time 37.5 min, A 0%; time 40 min, A 100%; time 52.5 min, A 100%. The biologically active HPLC separated peaks were collected for mass spectrometry and NMR analysis.

[0260] MS and NMR analysis Chemicals were used as received and without further purification unless otherwise stated. NMR analysis of all antigens was performed at 298 K on a Bruker Avance III NMR spectrometer operating at a proton frequency of 500 MHz equipped with a BBFO probehead or a Bruker Avance III HD NMR spectrometer operating at a proton frequency of 600 MHz equipped with a cryogenic QCI-F probe. Standard pulse sequences were used for cosy, tocsy, noesy, hsqc, hmqc, hmbc 2D-NMR experiments and spectra were processed using the topspin 4.0 software package. All new compounds were fully characterized by 2D-NMR and HiRes-ESI-MS. All compounds were characterized by: 1 H- and 1 H- 13 C-HSQC spectrum, as well as experimental and computational HiRes-ESI-MS spectra were obtained (not shown).

[0261] HRMS spectra were measured on a Bruker MaXis 4G high-resolution ESI mass spectrometer in direct injection mode using methanol containing 0.1% v / v formic acid.

[0262] Upregulation of cell surface MR1 THP-1 cells (10 5 cells / well) with synthetic compound: M 3 ADE (1 μM), OPdA (100 μM), M 1MR1 surface expression was examined after 6 h incubation at 37°C with G (13 μM) and OPdC (100 μM) or without synthetic compounds. Ac-6-FP (acetyl-6-formylpterin, 100 μM) (Schircks Laboratories, Cat. No. 11.418) was used as a positive control for MR1 surface upregulation. Cells were stained with anti-human MR1-APC mAb (clone 26.5) or APC-labeled mouse IgG2a,k isotype control antibody for 20 min at 4°C, then washed and analyzed by flow cytometry. For each condition, the net MFI was calculated by subtracting the isotype MFI from the anti-MR1 MFI, and the fold change of cells treated with synthetic molecules versus vehicle treatment was calculated.

[0263] Activation assay using live or immobilized APCs MR1T cells (5 × 10 unless otherwise noted) 4 / well) in triplicate with the indicated APCs (10 unless otherwise noted) for 18 h in a volume of 120 μL. 5 In some experiments, anti-MR1 mAb (clone 26.5, purified and endotoxin-free mouse IgG2a, (Lepore et al., 2014)) or mouse IgG2a isotype control mAb (LEAF, Biolegend, catalog no. 401504) (both at 30 μg / mL) were added and incubated at 37°C for 30 min before the addition of T cells.

[0264] Nucleobases, nucleosides or nucleotides (all at 250 μM) and synthetic compounds M 3 ADE, OPdA, M 1 When stimulating T cells with G or OPdC, THP-1 cells (10 5 The cells were incubated for 2 hours with 100 μM OPdA, 100 μM OPdC, and 13 μM M 1 G was used for all clones, except for 100 μM M 3ADE was used for clones other than DGB129, AC1A4, AC1B76, and AVA46, for which 1 μM was used.

[0265] In experiments using mycophenolic acid (10 μM), EHNA (25 μM), and Sp-bromobenzyl glutathione cyclopentyl diester (BBG, 20 μM) (all from Sigma-Aldrich), THP-1 cells (1 × 10 6 / mL) were treated with the indicated concentrations of drugs in complete medium for 18 h at 37 °C, then washed twice with PBS, counted, and used for T cell activation. 6 For experiments in which fixed A375-MR1 cells were used to activate MR1 T cells, APCs (4 × 10 5 100 μM / mL) were treated with apocynin (APO, 100 μM), L-glutathione reduced form (GSH, 4 mM), N-acetylcysteine ​​(NAC, 4 mM), L-buthionine sulfoximine (BSO, 400 μM), mercaptosuccinic acid (MSA, 3.3 μM), ML-210 (6 μM) or 1S,3R-RSL3 (RSL3, 1 μM), hydralazine hydrochloride (100 μM) or aminoguanidine hemisulfate (5 mM) for 18 h at 37°C, then washed twice with PBS, fixed with glutaraldehyde, counted and used for MR1T cell stimulation.

[0266] In some experiments, MAIT cells were stimulated with APC pulsed for 3 hours with 5-OP-RU as previously described, or with 30 μM 6,7-dimethyl-8-ribityrumazine (Cayman Chemical Catalog No. 23370).

[0267] To confirm that drugs that reduce MR1T stimulation do not affect MR1 presentation ability, A375-MR1 cells treated with various molecules were harvested before fixation and used to stimulate the MAIT clone MRC25 after pulsing with the indicated concentrations of freshly prepared 5-OP-RU or 6,7-dimethyl-8-ribitylmazine (Cayman Chemicals) for 2 h at 37°C.

[0268] Activation assay with plate-bound soluble MR1 Recombinant human β2m-MR1-Fc was produced in CHO-K1 cells as previously described (Lepore et al., 2017) and coated at 4 μg / mL onto 96-well plates (Nunc, Cat. No. 439454) for 18 h at 4 °C. Plate-bound MR1 was then washed twice with wash buffer (150 mM NaCl, 20 mM Tris, and 2% glycerol, pH 5.6) to remove bound antigen. Synthetic antigen (M 3 ADE, OPdA, M 1 G, OPdC) was added at the indicated concentrations and incubated for 6 h at room temperature. Unbound antigen was washed twice with PBS before the addition of excess PBS. In some experiments, bacterially produced and refolded MR1-M was used. 3 ADE proteins were serially diluted in PBS and added to high protein binding plates (Nunc, Cat. No. 439454) for 2 h at 37°C, washed twice, and used for stimulation assays. 5 / 100 μl / well) was added to the wells and the supernatant was collected after 18 hours. The released cytokines were detected by ELISA. Recombinant human β2m-MR1-Fc was produced in CHO-K1 cells as described previously and 4 μg / mL was coated onto 96-well plates. The antigen produced by the CHO-K1 cells was removed by washing twice with washing buffer (150 mM NaCl, 20 mM Tris and 2% glycerol, pH 5.6). The synthetic antigen was diluted in the washing solution and incubated for 3 hours at room temperature. MR1T cell clones (10 5The antigen was washed with wash buffer before the addition of 100 μg / well. Supernatants were harvested after 18 h and subjected to cytokine analysis by ELISA.

[0269] Cytokine analysis The following human cytokines were assessed by ELISA using specific mAbs: GM-CSF (purified clone BVD2-23B6 and biotinylated clone BVD2-21C11, Biolegend catalog numbers 502202 and 502304, respectively), IFN-γ (purified clone MD-1 and biotinylated clone 4S.B3, Biolegend catalog numbers 507502 and 502504, respectively), and IL-13 (purified clone JES10-5A2 and biotinylated clone SB126d, SouthernBiotech catalog numbers 10125-01 and 15930-08, respectively).

[0270] Generation and tetramerization of MR1 protein Soluble recombinant MR1 monomer was produced as previously described (Kjer-Nielsen, L., et al., Nature 491(7426):717-723). Briefly, nucleotide sequences encoding the soluble portions of mature human MR1 (GenBank Accession No. NM_001531) and mature human β2m (GenBank Accession No. NM_004048.3) were cloned into the bacterial expression vector pET23d (Novagen, Catalog No. 69748-3). The transformed E. coli BL21(DE3)pLysS was then cultured at OD 600nm The cells were grown to 0.4–0.6 and then induced with 0.6 M isopropyl β-D-1-thiogalactopyranoside (Sigma-Aldrich, catalog no. 10724815001). After an additional 4 h of incubation, the cells were lysed and the inclusion bodies were washed, purified, and completely denatured with 8 M urea, 10 mM EDTA, 0.1 mM DTT, and then stored at −80°C.

[0271] Protein refolding was performed by adding MR1 heavy chain (4 mM), β2m (2 mM) and compound (15 mM) to 1 L of refolding buffer containing 0.4 M L-arginine, 100 mM Tris pH 8.0, 2 mM EDTA, cooled to 4 °C and pre-added with 5 mM reduced glutathione and 0.5 mM oxidized glutathione. After 3 days, the refold mixture was concentrated to 1 mL and purified by HPLC using Superdex75 10 / 300GL (GE Healthcare, Catalog No. 17517401) and MonoQ 5 / 50 (GE Healthcare, Catalog No. 17516601) columns to obtain MR1 retaining the refolded compound.

[0272] The correct conformation of the protein was confirmed by performing plate binding activity of MR1T cell clone DGB129. Refolded MR1-compound protein was serially diluted in PBS (1.5–100 μg / mL) and added to a high protein binding plate (Nunc, Cat. No. 439454) for 2 h at 37°C. The wells were washed extensively with PBS and then 5 × 10 4 Cells were added and incubated overnight at 37°C. ELISA for IL-13 was then used as an activity readout. Functional monomers were then biotinylated overnight at 4°C using the BirA-500 Biotinylation Kit (Avidity, Cat. No. Bulk BirA). Excess biotin was removed by S75 10 / 30 (GE Healthcare, 29148721) gel filtration before tetramerization with phycoerythrin (PE)-streptavidin (Prozyme, Cat. No. PJRS25) at a 4:1 molar ratio.

[0273] As control MR1 tetramers, human MR1-5-OP-RU and human MR1-6-FP labeled with APC, PE or AlexaFluor488 were used. (This MR1 ​​tetramer technology was jointly developed by Drs. James McCluskey, Jamie Rossjohn and David Fairlie and produced by the NIH Tetramer Core Facility under license from the University of Melbourne.)

[0274] Immunofluorescence staining Cell surface labeling was performed using standard protocols. Intracellular labeling was performed using the True-Nuclear™ Transcription Factor Buffer set (Biolegend, Cat. No. 424401) according to the manufacturer's instructions. All mAbs used for staining were titrated on the appropriate cells before use. Biotinylated mAbs were revealed with Streptavidin-PE (Biolegend, Cat. No. 405204), -Alexa Fluor488 (Biolegend, Cat. No. 405235), or -Brilliant violet421 (Biolegend, Cat. No. 405226), all at 2 μg / mL.

[0275] When staining with tetramers, cells were pretreated with dasatinib (50 nM, Sigma-Aldrich, Cat. No. CDS023389) for 30 min, after which anti-CD8 mAb (Biolegend, clone RP8-TA BV711) was added first for 20 min at room temperature (RT), then 2.5 μg / mL tetramer was added for another 20 min at RT without washing. All remaining mAbs were then added without washing for another 20 min at RT. Cells were then washed with PBS before acquisition on the flow cytometer.

[0276] Samples were acquired on an LSR Fortessa flow cytometer equipped with FACS Diva software (Becton Dickinson). Cell sorting experiments were performed using Influx or FACSaria (Becton Dickinson). Dead cells and doublets were excluded based on forward scatter area and width, side scatter, DAPI (Sigma) or Live / Dead (Thermo Fisher Scientific) staining, as shown in Figure 6. When analyzing PBMCs, CD14 + Cells and CD19 + Cells were excluded. All data were analyzed using FlowJo (LLC).

[0277] ROS production measurement CM-H 2 The production of reactive oxygen species (ROS) during cell treatment with doxorubicin and paclitaxel was evaluated using DCFDA (Thermo Fisher Scientific). THP-1 cells (10 7 / mL) at 10 μM CM-H 2 The cells were labeled with DCFDA for 30 minutes at 37°C in the dark, then washed with PBS and resuspended in complete medium. 5 Cells were seeded and treated with 75 nM doxorubicin, 5 μM paclitaxel or vehicle for 18 h at 37° C. Phorbol 12-myristate 13-acetate (PMA, 50 ng / mL) was used as a positive control.

[0278] Quantification and statistical analysis Data processing and analysis of sgRNA-sequences Raw sequencing data were demultiplexed using bcl2fastq (v2.17.1.14) and read quality was checked using FastQC (v0.11.4). Reads were then trimmed to remove homologous regions flanking the sgRNA sequence using options HEADCROP:42 CROP:20 in Trimomatic v0.36. These trimmed reads were run through FastQC again to ensure that the average phred33 quality of sgRNA sequences was above 30. These reads were aligned to the GeCKO v2 sgRNA reference index using option -very-sensitive-local in Bowtie2 (v2.2.9). Read counts were then extracted from the resulting SAM files using a custom perl script map_count.pl (Cox, M. available upon request) and imported into R (R Development Core Team, 2018) for analysis using edgeR.

[0279] Because the heterogeneity of sgRNA activity in the GeCKO library precludes hit selection by rank-based methods, we performed differential enrichment analysis with the edgeR package (Dai et al., 2014) using the GLM Robust method to estimate variance after removing guides targeting known essential genes. The random enrichment and depletion levels of guides were calculated as log 1 of the top and bottom 1% of negative control guides in the GeCKO library. 2 The fold change was estimated using FDR < 0.05 and log 2 Guides with fold changes higher or lower than the top 1% and bottom 1% of negative control guides, respectively, were considered significantly enriched or depleted by our screen. GO-term and KEGG-pathway enrichment analysis was performed using binomial tests on significant unique gene targets identified by differential enrichment analysis (Carlson, 2016 #2707). Genes were annotated using biomaRt version 2.42.0.

[0280] CRISPR screening analysis Data analysis, statistical tests, and visualization were performed in R and GraphPad Prism. After removing guides targeting known essential genes, differential enrichment analysis of CRISPR array data was performed using edgeR (v3.24) using the GLM Robust method to estimate variance. The levels of random enrichment and depletion of guides were calculated as log 1 of the top and bottom 1% of negative control guides in the GeCKO library. 2 The fold change was estimated using FDR < 0.05 and log 2 Guides with fold changes higher or lower than the top 1% and bottom 1% of the negative control guides were considered significantly enriched or depleted by our screen, respectively. GO-term and KEGG-pathway enrichment analysis was performed using binomial tests on significant unique gene targets identified in the differential enrichment analysis. Genes were annotated using biomaRt version 2.42.0.

[0281] Statistical analysis was performed with Prism (GraphPad Software, Inc.) using multiple t-tests, one-way or two-way analysis of variance as indicated for each assay in the figure legends.

[0282] A p value <0.05 was considered statistically significant. * p<0.05, ** p ≤ 0.01, *** p ≤ 0.005.

[0283] result Compounds that stimulate MR1 T cells Previous studies by the present inventors have revealed that MR1 T cells recognize MR1 molecules complexed with ligands present on tumor cells. Compounds defined as modified nucleobases and nucleobase adducts were identified by purification of cell extracts of THP-1 cells.

[0284] The inventors screened commercially available compounds using three biological assays. All three assays are based on the ability of the compounds to bind to MR1 and i) modulate the surface expression level of MR1, ii) activate at least one MR1 T cell clone in a specific manner, or iii) compete with a stimulatory compound, thereby affecting the response of the MR1 T cell clone. The biologically active compounds are listed in Table 1.

[0285] Examples of compound reactivity in each of the above three functional assays are shown in Figures 1-20.

[0286] Detection and sorting of MR1 T cells by MR1 tetramer staining As a proof of concept to demonstrate the use of these novel compounds for the detection and capture of MR1 T cells, we used compound M 3 I chose ADE. 3 ADE-containing MR1 molecules were generated by in vitro refolding of bacterially produced human recombinant soluble MR1. 3 Correctly refolded MR1 monomer loaded with ADE was purified by gel filtration chromatography (Figure 4a) and its ability to stimulate MR1 T cells in the absence of APC was tested in a plate binding assay (Figure 4b). 3 The ADE complex was biotinylated and tetramerized using streptavidin. 3 The specificity of the ADE tetramer was verified by ex vivo capture of reactive T cells from PBMCs, followed by expansion and generation of T cell clones that maintained the original properties of tetramer reactivity and specificity for the desired compound. For example, one such clone, named AVA34, expressed MR1-M 3 This was confirmed by positive staining with the ADE tetramer but negative staining with the MR1-5-OP-RU tetramer (Figure 5a). 3The TCR specificity of this tetramer staining was further confirmed by inhibition of ADE tetramer binding (Figure 5a). Clone AVA34 also binds to M 3 Specific activation upon re-exposure to ADE, whereas MR1 + Other compounds presented by APCs were not activated (Figure 5b). 3 Using the ADE tetramer, we detected reactive MR1 T cells in freshly isolated PBMCs from healthy donors (Figure 6). 3 ADE tetramer-positive cells were easily detected, and their frequency was higher than that of CD3 + The frequency ranged from approximately 0.005% to 0.097% of cells (mean 0.027%), similar to previously reported frequencies for conventional HLA-restricted T cells and MR1 T cells using different strategies. To promote the accumulation of carbonylated nucleosides, in multiple experiments, THP-1 cells were treated with agents that increase the amount of carbonyls in cells. These agents (daidzin, disulfiram, oleic acid, and ellagic acid) induced strong T cell responses in MR1 T cell clones (Figure 7, panels a, b, c, d, and e). In further experiments, THP-1 cells were treated with the adenosine deaminase inhibitor EHNA to induce accumulation of adenosine-containing adducts or with mycophenolic acid, which inhibits the enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) and induces an increase in inosine- and adenosine-containing nucleoside adducts. All of these agents induced strong stimulation of MR1 T cells (FIG. 9, panels g and h).

[0287] Single gene knockout of metabolic pathways contributes to the efficiency of T cell-mediated killing In a previous study, we isolated human T cells that recognized tumor cells expressing low levels of MR1 under sterile conditions. These MR1 T cells recognized tumor cell lines grown in vitro or in vivo, indicating that stimulatory antigens preferentially accumulate in tumor cells according to environmental conditions. Furthermore, individual MR1 T cell clones showed tumor recognition patterns, suggesting that tumors harbor shared and unique antigens (Ags) that we hypothesize are of metabolic origin. To identify these Ags, we used multiple approaches.

[0288] To identify global metabolic key points that contribute to the production of metabolite autoantigens, we performed a genome-wide CRISPR knockout screen.

[0289] The A375 melanoma tumor cell line (A375-MR1 cells), into which the inventors had previously transfected the MR1 gene and CAS9 gene, was used as a target for the cytotoxic MR1T cell clone TC5A87. After transfecting a library of sgRNAs covering the entire human genome and killing three times in succession, the surviving A375-MR1 cells were subjected to deep sequencing to evaluate the enriched or depleted gRNAs.

[0290] Prior to differential enrichment analysis, genes essential for A375-MR1 cell proliferation were removed from the raw data to prevent results from being confounded by hits to gene targets that may be depleted in a non-T cell-dependent manner. In addition, many guides included in the library will show random enrichment and depletion since they do not affect the efficiency of T cell-mediated killing. The GecKO v2 library contains non-targeting negative control guides that should not show significant enrichment or depletion in response to T cell-mediated killing, i.e., they should show random abundance changes. Therefore, we calculated the Log of the negative control guides. 2 Log based on top and bottom 1% of fold changes 2A fold change cutoff was used to identify guides that were significantly enriched or depleted (FDR<0.05) above background enrichment or depletion (i.e., random). Because gRNA activity in GeCKO libraries is known to be uneven, which would prevent hit selection by rank-based methods, differential enrichment analysis was performed using the edgeR package. We hypothesized that knocking out genes involved in upstream steps of antigen biosynthesis could enhance the ability of A375-MR1 cells to escape T cell-mediated killing by reducing the production of antigenic compounds. Conversely, knocking out genes involved in downstream steps of antigen biosynthesis would increase the accumulation of antigenic compounds and enhance T cell-mediated killing. Indeed, the results of the differential enrichment analysis showed a fraction of enriched guides (n=243) that targeted 237 unique genes, including MR1 and beta2-microglobulin (B2M), two positive control genes that would otherwise not present antigens to MR1-restricted T cells. Among these genes, the inventors also found enrichment for guides specific for adhesion molecules including CD58 (LFA-3) and ICAM-1, which are ligands for CD2 and CD11a on T cells, respectively. The latter interaction is important for T cells to recognize target cells and was also found in a previous CRISPR screen for immune cell killing of tumor cells.

[0291] Among the depleted hits, 5331 guides targeting 4705 unique genes were significantly depleted compared to control cells transfected with gRNA libraries not subjected to TC5A87 killing. Since gene essentiality is context-dependent and sensitive to many experimental parameters, it is expected that not all essential genes were successfully ablated using the Hart A375-MR1 essential gene-set. Furthermore, the number of genes required for tumor cell survival should far exceed the number of genes that can escape T cell-mediated killing. Therefore, it is not surprising that we observed more significantly depleted guides than enriched. Binary enrichment analysis of gene-ontology (GO) terms annotated to the significant hits revealed that many depleted gene targets shared GO terms enriched in metabolic processes, suggesting that metabolic processes important for MR1 T cell stimulation require the coordinated activity of multiple genes. This was less evident for enriched gene targets. Furthermore, these significant hits showed enrichment in nucleobase and nucleic acid metabolic processes, suggesting that these metabolic pathways may be involved in MR1 T cell stimulation.

[0292] To explore the effects on global metabolism of the significantly enriched and depleted gene targets identified in the screen, we used structural sensitivity analysis, which has recently been extended from reaction-level to gene-level perturbations, to predict metabolic network responses to single gene knockouts in Recon3D, a genome-scale model of human metabolism, after appropriate model preprocessing.

[0293] Next, we used Pearson correlation between genome-wide reaction sensitivities of each modeled knockout to identify sets of two or more knockouts with similar (Pearson score ≥ 0.6) global effects on metabolic reactions. This analysis resulted in the selection of 125 genes among the significantly enriched or depleted genes detected in the CRISPR screen. When these selected genes were correlated with the corresponding KEGG pathways, it became clear that oxidative phosphorylation and purine metabolism could be perturbed by single-gene knockouts of a number of different gene targets. Furthermore, all but two of these genes were among the significantly depleted hits identified in the screen. Binomial enrichment analysis of KEGG pathways represented by 4705 gene targets depleted in the CRISPR screen or 125 genes selected from the in silico metabolic models also identified purine metabolism pathways as significantly enriched in both the entire CRISPR screen hits and the subset of correlated Recon3D models (binomial p-value = 2.00e -3 and 1.67e -4 Although not identified as significantly enriched pathways in the hits from the CRISPR screen, both the oxidative phosphorylation and glycerolipid pathways were significantly enriched in the metabolic perturbation model (binomial p-value = 6.58e -13 and 4.08e -4 ).

[0294] Nucleobase and nucleoside antigens presented by tumor cells can stimulate MR1 T cells We first focused on the purine pathway, where we identified several genes that may be involved in antigen accumulation in MR1T cells: adenosine deaminase (ADA), adenylosuccinate synthase 1 (ADSSL1), laccase domain containing 1 (LACC1), cGMP-specific 3',5'-cyclic phosphodiesterase (PDE5A), aldehyde dehydrogenase 16 family member A1 (ALDH16A1), and hypoxanthine phosphoribosyltransferase 1 (HPRT1). ADA converts adenosine to inosine, and ADSSL1 is required for the de novo production of adenosine monophosphate (AMP) from inosine monophosphate (IMP); whereas LACC1 enables the purine nucleoside cycle, and PDE5A catalyzes the specific hydrolysis of cGMP to 5'-GMP. Finally, ALDH16A1 and HPRT1 proteins form a complex to generate purine nucleotides via the purine salvage pathway. Overall, these findings further support purines as possible molecules involved in target recognition by MR1T cells.

[0295] To verify the relevance of these genes, we generated knockout lines and tested them individually for their ability to stimulate MR1 T cells. ADA- and LACC1-deficient cells induced higher stimulation than the parental A375-MR1 cells, as measured by IFN-γ release (Figures 8A,B and 8D,E). On the other hand, ADSSL1-deficient cells induced a slight but significant decrease in stimulation (Figures 8C and 8F). This experiment was performed with two MR1 T cell clones that were selected because they showed different tumor recognition patterns in preliminary experiments. In control experiments, all gene knockout cell lines equally stimulated MR1-restricted MAIT cells in the presence of 5-OP-RU antigen (Figure 15) and showed similar MR1 expression on the cell surface, indicating that the observed changes in MR1 T cell stimulation were not due to a general change in antigen presentation ability. These findings led us to focus on the possibility that purines are the antigens.

[0296] To investigate the possible role of purines in MR1 T cell stimulation, we incubated human acute monocytic leukemia THP-1 cells with synthetic nucleotides, nucleosides or nucleobases, then added MR1 T cell clones and measured IFN-γ production. THP-1 cells were chosen as targets because they constitutively express low levels of MR1 surface, induce some spontaneous MR1 ​​T cell stimulation, and show the ability to properly process and present MR1 T cell antigens. A375-MR1 cells, which express very high levels of MR1 surface, were also included as a positive control to stimulate cytokine production in MR1 T cells. We found that three different MR1T cell clones responded to different groups of compounds: TC5A87 did not respond significantly to the test compounds (Fig. 8G, left), DGB129 responded to adenine, adenosine, deoxyadenosine (dAdo), and inosine (Fig. 8G, middle), and MCA3C3 was activated by ADP, guanine, guanosine, deoxyguanosine, and xanthosine (Fig. 8G, right). THP-1 cells incubated with synthetic compounds did not stimulate MAIT cells (Fig. 16A). Interestingly, despite the use of high concentrations of the compounds, their stimulatory effect was minor compared to MR1T stimulation by A375-MR1, suggesting that these molecules may be intermediate precursors of antigens.

[0297] These results indicate that human MR1T cell clones can recognize nucleobase / nucleoside antigens processed and presented in cancer cell lines and that cross-reactivity may exist between different MR1T cell clones.

[0298] Methylglyoxal and purine metabolic pathways in tumor cells cooperate in MR1 T cell stimulation To understand which metabolic pathways may be involved in the recognition of nucleobase / nucleoside antigens on tumor cells by MR1T cells, we started by investigating genome-wide gene disruption screening data. We observed that some of the significantly depleted sgRNAs were associated with genes involved in glycolysis (TPI1) and methylglyoxal (MG) degradation, including glyoxalase 1 (GLO1) and glyoxalase domain containing 4 (GLOD4). TPI1 encodes triosephosphate isomerase within the glycolysis pathway and is responsible for the enzymatic conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (G3P), an otherwise spontaneous reaction that accompanies the generation of MG (Figure 9). Conversely, in GLO1-deficient cells, the degradation of MG (a highly reactive carbonyl) is inhibited, leading to its accumulation. As MG forms adducts with some nucleobases, these data suggested that MG may be involved in the generation of MR1T cell antigens.

[0299] Thus, we elucidated the possible role of glycolysis and MG degradation in MR1T stimulation by generating single gene KO (knockout) cell lines. Deletion of TPI1 in A375-MR1 cells significantly increased IFN-γ production by both MR1T cell clones (Fig. 9A, B). Furthermore, A375-MR1 cells pulsed with glucose and then fixed showed increased MR1T cell stimulatory capacity (Fig. 9C, D): this effect was abolished when the same cells were incubated with deoxyglucose (Fig. 9C, D), which does not enter the glycolytic pathway and therefore does not produce MG. We also confirmed that GLO1-deficient and GLO1-overexpressing A375-MR1 cells showed increased and decreased MR1T cell stimulatory capacity, respectively (Fig. 9E, F). Taken together, these results suggest that MG accumulation in target cells is important for the stimulation of MR1T cell clones.

[0300] To investigate the possible synergistic effect between the MG and purine metabolic pathways in the tumor cell stimulating ability of MR1T cells, we investigated the effect of individual and combined pharmacological inhibition of the key enzymes of these pathways. We tried to inhibit GLO1 with Sp-bromobenzylglutathione (BBG); inhibit inosine monophosphate dehydrogenase (IMPDH1, 2) with mycophenolic acid (MPA) to cause IMP accumulation; and inhibit ADA and phosphodiesterase 2 (PDE2) with erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA) to induce adenosine, dAdo and cGMP accumulation. To sensitively detect the effect of the inhibitors, we again used THP-1 cells as target cells: we found that the combination of BBG with each of the other two drugs significantly enhanced the IFN-γ release of both MR1T cell clones against THP-1 cells (Fig. 9G, H). The DGB129 clone was more sensitive to these treatments and also responded to THP-1 cells treated with EHNA, BBG, or MPA alone (Fig. 9H).

[0301] We tested the IFN-γ response of MR1T cells to GLO1-modified THP-1 cells and various doses of MG or dAdo. We found that MG treatment significantly increased MR1T cell stimulation by GLO1 deficiency compared to wild-type THP-1 cells (Figure 9I). Conversely, MG did not induce MR1T cell stimulation when administered to GLO1-overexpressing cells (Figure 9J). Similarly, MR1T responsiveness to dAdo was increased when GLO1-deficient THP-1 cells were used as antigen-presenting cells (APCs) (Figure 9K) and decreased when GLO1-overexpressing THP-1 cells were used (Figure 9L). Taken together, these findings suggest that nucleosides / nucleobases and MG cooperate to generate a possible MR1T cell antigen.

[0302] Multiple oxidative stress-related carbonyl species accumulate in tumor cells and contribute to the stimulation of MR1T cells In addition to the purine pathway, our model-based analysis showed genes related to oxidative phosphorylation, whose protein products are involved in ATP generation in mitochondria and whose changes promote the accumulation of reactive oxygen species (ROS). At the same time, this analysis also showed genes involved in the coupling of proton transport and ATP hydrolysis, which contribute to maintaining the physiological environment of intracellular organelles, including mitochondria. + We demonstrated the involvement of the transporter subunits ATP6V1C2, TCIRG1, and ATP6V0D2. Therefore, we next investigated the role of ROS in the stimulation of MR1T cells by tumor cells.

[0303] First, we focused on genes involved in oxidative phosphorylation. Our initial MR1T cell killing screen revealed significant depletion of sgRNAs specific for GSTM1, GSTA4, GSTA1, GSTM5, GSTA2, GSTA3, GSTM3, and GSTO1; these genes are involved in detoxifying electrophilic compounds and ROS by conjugation to the ROS scavenger glutathione (GSH). We therefore wondered whether tumor cells would accumulate MR1T cell stimulatory compounds in the absence of these GSTs and in the accumulation of ROS and electrophilic molecules. We therefore investigated whether sgRNAs specific for GSTM1, GSTA4, GSTA1, GSTM5, GSTA2, GSTA3, GSTM3, and GSTO1 were significantly depleted; these genes are involved in detoxifying electrophilic compounds and ROS by conjugation to the ROS scavenger glutathione (GSH). 2 - and H 2 O 2 We examined the effects of paclitaxel and doxorubicin, two drugs that induce the intracellular accumulation of ROS. When THP-1 cells were incubated with nucleoside compounds (Figure ​(Figure10A–C), both drugs significantly increased the accumulation of ROS and promoted the activation of all three MR1T cell clones. This mirrors the additive effect observed when combining purine-modifying drugs with carbonyl degradation inhibitors (Figure9G,H).

[0304] Next, the present inventors transformed A375-MR1 into 2 - Apocynin, a scavenger and NADPH oxidase inhibitor, or H 2 O 2After treatment with GSH or N-acetylcysteine ​​(NAC), which prevents accumulation, the cells were fixed and incubated with three MR1T cell clones. We found that A375-MR1 cells treated with either inhibitor stimulated significantly less IFN-γ production from MR1T cells, with apocynin being effective in one T cell clone (Figure 10D-F). We also treated A375-MR1 cells with buthionine sulfoximine (BSO), an inhibitor of GSH synthase, and observed a significant increase in stimulation of all MR1T clones tested (Figure 10D-F). Taken together, these data indicate that ROS are involved in MR1T antigen accumulation, but require concomitant changes in nucleobase metabolism.

[0305] Peroxides accumulate in many tumor types and are involved in various signaling pathways and cell fate decisions. Peroxides are also required for lipid peroxidation, a pathway that generates two highly reactive carbonyls, malondialdehyde (MDA) and 4-OH-nonenal (4-HNE). Both compounds form stable adducts with proteins, lipids, and nucleobases and accumulate in tumor cells. In addition to our findings that inhibiting ROS accumulation prevents tumor cell stimulation of MR1T cells (Figure 10D-F), we inferred a role for lipid peroxidation from the results of our CRISPR / Cas9 screen, which showed significant depletion of sgRNAs for glutathione peroxidase 4 (GPX4) and glutathione peroxidase 1 (GPX1).

[0306] GPX1 protein converts organic hydroperoxides and H2O3 into glutathione-catalyzed 2 O 2GPX4 catalyzes the reduction of , but has a higher preference for lipid hydroperoxides, protecting cells from membrane lipid peroxidation and cell death. Accordingly, when we pretreated A375-MR1 cells with mercaptosuccinic acid (MSA), a selective GPX1 inhibitor, or two GPX4 inhibitors, RSL3 and ML-210, they showed a significant increase in MR1T cell stimulatory activity (Figure 10G-I). None of these compounds affected MAIT cell responses to microbial antigens in control experiments, except for paclitaxel, which could induce a small but significant stimulation in the presence of nucleosides (Figure 16C-D). These results suggest that peroxides and lipid peroxidation contribute to the stimulation of MR1T cells by tumor cells.

[0307] To further evaluate the involvement of carbonyls in the generation of MR1 T antigen, we tested the ability of carbonyl scavengers to block MR1 T cell activation. A375-MR1 cells were incubated with aminoguanidine and hydralazine, which show preferential scavenging activity for different carbonyls, followed by fixation and washing before adding MR1 T cell clones. We found that both scavengers significantly suppressed IFN-γ production by MR1 T cell clones (Figures 10J-L) and had no effect on MAIT cell activation (Figure 16E).

[0308] Taken together, our data suggest that multiple oxidative stress-related reactive carbonyl species that accumulate in cells following metabolic changes conjugate with nucleobases to generate MR1-presenting antigens that stimulate MR1 T cells.

[0309] Evaluating the biological activity of compounds After incubation at 37°C for 3 hours in the presence or absence of the compounds (each at three doses), THP-1 MR1 cells (10 5"Cell surface MR1 regulation" was measured using 100 μM cells / well. Ac-6-FP (100 μM, Schircks Laboratories Cat. No. 11.418) was used as a positive control compound for MR1 surface upregulation. MR1 expression was assessed by staining with anti-human MR1 APC-conjugated mouse mAb (IgG2a,k clone 26.5, Biolegend Cat. No. 361108) and subtracting background staining (always below 300 MFI) with APC-conjugated mouse IgG2a,k isotype control antibody (Biolegend Cat. No. 400220).

[0310] The "competition assay" was performed using APC (10 5 Cells / well) and compounds (three doses each) were incubated at 37°C for 2 h, and then the optimal concentration (≥EC 50 ) and add antigen for each clone of interest for an additional 2 hours before incubating with T cells (10 4 The experiment was carried out by adding 100 μM Ac-6-FP (100 μM) to the wells of the plate (cells / well). As a positive control for competition with the antigen, Ac-6-FP was used (100 μM). After 24 hours, the supernatants were collected and subjected to cytokine analysis measured by ELISA.

[0311] Nucleobase adducts stimulate MR1 T cells Biochemical condensation of carbonyl species with nucleobases is a hallmark of many cancer cell types, leading to the generation of adducts. The inventors investigated the possibility that compounds containing nucleobase adducts could serve as MR1 ​​T cell antigens. To test this hypothesis, the inventors investigated the mechanism of action of nucleobase adducts by using four previously described adducts: the purine adduct 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarboxaldehyde (M 3 ADE), N 6 -(3-oxo-1-propenyl)-2'-deoxyadenosine (OPdA), and pyrimido[1,2-α]purin-10(3H)-one (M 1 G), and pyrimidine adduct N 4We synthesized 3-(3-oxo-1-propenyl)-2'-deoxycytidine (OPdC) and investigated their antigenic activity (Figure 11). We confirmed the identity of the adducts by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy.

[0312] Initial experiments showed that, except for MGG, these compounds were able to induce upregulation of MR1 on pulsed APCs (FIG. 11).

[0313] In T cell activation experiments using APCs expressing low levels of MR1, each compound exhibited different stimulatory properties for MR1 T cells (Figure 11). Dose-response studies revealed that individual MR1 T cells were sensitive to different degrees of sensitivities. In some cases, the EC 50 (1-10 nM) was observed, while this same antigen showed weak or no stimulation to other MR1T clones. A second important finding was the antigen cross-reactivity of some MR1T clones. However, these clones were shown to preferentially respond to one tested antigen, in agreement with the type of antigen cross-reactivity described for T cells specific for peptide or lipid antigens presented by MHC or CD1 molecules, respectively. These data are consistent with the cross-reactivity observed in the experiment in "Figure 8G". In THP-1 cells pulsed with various doses of the adduct, individual compounds differentially stimulated IFN-γ production by different MR1T cell clones, with some clones showing cross-reactivity to multiple compounds (Figure 11). In both cases, activation of MR1T cells by adduct-loaded THP-1 cells was completely inhibited by the addition of a blocking anti-MR1 monoclonal antibody (mAb), confirming the MR1-restricted nature of the recognition of the adduct (Figure 11).

[0314] We found that when we incubated THP-1 cells with the adducts, each induced a 1.5- to 5-fold increase in the average expression level of MR1 on the cells, as seen with other MR1-binding compounds (Figure 11A-E). In THP-1 cells pulsed with various doses of the adducts, each compound differentially stimulated IFN-γ production by different MR1 T cell clones, with some clones showing cross-reactivity to multiple compounds (Figure 11A-E). In each case, activation of MR1 T cells by adduct-loaded THP-1 cells was completely inhibited by the addition of a blocking anti-MR1 monoclonal antibody (mAb), confirming the MR1-restricted nature of adduct recognition (Figure 11A-E).

[0315] We next investigated whether these synthetic antigens were stimulatory without modification inside APCs. Plate-bound recombinant MR1 protein loaded with synthetic antigens efficiently stimulated specific MR1T clones, eliminating the need for intracellular processing (Figure 12A). Thus, carbonyl nucleobase adducts directly bind to MR1 without further modification and stimulate MR1T cells. These stimulations were completely inhibited by the addition of blocking anti-MR1 mAb, confirming the role of MR1 protein.

[0316] We further confirmed the stimulatory capacity of these antigens by extending the activation assay to include additional MR1 T cell clones expressing different TCRs: 14 randomly selected MR1 T clones were tested for TCR, and 8 responded to at least one ligand. 3 ADE was 6 clones, OPdA was 8 clones, M 1 G and OPdC each significantly stimulated three clones (Fig. 12B). Again, due to the different recognition patterns of the adducts, the levels of IFN-γ production by MR1T cells varied widely, along with the cross-reactivity of the MR1T clones. 1 G or M 3We found that the addition of ADE to clones QY1A16 and QY1C3, respectively, significantly reduced the response to THP-1 cells, suggesting that both adducts compete with endogenous stimulatory molecules in THP-1 cells. These findings also suggest that some MR1T clones recognize antigens different from those tested. Of note, THP-1 cells pulsed with synthetic compounds did not stimulate MAIT cell clone MRC25 (Figure 16F).

[0317] We next assessed whether processing of the adduct in THP-1 cells was required for MR1 T cell recognition using recombinant MR1 molecules bound to synthetic antigen-loaded plastic and found that this was not the case (Figure 12A). Thus, the carbonyl nucleobase adducts directly bind MR1 without further modification and stimulate MR1 T cells.

[0318] We next investigated whether MR1T clones that recognize different synthetic compounds differ in their ability to respond to different tumor cell lines expressing physiologically low levels of MR1. This question has relevance to the fact that preferential accumulation of unique carbonyl adducts in each tumor cell line may result in preferential stimulation of individual MR1T cells. 3 Clone AVA34 activated by ADE reacted specifically with KMOE-2 and HEL tumor cell lines; clone QY1A16 activated by OPdA and OPdC reacted selectively with H460, Juso, and KMOE-2 tumor cell lines; 3 ADE, OPdA, and M 1Clone AC1A4, activated by OPdA, MGG, and OPdC, reacted with all six tumor cell lines tested, while clone TC5A87, activated by OPdA, MGG, and OPdC, reacted with all tumor cell lines (Fig. 12C). Importantly, all reactivities were inhibited by the addition of anti-MR1 mAb, thus demonstrating the restricted nature of tumor recognition by MR1 (Fig. 12C). It is also noteworthy that the tumor lines tested were derived from different tissues. None of the MR1T clones tested reacted with leukocytes from healthy individuals, except for AVA34 and TC5A87 clones, which released significant but very low amounts of IFN-γ upon incubation with monocytes (Fig. 12C).

[0319] In conclusion, MR1T cells recognize compounds containing intact carbonyl adducts of nucleobases presented on the MR1 molecule. Some MR1T clones appear to be specific for one or another adduct: others show some degree of cross-reactivity. The broad recognition and wide distribution of nucleobase adducts in many cancers justifies the broad reactivity of MR1T cells against tumors originating from different tissues.

[0320] MR1 tetramer loaded with nucleobase adduct-containing metabolites detects MR1 ​​T cells To identify and characterize ex vivo MR1T cells that respond to nucleobase adduct-containing metabolites, we generated MR1 tetramers loaded with synthetic adducts. 3 We focused on ADE because it was the most efficient in increasing MR1 surface expression, showed the highest potency, and was recognized by 6 / 14 of the MR1 T cell clones tested. 3 Several experiments were performed to confirm ADE tetramer-specific MR1 T cell staining. 3 ADE monomer stimulated clone DGB129 in plate-binding assays (Figure 4A-B), whereas fluorescently labeled tetramerized MR1-M 3 ADE monomer stained the MR1T cell clone AC1A4 (Fig. 5A), and its M 3Consistent with ADE reactivity (Fig. 12A); in contrast, this tetramer failed to bind to a standard MAIT clone (MRC25; Fig. 13A).

[0321] For further verification, MR1-M 3 MR1T cells were isolated from peripheral blood mononuclear cells (PBMCs) using the ADE tetramer, and new clonal lineages were established. These clones were named MR1-M 3 A representative clone, AVA34, was demonstrated to be able to bind the ADE tetramer but not the MR1-5-OP-RU or MR1-6-FP tetramers (Figure 13B). The TCR specificity of this tetramer staining was further confirmed by inhibition of binding with an anti-TCRVβ monoclonal antibody (Figure 13C). Additionally, the MAIT clone MRC25 was labeled with the MR1-5-OP-RU tetramer but not with the MR1-M tetramer. 3 The ADE tetramer did not label the mAb (Figure 5). Importantly, clone AVA34 did not label the mAb. 3 The results of these experiments demonstrated that MR1-M reacted with ADE-loaded THP-1 cells but not with other tested antigens (Figure 13C). 3 ADE tetramer M 3 The ability to bind to ADE-specific T cells was demonstrated.

[0322] Next, MR1-M 3 We investigated whether ADE tetramers could bind and identify specific MR1 T cells ex vivo in the blood of healthy donors. Screening of peripheral blood mononuclear cells (PBMCs) from nine healthy donors revealed that total CD3 + Tetramer-positive cells were found in all individuals, with frequencies ranging from 0.006% to 0.077% of cells (median = 0.01%) (Figures 13D, E and 6).

[0323] MR1-M 3In addition to the striking interindividual differences in the frequency of ADE tetramer-binding T cells, we also uncovered notable phenotypic differences in the tetramer-positive population. While most cells expressed CD8α (ranging from 38% to 97%), some donors expressed distinct CD4 + In another donor, CD4 - / CD8 - According to the expression of CD45RA and CCR7, the tetramer-positive population was classified as naive (range 8%-55%, median 29%), central memory (range 7%-48%, median 22%), effector memory (range 5%-81%, median 23%), and T EMRA Large donor-specific differences were also observed in the frequency of (range <1%–42%, median 3.8%) ( Fig. 13G,H ).

[0324] Thus, MR1 T cells that recognize carbonyl nucleoside adducts exist in the blood of healthy individuals, exhibit a heterogeneous phenotype, and can undergo phenotypic differentiation in a donor-specific manner.

[0325] MR1T cells responding to metabolites containing nucleobase adducts infiltrate tumor tissues The subjects were asked to identify potential M 3 Since they possess ADE-reactive T cells, the inventors next 3 We investigated whether ADE-reactive T cells could be detected in tumor samples. We isolated TILs from non-small cell lung cancer biopsies of two patients and cultured them in mice to expand MR1 T cells. 3 After expansion, we found that MR1-M cells in TIL co-cultures from both patients were significantly higher in TILs than in control patients. 3 ADE tetramer-binding cells could be detected (Fig. 14A, B). In donor number 840, these cells were CD4 + (52.6%) or CD8 + (42%), whereas donor number 895 was mostly CD8 +(93.5%) (Figure 14A, B). To confirm its antigen specificity, the present inventors 3 ADE tetramer-positive cells were enriched by sorting and then cultured in A375-β2mKO cells, A375-MR1 or M 3 The present inventors measured the degree of MR1 T cell activation via TCR downregulation. This TCR downregulation was observed in A375-MR1 cells and M 3 This occurred only in the presence of ADE-loaded A375-MR1 cells and was blocked by anti-MR1 blocking mAb (Fig. 14C, donor 840, and Fig. 14D, donor 895). We further confirmed MR1-dependent activation of tetramer-positive T cells by measuring IFN-γ release (Fig. 14E). Cells from donor #840 were activated by M 3 The cells from donor 895 reacted with A375-MR1 cells regardless of the presence of ADE (Fig. 14C, E). 3 ADE-loaded A375-MR1 cells only reacted with ADE-loaded A375-MR1 cells (Fig. 14D, E). In both cases, the reaction was inhibited by anti-MR1 mAb (Fig. 14C, E). These data indicate that M 3 The presence of ADE-reactive T cells was confirmed, suggesting a possible role for MR1 T cells in tumor immunity.

[0326] Discussion In this study, we identified nucleobase adduct-containing metabolites as autoantigens capable of stimulating human T lymphocytes that recognize MR1-expressing tumor cells. Previous studies have shown that MR1 T cells respond to unique compounds fractionated from tumor cells, suggesting distinct antigen specificity. Here, we confirm these data and further show that structurally diverse nucleobase adduct-containing compounds bind to MR1 and stimulate individual MR1 T cells. Both purines and pyrimidines form antigen adducts, and various carbonyls are involved in their generation, confirming that MR1 is a molecule with diverse antigen-binding capabilities.

[0327] Carbonyls accumulate as a result of various metabolic changes, during glycolysis and lipid peroxidation, during the metabolism of biogenic amines, vitamins, and steroids, and during the biotransformation of environmental substances and drugs. How many carbonyls are involved remains to be investigated: this is an important question, since the number and diversity of carbonyl species involved in the generation of MR1-presented nucleobase adducts may determine the size and diversity of the MR1 T cell antigen repertoire. Of note, some MR1T clones did not react to any of the antigens tested here, but still reacted to A375-MR1 cells, suggesting that the MR1 T cell antigen repertoire may be highly heterogeneous.

[0328] Although carbonyl accumulation is important, it is not sufficient to stimulate MR1 T cells, because it requires the simultaneous availability of free purines and pyrimidines in the target cells. The structures of the modified nucleobases are composed of different modified heterocyclic compounds, making them suitable for MR1 binding and similar to those of other MR1 ligands.

[0329] ROS, which are by-products of oxidative phosphorylation, play an important role in the generation of immunogenic nucleobase adducts and promote tumor induction and tumor cell proliferation. ROS also promote lipid peroxidation, which promotes the generation of carbonyls, and indirectly affect the accumulation of MR1T cell antigens. In fact, treatment with ROS-inducing drugs enhanced the stimulatory capacity of MR1T target cells, which was then attenuated by the addition of ROS scavengers. Thus, MR1T cell antigens are induced by the combined changes of multiple metabolic pathways, leading to the accumulation of nucleobases, carbonyls, and ROS.

[0330] An important open question in this field was based on the specificity of tumor cell recognition by MR1T cells. Our data provide a plausible answer, since most normal cells in steady state physiologically regulate different metabolic pathways that contribute to the generation and accumulation of nucleobase adducts. Tumor cells, on the other hand, often have alterations in many of these pathways that sustain cell proliferation, including altered glucose and glutamine uptake and a high cellular demand for reducing nitrogen. Indeed, many tumors show increased transcription of key genes involved in de novo purine synthesis, suggesting a key role for the purinosome. Furthermore, tumor cells are susceptible to DNA damage by the generation of nucleic acid adducts formed by DNA oxidation and by interaction with end products of lipid peroxidation.

[0331] Importantly, in normal cells, several mechanisms are involved in trapping highly reactive carbonyls, which are then oxidized to carbonic acid, conjugated with glutathione, or reduced to less toxic alcohols. In our CRISPR / Cas9 screen, we identified several genes involved in these conserved processes, suggesting that they contributed to the evasion of MR1T cell recognition. The importance of these control mechanisms is further supported by the negative effect of carbonyl scavengers on the MR1T stimulating ability of tumor cells.

[0332] The recognition of MR1 T cells of nucleobase adduct-containing metabolites raises the question of the physiological role of these cells. One can consider their possible role in the investigation of cells that abnormally accumulate compounds that cause DNA changes and thus predispose to dangerous genetic mutations. The ubiquitous expression of MR1 may serve this function of cellular metabolic integrity control. Taken together, these properties of MR1 T cells make them an attractive target for the use of immunotherapy in cancer. We envision that the specificity of cancer patient T cells could be converted to these novel tumor-associated metabolite antigens using selected MR1T TCR genes, thereby equipping them with tumor-targeting capabilities. The detection of MR1 T cells within the tumor microenvironment of two lung cancer patients is promising evidence supporting the potential value of this strategy. Another possible application is the use of nucleobase adduct-containing metabolites as components of innovative antitumor vaccines. Importantly, the monomorphism of MR1 may offer the possibility to design T cell-based immunotherapies that circumvent HLA polymorphisms and are universally and independently of genetic background applicable to the entire population of cancer patients.

[0333] In conclusion, the immune system continues to amaze us with its ability to detect a broad repertoire of structurally variable antigens, and T cell recognition of nucleobase adduct-containing metabolites is the most recent evidence of this enormous flexibility.

[0334] References Geacintov, NE and S. Broyde (2010). The chemical biology of DNA damage. Weinheim, Wiley-VCH. Ishiwata et al. (1995). “Comparison of serum and urinary levels of modified nucleoside,1-methyladenosine, in cancer patients using a monoclonal antibody-based inhibition ELISA.” Tohoku J Exp Med 176(1):61-68. Kawai, Y. and E. Nuka (2018). "Abundance of DNA adducts of 4-oxo-2-alkenals, lipid peroxidation-derived highly reactive genotoxins." J Clin Biochem Nutr 62(1):3-10. Kim, C.S., S. Park and J. Kim (2017). "The role of glycation in the pathogenesis of aging and its prevention through herbal products and physical exercise." J Exerc Nutrition Biochem 21(3):55-61. Marnett, L.J. (2002). "Oxy radicals, lipid peroxidation and DNA damage." Toxicology 181-182:219-222. Richarme et al. (2017). "Guanine glycation repair by DJ-1 / Park7 and its bacterial homologs." Science 357(6347):208-211. Riggins et al. (2004). "Kinetic and thermodynamic analysis of the hydrolytic ring-opening of the malondialdehyde-deoxyguanosine adduct, 3-(2’-deoxy-beta-D-erythro-pentofuranosyl)- pyrimido[1,2-alpha]purin-10(3H)-one." J Am Chem Soc 126(26):8237-8243. Seidel,A.,S.Brunner,et al.(2006).「Modified nucleosides:an accurate tumor marker for clinical diagnosis of cancer,early detection and therapy control.」Br J Cancer 94(11):1726-1733. Stoneら(1990).「Investigation of the Adducts Formed by Reaction of Malondialdehyde with Adenosine」Chem.Res.Toxicol.3:33-38. Voulgaridouら(2011).「DNA damage induced by endogenous aldehydes:current state of knowledge.」Mutat Res 711(1-2):13-27. Wauchopeら(2015).「Nuclear Oxidation of a Major Peroxidation DNA Adduct,M1dG,in the Genome.」Chem Res Toxicol 28(12):2334-2342. Leporeら(2017).Functionally diverse human T cells recognize non-microbial antigens presented by MR1.ELife 6:e24476. Leporeら(2014).Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRbeta repertoire.Nat Commun 5,3866. Daiら(2014).edgeR:a versatile tool for the analysis of shRNA-seq and CRISPR-Cas9 genetic screens.F1000 Research 3,95. Hart et al. (2015). High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities. Cell 163, 1515-1526. Brunk et al. (2018). Recon3D enables a three-dimensional view of gene variation in human metabolism. Nature Biotechnology 36, 272-281. Kanehis et al. (2019). New approach for understanding genome variations in KEGG. Nucleic Acids Res 47, D590-D595. Schmaler et al. (2018). Modulation of bacterial metabolism by the microenvironment controls MAIT cell stimulation. Mucosal Immunology 11: 1060-1070. Langmead, B., and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-359. Sanson et al. (2018). Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun 9, 5416. Bolger, A. M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120. Durinck et al. (2009).Mapping identifiers for the integration of genomic datasets with the R / Bioconductor package biomaRt.Nature protocols 4,1184-1191.

[0335] [Table 2] JPEG0007680769000029.jpg79153

[0336] [Table 3A]

[0337] [Table 3B]

[0338] [Table 4]

Claims

[Claim 1] 1. A method for modulating an interaction between an MR1 polypeptide and an MR1-specific T cell receptor molecule, said method comprising: a. 1-Methyladenosine (1) b. 2-Methyladenosine (2) c. 2'-O-methyladenosine (3) d. N6,N6-Dimethyladenosine (4) e. N6-Threonylcarbamoyl adenosine (5) f. N6-Isopent-2-enyladenosine (6) g. N6-(cis-hydroxyisopent-2-enyl)adenosine (7) h. 2-Methylthio-N6-(cis-hydroxyisopent-2-enyl)adenosine (8) i. 2-Methylthio-N6-isopent-2-enyladenosine (9) j. N6-Methyl-N6-threonylcarbamoyl adenosine (10) k. 2'-O-ribosyladenosine phosphate (11) l. N6-(3-oxo-1-propenyl)-2'-deoxyadenosine (12) m. 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarboxaldehyde (13) n. 1-(3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3H-imidazo[2,1-i]purin-7-yl)heptan-2-one (14) o. 1-Methylguanosine (15) p. N2-methylguanosine (16) q. 7-methylguanosine (17) r. 2'-O-methylguanosine (18) s. N2,N2-dimethylguanosine (19) t. 2'-O-ribosylguanosine (20) u. 3-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,7-dihydroxy-6-methyl-6,7-dihydro-3H-imidazo[1,2-a]purin-9(5H)-one (21), 3-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,7-dihydroxy-7-methyl-6,7-dihydro-3H-imidazo[1,2-a]purin-9(5H)-one (22), or a mixture of the two; w. 3-((2R,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7-(2-oxoheptyl)-3H-imidazo[1,2-a]purin-9(5H)-one (25) aa. 2'-O-methylcytidine (29) bb. 3-Methyluridine (30) cc. 5-methyluridine (31) dd. 3,2'-O-dimethyluridine (32) ee. 6-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(2-oxoheptyl)-1,8a-dihydroimidazo[1,2-c]pyrimidin-5(6H)-one (37) ff. N4-(3-oxo-1-propenyl)-2'-deoxycytidine (38) ii. N6-methyladenosine (42) jj. 6-methylpurine (43) kk. 6-(dimethylamino)purine (44) ll. N6-(Δ2-isopentenyl)adenine (45) nn. 1-Methylguanine (47) oo. N2-methyl-2'-deoxyguanosine (48) ss. 5'-deoxy-5'-(methylthio)adenosine (52) tt. N6-methyl-2'-deoxyadenosine (53) uu. N6-(2-hydroxyethyl)-2'-deoxyadenosine (54) yy. Pyrimido[1,2-α]purin-10(3H)-one (M 1 G) (24) MR1 ligand compound selected from The method comprises contacting the

Images