This application claims the benefit of US Patent Application US 63224054 filed 21.07.2021 which is incorporated herein by reference.

Field

The present invention relates to ligands specifically presented by MR1 molecules to MR1 -specific T cells. These ligands are derivatives or analogues of nucleic acid forming bases, particularly ribonucleoside and deoxyribonucleoside adducts occurring in eukaryotic cells under certain conditions. The invention further relates to pharmaceutical preparations and methods for use of such ligands in treatment and research. The invention further relates to pharmaceutical preparations provided with the aim of modulating presentation of MR1 ligands in clinical situations where such modulation in presentation of MR1 ligands is of clinical benefit.

Background of the Invention

MR1 (Uniprot ID 95460) is a non-polymorphic MHC class l-like protein that is expressed at low levels on the surface of most cell types. MR1 is highly conserved across multiple species, with human and mouse MR1 sharing >90% sequence homology at the protein level.

The inventors recently published their work confirming the existence of human T cells that recognize tumor-associated antigens (TAAs) presented by MR1 (Lepore et al., ELIFE 6, DOI:10.7554/el_ife.24476). These novel T cells participate in tumor immune surveillance, thus representing novel tools for cancer immunotherapy. The antigens recognized by these MR1- specificT cells, however, remain unknown.

Adoptive therapy with donor- or patient-derived T cells engineered to express T-cell receptors (TCRs) specific for selected TAAs represents a promising and safe strategy to induce clinically relevant anti-tumor immune response in cancer patients. Targeting TAAs bound to MR1 non- polymorphic antigen presenting molecules might overcome this constraint and in principle be applicable to all patients bearing tumors expressing MR1. The use of tumor-reactive TCRs that recognize MR1 -presented antigens might also have the advantage of complementing anti-tumor responses mediated by MHC-presented peptide antigens, excluding cross-competition of TAAs for binding to the same type of presenting molecule. In addition, this strategy may provide the possibility of targeting antigens of different nature on the same tumor cells, thus minimizing the potential occurrence of tumor escape variants under selective immune pressure.

The absence of information about the nature of the presented antigens, and the lack of tools available to probe, analyze and modulate the presentation of MR1, its interaction with antigen and with cognate TCRs, presents an obstacle in the development of improved MR1 -centred immune- oncological treatments. Therefore, the identification of MR1-presented TAAs and the identification and isolation of MR1- restricted TCRs recognizing these antigens might have important implications for cancer immunotherapy.

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to modulate MR1-TCR interaction to enable and improve clinical applications of MR1 -based immunotherapy. This objective is attained by the subject-matter of the independent claims of the present specification.

Summary of the Invention

A first aspect of the invention relates to a method for modulating an interaction between an MR1 polypeptide and an MR1-specific T cell receptor molecule. The method employs the interaction of a specific nucleobase adduct specifically presented on MR1, which the TCR specifically interacts with in the context of MR1 representation.

A second aspect of the invention relates to an MR1 ligand compound as specified in the first aspect 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.

A third aspect of the invention relates to a method for identification of a T cell reactive to a MR1 ligand compound as specified in the first aspect.

A fourth aspect of the invention relates to an isolated T cell receptor (TCR) capable to specifically bind to an MR1 ligand compound as specified according to the first aspect of the invention, in association to an MR1 polypeptide.

A fifth aspect of the invention relates to a polynucleotide encoding a TCR as claimed in aspect four. A sixth aspect of the invention relates to an isolated T cell expressing, particularly expressing recombinantly, the TCR according to the fourth aspect.

A seventh aspect of the invention relates to an isolated T cell according to the fourth aspect, or a polynucleotide encoding a TCR according to the fifth aspect, 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.

An eighth aspect of the invention relates to certain compounds as specified herein.

In another embodiment, the present invention relates a pharmaceutical composition comprising at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.

Terms and definitions

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”

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

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

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

The term MR1 in the context of the present specification refers to either the MR1 gene (Entrez 3140) or the MR1 gene product (Uniprot Q95460), also referred to herein as “MR1 polypeptide” or “MR1 molecule”. By way of non-limiting example, an MR1 polypeptide may be present in an aspect or embodiment disclosed herein as an isolated MR1 polypeptide, for example in the context of an MR1 polypeptide tetramer (Gherardin, Immunol Cell Biol. 2018 May;96(5):507-525) or expressed on a patient’s cells either naturally or in response to the transfer of an MR1-encoding gene construct. The term MR1T cell in the context of the present specification refers to a T cell that expresses a T cell receptor capable of binding specifically to an MR1 molecule presenting an antigen molecule as specified herein.

The term MR1T cell receptor in the context of the present specification refers to a T cell receptor capable of binding specifically to an antigen presented, for example by a cancer cell, in association with an MR1 molecule.

The expression of a marker such as MR1 may be assayed via techniques such as fluorescence microscopy, flow cytometry, ELISPOT, ELISA or multiplex analyses.

A TCR sequence orTCR molecule described herein comprises, to be fully functional, a TCR alpha and a TCR beta polypeptide chain, or a TCR gamma and a TCR delta polypeptide chain. If reference is made to a TCR alpha or beta polypeptide having a particular sequence, it is understood that in order for this to be fully functional in the methods and cells described herein, it requires the presence of a complementary (beta or alpha, respectively) polypeptide chain. The same applies, mutatis mutandis, to the gamma delta pairing. Mention of a specific TCR alpha, beta, gamma or delta sequence implies the possibility that it is paired with the TCR sequence with which it is paired in the original clone as described herein, or a sequence of certain identity to the original pairing sequence, as specified herein. Mention of a specific TCR alpha, beta, gamma or delta sequence also implies the possibility that it is paired with another pairing TCR sequence.

The recognition of MR1 -presented cancer antigens is effected mainly through CDR3 sequences. Wherein a TCR sequence characterized only by a specific CDR3 sequence is mentioned herein, it is implied that the TCR sequence is a full alpha, beta, gamma or delta TCR sequence as provided herein, and a resulting TCR molecule is paired with an appropriate second sequence.

The term compound in association with MR1 in the context of the present specification refers to a compound which is non-covalently bound by an MR1 molecule. Binding may occur, for example, via Van-der-Waals forces and electrostatic interactions, including hydrogen bonds.

The compound and the MR1 molecule form a complex, which may be recognized by a specific T cell receptor. Recognition by a specific T cell receptor means that the T cell receptor can differentiate between an MR1 molecule without association with the compound, and the MR1- ligand compound-MR1 complex.

Sequences

Sequences similar or homologous (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTAand TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).

One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.-2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.

Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence).

Cell Biology, diagnostic method inventions: Markers, ligands

In the present specification, the term positive, when used in the context of expression of a marker, refers to expression of an antigen assayed by a fluorescent labelled antibody, wherein the fluorescence is at least 5% higher (³5%), in median fluorescence intensity in comparison to staining with an isotype-matched antibody which does not specifically bind the same target. Such expression of a marker is indicated by a superscript “plus” ( ⁺ ), following the name of the marker, e.g. MR1 ⁺ . In the present specification, the term positive, when used for determination of cytokine secretion by a T cell, when contacted with an antigen presenting cell expressing MR1 in the presence of an MR1 ligand compound, is considered as relative to the T cell’s cytokine secretion in the same setting in absence of an MR1 ligand. The determination being positive if a statistically significant positive effect is found for the MR1 ligand, particularly if the difference is at least 2-fold, more particularly if the difference is at least 10-fold. Non-limiting examples of cytokines known to be released upon T cell activation include, IL-2, IL-4, IL-9, IL-13, IL-17, IFN-gamma, GM-CSF, MIP-1 beta, TNF alpha, Granzyme B.

In the present specification, the term negative, when used in the context of expression of a marker, refers to expression of an antigen assayed by a fluorescent labelled antibody, wherein the median fluorescence intensity is less than 5% higher than the median fluorescence intensity of an isotype- matched antibody which does not specifically bind the same target. Such expression of a marker is indicated by a superscript minus ( ^' ), following the name of the marker, e.g. MR1\

Binding; Binders Ligands Antibodies:

The term specific binding in the context of the present invention refers to a property of ligands that bind to their target with a certain affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A specifically reactive ligand has a dissociation constant of < 10 ^'7 mol/L when binding to its target, but a dissociation constant at least three orders of magnitude higher in its interaction with a molecule having a globally similar chemical composition as the target, but a different three-dimensional structure.

(Cancer) Immunotherapy

In the context of the present specification, the term cancer immunotherapy, biological or immunomodulatory therapy is meant to encompass types of cancer treatment that help the immune system to fight cancer. Non-limiting examples of cancer immunotherapy include immune checkpoint inhibitors and agonists, T cell transfer therapy, cytokines and their recombinant derivatives, adjuvants, and vaccination with small molecules or cells.

In the context of the present specification, the term checkpoint inhibitory agent or checkpoint inhibitor antibody is meant to encompass a cancer immunotherapy agent, particularly an antibody (or antibody-like molecule) capable of disrupting an inhibitory signalling cascade that limits immune cell activation, known in the art as an immune checkpoint mechanism. In certain embodiments, the checkpoint inhibitory agent or checkpoint inhibitor antibody is an antibody to CTLA-4 (Uniprot P16410), PD-1 (Uniprot Q15116), PD-L1 (Uniprot Q9NZQ7), B7H3 (CD276; Uniprot Q5ZPR3), VISTA (Uniprot Q9H7M9), TIGIT (UniprotQ495A1), TIM-3 (HAVCR2, Uniprot Q8TDQ0), CD158 (killer cell immunoglobulin-like receptor family), TGF-beta (P01137).

In certain embodiments, the cancer immunotherapy agent is selected from the clinically available antibody drugs ipilimumab (Bristol-Myers Squibb; CAS No. 477202-00-9), nivolumab (Bristol-Myers Squibb; CAS No 946414-94-4), pembrolizumab (Merck Inc.; CAS No. 1374853-91-4), pidilizumab (CAS No. 1036730-42-3), atezolizumab (Roche AG; CAS No. 1380723-44-3), Avelumab (Merck KGaA; CAS No. 1537032-82-8), Durvalumab (Astra Zenaca, CAS No. 1428935-60-7), and Cemiplimab (Sanofi Aventis; CAS No.

1801342-60-8).

In the context of the present specification, the term checkpoint agonist agent or checkpoint agonist antibody is meant to encompass a cancer immunotherapy agent, particularly but not limited to an antibody (or antibody-like molecule) capable of enhancing an immune cell activation signalling cascade. The term checkpoint agonist agent further encompasses cytokines, recombinant immune stimulatory proteins, vaccines, adjuvants and agonist antibodies that promote immune activation. Non-limiting examples of cytokines known to stimulate immune cell activation include, IL-12, IL-2, IL-15, IL-21 and interferon-alpha. In certain embodiments, the checkpoint agonist agent or checkpoint agonist antibody is an antibody to CD122 (Uniprot P14784) and CD137 (4-1BB; Uniprot Q07011), ICOS (Uniprot Q9Y6W8), 0X40 (GP34, Uniprot P43489), or CD40 (Uniprot P25942).

In certain embodiments, the cancer immunotherapy is meant to encompass immune cell transfer cancer treatments wherein a patient’s immune cells are activated or expanded in vitro, and/or genetically modified, for example with the addition of a chimeric antigen receptor, before being infused back into the patient to inhibit neoplastic disease. Non-limiting examples of immune cell transfer therapy include chimeric antigen receptor T lymphocytes, and autologous activated T cells or dendritic cells.

In the context of the present specification, the term checkpoint inhibitory agent or checkpoint inhibitory antibody is meant to encompass an agent, particularly an antibody (or antibody-like molecule) capable of disrupting the signal cascade leading to T cell inhibition after T cell activation as part of what is known in the art the immune checkpoint mechanism. Non-limiting examples of a checkpoint inhibitory agent or checkpoint inhibitory antibody include antibodies to CTLA-4 (Uniprot P16410) such as exemplified by ipilimumab (Yervoy; CAS No. 477202-00-9), or antibodies to PD- 1 (Uniprot Q15116) or to PD-L1 (Uniprot Q9NZQ7), B7H3 (CD276; Uniprot Q5ZPR3), such as exemplified by the clinically available antibody drugs nivolumab (Bristol-Myers Squibb; CAS No 946414-94-4), pembrolizumab (Merck Inc.; CAS No. 1374853-91-4), pidilizumab (CAS No. 1036730-42-3), atezolizumab (Roche AG; CAS No. 1380723-44-3), and Avelumab (Merck KGaA; CAS No. 1537032-82-8).

The term having substantially the same biological activity in the context of the present specification relates, when used to define a TCR molecule capable of recognizing an MR1 ligand bound to an MR1 molecule, to the capability to recognize (or contribute in the recognition of) its cognate ligand (the MR1 ligand associated with MR1). Assays and methods to determine such interaction are described herein.

The term nucleic acid expression vector in the context of the present specification relates to a polynucleotide, for example a plasmid, a viral genome ora synthetic RNA molecule, which is used to transfect (in case of a plasmid or an RNA) or transduce (in case of a viral genome) a target cell with a certain gene of interest. In the case of a DNA expression construct, the gene of interest is under control of a promoter sequence and the promoter sequence is operational inside the target cell, thus, the gene of interest is transcribed either constitutively or in response to a stimulus or dependent on the cell’s status. In the case of an RNA expression construct, it is understood that the term relates to translation of the RNA and the construct can be employed by the target cell as an mRNA. In certain embodiments, the viral genome is packaged into a capsid to become a viral vector, which is able to transduce the target cell.

The term transgenic MR1 -reactive T cell in the context of the present invention relates to an autologous or allogeneic T cell expressing a T cell receptor (TCR) that specifically recognizes an MR1 molecule expressed on a patient’s cell. In certain embodiments, the TCR recognizes MR1- expressing tumour cells in the absence of any added foreign antigen and in MR1 -dependent manner. MR1 -restricted TCR sequences are disclosed in PCT/EP2019/074284 and US 20190389926 A1, both of which are incorporated herein by reference.

As used herein, the term pharmaceutical composition refers to a compound of the invention, ora pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.

As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow.

Detailed Description of the Invention

A first aspect of the invention relates to a method for modulating an interaction between an MR1 polypeptide and an MR1-specificT 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:

- R ^R is selected from H, 1 ’-ribosyl, 2’-deoxy-1’-ribosyl, 5’-phospho-1’-ribosyl, 5’- methylthio-1 ’-ribosyl, 1’-(2’-0-ribosyl-5”-phosphate)ribosyl, 1’-(2’-0-ribosyl)-ribosyl, and 1’-(2’-0-methyl)ribosyl, particularly R ^R is selected from H, 1 ’-ribosyl, and 2’- deoxy-1 ’-ribosyl;

- each R ^M is independently selected from H and methyl, particularly R ^M is methyl;

- R ^x is selected from

R ^z is selected from

In certain embodiments, R ^R is 1 ’-ribosyl.

In certain embodiments, the MR1 ligand compound is described by a formula selected from l-XXII. In certain embodiments, the MR1 ligand compound is described by a formula selected from l-XX. In certain embodiments, 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.

A second aspect of the invention relates to an MR1 ligand compound as specified in the first aspect 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.

In certain embodiments, the compound is co-administered with an anticancer drug, and/or checkpoint modulator agent.

In certain embodiments, 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 an MR1 -reactive T cell receptor or a polynucleotide expression vector encoding MR1.

In certain embodiments, the MR1 ligand and/or the polynucleotide expression vector encoding an MR1 -reactive TCR or an MR1 are administered into a tumour, into the vicinity of the tumour, or into a lymph node draining the tumour site.

A third aspect of the invention relates to a method for identification of a T cell reactive to a MR1 ligand compound as specified in the first aspect, said method comprising the steps a. providing a preparation of T cells reactive to / capable of specifically recognizing MR1 ; b. contacting said preparation of T cells with a complex comprising isolated MR1 associated to said compound, or a cell comprising MR1 associated to said compound; c. isolating (or characterizing or identifying) a T cell that is specifically reactive to said MR1 ligand compound in an isolation step.

A fourth aspect of the invention relates to an isolated T cell receptor (TCR), particularly a TCR comprising an a chain of TCR and a b chain of a TCR, or a TCR comprising a g chain of a TCR and a d chain of a TCR, particularly an a chain of TCR and a b chain of a TCR; wherein the TCR is capable to specifically bind to an MR1 ligand compound as specified in the first aspect 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, 61 and 62, 97 and 98, 101 and 102, 105 and 106, 109 and 110, 113 and 114, 117 and 118, 121 and 122, 125 and 126, and 129 and 130 are disclaimed.

In certain embodiments, the determination whether the TCR specifically binds to an MR1 ligand compound is made by determination of cytokine secretion, wherein the cytokine comprises and is not limited to at least one of the group comprising IFN gamma, GM-CSF, MIP-1 beta, TNF alpha, Granzyme B, IL-2, IL-4, IL-9, IL-13, and IL-17, of a T cell bearing said TCR when contacted with an antigen presenting cell expressing MR1 in the presence of said MR1 ligand compound, relative to the T cell’s cytokine secretion in the same setting in absence of MR1 ligand, the determination being positive if a statistically significant positive effect is found for the MR1 ligand, particularly if the difference is at least 2-fold, more particularly if the difference is at least 10-fold.

A fifth aspect of the invention relates to a polynucleotide encoding a TCR as claimed in aspect four, particularly wherein the polynucleotide is selected from b. a DNA expression vector; c. an RNA molecule, particularly a stabilized messenger RNA molecule d. a viral vector.

A sixth aspect of the invention relates to an isolated T cell expressing, particularly expressing recombinantly, the TCR according to the fourth aspect.

A seventh aspect of the invention relates to an isolated T cell according to the fourth aspect, or a polynucleotide encoding a TCR according to the fifth aspect, 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.

In certain embodiments, the disease is cancer characterized by MR1 expression.

In certain embodiments, the isolated T cell and/or the polynucleotide are co-administered with an MR1 ligand compound as specified in the first aspect.

In certain embodiments, the isolated T cell expressing the TCR and/or the polynucleotide is coadministered 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), 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.

In certain embodiments, the isolated T cell expressing the TCR and/or the polynucleotide is coadministered with a pharmaceutical compound selected from paclitaxel, doxorubicin, disulfiram, and MPA.

In certain embodiments, the co-administered pharmaceutical compound is selected from paclitaxel and doxorubicin.

An eighth aspect of the invention relates to a compound of any one of the following formulas: a. Formula VIII 5-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofu r-2-yl]-3- (2-oxoheptyl)-1,3a,5-triaza-5H-inden-4-one b. Formula IX (S)-2-{9-[(2R,4S,5R)-4-Hydroxy-5-(hydroxymethyl)tetrahydrofu r-2-yl]-N- adenineyljsuccinic acid c. Formula X 4-{9-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydr ofur-2-yl]-N- adenineyl}-1 ,2,3-butanetriol d. Formula XII 1-{3-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydr ofur-2-yl]- 1 ,3,4,5a,8-pentaaza-3H-as-indacen-6-yl}-2-heptanone e. Formula XI 4-{9-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydr ofur-2-yl]-2- (methylthio)-N-adenineyl}-1,2,3-butanetriol f. Formula XIII 10-{5-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahyd rofur-2-yl]- 4-oxo-1 ,3a,5-triaza-5H-inden-3-yl}-9-oxodecanoic acid g. Formula XIV 1-{9-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydr ofur-2-yl]- N-adenineyl}-2-hydroxy-1-ethanone h. Formula XV 3-{9-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydr ofur-2-yl]-N- adenineyl}propionaldehyde i. Formula XVI 10-{3-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahyd rofur-2-yl]- 1 ,3,4,5a,8-pentaaza-3H-as-indacen-6-yl}-9-oxodecanoic acid j. Formula XVII first structure ribose 1-{9-[(2R,3R,4S,5R)-3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-isopentan-1- on k. Formula XVII second structure ribose 5-{9-[(2R,3R,4S,5R)-3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}pentyl (E)-3-pentenoate

L. Formula XVII second structure deoxyribose 5-{9-[(2R,4S,5R)-4-Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}pentyl (E)-3-pentenoate m. Formula XVII third structure ribose 1-(2-{9-[(2R,3R,4S,5R)-3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-2-oxoethylam ino)-2-amino-1-ethanone n. Formula XVII third structure deoxyribose 1-(2-{9-[(2R ,4S,5R) -4-Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-2-oxoethylam ino)-2-amino-1-ethanone o. Formula XVII fourth structure ribose (S)-4-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-3-amino-4-ox obutyramide p. Formula XVII fourth structure deoxyribose (S)-4-{9-[(2R ,4S,5R) -4-Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-3-amino-4-ox obutyramide q. Formula XVII fifth structure ribose (S)-4-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-3-amino-4-ox obutyric acid r. Formula XVII fifth structure deoxyribose (S)-4-{9-[(2R ,4S,5R) -4-Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-3-amino-4-ox obutyric acid s. Formula XVII sixth structure ribose (S)-5-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-4-amino-5-ox ovaleramide t. Formula XVII sixth structure deoxyribose (S)-5-{9-[(2R ,4S,5R) -4-Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-4-amino-5-ox ovaleramide u. Formula XVII seventh structure ribose (S)-5-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-4-amino-5-ox ovaleric acid v. Formula XVII seventh structure deoxyribose (S)-5-{9-[(2R ,4S,5R) -4 Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-4-amino-5-ox ovaleric acid w. Formula XVII eighth structure ribose 1-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-5-guanidino- 1,2-pentanedione x. Formula XVII eighth structure deoxyribose 1-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-5-guanidino- 1 ,2-pentanedione y. Formula XVII nineth structure ribose (E)-1-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-5-guanidino- 2-penten-1-one z. Formula XVII nineth structure deoxyribose (E)-1-{9-[(2R ,4S,5R) -4 Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-5-guanidino- 2-penten-1-one aa. Formula XVII tenth structure ribose 1-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-6-guanidino- 1 ,2-hexanedione bb. Formula XVII tenth structure deoxyribose 1-{9-[(2R ,4S,5R) -4 Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-N-adenineyl}-6-guanidino- 1,2-hexanedione cc. Formula XVIIb first structure deoxyribose 9-[(2R ,4S,5R) -4 Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-2-isovalerylamino-1,9-dih ydropurin-6-one dd. Formula XVIIb second structure ribose 5-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin-2- ylamino}pentyl (E)- 3- pentenoate ee. Formula XVIIb second structure deoxyribose 5-{9-[(2R ,4S,5R) -4 Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin-2- ylamino}pentyl (E)- 3- pentenoate ff. Formula XVIIb third structure ribose 9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5-

(hydroxymethyl)tetrahydrofur-2-yl]-2-[2-(glycylamino)acet ylamino]-1,9-dihydropurin-6- one gg. Formula XVIIb third structure deoxyribose 9-[(2R ,4S,5R) -4 Hydroxy-5-

(hydroxymethyl)tetrahydrofur-2-yl]-2-[2-(glycylamino)acet ylamino]-1,9-dihydropurin-6- one hh. Formula XVIIb fourth structure ribose (S)-4-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin-2- ylamino}-3-amino-4- oxobutyramide ii. Formula XVIIb fourth structure deoxyribose (S)-4-{9-[(2R ,4S,5R) -4 Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin-2- ylamino}-3-amino-4- oxobutyramide jj. Formula XVIIb fifth structure ribose (S)-4-{9-[(2R ,3R?4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin-2- ylamino}-3-amino-4- oxobutyric acid kk. Formula XVIIb fifth structure deoxyribose (S)-4-{9-[(2R ,4S,5R) -4 Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin-2- ylamino}-3-amino-4- oxobutyric acid

II. Formula XVIIb sixth structure ribose (S)-5-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin-2- ylamino}-4-amino-5- oxovaleramide mm. Formula XVIIb sixth structure deoxyribose (S)-5-{9-[(2R ,4S,5R) -4 Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin-2- ylamino}-4-amino-5- oxovaleramide nn. Formula XVIIb seventh structure ribose (S)-5-{9-[(2R ,3R ,4S,5 R)-3,4-Dihydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin-2- ylamino}-4-amino-5- oxovaleric acid oo. Formula XVIIb seventh structure deoxyribose (S)-5-{9-[(2R ,4S,5R) -4-Hydroxy-5- (hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin-2- ylamino}-4-amino-5- oxovaleric acid pp. Formula XVIIb eighth structure ribose 1-{9-[(2R ,3R ,4S,5R) -3,4-Dihydroxy-5-

(hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin -2-ylamino}-5-guanidino-1,2- pentanedione qq. Formula XVIIb eighth structure deoxyribose 1-{9-[(2R ,4S,5R) -4-Hydroxy-5-

(hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin -2-ylamino}-5-guanidino-1,2- pentanedione rr. Formula XVIIb nineth structure ribose 2-[(E)-5-Guanidino-2-pentenoylamino]-9- [(2R ,3R ,4S,5R) -3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofur-2-yl]-1 ,9-dihydropurin-6- one ss. Formula XVIIb nineth structure deoxyribose 2-[(E)-5-Guanidino-2-pentenoylamino]-9- [(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofur-2-yl]-1 ,9-dihydropurin-6-one tt. Formula XVIIb tenth structure ribose 1-{9-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-

(hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin -2-ylamino}-6-guanidino-1,2- hexanedione uu. Formula XVIIb tenth structure deoxyribose 1-{9-[(2R,4S,5R)-4-Hydroxy-5-

(hydroxymethyl)tetrahydrofur-2-yl]-6-oxo-1,9-dihydropurin -2-ylamino}-6-guanidino-1,2- hexanedione vv.

(11 S,13R)-6-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetra hydrofur-2-yl]-13- hydroxy-11 -methyl-1 ,4,6,8,10-pentaazatricyclo[7.4.0.0 ³ ' ⁷ ]trideca-3(7),4,8-trien-2-one (11S,13S)-6-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)te trahydrofur-2-yl]-13- hydroxy-11 -methyl-1 ,4,6,8,10-pentaazatricyclo[7.4.0.03,7]trideca-3(7),4,8-trien -2-one (11 R,13S)-6-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetra hydrofur-2-yl]-13- hydroxy-11 -methyl-1 ,4,6,8,10-pentaazatricyclo[7.4.0.03,7]trideca-3(7),4,8-trien -2-one (11R,13R)-6-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)te trahydrofur-2-yl]-13- hydroxy-11 -methyl-1 ,4,6,8,10-pentaazatricyclo[7.4.0.03,7]trideca-3(7),4,8-trien -2-one ww

4-{9-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrah ydrofur-2-yl]-N- adenineyl}-1 ,2,3-butanetriol

1-{3-[(2R ,,3R ,,4S, 7R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofur-2-yl]-

1,3,4,5a,8-pentaaza-3H-as-indacen-6-yl}-2-heptanone

1-{3-[(2R ,,3R ,,4S, 7R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofur-2-yl]-

1,3,4,5a,8-pentaaza-3H-as-indacen-6-yl}-2-propanone

4-{9-[(2R ,3R ,4S,55R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofur-2-yl]- 2- (methylthio)-N-adenineyl}-1 ,2,3-butanetriol aaa.

1-{9-[(2R ,,3R ,,4S,57R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofur-2-yl] -N- adenineyl}-3-methyl-2-buten-1-one

(2R ,,3R ,,4S,57R)-5-(Hydroxymethyl)-2-[6-(2-hydroxyprc>pyl)-1,3,4 ,5a,8-pentaaza-

3/-/-as-indacen-3-yl]tetrahydrofuran-3,4-diol ccc.

5-[(2ft, 3R ,, 4S,57R)-3,4-DihydiOxy-5-(hydiOxymethyl)tetrahydrofur-2-yl]-1 0-methyl- 3,5,7,9,13-pentaazatricyclo[7.4.0.0 ² ' ⁶ ]trideca-1(13),2(6),3,7,11-pentaene-11- carbaldehyde (2R,3R,4S,5R)-5-(Hydroxymethyl)-2-[6-(1-hydroxyprc>pyl)-1 ,3,4,5a,8-pentaaza-3/-/- as-indacen-3-yl]tetrahydrofuran-3,4-diol eee. (6R,7R)-3-[(2R,4S,5R)-4-Hydroxy-5-

(hydroxymethyl)tetrahydrofur-2-yl]-6,7-dihydroxy-6-methyl -1 , 3,4,5, 7a-pentaaza- 3,5,6,7-tetrahydro-s-indacen-8-one

- (6S,77R)-3-[(2R ,,4S,57R)-4-Hydroxy-5-(hydroxymethyl)tetrahydrofur-2-yl]-6,7 - dihydroxy-6-methyl-1,3,4,5,7a-pentaaza-3,5,6,7-tetrahydro-s- indacen-8-one

- (6R ,,7S)-3-[(2R ,,4S,57R)-4-Hydroxy-5-(hydroxymethyl)tetrahydrofur-2-yl]-6,7 - dihydroxy-6-methyl-1,3,4,5,7a-pentaaza-3,5,6,7-tetrahydro-s- indacen-8-one

- (6S,7S)-3-[(2R,4S,5R)-4-Hydroxy-5-(hydroxymethyl)tetrahydrof ur-2-yl]-6,7- dihydroxy-6-methyl-1,3,4,5,7a-pentaaza-3,5,6,7-tetrahydro-s- indacen-8-one fff. -095

(2R,3S,5R)-5-{N-[(Z)-4-Hydroxy-3-methyl-2-butenyl]-9-aden ineyl}-2-

(hydroxymethyl)tetrahydrofuran-3-ol

5-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydr ofur-2-yl]-3-(2- oxoheptyl)-1,3a,5-triaza-5H-inden-4-one

1-{9-[(2f?,3f?,4S,5fR)-3,4-Dihydroxy-5-(hydroxymethyl)tet rahydrofur-2-yl]-N- adenineyl}-2-hydroxy-1 -ethanone

3-{9-[(2R ,3R ,,4S,5fR)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofur-2-yl] -N- adenineyl}propionaldehyde

1-{9-[(2R,3R,,4S,5fR)-3,4-Dihydroxy-5-(hydroxymethyl)tetr ahydrofur-2-yl]-N- adenineyl}-5-guanidino-1 ,2-pentanedione qqq.

(E)-1-{9-[(2R,3R,4S,5 R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofur-2-yl]-

N-adenineyl}-5-guanidino-2-penten-1-one

1-{9-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrah ydrofur-2-yl]-N- adenineyl}-6-guanidino-1 ,2-hexanedione sss. -146

5-{9-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrah ydrofur-2-yl]-N- adenineyl}pentyl (E)-3-pentenoate ttt.

(2R,3R,4S,5R)-2-(N-lsopentyl-9-adenineyl)-5-

(hydroxymethyl)tetrahydrofuran-3,4-diol

Methods to select T cells, TCR, B cells or antibodies reactive to MR1 -presented MR1 ligands

In one set of embodiments, a method of modulating the MR1-ligand interaction can be practiced in-vitro, for example to search for and identify novel binders, particularly T cells, B cells or antibodies reactive to the MR1 -ligand complex. Such binding molecules, or polynucleotide sequences encoding them, can subsequently be developed as pharmaceutical agents or diagnostic reagents, ora particularly useful reagent can be selected from a pre-existing repertoire.

These sequences then provide access to a variety of tools that will lend themselves to their use in pharmaceutical and diagnostic applications.

For example, an MR1 presenting cell is contacted with a ligand as disclosed herein in the presence of a T cell library or a B cell library (cells expressing a variety of TCR or B cell receptor (BCR) sequences), and cells bearing TCR or BCR sequences reactive to the MR1-ligand complex are identified and isolated by methods known in the art.

Accordingly, one aspect of the invention relates to a method for identification, isolation or selection of a T cell reactive to a MR1 ligand compound as specified herein, as presented on MR1. This method comprises the steps of providing a preparation of T cells reactive to /capable of specifically recognizing MR1; contacting said preparation of T cells with a complex comprising isolated MR1, or an MR1 on an MR1 presenting cell, associated to said MR1 ligand compound and then isolating a T cell that is specifically reactive to said MR1 ligand compound in the context of MR1 presentation, in an isolation step. A number of ways are available to the skilled person to detect TCR engagement with and recognition of a cognate antigen presented by MHC.

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

An alternative of this aspect of the invention relates to a method for identification, isolation or selection of a B cell or antibody reactive to a MR1 ligand compound as specified herein. This method comprises the steps of providing a preparation of B cells reactive to /capable of specifically recognizing MR1; contacting said preparation of B cells with a complex comprising isolated MR1 associated to said MR1 ligand compound and then isolating a B cell that is specifically reactive to said MR1 ligand compound in the context of MR1 presentation, in an isolation step. A number of ways are available to the skilled person to detect BCR engagement with and recognition of a cognate antigen presented by MHC.

According to another aspect of the invention, the method for modulating an interaction between an MR1 polypeptide and an MR1 -specific TCR molecule can be employed as part of a method of diagnosis to classify metabolically altered cells, including but not limited to tumor cells, according to the presence of the compounds, wherein a sample obtained from a patient is analyzed with regard to the presence of an MR1 ligand compound as identified herein.

This aspect is of particular use in selecting TCR molecules, or transgenic T cells or vectors for obtaining transgenic T cells from an autologous T cell population (“MR1 -specific T cell reagents”), in a patient with a disease, particularly a tumour, that is characterized by a metabolic profile suggestive of certain of the MR1 ligands provided herein. It is even better suited to selecting MR1- specific T cell reagents for a patient whose tumour has been directly analysed and found to present certain of the MR1 ligand compounds disclosed herein. Selection of the best fitting/most specific MR1 -specific T cell reagents will provide the best suited therapy for the patient, from a panel of MR1-specificT cell reagents available to the clinician.

Alternatively, the method can use an unbiased selection of T cells from which (a much smaller population) of T cells may be identified to increase the MR1-specificT cell reagent repertoire.

Use of MR1 ligand compounds in treatment or prevention of MR1 associated diseases

The invention facilitates the use of T cells capable of specifically recognizing and reacting to an MHC-presented ligand that is presented by the invariant MR1 molecule, thereby providing a therapeutic option that is not restricted by the MHC genotype of the patient.

One aspect of the invention provides an MR1 ligand compound as specified above, 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.

The inventors have first found evidence to prove that the compounds disclosed herein are specifically presented, and recognized, by T cells in the context of cancer. Hence, the MR1 ligand compounds disclosed herein lend themselves to use as “cancer vaccines” in the sense that their presence will enhance therapeutic approaches enabled by the MR1-T cell interaction.

In certain embodiments, the MR1 ligand compound is co-administered with an anticancer drug. As disclosed herein, the inventors have been able to demonstrate that the administration of paclitaxel or doxorubicin, established anti-tumour drugs, increase the presentation of MR1 ligand compounds as disclosed herein, presumably by increasing the metabolic stress inside cancer cells. Such combination is expected to provide a synergistic effect as it increases the load of MR1 ligands in the tissue.

In certain embodiments, the MR1 ligand compound is co-administered with a checkpoint modulator or checkpoint inhibitory agent. Such combination is expected to provide a synergistic effect as the downstream immune effect of MR1-ligand engagement with an MR1 specific T cell, physiologically present in the patient or possibly administered as part of an additional MR1 specific T cell therapy, is expected to be increased by removing inhibitory signals.

In particular embodiments, 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/ora (transgenic) MR1 -reactive T cell receptor polynucleotide construct (for example, a DNA expression construct or an RNA construct encoding an MR1-TCR ora viral vector having the same function).

Optionally, a polynucleotide expression vector encoding MR1 can be provided. Transgene expression of MR1 is a way to increase MR1 expression of tissue should affected disease tissue be found to down-regulate MR1 expression. The MR1 ligand and/or the polynucleotide expression vector encoding MR1TCR or MR1 can be administered into a tumour, into the vicinity of the tumour, or into a lymph node draining the tumour site. It is expected that local increase of either agent is beneficial in comparison to systemic administration.

Specifically, the invention enables the administration of recombinant (allogeneic or autologous) T cells carrying a transgene TCR specifically capable of recognizing and reacting to MR1 presented disease specific MR1 ligand compounds.

The invention further enables the analysis of a T cell sample obtained from a patient for the presence of T cells capable of recognizing MR1 ligand compounds presented by MR1, to selectively stimulate and amplify, or to engineer de novo, such MR1 patient T cells for subsequent therapeutic administration.

The invention further enables the targeted administration of MR1 ligand compounds as specified herein to a patient to facilitate or amplify an MR1 specific T cell response or an MR1 targeted T cell therapy.

The compounds identified herein can be employed in methods pertaining to: modulating an interaction between an MR1 (major histocompatibility complex class l-related gene protein 1) polypeptide and an MR1 -specific T cell receptor molecule, wherein the MR1 polypeptide is contacted with the MR1 ligand compound; generating a panel of MR1 multimeric reagents to use for identification and sorting of T cells reacting to MR1 ligand compounds presented on non-polymorphic MHC l-related MR1 antigen-presenting molecules; identifying a TCR gene specifically reactive to said compounds to be used in a personalized cellular immunotherapy. This may be achieved by providing a preparation of tumor cells isolated from a patient, and submitting it to mass-spectrometry-based determination for assessing the presence and identity of the compounds.

In certain embodiments identifying a TCR gene specifically reactive to the compounds identified herein for use in a personalized cellular immunotherapy can be effected by the following general sequence of processes: 1) compound detection in a tumor biopsy (the tumor is classified based on the TAA presence) and then 2) guided TCR-mediated cellular immunotherapy based on the TCR best reacting to the identified TAA in complex with MR1.

Combination of MR1 therapy with pharmaceutical drugs

An MR1 specific T cell response or an MR1 targeted T cell therapy may be further facilitated by combination with a pharmaceutical drug that increases the production of MR1 ligand compounds as specified herein.

The following pathways are targets of pharmaceutical drugs useful in this context: glutathione-S- transferase (GST), aldehyde reductases and aldehyde-keto reductases (AKR), aldehyde dehydrogenases (ALDH), aldehyde oxidase (AOX), xanthine oxidase (XO), Short chain reductase/oxidase (SDR).

Another aspect of the invention relates to the use of pharmaceutical drugs which facilitate the accumulation of nucleobases, thus increasing antigen availability. These drugs include, but are not limited to, the inhibitors of adenosine deaminase 1 (ADA1), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), and inhibitors of both ADA1 and ADA2, pentostatin (CAS No. 53910-25-1) and 1- deazaadenosine. They also include inhibitors of dehydrogenase 1 (IMPDH1) such as, but not limited to, mycophenolic acid (CAS No. 24280-93-1). The results shown in Fig. 7 support the compounds’ usefulness in administration as part of a MR1T targeted treatment.

A further aspect of the invention relates to the MR1 ligand compounds identified herein according to the first aspect of the invention, particularly an MR1 ligand identified by any one of the formulas of claim 1 or their specific embodiments given in the dependent claims, for use as a vaccine to elicit or boost an MR1T cell response, particularly in treatment or prevention of recurrence of cancer. In particular embodiments, the MR1 ligand compounds identified herein may be employed as a combination medicament to stimulate and augmenting antitumor activity of MR1T cells used in cellular therapy, and/or may be used in combination with immunostimulatory compounds and/or checkpoint modulator agents.

The MR1 ligand compounds identified herein may also be used as a vaccine in a subject without overt disease, but with a predisposition to develop such disease. Such as a human subject can be treated with preventative vaccination in advance of each of the maladies described herein.

Likewise, an alternative of this aspect of the invention relates to the MR1 ligand compounds identified herein according to the first aspect of the invention, particularly an MR1 ligand identified by any one of the formulas of claim 1 or their specific embodiments given in the dependent claims, for use in a combination medicament in combination with oncotherapeutic agents.

One important advantage of the methods and compounds provided herein relates to the ability to test and provide TAAs to patients regardless of their MHC haplotype. TAA are the targets of clinically relevant anti-tumor immune response in cancer patients. Nevertheless, the majority of the so far identified TAAs are peptides presented by polymorphic MHC molecules. The extreme polymorphism of MHC genes limits the TAA targeting to those patients expressing certain MHC alleles. Targeting TAAs bound to MR1 non-polymorphic antigen-presenting molecules overcomes this constraint and is applicable to all patients bearing tumors expressing MR1. In addition, because tumor cells may express different non-peptidic TAAs, this strategy provides the possibility of targeting multiple antigens displayed by the same tumor cells, thus minimizing the potential occurrence of tumor escape-variants under selective immune pressure. Therefore, the identification of MR1 -presented TAAs, matched with the specific MR1 -restricted TCRs recognizing these antigens, has important implications for cancer immunotherapy. In certain embodiments, the MR1 ligand compounds identified herein are provided for use in combination with an immune checkpoint modulator, particularly in combination with an immune checkpoint inhibitor agent.

In certain embodiments, the immune checkpoint inhibitor agent is ipilimumab (Yervoy; CAS No. 477202-00-9).

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

In certain embodiments, the MR1 ligand compounds identified herein are provided for use in combination, wherein any of the MR1 ligand compounds identified herein is a first combination partner, and a) a modified T cell reactive to an MR1 molecule presenting the MR1 ligand compound and/or a nucleic acid expression vector encoding MR1 is a second combination partner, and b) an immune checkpoint modulator, particularly an immune checkpoint inhibitor agent is a third combination partner.

Such combination is likely to be in a form where the combination partners are applied at different times and in different administration forms during treatment.

The identification of the compounds identified herein, which are presented by tumor cells in each tumor patient, represents a novel method to classify tumors. This classification is of relevance to select the appropriate MR1T-derived TCR to be used in a personalized TCR gene therapy.

Tumor patients can be also vaccinated with selected compounds previously detected in the tumor cells from the same patient. This treatment will have the goal of eliciting and/or stimulating MR1T cells specific for the compound and thus capable of recognizing and killing tumor cells.

Some of the MR1 ligand compounds described herein are also present in tissues of patients with autoimmune and metabolic diseases, including rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, atherosclerosis, inflammatory bowel disease and multiple sclerosis. In general, disease with abnormal generation of Reactive Oxygen Species (ROS) are characterized by an accumulation of the MR1 -binding compounds described below. In these cases, autoreactive MR1T cells that are stimulated during these diseases represent large T cell populations that may be inhibited in a therapeutic setting.

The inventors predict that appropriate therapeutic intervention forms can be found using those described for tumor immunotherapy as a template. The types of diseases which could be similarly treated because of the accumulation of the same types of MR1 ligands may thus be expanded. Yet another aspect of the invention relates to the detection of MR1 ligand compounds identified herein as part of a method of disease classification, wherein the presence of at least one of the MR1 ligand compounds according to the invention is identified in samples from patients. In case of cancer patients, the identification of these compounds extracted from fresh tumor samples is considered a diagnostic marker. In certain embodiments, the TAA MR1 ligand compounds identified herein can be used for guiding cellular immunotherapy and also vaccination in combination with other therapeutic interventions, in other words, once the MR1 ligand is identified as present in the tumor, patients could be treated with personalized immunotherapy interventions including i) administration of pharmaceutical drugs capable of facilitating the accumulation of MR1T cell-stimulatory compounds as identified herein, ii) administration of selected TAA MR1 ligand compounds, iii) MR1 TCR-mediated cell therapy alone or in combination with other oncotherapeutic agents.

The invention also relates to a research method directed at the identification of T cells reactive to MR1 -expressing cells. This encompasses a method to isolate from peripheral blood of normal donors or from patients suffering from cancer, metabolic or autoimmune diseases, MR1 -restricted T cells sorted by using compound-loaded onto MR1 multimeric molecules. The cell source encompasses, but is not limited to, T cells isolated from tissue biopsies.

The invention also relates to the possibility to readily identify the TCR gene and protein sequences expressed by the above-mentioned T cells.

In certain aspects, the current invention is centered on the identification of novel classes of compounds which bind to non-polymorphic MR1 molecules. Some of the compounds identified herein modulate the surface expression of MR1. Some of the compounds identified herein (not necessarily the same compounds as those found to modulate the surface expression of MR1) are antigens, stimulating specific human T cells restricted to MR1. Some of the compounds identified herein were isolated from tumor cells, they were purified, identified and synthetic analogs were produced. The compounds showing antigenic activity, when presented in association with MR1 molecules, stimulate a population of human T cells discovered by the inventors and termed MR1T cells. Applications of this invention encompass, but are not limited to, the following methods. i) a method to stimulate compound-specific T cells, to induce a prophylactic immune response, ii) a method to stimulate compound-specific T cells, to induce a therapeutic immune response, iii) a method for the identification and isolation of T cells reactive to the compounds, iv) a method to modulate (increasing or decreasing) the quantity of defined compounds in the cells or presented by the cells, v) a method to classify metabolically altered cells, including but not limited to tumor cells, according to the presence of the compounds. Thus, in certain aspects and embodiments, the invention relates to the use of the MR1 -associated compounds identified herein, for guiding personalized intervention of immunotherapy, of cellular immunotherapy, of vaccination strategies in people at risk and for diagnostics tests of several diseases, including cancer.

The invention further provides an isolated T cell and/or the polynucleotide for use as specified in the preceding paragraph, 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).

In a particular embodiment, the isolated T cell expressing the TCR and/or the polynucleotide is coadministered with a pharmaceutical compound selected from paclitaxel, doxorubicin, disulfiram, and MPA, 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.

In a more particular embodiment, the isolated T cell expressing the TCR and/or the polynucleotide is co-administered with a pharmaceutical compound 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.

Medical treatment, Dosage Forms and Salts

Similarly, within the scope of the present invention is a method or treating a condition associated with a lack of MR1-specific T cell responses, or with too much of an MR1-specific T cell response, in a patient in need thereof, comprising administering to the patient a compound as specified in detail above.

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

The skilled person is aware that any specifically mentioned drug may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Nonlimiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.

Dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. Alternatively, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present.

Pharmaceutical Compositions and Administration

Another aspect of the invention relates to a pharmaceutical composition comprising a compound as specified herein in the context of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

In certain embodiments of the invention, the compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant product, which is easy to handle.

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

The dosage regimen for the compounds of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

In certain embodiments, the pharmaceutical composition or combination of the present invention can be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg.

The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease. The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

“Swiss-type” method of manufacture: An alternative aspect of the invention relates to the use of an MR1 ligand compound as described in the first aspect in the manufacture of a medicament for 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, optionally in combination with a drug recited in claim 17. Another embodiment relates to the use of an isolated T cell receptor as described in claim 10 to 11 in the manufacture of a medicament for 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, optionally in combination with a drug recited in claim 17. Another embodiment relates to the use of an isolated T cell as described in claim 13 to 15 in the manufacture of a medicament for 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, optionally in combination with a drug recited in claim 17. Another embodiment relates to the use of a polynucleotide encoding a TCR as described in claim 10 to 11. in the manufacture of a medicament for 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, optionally in combination with a drug recited in claim 17.

Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Description of the Figures Fig. 1 shows glycolysis and methylglyoxal lead to MR1T antigen accumulation. Schematic representation of methylglyoxal generation. Dihydroxyacetone phosphate (DHAP), glyceraldehyde 3-phosphate (G3P). (A and B) Stimulation of MR1T cell clone TC5A87 (A) and DGB129 (B) with A375-MR1 cells transduced with sgRNAs targeting TPI1 (·) or scrambled control (O). (C and D) Stimulation of MR1T cell clone TC5A87 (C) and DGB129 (D) in response to fixed A375-MR1 cells incubated for 6 h with different concentrations of D-(+)-Glucose (O) or 2-deoxy-D-Glucose (■) before fixation. (E and F) Stimulation of MR1T cell clone TC5A87 (E) and DGB129 (F) with A375-MR1 cells transduced with sgRNAs against GL01 (■), scrambled sgRNAs control (O) or a vector to overexpress GL01 (▼). (G and H) Stimulation of MR1T cell clone TC5A87 (G) and DGB129 (H) with THP-1 cells pre-treated with 25 mM erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA), 10 pM mycophenolic acid (MPA) and 20 pM S- bromobenzylglutathione (BBG), alone or in combination. (I-L) MR1T clone DGB129 activation in response to THP-1 cells (O), GL01 -overexpressing (▼) and GL01 ko (■) THP-1 cells, in the presence of Methylglyoxal (I, J) or deoxyadenosine (K, L). IFN-y released is presented as mean ± SD of triplicate cultures. The data shown are representative of at least three independent experiments. (A, L) Mean ± SD, n=3, *p<0.05 ^** p<0.01 and ^*** p<0.001. (A, B and l-L) Multiple t-test, (C and D) one-way Anova with Dunnett’s multiple comparison, (E and F) two-way Anova with Dunnett’s multiple comparison, (G and H) one-way Anova with Tukey’s multiple comparison.

Fig. 2 shows aldehyde contributing and scavenging MR1T antigen accumulation (A-C)

Stimulation of MR1T cell clone TC5A87 (A), DGB129 (B) and MCA3C3 (C) with THP-1 cells pre-treated with Doxorubicin (75 nM) or Paclitaxel (5 pM) in the absence or presence of nucleosides (dAdenosine and Guanosine, both 150 pM). (D-F) Stimulation of MR1T cell clone TC5A87 (D), MCA2B1 (E) MCA3C3 (F) with fixed A375-MR1 cells treated with buthionine sulfoximine (400 pM, BSO), glutathione (4 mM, GSH), N-acetylcysteine (4 mM, NAC) and apocynin (100 pM, APO). (G-l) Stimulation of MR1T cell clone TC5A87 (G), DGB129 (H) MCA2B1 (I) with fixed A375-MR1 cells treated with ML-210 (6 pM), RSL-3 (1 pM) and mercaptosuccinic acid (3.3 pM, MSA). (J-L) Stimulation of MR1T cell clone TC5A87 (J), DGB129 (K) and MCA2B1 (L) with fixed A375-MR1 cells treated with hydralazine (100 pM) oraminoguanidine (5 mM). IFN-g release is presented as the mean ± SD of triplicate cultures. The data shown are representative of at least three independent experiments. (A-L) Mean ± SD, n=3, ^* p<0.05, ^** p<0.01 and * ^** p<0.001. (A-C) two-way Anova with Tukey’s multiple comparison, (D-L) oneway Anova with Dunnett’s multiple comparison.

Fig. 3 shows stimulation of MAIT clone MRC25 with nucleobases, inhibitory drugs and synthetic antigens. (A) MAIT clone MRC25 was stimulated with THP-1 cells in the presence of different nucleobases (250 pM), Methylglyoxal (250 pM) or 5-OP-RU (30 nM). (B and D) MRC25 cells were stimulated with THP-1 cells treated with indicated drugs. (C) MRC25 cells were stimulated with A375-MR1 cells treated with GSH, NAC, APO, BSO or GPX inhibitors and fixed or with THP-1 cells pulsed with 5-OP-RU (10 nM). (E) MRC25 cells were stimulated with A375-MR1 cells treated with carbonyl scavengers at indicated concentrations and fixed before T cell addition (empty bars). As controls, the same experiment was performed with the same carbonyl scavengers in the presence of 6,7-dimethyl-8-ribityllumazine (20 pM, black bars). (F) MRC25 cells were stimulated with THP-1 cells in the presence of M ₃ ADE, OPdA, OPdC (all 100 pM), MiG (13 pM) or 5-OP-RU (10 nM). n.d.=not determined ^*** p< 0.001 compared to vehicle- treated cells using One-way ANOVA (A, B, C, F) or Two-way ANOVA (D and E) with Dunnett’s multiple comparison. IFN-y is expressed as mean ± SD of triplicate independent cultures. The experiments were repeated at least twice and one representative experiment is shown.

Fig. 4 (A) Quantification of ROS produced in THP-1 cells treated with Doxorubicin,

Paclitaxel or Phorbol 12-myristate 13-acetate (PMA). Results are expressed Median Fluorescence Intensity (MFI) of live cells ± SD of triplicate independent cultures. (B) Surface expression in indicated tumor cell lines of MR1 (black line) or HLA A,B,C (grey dotted line). Isotype matching staining control is depicted as grey shade. (C) Table reporting tissue origin and diagnosed disease of each cell line. Each experiment was repeated at least twice and one representative experiment is shown. ^** p 0.01 and ^*** p 0.001 using one-way Anova with Dunnett’s multiple comparison.

Fig. 5 MR1 -eluted metabolite identification process.

Figs. 6 to 25 MR1 upregulation assay. MR1 surface expression of THP-1 MR1 cells following 4- 6 h incubation with synthetic compounds at the indicated concentrations (O), or with vehicle DMSO (·) or medium alone (A). Median fluorescence intensity (MFI) of the staining with anti-MR1 mAbs are shown. Control staining with irrelevant isotype-matched control mAbs is also shown (■). The data shown are representative of at least three independent experiments. Figs 6 to 20 show MR1 upregulation, Figs 21 to 25 show MR1 downregulation.

Figs. 26 to 29 Activation assay. IFN-g release response of the indicated MR1T cell clones following co-culture with THP-1 cells in the presence or absence of compounds (50 mM). Blocking of T-cell reactivity is shown using anti-MR1 mAbs, clone 26.5 (black columns). The data shown are representative of at least two independent experiments. Mean ± SD, n>3. ^* p<0.05, ^** p<0.01 , ^*** p<0.001.

Fig. 30 MS-confirmed compounds. Fig. 31 Mass found in MS, structure proposals synthesized. Fig. 32 Mass found in MS.

Fig. 33 MS Approach: Overview of the untargeted DNA adductomics approach which was modified to include detection of modified RNA nucleosides; A) Typical fragmentation used for the identification of DNA adducts B) Example of a chromatogram illustrating the signal resulting from the detection of the neutral loss in the MS ² fragmentation indicating the presence of an adduct whose peak can then be identified and extracted in the full scan.

Examples

Example 1: Compounds stimulating MR1T cells

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.

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 Figures 6-29.

Example 2: Methylglvoxal and purine metabolism pathways within tumor cells cooperate for

MR1T-cell stimulation

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 ( GL01 ), and Glyoxalase Domain Containing 4 ( GLOD4 ). TPI1 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 (Fig. 1). Conversely, GLO ^' /-deficient cells are impaired in MG (a highly reactive carbonyl) degradation, which therefore accumulates. As MG forms adducts with several nucleobases, these data suggest a potential involvement of MG in generating MR1T-cell antigens.

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-y production by both MR1T-cell clones (Fig. 1A, B). Furthermore, A375-MR1 cells pulsed with glucose and then fixed showed increased MR1T-cell stimulatory capacity (Fig. 1C, D); this effect was abolished when the same cells were incubated with deoxyglucose (Fig. 1C, D), which does not enter the glycolytic pathway and so does not generate MG. The inventors also saw that GL01 -deficient and GL01 -overexpressing A375-MR1 cells showed increased and reduced MR1T-cell stimulatory capacity, respectively (Fig. 1E, F). Altogether these results suggest that MG accumulation in target cells is important for the stimulation of MR1T-cell clones. To investigate the potential synergism between MG and purine metabolic pathways in MR1T cell stimulatory capacity of tumor cells, the inventors explored the effects of individual and combined pharmacologic inhibition of key enzymes in these pathways. The inventors used S-p- bromobenzylglutathione (BBG) to inhibit GL01; mycophenolic acid (MPA) to inhibit inosine monophosphate dehydrogenases (IMPDH1, 2), leading to IMP accumulation; and erythro-9-(2- Hydroxy-3-nonyl) adenine hydrochloride (EHNA) to inhibit ADA and phosphodiesterase 2 (PDE2), inducing adenosine, dAdo and cGMP accumulation. To sensitively detect the effects of the inhibitors, the inventors again used THP-1 cells as target cells: the inventors found that BBG in combination with each of the other two drugs significantly enhanced the IFN-y release of both MR1T cell clones to THP-1 cells (Fig. 1G, H). The DGB129 clone was more sensitive to these treatments and also reacted to THP-1 cells treated with EHNA, BBG, or MPA alone (Fig. 1H).

The inventors tested the IFN-g response of MR1T cells to GL01-modified THP-1 cells and various doses of MG or dAdo. The inventors found that MG treatment significantly increased MR1T-cell stimulation by GL01 -deficient compared to wild-type THP-1 cells (Fig. 11). Conversely, MG failed to induce MR1T-cell stimulation when administered to GL01 -overexpressing cells (Fig. 1J). Similarly, MR1T reactivity to dAdo was increased using GL01 -deficient THP-1 cells as antigen- presenting cells (APCs) (Fig. 1K) and decreased using GL01 -overexpressing THP-1 cells (Fig. 1L). Together, these findings suggest that nucleosides/nucleobases and MG cooperate in generating potential MR1T-cell antigens.

Example 3: Multiple oxidative stress-related carbonyl species accumulate within tumor cells and contribute to MR1T cell stimulation

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 ⁺ 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.

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 GST01 ; 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 Of and H2O2. Both drugs significantly increased ROS accumulation (Fig.4A) and promoted activation of all three MR1T-cell clones when incubated with THP-1 cells and nucleoside compounds (Fig. 2A-C), mirroring the additive effects observed when purine-modifying drugs and carbonyl-degradation inhibitors were combined (Fig. 1G, H).

Next, the inventors treated A375-MR1 with apocynin, an O2 ^' scavenger and NADPH oxidase inhibitor, or with GSH or N-acetylcysteine (NAC), which prevent H2O2 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-y production from MR1T cells, with apocynin being effective with one T-cell clone (Fig. 2D-F). The inventors also treated A375- MR1 cells with buthionine sulfoximine (BSO), an inhibitor of GSH synthase, and a significant increase was observed in the stimulation of all the tested MR1T clones (Fig.2D-F). Together, these data show that ROS participate in MR1T antigen accumulation, although requires concomitant alteration of nucleobases metabolism.

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 (Fig. 2D-F), the inventors inferred a role for lipid peroxidation from the results of the inventors CRISPR/Cas9 screen, which showed significant depletion of the glutathione peroxidase 4 (GPX4) and glutathione peroxidase 1 (GPX1) sgRNAs. While GPX1 protein catalyzes the reduction of organic hydroperoxides and H2O2 by glutathione, GPX4 has a high preference for lipid hydroperoxides and protects cells against membrane lipid peroxidation and death. Accordingly, when the inventors pre-treated A375-MR1 cells with the selective GPX1 inhibitor mercaptosuccinic acid (MSA), or with two GPX4 inhibitors RSL3 and ML-210, they showed significantly increased MR1T-cell stimulatory activity (Fig. 2G-I). None of these compounds influenced the MAIT-cell response to microbial antigens in control experiments, except paclitaxel that in presence of nucleosides could induce a little but significant stimulation (Fig. 3C-D). Taken together, these findings indicate that peroxides and lipid peroxidation contribute to MR1T-cell stimulation by tumor cells.

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-g production by MR1T cell clones (Fig. 2J-L) and had no effect on MAIT cell activation (Fig. 3E).

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. Materials and Methods

Cell adaptation and expansion for protein production

CH0-K1 and A375 cells stably expressing MR1 wild-type or K43A mutant as soluble recombinant p2m-MR1-lgG1-Fc fusion protein (obtained as described in Lepore et al. eLife 2017 doi:10.7554/el_ife.24476) are grown in RPMI-1640 supplemented with 1 mM sodium Pyruvate, 2 mM stable Glutamine (Ala-Gin), 1% NEAA and 50 pg/mL Kanamycin (complete medium) and with 3% heat-inactivated FCS. Cells are maintained splitting them 1:10 when reaching 80-90% confluence.

For protein production, cells are grown in serum-free medium (MAM-PF2 from Bioconcept 10- 02F25-I, supplemented with 1 mM sodium Pyruvate, 2 mM stable Glutamine (Ala-Gin), 1% NEAA and 50 pg/mL Kanamycin). Medium containing soluble MR1-lgG is collected every 48h and replaced each time with new pre-warmed serum-free medium for two additional production batches from the same cells. Collected supernatants are centrifuged 5 min at 600g, sterile-filtered through a 0.22 pM filter and stored at 4°C until further steps.

Soluble MR1 quantification and purification

Supernatant from cells is concentrated with Vivaspin 20 centrifugal concentrator with molecular cut-off at 100 kDa (MR1-lgG is ~160 kDa) (Sigma Aldrich Z614661). Tubes are repeatedly centrifuged at 3000g for 15-20 min until the MR1-lgG1 reaches a final concentration of at least 30 pg/mL. Concentrated protein is sterilized through a 0.22 pM syringe-filter.

Protein is quantified by ELISA using capture antibodies against b2hh (clone HB28) and a detection antibodies against MR1 (clone 26.5). As a standard, soluble MR1 monomers made in house is used.

Protein is also quantified by SDS-PAGE on 7.5% acrylamide gel in non-reducing conditions in parallel with known amounts of purified IgG antibodies. MR1-lgG concentration is calculated based on gel staining with SimplyBlue Safestain (ThermoFisher scientific, cat# LC6060) and band densitometry interpolation of IgG standards.

MR1-lgG is purified with Protein A MagBeads (Genscript L00273), according to manufacturer’s instruction (for purification of 1 mg of MR1-lgG1, 400 pL of 25% beads slurry is used). Beads are mixed with the supernatant and incubated overnight at 4°C under constant mixing. Beads are then extensively washed with PBS to remove contaminants and are transferred to a silanized glass tube (Thermo Fisher scientific CTS-1275).

Elution of MR1 -bound compounds and HPLC fractionation

To elute compounds from MR1, beads are resuspended in mildly acidic buffer. For this purpose, beads are incubated sequentially twice in ddhhO for 5 min, and then twice in 30% methanol (diluted in ddhhO) for 5 min, with collection of the supernatant after each step. All the eluted material is pooled in silanized glass vials (Thermo Fisher Scientific SAA-SV2-2) and volume is reduced under N2 stream. The material is dried or used directly for HPLC separation.

Separation is performed on a NucleodurC18 Pyramid column (Macherey Nagel) with a flowrate of 1 mL/min.

Solvent A: H2O. Solvent B: 100% acetonitrile Gradient:

Minutes: 0 15 25 35 50 51 61

B%: 5 5 35 100 100 5 5

Fractions are collected each minute in silanized glass tubes, transferred to silanized glass vials and stored at -80°C until biological assays and Mass Spectrometry are performed.

Cell lines

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, 1x MEM NEAA and 50 pg/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.

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 pg/mL puromycin resistance.

Cell surface MR1 expression determination

THP-1 cells and THP-1 MR1 cells (10 ⁵ cells/well) were tested for MR1 surface expression after incubation with several doses of synthetic compounds for 4 to 6 h at 37°C. After FcR blocking with TruStain FcX, Biolegend Cat#422302, the cells were stained with anti-human-MR1-APC mAbs (clone 26.5 Biolegend Cat#361108) or with APC-labeled mouse lgG2a, k irrelevant isotype control antibodies (Biolegend Cat#400220) for 20 min at 4°C, then washed and analyzed by flow cytometry. For each condition, median fluorescence intensity (MFI) of MR1 expression was calculated and compared to the MFI obtained with isotype control mAbs. MFI resulting from treatment of cells with medium or with DMSO vehicle control at the indicated concentrations were also included in all experiments. T cell activation assays

MR1T cells (5x10 ⁴ /well) were co-cultured with the indicated APCs (10 ⁵ cells/well) for 18 h in 120 mI_ volume in triplicate. For blocking MR1 interaction, anti-MR1 mAbs (clone 26.5, purified and endotoxin-free) were added at 20 pg/mL and incubated for 30 min at 37°C prior to the addition of T cells.

When synthetic compounds were used to stimulate T cells, the THP-1 cells (10 ⁵ /well) were cultured 3 h with the indicated molecules or medium only, prior to T-cell addition.

In control experiments, the MAIT cell clone MRC25 was stimulated by APCs pulsed 3 h with 5-OP- RU as previously described.

T cell activation was assessed by secretion of the following human cytokines by ELISA using specific mAbs: IFN-y (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). Results are expressed as mean of triplicate cultures ±SD.

ROS production measurement

CM-H2DCFDA (Thermo Fisher Scientific) was used to assess ROS production in cell upon cell treatment with Doxorubicin and Paclitaxel. THP-1 cells (10 ⁷ /mL) were labelled with 10 mM CM- H2DCFDA for 30 min at 37°C in the dark, then washed with PBS and resuspended in complete medium. 10 ⁵ cells were seeded per well and treated with 75 nM Doxorubicin, 5 mM Paclitaxel or vehicle for 18 h at 37°C. Phorbol 12-myristate 13-acetate (PMA, 50 ng/mL) was used as positive control.

MS: Overview of Technical Approach

The detection and identification of modified RNA nucleosides was performed using a high resolution/accurate mass data dependent-constant neutral loss-MS ³ methodology developed in our lab for DNA adductomics. ¹ The comprehensive approach identifies modified nucleosides upon observation of relevant neutral losses between parent ion masses and their corresponding fragment ion masses. Originally the method was developed to investigate DNA adducts using the neutral losses of the 2'-deoxyribose moiety or one of the four DNA bases. ¹ This method was modified to include modified RNA nucleosides by addition of the neutral losses of ribose and uracil (Figure 33). The accurate mass measurements of the observed DNA or RNA adducts allows determination of their molecular formulas and the triggered MS ³ fragmentation spectrum provides structural information, as shown in Figure 33.

The method uses data dependent scanning performed in “real time” by the instrument software, where full scan analysis followed by software-triggered fragmentation of the most abundant ions is repeated throughout the entire chromatographic run with HRAM detection of both the full scan and fragmentation spectra. In “real time” the software compares the mass of the parent ion and ions in the triggered fragmentation spectrum. Upon observation of the neutral loss of the exact mass of diagnostic fragments of interest, an additional fragmentation event is triggered which increases the specificity of the fundamental adduct identification step.

This LC-NSI-HRMS ⁿ approach is unique and its use of powerful Orbitrap Tribrid instrumentation provides for the ability to detect all modified nucleosides and nucleobases including previously uncharacterized modified nucleosides by combining sensitivity, specificity, and generation of chemical information that is absent from previous MS techniques. The Orbitrap Tribrid technology allows for the use of the “AcquireX” feature whereby an initial injection of a matrix blank or negative control is performed to generate a time-scheduled ion exclusion list of thousands of ions which is incorporated into the methods used for subsequent sample injections.

This approach has been successfully used to identify novel DNA adducts of the genotoxins produced by the microbiome, ² ' ³ to investigate the genotoxicity of nanoscale battery cathode materials, ⁴ to characterize the DNA damage resulting from endogenous electrophiles ⁵ as well as to profile the DNA damage resulting from exposures to alcohol, ⁶ tobacco ⁷ and alkylating drugs ¹ .

Untargeted Adductomics Technical Details

LC-MS experiments are performed on an Orbitrap Lumos mass spectrometer (Thermo Scientific, Waltham, MA) coupled to a Dionex RSLCnano UPLC (Thermo Scientific, Sunnyvale, CA). Reverse phase chromatography is performed with a hand-packed Luna C18 column (5 pm, 120 A, 200 mm x 75 pm ID, Phenomenex, Torrance, CA) at room temperature and flow rate of 0.3 pL/min using 0.05% formic acid as mobile phase A and acetonitrile as mobile phase B. The LC gradient starts at 2% B for the first 5.5 min with a flow rate of 1.0 pL min-1 , followed by switching of the injection valve to remove the 5 pL loop from the flow path, and reducing the flow rate to 0.3 pL min-1 over 0.5 minute. A linear gradient from 2% to 50% B over 39 min is used, then ramped to 95% B over 1 minute. The mobile phase composition is allowed to stay at 95% B for 2 min. Finally, re-equilibration was performed by changing the mobile phase composition from 95% to 2% B over 2 minute and increasing the flowrate to 1.0 pL/min over 1 minute, then held at 2% B for 2 min. The total run time is 52 min.

CNL-MS ⁿ DDA is performed with the instrument operating in positive ionization mode, by repeated full scan detection followed by MS ² acquisition and constant neutral loss triggering of MS ³ fragmentation over a cycle time of 3s. Full scan (range 100-1000 Da) detection is performed by setting the Orbitrap detector at 120,000 resolution, using EASY-IC internal mass calibration, normalized automatic gain control (AGO) target settings of 1 E6, and maximum ion injection time set at 50 ms. The MS ² fragmentation parameters are as follows: quadrupole isolation window of 1.5 amu, HOD collision energy of 25%, Orbitrap detection at a resolution of 15,000, AGO of 2E5, and maximum injection time of 50 ms. Data-dependent conditions are as follows: triggering intensity threshold of 2E4, repeat count of 1, and exclusion duration of 15 s. The MS ³ fragmentation parameters are as follows: 2 amu isolation window, HCD collision energy of 30%, Orbitrap detection at a resolution of 15,000, AGC of 2.0E5, and maximum injection time of 200 ms. Fragmentation is triggered upon the observation of neutral loss of deoxyribose (-116.0474), ribose (-132.0423), adenine (-135.0545), guanine (-151.0494), cytosine (-111.0433), thymine (-126.0429), uracil (- 111.0433), adenine+water (-152.0651) and guanine+water (-169.0600) between the parent ion from the full scan and one of the product ions. The “AcquireX” feature was used with a blank injection used to generate timed ion exclusion lists of approximately 4500 ions with mass tolerances of 5 ppm.

Targeted Adductomics Method:

Targeted MS/MS was also performed using the same LC conditions as for the untargeted analysis and performed on a list of suspected modified nucleosides, ⁵ with the following parameters: Orbitrap detection at a resolution of 30,000, HCD collision energy of 25%, quadrupole isolation window of 1.6 amu, AGC of 5.0E4, and a maximum injection time of 100 ms. Product ion detection was based on extracted ion chromatograms using mass accuracy tolerances of 5 ppm for all masses.

DNA Adductomics Data Analysis: For modified nucleoside screening data analysis, Freestyle 1.7 (Thermo Scientific, Waltham, MA) software was used to manually interrogate all MSMriggering analytes (not observed in negative controls) to identify all unknown modified nucleosides. Extracted ion chromatograms for all putative modified nucleosides were generated at 5 ppm mass tolerance. MS ² and MS ³ spectra of each putative DNA adduct is subsequently evaluated for structural information and the peak area of the precursor mass is determined.

Table 1: Designation of sequence ID Nos ofMR1 specific TCRs (first half disclosed in PCT/EP2019/074284)

References

1. Stornetta A., Villalta P.W., Hecht S.S., Sturla S.J., Balbo S. Screening forDNA Alkylation Mono and cross-linked adducts with a comprehensive LC-MS3 adductomic approach. Anal Chem. 2015 Dec 1;87(23):11706-13. doi: 10.1021/acs.analchem.5b02759. PMID: 26509677 PMCID: PMC5126974

2. Wilson, M.R, Jiang, Y., Villalta, P.W., Stornetta, A., Boudreau, P.D., Carra, A., Brennan, C.A., Chun, E., Ngo, L, Samson, L.D., Engelward, B.P., Garrett, W.S., Balbo, S., and Balskus, E.P. (2019) The Human Gut Bacterial Genotoxin Colibactin Alkylated DNA. Science, 363(6428), PMID: 30765538

3. Alexander, E.M., Kreitler, D.F., Guidolin, V., Hurben, A.K., Drake, E., Villalta, P.W., Balbo, S., Gulick, A.M., Aldrich, C.C. (2020) Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca. ACS Infect Dis., 6(7), 1976-1997, PMID: 32485104

4. Qiu T.A., Guidolin V., Hoang K.L.N., Pho T, Carra’ A., Villalta P.W., He J., Yao X., Hamers R.J., Balbo S. Feng Z.V., Haynes C.L. (2020) Nanoscale Battery Cathode Materials Induce DNA Damage in Bacteria, Chemical Science, 11 11244-11258

5. 5. Carra, A., Guidolin, V., Dator, R.P., Upadhyaya, P., Kassie, F., Villalta, P.W., Balbo, S. (2019) Targeted High Resolution LC/MS3 Adductomics Method for the Characterization of Endogenous DNA Damage. Front. Chem., 7(658) PMID: 31709223 6. Guidolin V., Erik S. Carlson E.S., Carra’A., Villalta P.W., Maertens L.A., HechtS.S. and Balbo S. (2021) Identification of new markers of alcohol-derived DNA damage in humans using an ultrasensitive DNA adductomic approach. Biomolecules, accepted for publication

7. Balbo S., Hecht S.S., Upadhyaya P., Villalta P.W. (2014) Application of a high-resolution mass-spectrometry-based DNA adductomics approach for identification of DNA adducts in complex mixtures”. Anal Chem. 86(3): 1744-52. doi: 10.1021/ac403565m. PMID: 24410521 PMCID: PMC3982966

8. Geacintov, N. E. and S. Broyde (2010). The chemical biology of DNA damage. Weinheim, Wiley- VCH.

9. 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.

10. Kawai, Y. and E. Nuka (2018). "Abundance of DNA adducts of 4-oxo-2-alkenals, lipid peroxidation-derived highly reactive genotoxins." J Clin Biochem Nutr62(1): 3-10.

11. 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.

12. Marnett, L. J. (2002). "Oxy radicals, lipid peroxidation and DNA damage." Toxicology 181- 182: 219-222.

13. Richarme et al. (2017). "Guanine glycation repair by DJ-1/Park7 and its bacterial homologs." Science 357(6347): 208-211.

14. 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.

15. 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.

16. Stone et al. (1990). "Investigation of the Adducts Formed by Reaction of Malondialdehyde with Adenosine" Chem. Res. Toxicol. 3: 33-38.

17. Voulgaridou et al. (2011). "DNA damage induced by endogenous aldehydes: current state of knowledge." Mutat Res 711 (1 -2): 13-27.

18. Wauchope et al. (2015). "Nuclear Oxidation of a Major Peroxidation DNA Adduct, M1dG, in the Genome." Chem Res Toxicol 28(12): 2334-2342.

19. Lepore et al. (2017). Functionally diverse human T cells recognize non-microbial antigens presented by MR1. ELife 6: e24476.

20. Lepore et al. (2014). Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRbeta repertoire. Nat Commun 5, 3866. 21. Dai et al. (2014). edgeR: a versatile tool for the analysis of shRNA-seq and CRISPR-Cas9 genetic screens. F1000 Research 3, 95.

22. Hart et al. (2015). High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype- Specific Cancer Liabilities. Cell 163, 1515-1526. 23. Brunk et al. (2018). Recon3D enables a three-dimensional view of gene variation in human metabolism. Nature Biotechnology 36, 272-281.

24. Kanehis et al.. (2019). New approach for understanding genome variations in KEGG. Nucleic Acids Res 47, D590-D595.

25. Schmaler et al. (2018). Modulation of bacterial metabolism by the microenvironment controls MAIT cell stimulation. Mucosal Immunology 11: 1060-1070.

26. Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-359.

27. Sanson et al. (2018). Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun 9, 5416. 28. Bolger, A.M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for lllumina sequence data. Bioinformatics 30, 2114-2120.

29. Durinck et al. (2009). Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nature protocols 4, 1184-1191.