MR1 LIGANDS AND PHARMACEUTICAL COMPOSITIONS FOR IMMUNOMODULATION
20230099822 · 2023-03-30
Assignee
Inventors
- Gennaro DE LIBERO (Basel, CH)
- Lucia MORI (Basel, CH)
- Alessandro VACCHINI (Basel, CH)
- Andrew CHANCELLOR (Basel, CH)
- Julian SPAGNUOLO (Basel, CH)
- Qinmei YANG (Basel, CH)
Cpc classification
C07D239/70
CHEMISTRY; METALLURGY
International classification
G01N33/50
PHYSICS
C07D239/70
CHEMISTRY; METALLURGY
Abstract
The invention relates to a method for modulating an interaction between an MR1 polypeptide and an MR1-specific T cell receptor molecule, whereby a MR1 polypeptide is contacted with a MR1 ligand compound that is a nucleobase adduct product reflecting a state of metabolic distress of a cell.
The invention further relates to the use of compounds identified as MR1 ligands in vaccination or modulation of an MR1-restricted immune response.
Claims
1. A method for modulating an interaction between an MR1 polypeptide and an MR1-specific T cell receptor molecule, said method comprising contacting said MR1 polypeptide with a. An MR1 ligand compound described by any one of the following general formulas: ##STR00072## ##STR00073## wherein R.sup.1A is H or methyl, R.sup.1G is H or methyl; R.sup.N3 is H or methyl; R.sup.2 is selected from H, methyl and —S-methyl; R.sup.3U and R.sup.5U are selected from H and methyl; R.sup.5C is selected from H and methyl; R.sup.N1 and R.sup.N2 are both H or C.sub.1 to C.sub.3 alkyl, or R.sup.N1 is H or C.sub.1 to C alkyl (particularly R.sup.N1 is H or methyl) and R.sup.N2 is selected from a C.sub.1-C.sub.6 alkyl and a C.sub.2-C.sub.6 alkylene and a C.sub.1-C.sub.6 alkyl substituted carbamoyl, wherein the alkyl or alkylene is unsubstituted or substituted with carbonyl, carboxyl and/or hydroxyl, particularly R.sup.N1 is H and R.sup.N2 is selected from a methyl, 2-hydroxy-ethyl, 1-carboxethyl, 1,2-dicarboxy-ethyl, threonylcarbamoyl, isopent-2-enyl, cis-hydroxyisopent-2-enyl, 3-oxo-1-propenyl, and hexa-1,3,5-triene-1,1,3-tricarbaldehyde; or R.sup.N1, R.sup.N2 and the nitrogen together form a 2-oxa-8-azabicyclo [3.3.1] nona-3,6-diene-4,6-dicarbaldehyde bi-annular system; or R.sup.N3 and R.sup.1A together, or R.sup.N2 and R.sup.3C together, or R.sup.N1 and R.sup.1G together form an unsubstituted or C.sub.4 to C.sub.16-2-oxo-alkyl-substituted or □-carboxy-2-oxo-alkyl-substituted imidazole ring, or R.sup.N3 and R.sup.1A together, or R.sup.N2 and R.sup.3C together, or R.sup.N1 and R.sup.1G together are —C(CH.sub.3)OH—CHOH— or C(R′)OH— CH.sub.2—CHOH— or oxy-cyclopropylidene-malonaldehyde-substituted prop-2-ene with R′ being selected from H, CH.sub.3, CH(OH)C.sub.2H.sub.5, C.sub.2H.sub.5 and C.sub.4H.sub.9; or R.sup.N1 and R.sup.1G form a pyrimidine, or R.sup.N1 and R.sup.1G or R.sup.N3 and R.sup.1A form a 12-oxo-5,6,10,12-tetrahydro-3H-6,10-methano[1,3,5]oxadiazocine ring system, or R.sup.N1 and R.sup.1G form a 2-oxa-6,8-diazabicyclo [3.3.1] nona-3-ene-4-carbaldehyde bi-annular system; and R.sup.O is selected from H, unsubstituted or hydroxyl-substituted C.sub.1-C.sub.5 alkyl or C.sub.2-C.sub.5 alkylene, R.sup.X is selected from SH, C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkylene, and C.sub.1-C.sub.5 S-alkyl; R.sup.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; with the proviso that adenine, adenosine, deoxyadenosine, guanine, guanosine, deoxyguanosine, uracil, uridine, deoxyuridine, thymine, thymidine, deoxythymidine (5-methyluridine), cytosine and cytidine and deoxycytidine are not encompassed by the general formulas I, II, III and IV, b. or with an MR1 ligand compound selected from 3-methyladenine (41), 7-methyl-7-deaza-2′-deoxyguanosine (49), queuosine (33), wybutosine (34), hydroxywybutosine (35) or pseudouridine (36), or with (2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-(methylthio)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (40).
2. The method according to claim 1, wherein R.sup.R is described by the general formula (V) or (V-S) ##STR00074## wherein R.sup.B is the bond connecting the moiety to the N.sup.9 nitrogen of I, I-1, II, II-plus, II-1, II-1-plus, Ix, or to the N.sup.1 nitrogen of III, IV or IV-1, R.sup.2′ is selected from H, OH, OCH.sub.3, O-ribosyl and O-ribosyl-5″-phosphate, and R.sup.5′ is selected from H and PO.sub.3.sup.2−; particularly wherein R.sup.R is described by the general formula (V), more particularly wherein R.sup.R is described by the general formula (Va) ##STR00075## even more particularly wherein R.sup.R is described by the general formula (Vb: R.sup.2′ is OH) or (Vc: R.sup.2′ is 5′-phosphoribosyl) or (Vd: R.sup.2′ is H) or (Ve: R.sup.2 is ribosyl): ##STR00076##
3. The method according to claim 1, wherein the MR1 ligand compound is described by I-1 or the general formula IIa ##STR00077## wherein R.sup.N1, R.sup.R, R.sup.N2, R.sup.1A, R.sup.1G, R.sup.2′ and R.sup.5′ can have the meaning indicated above.
4. The method according to claim 1, wherein the MR1 ligand compound is described by formula I and R.sup.N1, R.sup.N2 and the nitrogen together form a 2-oxa-8-azabicyclo [3.3.1] nona-3,6-diene-4,6-dicarbaldehyde bi-annular system.
5. The method according to claim 1, wherein the MR1 ligand compound is described by a. formula (I) wherein R.sup.2 is S-methyl and R.sup.N1 and R.sup.N2 are both H; b. formula (I) wherein R.sup.2 is methyl and R.sup.N1 and R.sup.N2 are both H; or c. formula (I-1) wherein R.sup.1A is methyl, R.sup.2 is H and R.sup.N3 is H; or d. formula (I-1) wherein R.sup.1A is methyl, R.sup.2 is methyl and R.sup.N3 is H; or e. formula (I-1) wherein R.sup.1A is methyl, R.sup.2 is S-methyl and R.sup.N3 is H; or f. formula (I) wherein R.sup.2 is H, one of R.sup.N1 and R.sup.N2 is selected from H and methyl, and the other one of R.sup.N1 and R.sup.N2 is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl and threonylcarbamoyl; or g. formula (I) wherein R.sup.2 is S-methyl and one of R.sup.N1 and R.sup.N2 is selected from H and methyl, and the other one of R.sup.N1 and R.sup.N2 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.sup.2 is methyl and one of R.sup.N1 and R.sup.N2 is selected from H and methyl, and the other one of R.sup.N1 and R.sup.N2 is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl and threonylcarbamoyl; i. formula (II) wherein R.sup.1G is methyl, and R.sup.N1 and R.sup.N2 are both H; or j. formula (II-plus) wherein R.sup.1G is methyl, and R.sup.N1 and R.sup.N2 are both H; or k. formula (II-plus) wherein R.sup.1G is H, and R.sup.N1 and R.sup.N2 are both H; or l. formula (II) wherein R.sup.1G is methyl, and one of R.sup.N1 and R.sup.N2 is selected from H and methyl, and the other one of R.sup.N1 and R.sup.N2 is selected from methyl, ethyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl and threonylcarbamoyl; or m. formula (II-plus) wherein R.sup.1G is methyl, and one of R.sup.N1 and R.sup.N2 is selected from H and methyl, and the other one of R.sup.N1 and R.sup.N2 is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl and threonylcarbamoyl; or n. formula (II-plus) wherein R.sup.1G is H, and one of R.sup.N1 and R.sup.N2 is selected from H and methyl, and the other one of R.sup.N1 and R.sup.N2 is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl and threonylcarbamoyl; or o. formulas (I-IM or II-IM), with R.sup.IM selected from H, CH.sub.2COC.sub.nH.sub.(2n+1) with n from 3 to 7 (particularly n=5), and CH.sub.2CO(CH.sub.2).sub.mCOO.sup.− with m from 3 to 9 (particularly n=7) ##STR00078## or p. formulas (II-c), (II-d), (II-e) (II-f) ##STR00079## q. formulas (II-g) or (II-h), wherein R.sup.2 is selected from H, methyl and S-methyl: ##STR00080## r. formulas (II-i) (II-j), wherein R.sup.N1 is selected from H and methyl and wherein R.sup.2 is selected from H, methyl and S-methyl: ##STR00081## s. formula (III), wherein R.sup.3U is H and R.sup.5U is methyl, t. formula (III), wherein R.sup.3U is methyl and R.sup.5U is H, u. formula (III), wherein R.sup.3U and R.sup.5U are both methyl, v. formula (IV), wherein R.sup.5C is H, one of R.sup.N1 and R.sup.N2 is selected from H and methyl, and the other one of R.sup.N1 and R.sup.N2 is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl and threonylcarbamoyl; w. formula (IV-1), wherein R.sup.N2 is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl and threonylcarbamoyl; or x. formula (IV), wherein R.sup.5C is methyl, one of R.sup.N1 and R.sup.N2 is selected from H and methyl, and the other one of R.sup.N1 and R.sup.N2 is selected from methyl, 3-methylbut(2)enyl, 3-hydroxymethylbut(2)enyl and threonylcarbamoyl; y. formula (Ix), wherein R.sup.2 is H, and R.sup.X is selected from methyl and S-methyl; z. formula (I), wherein R.sup.2 is H, R.sup.N1 is H, R.sup.N2 is 3-oxo-1-propenyl; aa. formula (I-a), ##STR00082## bb. formula (II-k), ##STR00083## cc. formula (I-2), wherein R.sup.1A, R.sup.N1 and R.sup.2 are H, and R.sup.N2 is isopent-2-enyl, or cis-hydroxyisopent-2-enyl, dd. formula (I), wherein R.sup.N1 and R.sup.2 are both H, and R.sup.N2 is isopent-2-enyl, or cis-hydroxyisopent-2-enyl, ee. formula (II-1), wherein R.sup.O is methyl or ethan-2-ol, and R.sup.N1 and R.sup.2 are both H; ff. formula (I-b), ##STR00084##
6. (canceled)
7. (canceled)
8. (canceled)
9. The method according to claim 1, wherein 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-threonylcarbamoyladenosine (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-threonylcarbamoyladenosine (10) k. 2′-O-ribosyladenosinephosphate (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-dicarbaldehyde (13) n. 1-(3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-3H-imidazo[2,1-r] 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. 3-((2R,5R)-4-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-7-(2-oxoheptyl)-3H-imidazo[1,2-a]purin-9(5H)-one (25) x. 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one (26) y. N2-oxopropenyl-deoxyguanosine (27) z. 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) 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) gg. 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) hh. 3-methyladenine (41) ii. N6-methyladenosine (42) jj. 6-methylpurine (43) kk. 6-(dimethylamino)purine (44) ll. N6-(A2-isopentenyl) adenine (45) mm. N1-methyl-2′-deoxyguanosine (46) nn. 1-methylguanine (47) oo. N2-methyl-2′-deoxyguanosine (48) pp. 7-methyl-7-deaza-2′-deoxyguanosine (49) qq. 06-methyl-2′-deoxyguanosine (50) rr. N2-ethyl-2′-deoxyguanosine (51) ss. 5′-deoxy-5′-(methylthio)adenosine (52) tt. N6-methyl-2′-deoxyadenosine (53) uu. N6-(2-hydroxyethyl)-2′-deoxyadenosine (54) vv. 06-(2-hydroxyethyl)-2′-deoxyguanosine (55) ww. N6-succinyl adenosine (56) xx. 2-(2-((3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3,7-dihydropyrimido[2,1-,]purin-7-yl)oxy)cyclopropylidene)malonaldehyde (57); yy. pyrimido[1,2-a]purin-10(3H)-one (M.sub.1G) (24).
10. The method according to claim 1, wherein the method is selected from a. a method for the identification and isolation of T cells or T cell receptor molecules reactive to the MR1 ligand compound when the MR1 ligand compound is presented by an MR1 molecule, and b. a method for the identification and isolation of an antibody reactive to the MR1 ligand compound when the MR1 ligand compound is presented by an MR1 molecule, and c. a method of diagnosis, wherein a sample obtained from a patient is analyzed with regard to the presence of an MR1 ligand compound as identified herein, particularly wherein the MR1 ligand compound is presented by or associated with an MR1 molecule on a patient's cell, more particularly wherein the MR1 ligand compound is presented by or associated with an MR1 molecule on a patient's cancer cell d. a method of diagnosis, wherein a sample obtained from a patient is analyzed with regard to the presence of a T cell reactive towards an MR1 ligand compound as identified herein, wherein the MR1 ligand compound is presented by or associated with an MR1 molecule on a cell, particularly a patient's cell, more particularly wherein the MR1 ligand compound is presented by or associated with an MR1 molecule on a patient's cancer cell.
11. An MR1 ligand compound as specified in claim 1 for use in prophylaxis or treatment of a disease associated with an aberrant or absent MR1-specific T cell response, particularly in treatment of cancer characterized by tumor cells expressing MR1.
12. (canceled)
13. The MR1 ligand compound for use according to claim 11, wherein the compound is administered in association with (administered prior to, concomitant with or after administration of) a preparation comprising (transgenic) MR1-reactive T cells and/or a polynucleotide expression vector encoding MR1.
14. (canceled)
15. A method for identification of a T cell reactive to a MR1 ligand compound as specified in claim 1, said method comprising the steps providing a preparation of T cells reactive to/capable of specifically recognizing MR1; a. contacting said preparation of T cells with a complex comprising isolated MR1 associated to said compound; b. isolating a T cell that is specifically reactive to said MR1 ligand compound in an isolation step.
16. An isolated T cell receptor (TCR), particularly a TCR comprising an α chain of TCR and a β chain of a TCR, or a TCR comprising a γ chain of a TCR and a δ chain of a TCR, particularly an α chain of TCR and a β chain of a TCR; wherein the TCR is capable to specifically bind to an MR1 ligand compound as specified in claim 1 in association to an MR1 polypeptide, with the proviso that the TCR formed by association of SEQ ID NO 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 disclaimed; and wherein the TCR recognizes an MR1 ligand compound in association with MR1, and wherein the MR1 ligand compound is selected from: a. 1-methyladenosine (1); b. 2-methyladenosine (2); c. 2′-O-methyladenosine (3), with the proviso that the TCR composed of SEQ ID NO 22 and 34 is disclaimed; d. N.sup.6, N.sup.6-dimethyladenosine (4), with the proviso that the TCRs composed of SEQ ID NO 1 and 2, and of 22 and 34 are disclaimed; e. N.sup.6-threonylcarbamoyladenosine (5), with the proviso that the TCR composed of SEQ ID NO 22 and 34 is disclaimed; f. N.sup.6-isopentenyladenosine (6), with the proviso that the TCRs composed of SEQ ID NO 1 and 2, 16 and 28, and of 22 and 34 are disclaimed; q. N6-(cis-hydroxyisopentenyl) adenosine (7); h. 2-methylthio-N.sup.6-(cis-hydroxyisopentenyl) adenosine (8), with the proviso that the TCRs composed of SEQ ID NO 1 and 2, 16 and 28, and of 22 and 34 are disclaimed; i. 2-methylthio-N6-isopentenyladenosine (9); i. N6-methyl-N.sup.6-threonylcarbamoyladenosine (10), with the proviso that the TCR composed of SEQ ID NO 22 and 34 is disclaimed; k. 2′-O-ribosyladenosine (phosphate) (11); l. NV-(3-Oxo-1-propenyl)-2′-deoxyadenosine (12), with the proviso that the TCRs composed of SEQ ID NO 1 and 2, 16 and 28, and of 22 and 34 are disclaimed; m. 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarbaldehyde (13), with the proviso that the TCR composed of SEQ ID NO 16 and 28 is disclaimed; 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), with the proviso that the TCR composed of SEQ ID NO 22 and 34 is disclaimed; q. 2′-O-methylguanosine (18); r. N2, N2-dimethylguanosine (19), with the proviso that the TCR composed of SEQ ID NO 1 and 2 is disclaimed; s. 2′-O-ribosylguanosine phosphate (20), with the proviso that the TCR composed of SEQ ID NO 22 and 34 is disclaimed; 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), with the proviso that the TCR composed of SEQ ID NO 16 and 28 is disclaimed; 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), with the proviso that the TCR composed of SEQ ID NO 16 and 28 is disclaimed; v. 1-methylguanosine (24), with the proviso that the TCR composed of SEQ ID NO 22 and 34 is disclaimed; 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); v. 3-methyluridine (30), with the proviso that the TCR composed of SEQ ID NO 22 and 34 is disclaimed; z. 5-methyluridine (31); aa. 3,2′-O-dimethyluridine (32); bb. queuosine (33); cc. wybutosine (34); dd. hydroxywybutosine (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); qq. N4-(3-oxo-1-propenyl)-2′-deoxycytidine (38), with the proviso that the TCR composed of SEQ ID NO 22 and 34 is disclaimed; hh. 6-methylmercaptopurine (40), with the proviso that the TCRs composed of SEQ ID NO 16 and 28, 22 and 34, and of 24 and 36 are disclaimed; ii. N6-methyladenosine (42); jj. 6-Methylpurine (43); kk. 6-(Dimethylamino)purine (44); ll. N6-(A2-Isopentenyl) adenine (45); mm. 1-Methylguanine (47); nn. N2-Methyl-2′-deoxyguanosine (48); oo. 5′-Deoxy-5′-(methylthio)adenosine (52); pp. N.sup.6-Methyl-2′-deoxyadenosine (53), with the proviso that the TCR composed of SEQ ID NO 3 and 4 is disclaimed; and qq. N.sup.6-(2-Hydroxyethyl)-2′-deoxyadenosine (54), with the proviso that the TCR composed of SEQ ID NO 3 and 4 is disclaimed.
17. (canceled)
18. (canceled)
19. The isolated T cell receptor (TCR) according to claim 16, wherein the TCR recognizes the following compound in association with MR1: a. N6-isopentenyladenosine (6), particularly wherein the TCR comprises the CDRs comprised of SEQ ID 99 and 100, particularly wherein the TCR comprises the polypeptide chains characterized by SEQ ID 97 and 98; b. NP-(3-Oxo-1-propenyl)-2′-deoxyadenosine (12), particularly wherein the TCR comprises the CDRs comprised of SEQ ID 99 and 100, or 103 and 104, or 108 and 108, or 111 and 112, or 123 and 124, particularly wherein the TCR comprises the polypeptide chains characterized by SEQ ID 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-dicarbaldehyde (13), particularly wherein the TCR comprises the CDRs comprised of SEQ ID 99 and 100, or 103and 104, or 107and 108, or 111 and 112, or 123and 124, particularly wherein the TCR comprises the polypeptide chains characterized by SEQ ID 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), particularly wherein the TCR comprises the CDRs comprised of SEQ ID 99 and 100, or 103 and 104, or 111 and 112, particularly wherein the TCR comprises the polypeptide chains characterized by SEQ ID 97 and 98, or 101 and 102, or 109 and 110; e. N.sup.4-(3-oxo-1-propenyl)-2′-deoxycytidine (38), particularly wherein the TCR comprises the CDRs comprised of SEQ ID 111 and 112, or 123 and 124, particularly wherein the TCR comprises the polypeptide chains characterized by SEQ ID 109 and 110, or 121 and 122; f. 6-Methylmercaptopurine (40), particularly wherein the TCR comprises the CDRs comprised of SEQ ID 107 and 108, particularly wherein the TCR comprises the polypeptide chains characterized by SEQ ID 105 and 106.
20. An isolated T cell receptor (TCR) protein heterodimer comprising a TCR α chain and a TCR β chain, the TCR α chain and the TCR β chain each being characterized by a CDR3 sequence and the TCR protein heterodimer being 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; particularly wherein the TCR α chain and the TCR β chain are selected from the pairs of α chain and β 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 of 129 and 130, or a sequence at least 85% (≥90%, 95%, 98%) identical to said pair of α chain and β chain amino acid sequences, and having the same biological activity.
21. A polynucleotide encoding a TCR as claimed in claim 16, particularly wherein the polynucleotide is selected from a. a DNA expression vector; b. an RNA molecule, particularly a stabilized messenger RNA molecule c. a viral vector.
22. An isolated T cell expressing, particularly expressing recombinantly, the TCR according to claim 16.
23. The isolated T cell according to claim 22, for use in prophylaxis or treatment of a disease associated with an aberrant or absent MR1-specific T cell response, particularly for use in treatment of cancer.
24. The isolated T cell and/or the polynucleotide for use according to claim 23, wherein the disease is cancer characterized by MR1 expression, and wherein the isolated T cell and/or the polynucleotide are co-administered with the MR1 ligand compound.
25. (canceled)
26. An MR1 ligand compound as specified in claim 1 for use in the treatment of cancer, wherein the MR1 ligand compound is administered in association with (administered prior to, concomitant with or after administration of) an isolated T cell expressing an MR1 specific TCR comprising a pair of □ and □ CDR3 sequences identified by the same line of Table 3A, particularly an MR1 specific TCR constituted by SEQ ID NO 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.
27. The isolated T cell and/or the polynucleotide for use according to claim 23, wherein the isolated T cell expressing the TCR and/or the polynucleotide 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, tisopurine, topiroxostat, inositols (phytic acid and myo-inositol), particularly wherein the isolated T cell expressing the TCR and/or the polynucleotide is co-administered with a pharmaceutical compound selected from paclitaxel, doxorubicin, disulfiram, and MPA, more particularly wherein the co-administered pharmaceutical compound is selected from paclitaxel and doxorubicin, for treatment or prevention of a disease associated with aberrant or lacking MR1 expression, particularly treatment or prevention of recurrence of cancer disease associated with tumor cells expressing MR1.
28. A compound consisting of 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarbaldehyde.
Description
DESCRIPTION OF THE FIGURES
[0388]
[0389]
[0390]
[0391]
[0392]
[0394]
[0395]
[0396]
[0397]
[0398]
[0399]
[0400]
[0401]
[0402]
[0403]
[0404]
[0405]
[0406]
[0407]
[0408]
TABLE-US-00001 TABLE 1 List of exemplary active compounds with structures and references. Full name Short name CAS Number Molecular Formula Molecular Weight Ref. 1-methyladenosine (1) m1A 15763-06-1 C.sub.11H.sub.15N.sub.5O.sub.4 281.27 Ishiwata, Itoh et al. 1995 Seidel, Brunner et al. 2006 Cayman Chemical, Cat. 16937
[0409] All exemplary compounds were shown to interact with MR1, elicited MR1-restricted T cell responses and/or stabilized MR1 expression on cells as evidenced by results obtained by the assay methods as shown in the examples.
Examples
Material and Methods
Human Blood Samples
[0410] Blood and tissue specimens for T cell cloning, FACS analysis and antigen-presentation assays were obtained from the University Hospital Basel after informed consent, according to protocols EKNZ 2017-01888, which received ethical approval from the Swiss authorities (EKNZ, Ethics Committee North-West & Central Switzerland), and all patients and healthy donors consented in writing to the analysis of their samples.
Cell Lines
[0411] The cell lines used as antigen-presenting cells (APCs) in this study are A375 (ATCC CRL-1619), THP-1 (ATCC TIB-202), A375-MR1 and THP1-MR1, previously generated and described (Lepore et al 2017). The 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, 1×MEM NEAA and 50 μg/ml kanamycin (all from Bioconcept). The culture media for TF-1 cells was additionally 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 was previously characterized (Schmaler et al. (2018). Mucosal Immunology 11:1060-1070). Cells were free from Mycoplasma as evaluated by PCR analysis on DNA samples. When possible, cells were authenticated by staining with mAb for specific cell surface markers.
[0412] Lentiviral transductions were carried out as previously described. Transduced cells were selected by FACS sorting based on the expression of EGFP or mCherry reporters, or by 2 μg/mL puromycin resistance.
Human Knockout Library Screening
[0413] A375-MR1-Cas9 cells generated using the previously described cell line and Lenti Cas9-Blast plasmid (Addgene) were transduced at 0.3 MOI by both part A and B of the pooled Human GeCKO v2 CRISPR library (Addgene), and subsequently selected by 2 μg/mL puromycin (Calbiochem, Cat #540411) for 96 hours. Eight biological replicates of the resulting APCs, each with 64-fold representation of each guide within the library underwent 4 consecutive rounds of killing by TC5A87 cells at a E:T of 2:1, following which surviving cells were expanded for 24 hours and DNA extracted using the NucleoSpin Tissue kit (Macherey-Nagel, Cat #740952). An additional 8 biological replicates were similarly prepared but did not undergo killing by TC5A87 to act as controls. Illumina libraries were prepared as previously described. Briefly, primers JScrispr1 and JScrispr3 were used to amplify genomic gRNA from the extracted gDNA and attach common Illumina primer handles for attaching sequencing library indexes. Additionally, the former primer inserts an 8-nt degenerate sequence immediately downstream of the Illumina read 1 start site, decreasing issues of sequencing low-complexity libraries. Each replicate was barcoded by a unique pair of Nextera indexes (Illumina, Cat #15055290) in a second step PCR performed as described in the Nextera DNA library preparation protocol (Illumina). The high-fidelity Advantage HF2 PCR kit (Takara, Cat #639123) was used in each of the PCR steps involved in preparing the sequencing libraries. Libraries were quantified using the BioAnalyser high sensitivity DNA kit (Agilent, Cat #5067-4626) and Qubit high-sensitivity dsDNA kit (ThermoFisher, Cat #Q32851) and pooled to form an equimolar sequencing library that was denatured and diluted to 1.2 μM with 20% PhiX v3 control library (Illumina, Cat #FC-110-3001) as described in the Illumina Denature and Dilution protocol (Illumina) before sequencing on a NextSeq500 using the High-output 150-cycle v2 kit (Illumina, discontinued product). Both sets of sequencing libraries were sequenced using a dual-indexed single-end protocol (131 cycles on read 1, 8 cycles on each barcode) to a depth of 25 million reads per replicate, ensuring that guides depleted after T-cell mediated killing may be detected.
Isolation and Culture of Primary Cells from Human Blood and Tissue Samples
[0414] MR1T cells were isolated from the peripheral blood of healthy individuals. After PBMC separation by density gradient centrifugation, T cells were purified by negative selection using EasySep Human T Cell Enrichment Kit and stimulated with irradiated (80 Gray) A375-MR1 cells (ratio 2:1) and antigen once a week for three weeks. Human rIL-2 (5 U/mL) was added at day +2 and +5 after each stimulation. Twelve days after the final stimulation, cells were washed and co-cultured overnight with A375-MR1 cells (ratio 2:1) in the presence or absence of antigens.
[0415] CD3.sup.+ CD69.sup.+ CD137.sup.high cells were then FACS sorted and cloned by limiting dilution in the presence of phyto-haemagglutinin (1 μg/mL, Remel, Cat #30852801 HA16), human rIL-2 (100 U/mL,) and irradiated PBMC (5×10.sup.5 cells/mL). In some experiments, MR1T cells clones were isolated by limiting dilution of a FACS sorted CD3.sup.+, M.sub.3ADE-MR1-tetramer.sup.+ cells from a T cell line generated through expansion of purified T cells with A375-β2mKO-MR1 cells pulsed with synthetic Ag. T cell clones were periodically re-stimulated following the same protocol. PBMCs were isolated from peripheral blood by density gradient centrifugation and frozen in liquid N.sub.2 until use.
[0416] T cells, B cells, monocytes, myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) were purified from PBMCs using immunomagnetic separation with kits indicated in Key Resource table, according to manufacturer's protocol.
[0417] Tissue biopsy samples derived from small cell lung tumors that were digested with media containing Accutase (Innovative Cell; Cat #AT-104), Collagenase IV 200 U/mL (Worthington; Cat #LS004189), DNAse I 0.5 mg/mL (Sigma-Aldrich Cat #D5025) and Hyaluronidase 50 mg/mL (Sigma; Cat #H6254) for 1 hour at 37° C. Digested material was passed through a 70 μM cell strainer and erythrocytes were lysed before being frozen and stored in liquid nitrogen. After thawing, TILs were rested for 2 days prior to co-culture with A375-p2mKO-MR1 cells in a ratio of 1:1 and in the presence of 50 μM M.sub.3ADE. On day 5, human rIL-2 (5 U/mL) was added to the cultures for a further 5 days, and expansion in this manner was repeated 3 times. Cells were then stained with MR1-M.sub.3ADE tetramer, anti-CD3, anti-CD4 and anti-CD8 mAbs, and tetramer positive cells were sorted into a bulk line before functional experiments.
CRISPR-Cas9-Mediated Gene Disruption
[0418] Results obtained in the screening were confirmed by knock-out of selected genes in A375-MR1-Cas9 cells transduced with gRNAs different from the ones present in the library (Table 4). After lentiviral transduction and selection, A375-MR1-Cas9 cells were maintained for limited number of passages and used in activation assays as bulk population. THP-1 cells were cloned by limiting dilution and screened for GLO1 expression. Expression levels of target proteins was assessed by western blotting (
[0419] The Antigen presentation ability of different cell lines was tested by stimulation of MAIT clone MRC25 after pulsing APCs 2 h at 37° C. with indicated concentrations of freshly-prepared 5-OP-RU (
TCR Gene Transfer
[0420] The TCRα and β functional cDNA from MR1T clones were cloned into a modified version of the Lenti expression vector (Addgene, Cat #52962). Endogenous TCR-deficient SKW-3 or J76 cells were transduced with virus particle-containing supernatants generated as previously described (Lepore et al. 2017). Transduced cells were selected by FACS sorting based on CD3 expression, when necessary.
Preparation and Purification of Synthetic Antigens
[0421] Compounds M.sub.3ADE, OPdA, M.sub.1G, OPdC, M1dC, MGdA, MGG, m6,6A, io6A, m6t6A, Ar(p), M1dA, M3dA, ONEdA, m2,2G, Gr, CEdG, ONEdG, M1dG, N.sup.2OPdG, M2dG, m3U, m3Um, yW, OHyW, Psi, ONEdC, M3dC were synthesized and subsequently purified before use with cells. All other compounds were purchased from different vendors as indicated in Table 1.
Synthesis of 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarbaldehyde (M.SUB.3.ADE) (13)
[0422] M.sub.3ADE was synthesized as previously described (Stone et al., Chemical Research in Toxicology 3, 33-38 (1990)) with some modifications. 1,1,3,3-tetraethoxypropane (1.1 g, 5 mmol, 4.0 eq.) in aq. HCl (25 mL, 1 M) was stirred at 40° C. for 1 h. Subsequently, a solution of adenine (168.9 mg, 1.25 mmol, 1.0 eq.) in water (25 mL) was added. The mixture was adjusted to pH 4.0 with aq. NaOH (1 M) and stirred for 5 days at 37° C. M.sub.3ADE was purified by solid phase extraction over Sep-Pak C18 2 g cartridges (Waters Corp., Milford, Mass.). Cartridges were preconditioned with 10 mL water and 10 mL Acetonitrile. Raw M.sub.3ADE was washed with 20 mL water, 20 mL 10% Acetonitrile, then eluted with 20 mL 20% Acetonitrile.
[0423] M.sub.3ADE HPLC purification was performed on a JASCO RHPLC system equipped with an MD-4010 Photo Diode Array detector. Semi-preparative HPLC purification was performed using a 250×10 mm 5 μM NUCLEODUR C18 Pyramid HPLC column at a temperature of 23° C. where the mobile phases A and B were water and 95% methanol in water, respectively. Separation was performed with a flow rate of 6 mL/min with a linear gradient of 0-50% B from 0 to 15 min, 50-100% B from 15 to 38 min, 100% B from 38 to 43 min, 100-0% B from 43 to 44 min and 0% B until 50 min. M.sub.3ADE yield was 12.5 mg (42 μmol, 3.4%). Biologically active HPLC peaks were collected for mass spectrometric and NMR analyses.
[0424] .sup.1H-NMR (600 MHz, D.sub.2O, δ/ppm): 9.24 (s, 1H, H.sub.21), 9.11 (s, 1H, H.sub.19), 9.09 (s, 1H, H.sub.1s), 8.61 (s, 1H, H.sub.2), 8.38 (s, 1H, H.sub.8), 7.64 (s, 1H, H.sub.17), 7.33-7.29 (m, 1H, H.sub.11), 4.09-4.07 (m, 1H, H.sub.13), 2.21 (ddd, .sup.2J.sub.H12a-H12b=13.7 Hz, .sup.3J.sub.H12a-H11=2.8 Hz, .sup.3J.sub.H12a-H13=2.8 Hz, 1H, H.sub.12a), 2.03 (ddd, .sup.2J.sub.H12b-H12a=13.7 Hz, .sup.3J.sub.H12b-H11=2.2 Hz, .sup.3J.sub.H12b-H13=2.2 Hz, 1H, H.sub.12b).
[0425] .sup.13C-NMR (151 MHz, D.sub.2O, extracted from HSQC and HMBC, δ/ppm): 193.2 (C.sub.19), 192.2 (C.sub.21), 166.5 (C.sub.17), 154.3 (C.sub.4), 152.7 (C.sub.2), 150.3 (C.sub.6), 149.3 (C.sub.15), 144.8 (C.sub.8), 126.2 (C.sub.14), 125.7 (C.sub.16), 121.8 (C.sub.5), 79.9 (C.sub.11), 25.0 (C.sub.12), 17.4 (C.sub.13).
[0426] HR-ESI-MS: calcd. for [M+Na].sup.+ C.sub.14H.sub.11N5NaO.sub.3 m/z=320.0754, found 320.0758.
Synthesis of Pyrimido[1,2-a]purin-10(3H)-one (24) (M.SUB.1.G, CAS 103408-45-3
[0427] M.sub.1G was synthesized as previously described (Seto et al. Bulletin of the Chemical Society of Japan 58, 3431-3435 (1985).; Hadley and Draper, Lipids 25, 82 (1990).) with some modifications. 1,1,3,3-Tetraethoxypropane (1.4 g, 6.25 mmol, 5.0 eq.) in aq. HCl (25 mL, 1 M) was stirred at 40° C. for 1 hour. Subsequently, a solution of guanine (188.9 mg, 1.25 mmol, 1.0 eq.) in aq. 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 3 times with absolute ethanol at 2000×g for 10 min. The raw M.sub.1G was extracted 3 times from the precipitate with 65° C. water. The combined extracts were filtered with 0.22 μm filter. The mixture was adjusted to pH 7.0 with aq. NaOH (1 M).
[0428] HPLC analysis for M.sub.1G was performed on a JASCO RHPLC system. Semi-preparative HPLC purification was performed using a 250/10 NUCLEODUR C18 Pyramid HPLC column with a column temperature of 23° C. Solvent A was Milli-Q water, solvent B consisted of 95% methanol and 5% Milli-Q water. The total run was 55 min with a flow rate of 6 mL/min. The initial mobile phase was 100% Solvent A for 10 min. Solvent B increased linearly until the gradient reached 80% Solvent A and 20% Solvent B at 40 min. Solvent B was increased linearly again until it was briefly 100% at 41 min. Isocratic flow at 100% B for 5 min, a linear gradient to 100% Solvent A for 1 min and continuous for 8 min. Biologically active HPLC peaks were collected for mass spectrometric and NMR analyses (12.5 mg, 66.8 μmol, 5.3%).
[0429] .sup.1H-NMR (600 MHz, D.sub.2O, δ/ppm): 9.31 (d, .sup.3J.sub.H13-H12=7.2 Hz, 1H, H.sub.13), 8.97 (dd, .sup.3J.sub.H11-H12=4.1 Hz, .sup.4J.sub.H11-H13=2.0 Hz, 1H, H.sub.11), 8.22 (s, 1H, H.sub.8), 7.30 (dd, .sup.3J.sub.H12-H13=7.2 Hz, .sup.3J.sub.H12-H11=4.2 Hz, 1H, H.sub.12).
[0430] .sup.13C-NMR (151 MHz, D.sub.2O, extracted from HSQC and HMBC, δ/ppm): 162.9 (C.sub.11), 154.4 (C.sub.4), 154.1 (C.sub.6), 149.9 (C.sub.2), 146.0 (C.sub.8), 138.4 (C.sub.13), 116.7 (C.sub.5), 112.0 (C.sub.12).
[0431] HR-ESI-MS: calcd. for [M+H]+C.sub.8H.sub.6N.sub.5O m/z=188.0567, found 188.0571.
Synthesis of N.SUP.6.-(3-Oxo-1-propenyl)-2′-deoxyadenosine (12) (OPdA, CAS 178427-43-5)
[0432] OPdA was synthesized as previously described (Szekely et al., Nucleosides, Nucleotides and Nucleic Acids 27, 103-109 (2008)) with some modifications. 2′-deoxyadenosine monohydrate (219 mg, 0.813 mmol, 1 eq.) was dissolved in 2 mL anhydrous dimethyl sulfoxide under argon atmosphere. Propargyl aldehyde (12 μl, 11.0 mg, 0.203 mmol, 0.25 eq.) was added to the stirred solution and additional propargyl aldehyde (1.25 eq.) was added over a 72-h period. The reaction mixture was filtered and purified by preparative HPLC on a Shimadzu LC system (LC-20AT prominence liquid chromatograph, with an SPD-20A prominence UV/VIS detector (A=254 and 280 nm). Preparative HPLC purification was performed using a Reprosil-Pur 120 ODS 3, 5 μM, 150×20 mm column, where the mobile phases A and B were water and 90% acetonitrile in water, respectively. Separation was performed with 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 with a LC-20AD prominence liquid chromatograph combined with a Shimadzu LCMS-2020 liquid chromatograph mass spectrometer. Biologically active HPLC peaks were collected for mass spectrometric and NMR analyses. The OPdA yield was 13.5 mg (44.0 μmol, 5.4%).
[0433] .sup.1H-NMR (500 MHz, D.sub.2O, δ/ppm): 9.21 (d, .sup.3J.sub.H13-H12=8.7 Hz, 1H, H.sub.13), 8.49 (d, .sup.3J.sub.H11-H12=13.5 Hz, 1H, H.sub.11), 8.43 (s, 1H, H.sub.8), 8.38 (s, 1H, H.sub.2), 6.45 (dd, .sup.3J.sub.H1′-H2′a=6.8 Hz, .sup.3J.sub.H1′-H2′b=6.8 Hz, 1H, H.sub.1′), 5.89 (dd, .sup.3J.sub.H12-H11=13.5, .sup.3J.sub.H12-H13=8.7 Hz, 1H, H.sub.12), 4.65 (ddd, .sup.3J.sub.H3′-H2′a=6.1 Hz, .sup.3J.sub.H3′-H2′b=3.5 Hz, .sup.3J.sub.H3′-H4′=3.5 Hz, 1H, H.sub.3′), 4.19 (ddd, .sup.3J.sub.H4′-H5′b=3.8 Hz, .sup.3J.sub.H4′-H5′a=3.5 Hz, .sup.3J.sub.H4′-H3′=3.5 Hz, 1H, H.sub.4′), 3.86 (dd, .sup.2J.sub.H5′a-H5′b=12.5 Hz, .sup.3J.sub.H5′a-H4′=3.4 Hz, 1H, H.sub.5′a), 3.80 (dd, .sup.2J.sub.H5′b-H5′a=12.6 Hz, .sup.3J.sub.H5′b-H4′=4.3 Hz, 1H, H.sub.5′b), 2.80 (ddd, .sup.2J.sub.H2′a-H2′b=13.7 Hz, .sup.3J.sub.H2′a-H1′=7.1 Hz, .sup.3J.sub.H2′a-H3′=6.4 Hz, 1H, H.sub.2′a), 2.58 (ddd, .sup.2J.sub.H2′b-H2′a=14.0 Hz, .sup.3J.sub.H2′b-H1=6.3 Hz, .sup.3J.sub.H2′b-H3′=3.5 Hz, 1H, H.sub.2′b).
[0434] .sup.13C-NMR (126 MHz, D.sub.2O, extracted from HSQC and HMBC, δ/ppm): 195.7 (C.sub.13), 151.9 (C.sub.2), 151.1 (C.sub.11), 150.6 (C.sub.4), 148.9 (C.sub.6), 142.6 (C.sub.5), 120.8 (C.sub.5), 111.1 (C.sub.12), 87.5 (C.sub.4′), 84.7 (C.sub.1′), 71.1 (C.sub.3′), 61.6 (C.sub.5′), 39.1 (C.sub.2′).
[0435] HR-ESI-MS: calcd. for [M+Na].sup.+ C.sub.13H.sub.15N5NaO.sub.4 m/z=328.1016, found 328.1020.
Synthesis of N.SUP.4.-(3-Oxo-1-propenyl)-2′-deoxycytidine (38) (OPdC, CAS 129124-79-4
[0436] OPdC was synthesized as previously described (Szekely et al. ibid.) with some modifications. 2′-deoxycytidine (185 mg, 0.813 mmol, 1 eq.) was dissolved in 2 mL anhydrous dimethyl sulfoxide under argon atmosphere. Propargyl aldehyde (12.0 μL, 11.0 mg, 0.203 mmol, 0.25 eq.) was added to the stirred solution and additional propargyl aldehyde (1.25 eq.) was added over a 72-hour period. The reaction mixture was filtered and purified by prep.
[0437] HPLC purification was performed as described for OPdA. The OPdC yield was 7.00 mg (25.0 μmol, 3.1%). Biologically active HPLC peaks were collected for mass spectrometric and NMR analyses.
[0438] .sup.1H-NMR (500 MHz, D.sub.2O, δ/ppm): 9.31 (d, .sup.3J.sub.H10-H9=8.6 Hz, 1H, H.sub.10), 8.29 (d, .sup.3J.sub.H8-H9=13.7 Hz, 1H, H.sub.8), 8.19 (d, .sup.3J.sub.H6-H5=7.4 Hz, 1H, H.sub.6), 6.28 (d, .sup.3J.sub.H5-H6=7.4 Hz, 1H, H.sub.5), 6.24 (dd, .sup.3J.sub.H1′-H2′a=6.1 Hz, .sup.3J.sub.H1′-H2′b=6.1 Hz, 1H, H.sub.1′), 5.91 (dd, .sup.3J.sub.H9-H8=13.7 Hz, .sup.3J.sub.H9-H10=8.6 Hz, 1H, H.sub.9), 4.43 (ddd, .sup.3J.sub.H3′-H2′a=6.4 Hz, .sup.3J.sub.H3′-H2′b=4.3 Hz, .sup.3J.sub.H3′-H4′=4.3 Hz, 1H, H.sub.3′), 4.12 (ddd, .sup.3J.sub.H4′-H5′b=4.8 Hz, .sup.3J.sub.H4′-H5′a=4.1 Hz, .sup.3J.sub.H4′-H3′=4.1 Hz, 1H, H.sub.4′), 3.87 (dd, .sup.2J.sub.H5′a-H5′b=12.5 Hz, .sup.3J.sub.H5′a-H4′=3.5 Hz, 1H, H.sub.5′a), 3.77 (dd, .sup.2J.sub.H5′b-H5′a=12.5 Hz, .sup.3J.sub.H5′b-H4′=5.3 Hz, 1H, H.sub.5′b), 2.55 (ddd, .sup.2J.sub.H2′b-H2′a=14.1 Hz, .sup.3J.sub.H2′b-H1′=6.3 Hz, .sup.3J.sub.H2′b-H3′=4.3 Hz, 1H, H.sub.2′b), 2′0.32 (ddd, .sup.2J.sub.H2′a-H2′b=14.2 Hz, .sup.3J.sub.H2′a-H1′=6.5 Hz, .sup.3J.sub.H2′a-H3′=6.5 Hz, 1H, H.sub.2′a).
[0439] .sup.13C-NMR (126 MHz, D.sub.2O, extracted from HSQC and HMBC, δ/ppm): 196.2 (C.sub.1), 161.5 (C.sub.4), 156.8 (C.sub.2), 149.6 (C.sub.8), 144.3 (C.sub.6), 112.1 (C.sub.9), 96.8 (C.sub.5), 87.1 (C.sub.1′), 87.1 (C.sub.4′), 70.3 (C.sub.3′), 61.1 (C.sub.5′), 39.8 (C.sub.2′).
[0440] HR-ESI-MS: calcd. for [M+Na].sup.+ C.sub.12H.sub.15N.sub.3NaO.sub.5 m/z=304.0904, found 304.0902.
[0441] M.sub.1dC was produced by mixing 2′-deoxycytidine (25 mM, Sigma, Cat #D3897) with malonaldehyde tetrabutylammonium salt (100 mM). The mixture was incubated for 18 h at 70° C. under 400 rpm shaking. M.sub.1dC crude compound preparation was subjected to Solid Phase Extraction as above. M.sub.1dC was eluted with 20% acetonitrile.
[0442] Further HPLC purification was performed by reversed-phase HPLC with a C18 Pyramid column (Macherey-Nagel, Cat #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, A 100%; time 1 min, A 98%; time 43 min A 50%; time 46 min A 50%; time 47 min A 100%; time 56 min A 100%. Biologically active HPLC-separated compounds were collected for mass spectrometric and NMR analyses.
[0443] MGG was produced by mixing guanosine (100 mM, Sigma, Cat #G6752) with methylglyoxal solution (100 mM) in DMSO (33.3%, v/v in water, Sigma Cat #D4540). The mixture was incubated for 2 h at 70° C. under 400 rpm shaking. Further HPLC purification was performed by reversed-phase HPLC with a C18 Pyramid column (Macherey-Nagel, Cat #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%. Biologically active HPLC-separated compounds were collected for mass spectrometric and NMR analyses.
MS and NMR Analysis
[0444] Unless otherwise stated, chemicals were used as received without further purification. NMR analysis of all of the antigens was performed at 298 K on a Bruker Avance III NMR spectrometer operating at 500 MHz proton frequency equipped with a BBFO probehead or on a Bruker Avance III HD NMR spectrometer operating at 600 MHz proton frequency equipped with a cryogenic QCI-F probe. Standard pulse sequences were used for cosy, tocsy, noesy, hsqc, hmqc and hmbc 2D-NMR experiments and the spectra were processed using the topspin 4.0 software package. All new compounds were fully characterized by means of 2D-NMR and HiRes-ESI-MS. For all compounds .sup.1H- and .sup.1H-.sup.13C-HSQC spectra, as well as experimental and calculated HiRes-ESI-MS spectra were obtained (not shown) 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.
Cell Surface MR1 Upregulation
[0445] THP-1 cells (10.sup.5 cells/well) were tested for MR1 surface expression after incubation with or without synthetic compounds: M.sub.3ADE (1 μM), OPdA (100 μM), M.sub.1G (13 μM) and OPdC (100 μM) for 6 h at 37° C. Ac-6-FP (acetyl-6-formylpterin, 100 μM) (Schircks Laboratories, Cat #11.418) was used as positive control for MR1 surface upregulation. The cells were stained with an anti-human-MR1-APC mAb (clone 26.5) or with APC-labeled mouse IgG2a, k isotype control antibodies for 20 min at 4° C., then washed and analyzed by flow cytometry. For each condition, net MFI was calculated subtracting isotype MFI from anti-MR1 MFI and fold change of cells treated with synthetic molecules over cells treated with vehicle was calculated.
Activation Assay with Living or Fixed APCs
[0446] MR1T cells (5×10.sup.4/well unless otherwise indicated) were co-cultured with the indicated APCs (10.sup.5 cells per well unless otherwise indicated) for 18 h in 120 μL volume in triplicate. In some experiments, anti-MR1 mAbs (clone 26.5, purified and endotoxin-free mouse IgG2a, (Lepore et al., 2014)) or mouse IgG2a isotype control mAbs (LEAF, Biolegend, Cat #401504) (both at 30 μg/mL) were added and incubated for 30 min at 37° C. prior to the addition of T cells.
[0447] When nucleobases, nucleosides or nucleotides (all 250 μM) and synthetic compounds M.sub.3ADE, OPdA, M.sub.1G or OPdC were used to stimulate T cells, the THP-1 cells (10.sup.5/well) were cultured 2 h with the indicated molecules or medium only, prior to T-cell addition. When a single concentration of synthetic antigens was used, 100 μM OPdA, 100 μM OPdC and 13 μM M.sub.1G were used for all clones, while 100 μM M.sub.3ADE was used for all clones except DGB129, AC1A4, AC1B76 and AVA46 for which 1 μM was used.
[0448] In experiments with mycophenolic acid (10 μM), EHNA (25 μM), S-p-bromobenzylglutathione cyclopentyl diester (BBG, 20 μM) (all from Sigma-Aldrich), THP-1 cells (1×10.sup.6/mL) were treated with the indicated concentrations of drugs in complete medium at 37° C. for 18 h before being washed twice with PBS, counted and used for T-cell activation. In experiments where THP-1 cells (1×10.sup.6/mL) were treated with doxorubicin (75 nM) and paclitaxel (5 μM) (both from Sigma-Aldrich), the cells were incubated at 37° C. for 18 h before being washed twice with PBS, counted, and incubated for 2 h with vehicle or 150 μM dAdo and 150 μM guanosine (Sigma-Aldrich) prior to T-cell addition. In experiments where fixed A375-MR1 cells were used to activate MR1T cells, APCs (4×10.sup.5/mL) were treated with apocynin (APO, 100 μM), L-glutathione reduced (GSH, 4 mM), N-acetyl cysteine (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 salt (5 mM) for 18 h at 37° C. before being washed twice with PBS, fixed with glutaraldehyde, counted and used for MR1T cell stimulation.
[0449] In some experiments, MAIT cells were stimulated by APCs pulsed 3 h with 5-OP-RU as previously described or with 30 μM 6,7-dimethyl-8-ribityllumazine (Cayman Chemical Cat #23370).
[0450] To confirm that drugs reducing MR1T stimulation do not affect MR1 presentation ability, A375-MR1 cells treated with different molecules were collected before fixation and used to stimulate the MAIT clone MRC25 after pulsing for 2 h at 37° C. with the indicated concentrations of freshly-prepared 5-OP-RU or 6,7-dimethyl-8-ribityllumazine (Cayman Chemicals).
Activation Assay with Plate-Bound Soluble MR1
[0451] Recombinant human P2m-MR1-Fc was produced in CHO-K1 cells as previously described (Lepore et al., 2017) and 4 μg/mL were coated onto 96 wells plates (Nunc, Cat #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 antigens. Then, the synthetic antigens (M.sub.3ADE, OPdA, M.sub.1G and OPdC) were added at the indicated concentrations and incubated for 6 h at room temperature (RT). Unbound antigens were washed twice with PBS before the addition of excess PBS. In some experiments, bacteria-produced and refolded MR1-M.sub.3ADE protein was serially diluted in PBS and added to a high protein binding plate (Nunc, Cat #439454) for 2 h at 37° C. then washed twice and used in the stimulation assay. Indicated MR1T cell clones (105/100 μl/well) were added and supernatants were collected after 18 h. Released cytokines were detected by ELISA. Recombinant human P2m-MR1-Fc was produced in CHO-K1 cells as previously described and 4 μg/mL were coated onto 96 wells plates. Antigens produced by CHO-K1 cells were removed by washing twice with wash buffer (150 mM NaCl, 20 mM Tris and 2% Glycerol, pH 5.6). Synthetic antigens were diluted in wash buffer and incubated 3 h at RT. Antigens were washed out with wash buffer before the addition of MR1T cell clones (10.sup.5/well). Supernatants were collected after 18 h for cytokine analysis by ELISA.
Cytokine Analysis
[0452] The following human cytokines were assessed by ELISA using specific mAbs: GM-CSF (purified clone BVD2-23B6 and biotinylated clone BVD2-21C11, Biolegend Cat #502202 and 502304, respectively), IFN-γ (purified clone MD-1 and biotinylated clone 4S.B3, Biolegend Cat #507502 and 502504, respectively), IL-13 (purified clone JES10-5A2 and biotinylated clone SB126d, SouthernBiotech Cat #10125-01 and 15930-08, respectively).
MR1 Protein Production and Tetramerization
[0453] Soluble recombinant MR1 monomers were generated as previously described (Kjer-Nielsen, L., et al. (2012). Nature 491(7426): 717-723). Briefly, nucleotide sequences coding for the soluble portions of the mature human MR1 (GenBank accession number NM_001531) and mature human P2m (GenBank accession number NM_004048.3) were cloned into the bacterial expression vector pET23d (Novagen, Cat #69748-3). Transformed E. coli BL21(DE3)pLysS were then grown to ODeoonm 0.4-0.6 before induction with 0.6M isopropyl β-D-1-thiogalactopyranoside (Sigma-Aldrich, Cat #10724815001). After 4 h of further culture, cells were lysed and inclusion bodies were cleaned, purified and fully denatured in 8M Urea, 10 mM EDTA, 0.1 mM DTT for subsequent storage at −80° C.
[0454] For protein refolding, MR1 heavy chain (4 mM), β2m (2 mM) and compound (15 mM) were added to 1 L refolding buffer containing 0.4M L-arginine, 100 mM Tris pH 8.0, 2 mM EDTA that was cooled to 4° C. and supplemented with 5 mM reduced glutathione and 0.5 mM oxidized glutathione immediately beforehand. After 3 days, the refold mix was concentrated down to 1 mL and the refolded compound-bearing MR1 was purified by HPLC using Superdex 75 10/300 GL (GE Healthcare, Cat #17517401) and MonoQ 5/50 (GE Healthcare, Cat #17516601) columns.
[0455] Correct protein conformation was confirmed by performing a plate-bound activation of the 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 #439454) for 2 h at 37° C. The wells were washed thoroughly with PBS before addition of 5×10.sup.4 cells that were left overnight at 37° C. IL-13 ELISA was then used as an activation readout. The functional monomer was then biotinylated using BirA-500 biotinylation kit (Avidity, Cat #Bulk BirA) overnight at 4° C. Excess biotin was removed using S75 10/30 (GE Healthcare, 29148721) gel filtration before tetramerization with phycoerythrin (PE) streptavidin (Prozyme, Cat #P JRS25) in a 4:1 molar ratio. As control MR1 tetramers, human MR1-5-OP-RU and human MR1-6-FP labelled with APC, PE or AlexaFluor488 were used (The MR1 tetramer technology was developed jointly by Dr. James McCluskey, Dr. Jamie Rossjohn, and Dr. David Fairlie, and the material was produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne).
Immunofluorescence Staining
[0456] Cell surface labeling was performed using standard protocols. Intracellular labeling was performed using the True-Nuclear™ Transcription Factor Buffer Set (Biolegend, Cat #424401) according to the manufacturers' instructions. All mAbs for staining were titrated on appropriate cells before use. Biotinylated mAbs were revealed with streptavidin-PE (Biolegend, Cat #405204), -Alexa Fluor 488 (Biolegend, Cat #405235), or -Brilliant violet 421 (Biolegend, Cat #405226) all at 2 μg/mL.
[0457] When staining with tetramers, the cells were pre-treated with Dasatinib (50 nM, Sigma-Aldrich, Cat #CDS023389) for 30 min at 37° C. before first adding anti-CD8 mAbs (Biolegend, clone RP8-TA BV711) for 20 min at room temperature (RT), then 2.5 μg/mL tetramer for a further 20 min at RT without washing. All remaining mAbs were then added for a further 20 min at RT without washing. The cells were then washed in PBS before acquisition at the flow cytometer.
[0458] Samples were acquired on LSR Fortessa flow cytometer with the FACS Diva software (Becton Dickinson). Cell sorting experiments were performed using an Influx or FACSaria (Becton Dickinson). Dead cells and doublets were excluded on the basis of forward scatter area and width, side scatter, and DAPI (Sigma) or Live/Dead (Thermo Fisher Scientific) staining, as depicted in
ROS Production Measurement
[0459] CM-H.sub.2DCFDA (Thermo Fisher Scientific) was used to assess ROS production in cell upon cell treatment with Doxorubicin and Paclitaxel. THP-1 cells (107/mL) were labelled with 10 μM CM-H.sub.2DCFDA for 30 min at 37° C. in the dark, then washed with PBS and resuspended in complete medium. 10.sup.5 cells were seeded per well 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 positive control.
Quantification and Statistical Analysis
[0460] sgRNA-Seq Data Processing and Analysis
[0461] Raw sequencing data was demultiplexed using bcl2fastq (v2.17.1.14) and read quality checked using FastQC (v0.11.4). Reads were then trimmed to remove the homologous regions flanking the sgRNA sequences using Trimmomatic v0.36 using options HEADCROP:42 CROP:20. These trimmed reads were again passed through FastQC to check that average phred33 quality in the sgRNA sequences was >30. These reads were aligned to a GeCKO v2 sgRNA reference index using Bowtie2 (v2.2.9) with options—very-sensitive-local. Read counts were then extracted from the resulting SAM files using custom perl script map_count.pl (Cox, M. available on request) and imported into R (R Development Core Team, 2018) for analysis using edgeR.
[0462] Inequality of sgRNA activity in the GeCKO library hinders hit-selection using rank-based methodology, thus differential enrichment analysis was performed with the edgeR package (Dai et al., 2014) using the GLM Robust method to estimate dispersions, after removing guides targeting known essential genes. The level of random enrichment and depletion of guides was estimated using the log 2 fold-change in the top and bottom 1% of negative control guides in the GeCKO library. Thus, guides with an FDR<0.05 and log 2 fold-change greater or lower than the top and bottom 1% of negative control guides, respectively, were said to be significantly enriched or depleted by the inventors screen. GO-term and KEGG-pathway enrichment analysis was performed using a binomial test on the significant unique gene-targets identified in the differential enrichment analysis {Carlson, 2016 #2707}. Genes were annotated using biomaRt version 2.42.0.
CRISPR Screen Analysis
[0463] Data analysis, statistical tests and visualization were conducted in R and GraphPad Prism. After removing guides targeting known essential genes, differential enrichment analysis of CRISPR sequence data was performed using edgeR (v3.24) using the GLM Robust method to estimate dispersions. The level of random enrichment and depletion of guides was estimated using the log 2 fold-change in the top and bottom 1% of negative control guides in the GeCKO library. Thus, guides with FDR<0.05 and log 2 fold-change greater or lower than the top and bottom 1% of negative control guides, respectively, were said to be significantly enriched or depleted by the inventors screen. GO-term and KEGG-pathway enrichment analysis was performed using a binomial test on the significant unique gene-targets identified in the differential enrichment analysis. Genes were annotated using biomaRt version 2.42.0.
[0464] Statistical analysis was performed with Prism (GraphPad Software, Inc.) using multiple t-test, One- or Two-way Anova as indicated for each assay in Fig.legends.
[0465] A p value<0.05 was considered statistically significant. *p<0.05, **p 0.01, ***p 0.005.
Results
Compounds Stimulating MR1T Cells
[0466] The inventors' previous results showed that MR1T cells recognize MR1 molecules complexed with ligands present in tumor cells. The purification of cellular extracts of THP-1 cells, led them to the identification of compounds which can be defined as modified nucleobase and nucleobase adducts.
[0467] The inventors screened commercially available compounds using three types of biological assays. All three assays are based on the capacity of the compounds to bind MR1 and i) to modulate surface expression levels of MR1, ii) to activate in a specific manner at least one MR1T cell clone, or iii) to compete with the stimulatory compounds, thus affecting the response of MR1T cell clones. The biologically active compounds are reported in Table 1.
[0468] Examples of compound reactivity for each of the three functional assays indicated above, are illustrated in
Detection and Sorting of MR1T Cells by MR1 Tetramer Staining
[0469] As proof of concept to demonstrate the use of these novel compounds for the detection and capture of MR1T cells, the inventors selected the compound M.sub.3ADE. MR1 molecules containing the M.sub.3ADE were generated by in vitro refolding of human recombinant soluble MR1 produced in bacteria. Properly refolded MR1 monomers loaded with M.sub.3ADE were purified by gel filtration chromatography (
Single-Gene Knock-Outs in Metabolic Pathways Contribute to the Efficiency of T-Cell Mediated Killing
[0470] In previous studies the inventors isolated human T cells that recognize tumor cells expressing low levels of MR1 under sterile conditions. These MR1T cells, recognize tumor cells lines expanded in vitro or in vivo, indicating that stimulatory antigens preferentially accumulate in tumor cells, according to environmental conditions. Furthermore, individual MR1T cell clones showed patterns of tumor recognition suggesting the tumors bore shared and unique Ags that the inventors posited were of metabolic origin. Multiple approaches were used to identify these Ags.
[0471] In order to reveal key points within global metabolism contributing to the production of metabolite self-antigens, the inventors performed a genome-wide CRISPR knock-out screen.
[0472] An A375 melanoma tumor cell line that the inventors previously transfected with the MR1 and CAS9 genes (A375-MR1 cells), were used as a target for the cytotoxic MR1T cell clone TC5A87. After transduction with a library of sgRNAs covering the total human genome and three sequential killing rounds, surviving A375-MR1 cells were subjected to deep sequencing and enriched or depleted gRNAs were evaluated.
[0473] Prior to differential enrichment analysis, genes essential for growth of A375-MR1 cells were removed from the raw data to avoid confounding results with hits to gene-targets that are likely to be depleted in a non-T-cell dependent manner. It was also expected that many guides in the library would not affect the efficiency of T-cell mediated killing and therefore would display random enrichment and depletion. The GecKO v2 library contains non-targeting negative control guides, which should not display any significant enrichment or depletion in response to T-cell mediated killing, i.e. they should display random changes in abundance. Hence, a Log.sub.2 fold-change cutoff based on the top and bottom 1% of Log.sub.2 fold-changes in the negative control guides was used to identify guides that had been significantly enriched or depleted (FDR<0.05) more than background enrichment or depletion (i.e. random). The documented inequality of gRNA activity in the GeCKO library, hampers hit-selection using rank-based methodology, hence differential enrichment analysis was performed using the edgeR package. The inventors hypothesized knock-outs of genes involved in steps upstream of antigen biosynthesis will enhance the ability of A375-MR1 cells to escape T-cell mediated killing by reducing the generation of antigenic compounds. Conversely, knock-outs of genes involved in steps downstream of antigen biosynthesis will increase the accumulation of antigenic compounds and lead to increased T-cell mediated killing. Indeed, the results of differential enrichment analysis showed a fraction of enriched guides (n=243) that targeted 237 unique genes, including two-positive control genes, MR1 and beta-2-microglobulin (B2M) without which, antigens would not be presented to MR1-restricted T-cells. Among these genes the inventors also found enrichment of guides specific for adhesion molecules including CD58 (LFA-3), ICAM-1 that are the ligands of CD2 and CD11a on T cells, respectively. The latter interactions are important for T cell recognition of target cells and were also found in a previous CRISPR screening of tumor cell killing by immune cells.
[0474] Among the depleted hits, 5331 guides that targeted 4705 unique genes were significantly depleted relative to the control cells transduced with the gRNA library not subjected to TC5A87 killing. Since gene-essentiality is dependent on context and sensitive to many experimental parameters, it should be expected that not all essential genes were successfully removed using the Hart A375-MR1 essential gene-set. Furthermore, the number of genes required for tumor cell survival should far outweigh the number of genes that allow escape from T-cell mediated killing, thus, it is unsurprising that many more significantly depleted than enriched guides were observed. Binomial enrichment analysis of gene-ontology (GO) terms annotated to significant hits, revealed a large number of depleted gene targets sharing enriched GO terms in Metabolic Processes, suggesting that metabolic processes important for MR1T cell stimulation require coordinated activity of several genes. This was less evident in enriched gene targets. Furthermore, these significant hits showed enrichment in Nucleobase and Nucleic Acid Metabolic Processes, suggesting a potential role for these metabolic pathways in MR1T cell stimulation.
[0475] To explore the effect of the significantly enriched and depleted gene targets identified in the screen on global metabolism, the inventors used structural sensitivity analysis, which was recently extended from reaction-level to gene-level perturbations, to predict the metabolic network response to single-gene knock-outs of genes in Recon3D, a genome-scale model of human metabolism. The model was appropriately pre-processed.
[0476] Pearson correlations between the genome-wide reaction sensitivities of each modelled knock-out were then used to identify sets of 2 or more knock-outs that had similar (Pearson score>0.6) global effects on metabolic reactions. This analysis led to selection of 125 genes among the significantly enriched or depleted genes found in the CRISPR screening. When these selected genes were correlated to their corresponding KEGG pathways, it became apparent that Oxidative phosphorylation and Purine Metabolism can be perturbed by single-gene knockouts of a number of different gene targets. Additionally, all of these genes, except 2, were among the significantly depleted hits identified in the screen. Independently, binomial enrichment analysis of KEGG Pathways represented by the 4705 depleted gene targets in the CRISPR screen or by the 125 genes selected from the in silico metabolic model, identified that the Purine Metabolic Pathway is significantly enriched in both the CRISPR-screen hits as a whole and the subset of correlated Recon3D models (Binomial p-value=2.00e.sup.−3 and 1.67e.sup.−4. While not identified as a significantly enriched pathway in the hits from the CRISPR screen, both Oxidative phosphorylation and Glycerolipid pathways were significantly enriched in the metabolic perturbation model (Binomial p-value=6.58e.sup.−13 and 4.08e.sup.−4.
Nucleobase and Nucleoside Antigens Presented by Tumor Cells can Stimulate MRIT Cells
[0477] The inventors focused first on the purine pathway. Here the inventors identified several genes that might be involved in MR1T-cell antigen accumulation: 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 necessary for the de novo production of adenosine monophosphate (AMP) from inosine monophosphate (IMP); while LACC1 enables the purine nucleoside cycle, and PDE5A catalyzes the specific hydrolysis of cGMP to 5′-GMP. Lastly, ALDH16A1 and HPRT1 proteins form a complex that generates purine nucleotides through the purine salvage pathway. All together, these findings further support purines as molecules potentially involved in target recognition by MR1T cells.
[0478] To validate the relevance of these genes, the inventors generated knock-out lines, which were individually tested for their competence to stimulate MR1T cells. ADA- and LACC1-deficient cells induced an increased stimulation as compared to parental A375-MR1 cells as measured by IFN-γ release (
[0479] To investigate the possible role of purines in MR1T-cell stimulation, the inventors incubated human acute monocytic leukemia THP-1 cells with synthetic nucleotides, nucleosides or nucleobases before adding MR1T-cell clones and measuring IFN-γ production. THP-1 cells were selected as targets because they constitutively express low surface levels of MR1 and induce some spontaneous MR1T-cell stimulation, demonstrating their ability to appropriately process and present MR1T-cell antigens. A375-MR1 cells expressing very high surface levels of MR1 were also included as a positive control to stimulate MR1T-cell cytokine production. The inventors found that three distinct MR1T-cell clones reacted to different groups of compounds: TC5A87 did not significantly respond to tested compounds (
[0480] Taken together, these findings show that human MR1T cell clones can recognize nucleobase/nucleoside antigens processed by and presented on a cancer cell line, and that cross-reactivity might exist between different MR1T-cell clones.
Methylglyoxal and Purine Metabolism Pathways within Tumor Cells Cooperate for MR1T-Cell Stimulation
[0481] To understand which metabolic pathways might be involved in recognizing nucleobase/nucleoside antigens on tumor cells by MR1T cells the inventors began by interrogating the inventors whole-genome gene disruption screening data. The inventors observed that some of the significantly depleted sgRNAs were related to genes involved in glycolysis (TPI1) and methylglyoxal (MG) degradation, including Glyoxalase 1 (GLO1), and Glyoxalase Domain Containing 4 (GLOD4). TP/1 encodes a triosephosphate isomerase within the glycolytic pathway and is responsible for the enzymatic conversion of dihydroxyacetone phosphate (DHAP) into glyceraldehyde 3-phosphate (G3P)—a reaction that can otherwise occur spontaneously with the generation of MG (
[0482] The inventors then dissected the possible roles of glycolysis and MG degradation in MR1T stimulation by generating single gene KO cell lines. Loss of TPI1 in A375-MR1 cells significantly increased IFN-γ production by both MR1T-cell clones (
[0483] The inventors tested the IFN-γ response of MR1T cells to GLO1-modified THP-1 cells and various doses of MG or dAdo. The inventors found that MG treatment significantly increased MR1T-cell stimulation by GLO1-deficient compared to wild-type THP-1 cells (
Multiple Oxidative Stress-Related Carbonyl Species Accumulate within Tumor Cells and Contribute to MR1T Cell Stimulation
[0484] In addition to the purine pathway, the inventor's model-based analysis highlighted genes related to oxidative phosphorylation, whose protein products participate in ATP generation within mitochondria and whose alteration promotes accumulation of reactive oxygen species (ROS). Alongside, the analysis pointed towards the relevance of the H.sup.+ transporter subunits ATP6V1C2, TCIRG1 and ATP6V0D2 involved in coupling proton transport and ATP hydrolysis and thus contributing to maintaining the organelle physiological milieu in the cell, including mitochondria. Therefore, the inventors next investigated the roles of ROS in MR1T-cell stimulation by tumor cells.
[0485] First, the inventors focused on genes involved in oxidative phosphorylation. The inventors initial MR1T-cell killing screen uncovered a significant depletion of sgRNAs specific for GSTM1, GSTA4, GSTA1, GSTM5, GSTA2, GSTA3, GSTM3, and GSTO1; these genes are involved in the detoxification of electrophilic compounds and ROS by their conjugation to glutathione (GSH), a ROS scavenger. The inventors therefore hypothesized that in the absence of GSTs, and upon accumulation of ROS and electrophilic molecules, tumour cells may accumulate MR1T-cell stimulatory compounds. Accordingly, the inventors tested the effects of paclitaxel and doxorubicin, two drugs that induce cellular accumulation of O.sub.2.sup.− and H.sub.2O.sub.2. Both drugs significantly increased ROS accumulation (
[0486] Next, the inventors treated A375-MR1 with apocynin, an O.sub.2.sup.− scavenger and NADPH oxidase inhibitor, or with GSH or N-acetylcysteine (NAC), which prevent H.sub.2O.sub.2 accumulation before fixation and incubation with the three different MR1T cell clones. The inventors found that A375-MR1 cells treated with any of the inhibitors stimulated significantly less IFN-γ production from MR1T cells, with apocynin being effective with one T-cell clone (
[0487] Peroxide accumulates in many tumor types and is involved in various signal transduction pathways and cell fate decisions. Peroxide is also necessary for lipid peroxidation, a pathway that generates malondialdehyde (MDA) and 4-OH-nonenal (4-HNE), two highly reactive carbonyls. Both compounds form stable adducts with proteins, lipids and nucleobases and accumulate within tumor cells. Alongside the inventors findings that inhibiting ROS accumulation impedes tumor cell stimulation of MR1T cells (
[0488] To further assess the carbonyl involvement in the generation of MR1T antigens, the inventors tested the capacity of carbonyl scavengers to prevent MR1T-cell activation. A375-MR1 cells were incubated with aminoguanidine and hydralazine, which show preferential scavenging activity for different carbonyls, then fixed and washed before the addition of MR1T cell clones. The inventors found that both scavengers showed significant inhibition of IFN-γ production by MR1T cell clones (
[0489] Collectively, the inventor's data suggest that multiple oxidative stress-related reactive carbonyl species accumulating in cells following metabolic alterations combine with nucleobases to generate MR1-presented antigens that stimulate MR1T cells.
Evaluation of the Biological Activity of Compounds
[0490] Cell surface MR1 modulation was measured on THP-1 MR1 cells (10.sup.5 cells/well) after incubation for 3 h at 37° C. in the presence or absence of compounds (3 doses each). Ac-6-FP (100 μM, Schircks Laboratories Cat #11.418) was used as positive control compound for MR1 surface upregulation. MR1 expression was evaluated by staining with mouse mAbs anti-human-MR1 APC-labeled (IgG2a,k clone 26.5, Biolegend Cat #361108) and subtracting the background staining with APC-labeled mouse IgG2a,k isotype control antibodies (Biolegend Cat #400220), which was always below 300 MF.
[0491] Competition assays were performed by incubating APCs (10.sup.5 cells/well) with compounds (3 doses each) for 2 h at 37° C., then adding the antigen for each clone of interest at optimal concentration (≥EC.sub.50) and incubating for 2 additional h before the addition of T cells (10.sup.4 cells/well). As positive control for competition with antigens, Ac-6-FP was used (100 μM). Supernatants were collected after 24 h for cytokine analysis measured by ELISA.
Nucleobase Adducts Stimulate MR1T Cells
[0492] The biochemical condensation of carbonyl species with nucleobases is a unique feature of many cancer cell types, and leads to the generation of adducts. The inventors considered the possibility that nucleobase-adduct-containing compounds might be MR1T-cell antigens. To test this hypothesis, the inventors synthesized four previously described adducts: the purine adducts 8-(9H-purin-6-yl)-2-oxa-8-azabicyclo[3.3.1]nona-3,6-diene-4,6-dicarbaldehyde (M.sub.3ADE), N5-(3-oxo-1-propenyl)-2′-deoxyadenosine (OPdA), and pyrimido[1,2-a]purin-10(3H)-one (M.sub.1G), and the pyrimidine adduct, N.sup.4-(3-oxo-1-propenyl)-2′-deoxycytidine (OPdC), and investigated their antigenic activity (
[0493] Initial experiments showed that these compounds were capable of inducing MR1 upregulation on pulsed APC, with the exception of MGG (
[0494] T cell activation experiments using APC expressing low levels of MR1 showed that each compound was stimulatory for different MR1T cells (
[0495] When the inventors incubated THP-1 cells with the adducts the inventors found that they all induced a 1.5-5 fold increase in the average expression level of MR1 on the cell, as found with other MR1-binding compounds (
[0496] Next, the inventors sought to investigate whether these synthetic antigens were stimulatory without modifications inside APC. Plate-bound recombinant MR1 protein loaded with synthetic antigens efficiently stimulated specific MR1T clones, thus excluding a requirement for intracellular processing (
[0497] The inventors further confirmed the stimulatory capacity of these antigens by extending the activation assays to include additional MR1T-cell clones expressing different TCRs: of the fourteen randomly selected MR1T clones tested, eight reacted to at least one ligand. M.sub.3ADE significantly stimulated six clones, OPdA eight clones, M.sub.1G and OPdC three clones each (
[0498] The inventors then used plastic-bound recombinant MR1 molecules loaded with synthetic antigens to assess whether THP-1 cell processing of the adducts was needed for MR1T-cell recognition: the inventors found that it was not (
[0499] Next, the inventors asked how MR1T clones recognizing different synthetic compounds differ in their capacity to react to different tumor cell lines expressing physiological low levels of MR1. This question is relevant as preferential accumulation of unique carbonyl adducts in each tumor cell line may result in preferential stimulation of individual MR1T cells. The clone AVA34 activated by M.sub.3ADE, reacted specifically to KMOE-2 and HEL tumor cell lines; the clone QY1A16 that is stimulated by OPdA and OPdC selectively reacted to H460, Juso and KMOE-2 tumor cell lines; the clone AC1A4 that is activated by M.sub.3ADE, OPdA and M.sub.1G reacted to all six tested tumor cell lines, and the clone TC5A87 that is activated by OPdA, MGG, and OPdC reacted to all tumor cell lines (
[0500] In conclusion, MR1T cells recognize compounds containing intact carbonyl adducts of nucleobases presented on MR1 molecules. Some MR1T clones seem to be specific for one or another adduct; others show a degree of cross-reactivity. The broad recognition and the broad distribution of nucleobase adducts in many cancers may justify the broad reactivity of MR1T cells to tumors derived from different tissues.
MR1 Tetramers Loaded with Nucleobase Adduct-Containing Metabolites Detect MR1T Cells
[0501] To identify and characterize ex vivo MR1T cells reactive to nucleobase adduct-containing metabolites, the inventors generated MR1 tetramers loaded with the synthesized adducts. The inventors focused on M.sub.3ADE, as it was the most efficient in increasing MR1 surface expression, showed the highest potency, and was recognized by 6/14 tested MR1T-cell clones. The inventors performed several experiments to confirm proper protein refolding and MR1-M.sub.3ADE tetramer-specific MR1T-cell staining. MR1-M.sub.3ADE monomers stimulated clone DGB129 in plate-bound assays (
[0502] For further validation, the MR1-M.sub.3ADE tetramers were used to isolate MR1T cells from peripheral blood mononuclear cells (PBMCs) and establish a novel series of clones. These clones were able to bind MR1-M.sub.3ADE tetramers but not MR1-5-OP-RU or MR1-6-FP tetramers, demonstrated by the representative clone AVA34 (
[0503] The inventors next asked whether MR1-M.sub.3ADE tetramers could bind and identify specific MR1T cells ex vivo in the blood of healthy donors. Screening of peripheral blood mononuclear cells (PBMCs) from nine healthy individuals revealed tetramer-positive cells in all individuals with the frequencies ranging between 0.006% and 0.077% (median=0.01%) of total CD3.sup.+ cells (
[0504] Alongside the marked inter-individual differences in the frequency of MR1-M.sub.3ADE tetramer-binding T cells, the in inventors also uncovered notable phenotypic variations across the tetramer positive populations. While most cells expressed CD8α (range 38%-97%), one donor displayed a distinct CD4.sup.+ cell population that accounted for 57% of all tetramer-positive cells (
[0505] Thus, MR1T cells that recognize carbonyl-nucleoside-adducts are present in the blood of healthy individuals; they display a heterogeneous phenotype, and potentially undergo phenotypic differentiation in a donor-specific manner.
MR1T Cells Reactive to Nucleobase Adduct-Containing Metabolites Infiltrate Tumor Tissue
[0506] As all tested individuals possessed potentially M.sub.3ADE-reactive T cells in their blood, the inventors next asked whether M.sub.3ADE-reactive T cells could be detected in tumor samples. The inventors isolated TILs from non-small cell-lung-cancer biopsies from two patients and co-cultured them with A375-MR1 cells loaded with M.sub.3ADE, in order to expand MR1T cells. After expansion, the inventors were able to detect MR1-M.sub.3ADE tetramer-binding cells in TIL co-cultures from both patients (
Discussion
[0507] In this study, the inventors have identified nucleobase adduct-containing metabolites as self-antigens capable of stimulating human T lymphocytes recognizing MR1-expressing tumor cells. Previous studies showed that MR1T cells react to unique compounds fractionated from tumor cells, suggesting distinct antigen specificity. Here, the inventors confirm those data and extend them to show that structurally-diverse nucleobase adduct-containing compounds bind MR1 and stimulate individual MR1T cells. Both purines and pyrimidines form antigenic adducts and different carbonyls participate in their generation, confirming that MR1 is a molecule with versatile antigen-binding capacity.
[0508] Carbonyls accumulate as a consequence of different metabolic alterations in glycolysis and lipid peroxidation, during the metabolism of biogenic amines, vitamins and steroids, as well as upon biotransformation of environmental agents and drugs. How many carbonyls are involved warrants further investigation: this is an important question as the number and diversity of carbonyl species participating in the generation of MR1-presented nucleobase adducts may determine the size and variety of the MR1T cell antigen repertoire. It is of note that some MR1T clones did not react to any of the antigens tested here, whereas still responded to A375-MR1 cells, suggesting the MR1T cell antigen repertoire can be quite heterogeneous.
[0509] While carbonyl accumulation is important, it is not sufficient to stimulate MR1T cells, as concomitant availability of free purines and pyrimidines must occur within the target cells. The structures of modified nucleobases are suited to MR1 binding and resemble those of other MR1 ligands, as they are composed of differently modified heterocyclic compounds.
[0510] Important players in the generation of immunogenic nucleobase adducts are ROS—side products of oxidative phosphorylation that promote tumor induction and tumor cell proliferation. ROS also promote lipid peroxidation leading to carbonyl generation, thus also exerting indirect effects on accumulation of MR1T cell antigens. Indeed, treatment with ROS-inducing drugs enhanced MR1T-target cell stimulatory capacity, which in turn was dampened by the addition of ROS scavengers. Thus, MR1T-cell antigens are derived by combined alterations of multiple metabolic pathways, leading to the accumulation of nucleobases, carbonyls and ROS.
[0511] An important unanswered question in the field has been the basis of the specificity of tumor cell recognition by MR1T cells. The inventors' data provide a plausible answer to this, as most normal cells at steady state physiologically regulate the distinct metabolic pathways contributing to nucleobase adduct generation and accumulation. By contrast, tumor cells frequently become altered in many of these pathways sustaining cell proliferation, including alterations in glucose and glutamine uptake, and high cellular demand for reduced nitrogen. Indeed, many tumors increase the transcription of key genes involved in the de novo purine synthesis, suggesting a key role for purinosomes. Furthermore, tumor cells are prone to DNA damage through the generation of nucleic acid adducts formed upon DNA oxidation and interaction with end products of lipid peroxidation.
[0512] Importantly, in normal cells, several mechanisms are involved in scavenging highly reactive carbonyls, through which carbonyls are oxidized to carbonic acids, are conjugated with glutathione, or are reduced to less toxic alcohols. In the inventors' CRISPR/Cas9 screen, the inventors identified several genes involved in these processes that were conserved, suggesting that they contributed to escaping MR1T-cell recognition. The relevance of these controlling mechanisms is further supported by the negative effect of carbonyl scavengers on MR1T-stimulatory capacity of tumor cells.
[0513] MR1T-cell recognition of nucleobase adduct-containing metabolites raises the question of the physiological role of these cells. It is conceivable to attribute them a potential role in surveying cells abnormally accumulating compounds responsible for DNA alterations and therefore predisposed to dangerous genetic mutations. The ubiquitous expression of MR1 might be instrumental to this function of cellular metabolic integrity control. Together these properties of MR1T cells make them attractive targets for immunotherapeutic use in cancer. The inventors envisage the possibility of using selected MR1T TCR genes to redirect the specificity of cancer patient T cells toward these novel tumor-associated metabolite antigens, and therefore equip them with tumor-targeting capacity. The detection of MR1T cells within the tumor microenvironment in two lung cancer patients is promising evidence that supports the potential value of this strategy. Another possible application is the use of nucleobase adduct-containing metabolites as components of innovative anti-tumor vaccines. Importantly, the monomorphic nature of MR1 might offer the possibility to circumvent HLA-polymorphism and design T-cell-based immunotherapies applicable to the entire population of cancer patients on universal basis and independent of genetic background.
[0514] In conclusion, the immune system continues to surprise us with its capacity to detect a wide repertoire of structurally-variable antigens. T-cell recognition of nucleobase adduct-containing metabolites is the most recent evidence of this enormous flexibility.
REFERENCES
[0515] Geacintov, N. E. and S. Broyde (2010). The chemical biology of DNA damage. Weinheim, Wiley-VCH. [0516] 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. [0517] 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. [0518] 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. [0519] Marnett, L. J. (2002). “Oxy radicals, lipid peroxidation and DNA damage.” Toxicology 181-182: 219-222. [0520] Richarme et al. (2017). “Guanine glycation repair by DJ-1/Park7 and its bacterial homologs.” Science 357(6347): 208-211. [0521] 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. [0522] 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. [0523] Stone et al. (1990). “Investigation of the Adducts Formed by Reaction of Malondialdehyde with Adenosine” Chem. Res. Toxicol. 3: 33-38. [0524] Voulgaridou et al. (2011). “DNA damage induced by endogenous aldehydes: current state of knowledge.” Mutat Res 711(1-2): 13-27. [0525] Wauchope et al. (2015). “Nuclear Oxidation of a Major Peroxidation DNA Adduct, M.sub.1dG, in the Genome.” Chem Res Toxicol 28(12): 2334-2342. [0526] Lepore et al. (2017). Functionally diverse human T cells recognize non-microbial antigens presented by MR1. ELife 6: e24476. [0527] Lepore et al. (2014). Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRbeta repertoire. Nat Commun 5, 3866. [0528] Dai et al. (2014). edgeR: a versatile tool for the analysis of shRNA-seq and CRISPR-Cas9 genetic screens. F1000 Research 3, 95. [0529] Hart et al. (2015). High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities. Cell 163, 1515-1526. [0530] Brunk et al. (2018). Recon3D enables a three-dimensional view of gene variation in human metabolism. Nature Biotechnology 36, 272-281. [0531] Kanehis et al. (2019). New approach for understanding genome variations in KEGG. Nucleic Acids Res 47, D590-D595. [0532] Schmaler et al. (2018). Modulation of bacterial metabolism by the microenvironment controls MAIT cell stimulation. Mucosal Immunology 11: 1060-1070. [0533] Langmead, B., and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-359. [0534] Sanson et al. (2018). Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun 9, 5416. [0535] Bolger, A. M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120. [0536] Durinck et al. (2009). Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nature protocols 4, 1184-1191.
TABLE-US-00002 TABLE 2 Summary of compound functional activity determined to date Functional assay Cmpd MRI MRI Reactive T cells n° Cmpd short name upregulation downmodulation Competition isolated 1 m1A YES NO YES NO 2 m2A YES NO YES NO 3 Am YES NO NO YES 4 m6,6A or m6,2A YES NO NO YES 5 t6A YES NO YES YES 6 t6A YES NO YES YES 7 io6A NO NO YES NO 8 ms2io6A YES NO NO YES 9 ms2i6A NO NO YES NO 10 m6t6A YES NO YES YES 11 Ar(p) YES NO YES NO 12 M1dA or OPdA YES NO ND YES 13 M3ADE YES NO ND YES 14 ONEdA YES NO NO ND 15 me1G NO NO YES YES 16 m2G YES NO NO NO 17 m7G NO NO YES YES 18 Gm YES NO YES NO 19 m2,2G YES NO YES YES 20 Gr YES NO YES YES 21 MGG (6M) NO NO ND YES 22 MGG (7M) NO NO ND YES 24 M1G YES NO ND YES 25 ONEdG YES NO NO ND 29 Cm YES NO YES NO 30 m3U YES NO YES YES 31 m5U YES NO YES NO 32 m3Um YES NO YES NO 33 Q YES NO YES NO 34 yW YES NO YES NO 35 OHyW YES NO YES NO 36 Psi YES NO YES NO 37 ONEdC YES NO NO ND 38 M1dC or OPdC YES NO ND NO 40 6-MMPr YES NO NO YES 42 m6A YES NO YES NO 43 MeP NO YES YES NO 44 6DMAP YES NO YES NO 45 i6Ade NO YES YES NO 47 MeG NO YES NO NO 48 N2MedG YES NO NO NO 52 MTA NO NO YES NO 53 N6MedAdo NO NO ND YES 54 N6HEdA NO NO ND YES ND, not determined
TABLE-US-00003 TABLE 3A Designation of sequence ID Nos of MR1 specific TCRs of PCT/EP2019/074284 Protein Clone Clone α β Nucleic acid No Name α β CDR3 CDR3 α β 1-o MCA2E7 1 2 65 80 7 8 2-o MCA3C3 3 4 66 81 9 10 3-o CHO9A4 5 6 67 82 11 12 4-o DGA4 13 25 68 83 37 49 5-o DGA28 14 26 69 84 38 50 6-o DGB70 15 27 70 85 39 51 7-o DGB129 16 28 71 86 40 52 8-o CH9A3 17 29 72 87 41 53 9-o JM64-8 18 30 73 88 42 54 10-o JMA 19 31 74 89 43 55 11-o LMB1F3 20 32 75 90 44 56 12-o MCA3D9 21 33 76 91 45 57 13-o TC5A87 22 34 77 92 46 58 14-o SMC3 23 35 78 93 47 59 15-o TRA44 24 36 79 94 48 60 Clone Clone γ δ No Name γ δ CDR3 CDR3 γ δ 1-gdo MGDA1G5 61 62 95 96 63 64
TABLE-US-00004 TABLE 3B Designation of sequence ID Nos of new MR1 specific TCRs first disclosed herein Protein Clone Clone α β Nucleic acid No Name α β CDR3 CDR3 α β 1-N AC1A4 97 98 99 100 133 134 2-N AC1B76 101 102 103 104 135 136 3-N AVA34 105 106 107 108 137 138 4-N AVA46 109 110 111 112 139 140 5-N LMC1D1 113 114 115 116 141 142 6-N MCA2B1 117 118 119 120 143 144 7-N QY1A16 121 122 123 124 145 146 8-N QY1B42 125 126 127 128 147 148 9-N QY1C3 129 130 131 132 149 150
TABLE-US-00005 TABLE 4 gRNA target sequences and cloning vectors. Target SEQ gene gRNA sequence ID N Vector ADA CAGGCTTGATGGA 151 pLV-mCherry-U6 gRNA TCCGTCT ADA TCACCGTACTGTC 152 lentiGuide-Puro CACGCCG ADA GCGGTACAGTCCG 153 lentiGuide-Puro CACCTGC ADSSL1 TTCCAGGGGGGCA 154 lentiGuide-Puro ACAACGC ADSSL1 GCTGATGATGTCG 155 lentiGuide-Puro GCGTCCG ADSSL1 ACATACCGAAGTC 156 lentiGuide-Puro AATGTCG LACC1 CGTAGGTTGGCGA 157 lentiGuide-Puro ATGCTGC LACC1 TACCTTGGGATCT 158 lentiGuide-Puro CTCCGTT LACC1 TCAAGAAAATCTG 159 lentiGuide-Puro CGTAGGT GLO1 GAACCGCAGCCCC 160 pRP[gRNA]-EGFP: CGTCCGG P2A:Puro-U6 GLO1 GTCCGGCGGCCTC 161 pRP[gRNA]-EGFP: ACGGACG P2A:Puro-U6 TPI1 CGGCGAGGGCTTA 162 lentiGuide-Puro CCGGTGT TPI1 ACCGGTGTCGGCC 163 lentiGuide-Puro GGCACCT TPI1 CGAAGTCGATATA 164 lentiGuide-Puro GGCAGTA Scrambled GTGTAGTTCGACC 165 lentiGuide-Puro sequence ATTCGTG Scrambled GTTCAGGATCACG 166 lentiGuide-Puro sequence TTACCGC Scrambled AAATGTGAGATCA 167 lentiGuide-Puro sequence GAGTAAT