Treatment for cancer

Abstract

Novel nucleic acid compositions, TMBIM6 variant peptide binding proteins, vector systems, modified cells and pharmaceutical compositions that encode or express T cell receptor components directed against a TMBIM6 variant peptide comprising a W to F substitution are provided herein. These novel components may be used to enhance an immune response in a subject diagnosed with a disease or condition, such as cancer. Associated methods for treating such subjects are therefore also provided herein. Also provided are methods of producing such binding proteins and a kit of parts including the novel nucleic acid compositions, TMBIM6 variant peptide binding proteins, vector systems, modified cells or pharmaceutical compositions.

Description

[0001] Treatment for Cancer

[0002] The invention provides novel nucleic acid compositions, vector systems, modified cells and pharmaceutical compositions that encode or express T cell receptor components directed against a peptide comprising an amino acid sequence of an antigen or epitope of a W to F substitutant peptide derived from TMBIM6. Also provided herein are nucleic acid compositions, vector systems, modified cells and pharmaceutical compositions for use in methods of treating diseases and conditions is a subject, such as cancer.

[0003] Eukaryotic mRNA translation is a highly regulated and conserved process56. Yet, tumor initiation, progression, and metastasis promote dysregulation of mRNA translation. Here, oncogenic pathways (e.g., RAS-MAPK, mTOR, YAP1 , and Myc) that stimulate mRNA translation initiation via the eukaryotic initiation factor 4F (elF4F) protein complex, play an important role7-13. In addition, ribosome concentration and tRNA modifications represent key processes in cancer cell behaviour14 15. However, while dysregulated mRNA translation is key to oncogenesis, it also impacts the quality of proteins produced during periods of amino acid shortages, such as tryptophan depletion, which occurs following immune cell activation by cancer cells16-18.

[0004] T cells activated by cancer-specific antigens (neoantigens) secrete interferon-gamma (IFNy), which induces I DO119in the target cancer cells. IDO1 catabolizes tryptophan to metabolites along the kynurenine pathway, generating intracellular tryptophan shortage if the exposure is persistent20. While an increased kynurenine level in the tumor microenvironment benefits cancers by suppressing T-cell activity, IDO1 creates an intracellular shortage of tryptophan in the cancer cells, which stimulates ribosomal frameshifting and codon reassignments at the “starved” tryptophan codons2. In particular, tryptophan to phenylalanine codon reassignments (W>F substitutants) were identified as the major event that facilitates in-frame protein synthesis in IFNy-treated and tryptophan-depleted cancer cells2,16,18. It has recently been demonstrated that substitutant proteins are enriched in cancer specimens and have the potential to produce Human Leukocyte Antigen (HLA)-bound cancer-specific epitopes (neoepitopes) that can trigger an anti-tumor immune response.

[0005] In recent years, adoptive transfer of T cells genetically engineered to express artificial immune receptors called chimeric antigen receptors (CAR T cells)19has revolutionized the treatment of IB- cell malignancies20,21. CAR T cells are, however, antibody-based and can, therefore, recognize only cell-surface molecules, and it has proven difficult to identify cell type-specific cell surface molecules that can be safely targeted in solid cancers22. T-cell receptors can, on the other hand, recognize peptides derived from cellular proteins with any subcellular location, greatly increasing the number of potential therapeutic targets. Recently, the first case reports on TCR-T cell therapy targeting shared neoantigens were published, showing promising responses in solid cancer patients2324. However, TCR-T cell therapy directed against tumor-associated antigens, such as MARTI , gp100 or MAGE-A3 in melanoma and CEA in metastatic colorectal cancers, can suffer from severe on-target toxicities, limiting their usage25-30.

[0006] The identification of new genetically encoded neoantigens derived from shared, somatic, non- synonymous mutations remains a major challenge, as 99% of mutations are unique to the individual patient and tumor31 32. In addition, neoantigenic peptides only represent a minute fraction of the tumor immunopeptidome, estimated at one mutated peptide per -2,000 non- synonymous mutations among over 10,000 unique HI_A class I peptides33. Moreover, classical neoantigens are rarely strongly immunogenic due to counter-selection during tumor evolution34-36. In contrast, W>F-substitutant-derived neoepitopes are treatment-inducible, with broad and shared expression - only restricted by IDO1 expression and the antigen-presenting capacity of cancer cells.

[0007] There is a need for an improved TCR based therapeutics.

[0008] There is a need for improved TCR based therapeutics for use in treating diseases such as cancer. There is need for improved TCR based combination therapies for use in treating diseases such as cancer.

[0009] Brief summary of the disclosure

[0010] The inventors have identified a reactive TCR, and demonstrated its specificity and efficiency in tumour cell killing both in vitro and in vivo. The inventors have also shown that TMBIM6W>F TCR transduced T cells have the capacity to enhance the killing efficacy of other therapeutics such as anti-MART1 antigen-driven TCR T cells, opening new avenues for T-cell transfer therapy.

[0011] Furthermore, the inventors have surprisingly found that the TCR recognizes the substitutant TMBIM6 W>F peptide with improved properties as compared with the WT peptide, even though both TMBIM6 W>F and wild-type peptides bind HLA-A24 quite well, and the substitutant F residues of TMBIM6 W>F face downwards towards the HI_A molecule rather than upwards towards the TCR.

[0012] Local IFNy injections have limited clinical relevance in metastatic cancer, and systemic application has not been successfully applied in cancer therapy due to dual effects and toxicity2. A more clinically relevant scenario could, therefore, be to induce local secretion of IFNy in the tumor microenvironment by cancer antigen-targeting TCRs, such as TCRMART1in melanoma patients, combined with treatment with TCR T cells targeting W>F neoepitopes. Such combination therapies may improve efficacy while potentially reducing side effects of high doses of TCR T cells targeting tumor-associated antigens or cancer-testis antigens34. In light of this, the inventors show the potential of T cells including encoding and expressing TCRs disclosed herein to improve the efficacy of TCRMART1T cells in a combined novel immunotherapy approach. This represents a relevant, and beneficial alternative approach to improving conventional adoptive cancer immunotherapy in patients while reducing side effects. In one aspect of the invention there is provided, an isolated nucleic acid composition that encodes a TMBIM6 variant peptide binding protein having a TCR a chain variable (Va) domain and a TCR P chain variable (VP) domain, wherein the variant peptide comprises a W to F substitution, the composition comprising:

[0013] (a) an isolated nucleic acid molecule that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:1 , or a functional fragment thereof; and

[0014] (b) an isolated nucleic acid molecule that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 27, or a functional fragment thereof.

[0015] In a second aspect of the invention there is provided, a TMBIM6 variant peptide binding protein having a TCR a chain variable (Va) domain and a TCR p chain variable (VP) domain, wherein the variant peptide comprises a W to F substitution, the composition comprising: a) a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:1 , or a functional fragment thereof; and b) a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 27, or a functional fragment thereof

[0016] In certain embodiments, the encoded binding protein is capable of binding to a peptide:HLA complex, wherein the peptide comprises the TMBIM6 variant peptide.

[0017] In certain embodiments, the TMBIM6 variant peptide comprises or consists of an amino acid sequence according to SEQ ID NO 58.

[0018] In certain embodiments, the CDR3 of (a) has an amino acid sequence having at least 90% sequence identity to SEQ ID NO 1.

[0019] In certain embodiments, the CDR3 of (a) is encoded by a nucleic acid sequence according to SEQ ID NO 3.

[0020] In certain embodiments, the CDR3 of (b) has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 27.

[0021] In certain embodiments, the CDR3 of (b) is encoded by the nucleic acid sequence according SEQ ID NO: 29.

[0022] In certain embodiments, (a) further comprises a TCR a chain constant region.

[0023] In certain embodiments, the TCR Va domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10.

[0024] In certain embodiments, the TCR Va domain is encoded by the nucleic acid sequence according to SEQ ID NO: 12.

[0025] In certain embodiments, (b) further comprises a TCR p chain constant region. In certain embodiments, the TCR Vp domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 36.

[0026] In certain embodiments, the Vp domain is encoded by the nucleic acid sequence according to any one of SEQ ID NO: 38.

[0027] In certain embodiments, the CDR3 of (a) is within a TCR Va domain having at least 90% sequence identity to SEQ ID NO: 10 or 24, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 1 ; and optionally wherein (a) comprises a TCR a chain constant domain.

[0028] In certain embodiments, the TCR Va domain CDR1 has an amino acid sequence of SEQ ID NO: 4 and the TCR Va domain CDR2 has an amino acid sequence of SEQ ID NO: 7.

[0029] In certain embodiments, the CDR3 of (b) is within a TCR Vp domain having at least 90% sequence identity to SEQ ID NO: 36 or 44, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 27; and optionally wherein (b) comprises a TCR p chain constant domain.

[0030] In certain embodiments, the TCR Vp domain CDR1 has an amino acid sequence of SEQ ID NO: 30 and the TCR Vp domain CDR2 has an amino acid sequence of SEQ ID NO: 33.

[0031] In certain embodiments, the TCR Va domain and / or TCR Vp domain each comprise a leader peptide positioned at the N-terminal; optionally wherein the TCR Va domain leader peptide comprises an amino acid sequence according to SEQ ID NO: 19 and / or wherein the TCR Vp domain leader peptide comprises an amino acid sequence according SEQ ID NO: 42.

[0032] In certain embodiments, the nucleic acid molecule or molecules encode or the TMBIM6 variant peptide binding protein comprises: a) a TCR a chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 21 or 24; and / or b) a TCR p chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 47 or 50.

[0033] In certain embodiments,

[0034] I. the TCR a chain is encoded by the nucleic acid sequence according to SEQ ID NO: 23 or 26; and / or

[0035] II. TCR p chain is encoded by the nucleic acid sequence according to SEQ ID NO: 49 or 52.

[0036] In certain embodiments, the nucleic acid molecule or molecules encode an amino acid sequence according to SEQ ID NO: 55.

[0037] In certain embodiments, the TMBIM6 variant peptide binding protein comprises or consists of an amino acid sequence according to SEQ ID NO: 55.

[0038] In certain embodiments, the nucleic acid molecule or molecules comprises or consists of a sequence according to SEQ ID NO: 57.

[0039] In certain embodiments, the nucleic acid molecule or molecules encodes a T cell receptor (TCR) or an antigen binding fragment thereof. In certain embodiments, the TMBIM6 variant peptide binding protein comprises or consists of a T cell receptor (TCR).

[0040] In certain embodiments, the encoded binding protein or TMBIM6 variant peptide binding protein comprises a TCR, an antigen binding fragment of a TCR, or a chimeric antigen receptor (CAR).

[0041] In certain embodiments, the antigen binding fragment of a TCR is a single chain TCR (scTCR) or a chimeric TCR dimer in which the antigen binding fragment of the TCR is linked to an alternative transmembrane and intracellular signalling domain.

[0042] In a third aspect of the invention there is provided, a vector system comprising a nucleic acid composition as described herein.

[0043] In certain embodiments, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.

[0044] In a fourth aspect there is provided, a modified cell transfected or transduced with a nucleic acid composition as described herein.

[0045] In certain embodiments, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line.

[0046] In certain embodiments, the modified cell is a human cell.

[0047] In certain embodiments, the modified cell expresses the TMBIM6 variant peptide binding protein. In a fifth aspect there is provided, a pharmaceutical composition comprising a nucleic acid composition, a TMBIM6 variant peptide binding protein, a vector, or a modified cell, as described herein and a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

[0048] In certain embodiments, the pharmaceutical composition further comprises IFNy and / or activators of IFNy.

[0049] In certain embodiments, the pharmaceutical composition further comprises a KYNase.

[0050] In a sixth aspect there is provided, a nucleic acid composition, a TMBIM6 variant peptide binding protein, a vector, a modified cell, or a pharmaceutical composition, as described herein for use a medicament.

[0051] In a seventh aspect there is provided method of treating or preventing cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid composition, a TMBIM6 variant peptide binding protein, a vector, a modified cell, or a pharmaceutical composition, as described herein.

[0052] In an eighth aspect there is provided, a nucleic acid composition, a TMBIM6 variant peptide binding protein, a vector, a modified cell, or a pharmaceutical composition, as described herein for use in treating or preventing cancer in a subject.

[0053] In certain embodiments, the cancer is associated with tryptophan depletion.

[0054] In certain embodiments, the cancer is associated with increased IDO1 activity and / or IFNy activity. In certain embodiments, the cancer is glioblastoma, prostate cancer, pancreatic cancer, nonsmall lung carcinoma cell, melanoma, breast cancer, gastric cancer; a head and / or neck cancer, a cancer related to viral infection or colorectal cancer.

[0055] In certain embodiments, the method induces or enhances a cell mediated immune response in the subject.

[0056] In certain embodiments, the pharmaceutical composition is for use in inducing or enhancing a cell mediated immune response in the subject.

[0057] In certain embodiments, the method or pharmaceutical composition for use as described herein further comprises administering at least one additional therapeutic.

[0058] In certain embodiments, the additional therapeutic is selected from: a) activators of I FNy; b) a further T-cell receptor; c) a modified cell comprising a further T-cell receptor or comprising a nucleic acid encoding the further T-cell receptor; d) an immune checkpoint inhibitor; e) nucleic acid based therapeutics comprising a nucleic acid sequence encoding one or more activators of I FNy; f) protein based therapeutics comprising an amino acid sequence encoding one or more activators of I FNy; g) immunofilaments comprising activators of I FNy; and / or h) a chimeric antigen receptor cell therapeutic.

[0059] In certain embodiments, the additional therapeutic increases a level of a of a TMBIM6 variant peptide, wherein the variant comprises a W to F substitution in the subject.

[0060] In certain embodiments, the additional therapeutic is administered, prior to, subsequently to and / or concurrently with the pharmaceutical composition.

[0061] In certain embodiments, the subject is HLA-A*24 positive or HLA-C*02:02 positive; optionally HI_A-*24:02 positive or HI_A-C*02:02 positive.

[0062] In a ninth aspect there is provided, a pharmaceutical composition as described herein for use in treating or preventing cancer in a human subject, wherein the subject has been identified as having a cancer by the presence of a peptide in a sample isolated from the subject, wherein the peptide comprises or consists of SEQ ID NO: 58.

[0063] In a tenth aspect there is provided, use of a pharmaceutical composition as described herein in the manufacture of a medicament for treating or preventing cancer.

[0064] In a n eleventh aspect there is provided, a kit of parts comprising: a) a nucleic acid composition, a TMBIM6 variant peptide binding protein, a vector, a modified cell, or a pharmaceutical composition, as described herein; and one or more of: b) a KYNase; c) activators of I FNy d) a further T-cell receptor; e) a modified cell comprising a further T-cell receptor or comprising a nucleic acid encoding the further T-cell receptor; f) an immune checkpoint inhibitor; g) nucleic acid therapeutics comprising a nucleic acid sequence encoding one or more activators of IFNy; h) protein based therapeutics comprising an amino acid sequence encoding one or more activators of IFNy; i)immunofilaments comprising activators of IFNy; and / or j)a chimeric antigen receptor cell therapeutic.

[0065] In a twelfth aspect there is provided, an isolated nucleic acid composition that encodes a T cell receptor (TCR), wherein the TCR comprises:

[0066] (i) a TCR Va domain comprising a CDR3 amino acid sequence of SEQ ID NO: 1 , and a TCR Vp domain comprising a CDR3 amino acid sequence of SEQ ID NO: 27; or

[0067] (ii) a TCR Va domain having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 10 or 13; and (ii) a p domain having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 36 or 44.

[0068] In a thirteenth aspect there is provided, a method of generating a binding protein that is capable of specifically binding to a TMBIM6 variant peptide and does not bind to a peptide that does not contain the TMBIM6 variant peptide, comprising contacting a nucleic acid composition as described hereon with a cell under conditions in which the nucleic acid composition is incorporated and expressed by the cell, wherein TMBIM6 the variant peptide comprises a Wto F substitution.

[0069] In certain embodiments, the method is ex vivo.

[0070] In a fourteenth aspect there is provided, an isolated nucleic acid sequence comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs 3, 6, 9, 12, 15, 23, 26, 29, 32, 35, 38, 46, 49, 52, or 57.

[0071] In a fifteenth aspect there is provided, an isolated nucleic acid sequence comprising or consisting of the nucleotide sequence of SEQ ID NOs: 3, 6, 9, 12, 15, 23, 26, 29, 32, 35, 38, 46, 49, 52, or 57 for use in therapy.

[0072] In a sixteenth aspect there is provided, a T cell receptor (TCR), wherein the TCR comprises:

[0073] (i) a TCR Va domain comprising a CDR3 amino acid sequence of SEQ ID NO: 1 , and a TCR Vp domain comprising a CDR3 amino acid sequence of SEQ ID NO: 27; or

[0074] (ii) a TCR Va domain having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 10 or 13; and (ii) a Vp domain having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 36 or 44; or

[0075] (iii) an amino acid sequence comprising or consisting of SEQ ID NO: 55.

[0076] In certain embodiments, the T cell receptor (TCR) as described herein is for use in therapy. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0077] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0078] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0079] Various aspects of the invention are described in further detail below.

[0080] Brief description of the Figures

[0081] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0082] Figure 1 shows TMBIM6W>Fis a broadly expressed, inducible W>F substitutant neoepitope A. Peptide-binding consensus sequences of 4 of the most prevalent worldwide HLA-A alleles as determined by NetMHCpan4.161’62. Highlighted in red are the preferred positions of phenylalanine binding. B. A scheme outlining the identification of common W>F neoepitopes by immunopeptidomics. C. A matrix indicating the expression of the most common (present in 5 or more cell lines) HLA-A*24:02-bound W>F neoepitopes, as determined by immunopeptidomics. The presence of the W>F neoepitopes is indicated by black dots while absence is indicated by grey dots. Purple squares indicate the presence of the W>F neoepitopes in the mock-treated control condition. Grey squares indicate the absence of the neoepitopes in a dataset of immunopeptidomics from 25 benign tissues43. Finally, the 13 peptides were assessed for their capacity to bind HI_A-A*24:02 by NetMHCpan4.1 (red squares indicate strong binders; light red squares indicate weak binders). Cell lines marked in red are HI_A-A*24:02 positive, whereas those marked in black are HI_A-A*24:02 negative. The total number of W>F substitutant peptides per cell line is plotted on the right side of the matrix. Dots in red mark the TMBIM6W>Fneoepitope. D. A table indicating the sequences and expression characteristics of the most common HLA- A*24:02-bound W>F neoepitopes identified in this study. W>F residues are annotated with ‘f’. Average mRNA expression (Log2(TPM+1)) was extracted from the Depmap gene expression portal for cell lines (https: / / depmap.org / ).

[0083] Figure 2 shows Selection of HLA-A*24+ cancer cell lines. Related to Figure 1. A. Peptide- binding consensus sequences of the most worldwide abundant HLA-A, HLA-B, and HLA-C molecules, as determined by NetMHCpan4.161’62. B. A barplot depicting the relative intracellular level of tryptophan and of kynurenine in DLD-1 , Mi a- Paca-2, NCI-H1299, HepG2, and Colo-320 cells treated or not for 48 hrs with IFNy (IFN). Values represent the average of the 3 independent experiments ± SD. C. A list of cancer cell lines used in this study. Cell lines were classified according to their tissue origin. If the cells have been used for immunopeptidomics, the condition used to deplete tryptophan is mentioned. Finally, the table presents the HLA-A, HI_A-B and HLA- C molecules expressed by these cancer cells, as determined by the Cellosaurus (https: / / www.cellosaurus.org / ).

[0084] Figure 3 shows Validation of cancer cells HLA-type by immunopeptidomics. Related to Figure 1. A. HI_A peptide signatures for every cell line that has been used in the immunopeptidomics experiment. The signatures were obtained from the WT peptides detected in the immunopeptidomics. The 4 most common motifs for every cell line are displayed. The signatures that fit the consensus motif of HI_A-A*24:02 (Figure 1A) are highlighted in red. This analysis confirms the presence of HLA-A*24:02 in all the expected cell lines (Figure 2C). B. A boxplot representing the average number of peptides (Iog2 scale) detected by immunopeptidomics in the indicated cancer cell lines exposed to IFN© in combination or not with W-depleted medium. For the Dll-145 cells, HLA-A*24:02 was ectopically overexpressed using lentiviral transduction. C. A matrix indicating the expression of the WT peptides from the most common (present in 5 or more cell lines) HLA-A*24: 02- bound W>F neoepitopes as determined by immunopeptidomics (Figure 1C). Black dots mark the presence of WT peptide. The 13 WT peptides were computationally assessed for their capacity to bind HLA-A*24:02 by NetMHCpan4.1 (blue = strong binder; light blue = weak binder). The cell lines marked in red are HLA-A*24:02 positive, whereas the ones in black are HLA-A*24:02 negative. The cell line marked in light red is HLA-A*24:02 but has a mutation in one of the key proteins for peptide presentation. Next to the matrix, the total number of peptides detected in the immunopeptidomics analysis is displayed. The second column corresponds to the expression of the WT peptide of TMBIMG^ neoepitope

[0085] Figure 4 shows Identification and validation of a TCR against TMBIM6W>F. Related to Figure 5. A. Evaluation of transduction efficiency by flow cytometry analysis of TCRTMBIM6W>F 1transduced CD8 T cells visualized as living mTCRp+CD8+T cells. Transduction efficiency was compared to T cells that in parallel underwent the transduction procedure without the addition of TCR- containing retroviral supernatant (non-transduced TCR). B. Validation of TCR target specificity by flow cytometry analysis of TCR T cells that were transduced with TCR™BIM6W>F 1anc|stajnec|wjth either TMBIM6W>Fneoepitope specific or mock peptide tetramers. C. A flow cytometry analysis of the TCR™BIM6W>F 1-transduced T cells co-cultured with peptide-loaded RA cells. RA cells were loaded with different concentrations of synthetic peptides corresponding to TMBIM6w>For to its wild-type counterpart (TMBIM6wr). Then, RA cells and TCR-transduced T cells were co-cultured for 16 hrs and T cells were stained for live-dead marker, CD8, mTCRp and CD137. The black square represents the activated T-cell population. Above the square, the number indicates the percentage of reactive T cells. Figure 5a shows the quantification of this experiment.

[0086] Figure 5 shows The identification and characterization of TMBIM6w>F-targeting TCR T cells A. Graph representing the percentage of activated TCRTMBIM6W>F 1q-ce||S UpOn C0-culture with RA cells at an Effector : Target (E:T) ratio of 1 :2. Prior to co-culture, RA cells were loaded with the indicated concentrations of either TMBIM6W>For its corresponding WT synthetic peptide. TCRTMBIM6W>F^-transduced T cells were incubated overnight (16 hrs) with the peptide-loaded cells and subsequently examined by flow cytometry for activation by staining with anti-CD137 antibody. Dots represent the average of two technical replicates ± SD. B. A scheme of the setup of the coculture T cell recognition assays. C. A barplot depicting the percentage of activated TCR™BIM6W>F 1T cells measured by flow cytometry following anti-CD137 staining. RA glioblastoma cells were treated as indicated for 48 hrs and then co-cultured with TCRTMBIM6W>F 1-ce||S at an^.q-ratj0 Of 1 :2 for 16 hrs prior to analysis. As a positive control for activation, TCR™BIM6W>F 1q-ce||S weretreated with a combination of ionomycin and PMA. Bars represent the average ± SD, and dots show each of the 3 independent co-culture experiments. ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test. D. Immunoblot analysis for GCN2, pGCN2, pSTATI , WARS, IDO1 and Tubulin of RA KOs cells for IDO-1 or TMBIM6 and exposed or not for 48 hrs to IFNy. TMBIM6 KO was demonstrated using DNA sequencing and TIDE analysis63(Figure 6D). E. A barplot depicting the percentage of activated TCRTMBIM6W>F 1q-ce||Smeasured by flow cytometry using anti-CD137 antibody staining. Co-culture experiments of the cell lines presented in panel ‘d’ with the TCR™BIM6W>F.I q-ce||S wereperformed as indicated in panel ‘b’. Values represent the average of the 3 independent co-cultures ± SD. ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test. F. Dot plot depicting the peptide intensity of both TMBIM6m(gray dots) and of the substitutant counterpart TMB IM 6^ (red dots) in RA-IDO1KOcells using immunopeptidomics. As indicated, cells were treated for 48 hrs with IFNy combined or not with tryptophan depleted medium. Every dot represents an independent experiment, and the lines represent the median intensity. **p < 0.01 , ***p < 0.001 by 2-way ANOVA, followed by Bonferroni post hoc test. G. A scheme of the setup of the T cells / cancer cells co-culture killing assay. Note that the procedure is the same as in ‘b’ except that the co-culture period lasted either 16 hrs or 48 hrs. Cell viability is based on resazurin or crystal violet staining. H. A barplot depicting the percentage of activated TCRTMBIM6W>F 1or TCRMART1T cells as measured by flow cytometry using anti-CD137 antibody staining. RA cells overexpressing TMBIM6m, TMBIMe^, or MARTI (as depicted above the graph) were used for these assays. Prior co-culture of 16 hrs at an E:T ratio of 1 :2, RA cells were treated or not for 48 hrs with IFNy or loaded overnight with the TMBIMe^ peptide as indicated. Values represent the average of 3 independent co-culture experiments ± SD. ***p < 0.001 by 2- way ANOVA, followed by Bonferroni post hoc test. I. A barplot depicting the relative viability of RA cells overexpressing the TMBIM6wror TMBIM6w>Fepitopes upon 16 hrs co-culture with either TCRMART1or TCR™BIM6W>F.I q-ce||S at an^.q-ratj0 Of 2:1 Viability was normalized to the control situation (normal medium and no TCR T cell). Values represent the average of 3 independent cocultures ± SD. ***p < 0.001 by 2-way ANOVA, followed by Bonferroni post hoc test. J. A barplot depicting the relative viability of RA cells expressing a control sgRNA(NTI) or KOs for IDO1 or TMBIM6 using 2 different sgRNA (#1 and #2) treated or not for 48 hrs with IFNy and then coculture for 16 hrs with either TCRMART1or TCRTMBIM6W>F 1-ce||S at an^. -ratj0 Of 2:1. Viability was normalized to the control situation (normal medium and no TCR T cell). Values represent the average of 3 independent co-cultures ± SD. *p < 0.05, **p < 0.01 , and ***p < 0.001 by 2-way ANOVA, followed by Bonferroni post hoc test.

[0087] Figure 6 shows TCRTMBIM6W>F 1activation is IDO-1 and TMBIM6-dependent in RA cells. Related to Figure 5. A. A barplot depicting the percentage of activated TCRTMBIM6W>F 1q-ce||Smeasured by flow cytometry using anti-CD137 antibody staining following co-culture with RA cells at an E:T ratio of 1 :2. RA cells were treated with IFNy for 48 hrs in the presence or absence of KYNase, and then subsequently co-cultured with TCRTMBIM6W>F 1q-ce||S asindicated in Figure 5b. KYNase treatment was thus limited to the period of IFNy treatment. Values represent the average of the 3 independent co-cultures ± SD. ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test. B. Same as Figure 5c using another CD8 T cell donor. A barplot depicting the percentage of activated TCRTMBIM6W>F 1q-ce||Smeasured by flow cytometry following anti-CD137 staining. RA glioblastoma cells were treated as indicated for 48 hrs and then co-cultured with TCRTMBIM6W>F 1T cells or with untransduced TCR T cells for 16 hrs prior to analysis. As a positive control for activation, TCRTMBIM6W>F 1q-ce||S weretreated with a combination of ionomycin and PMA. Bars represent the average ± SD, and dots show each of the 3 independent co-culture experiments ***p < 0 001 by 1-way ANOVA, followed by Bonferroni post hoc test. C. Sanger sequencing images of RA cells expressing a control sgRNA (NT1) or TMBIM6 KOs using two independent sgRNA per gene. The list of primers used for the PCR is listed in Table 2. The sgRNA sequences are marked by the black line. A red star annotates the nucleotide where the errors start to accumulate. The samples were also submitted to TIDE analysis to evaluate the efficiency of the Kos (TMBIM6 KO#1 = 33,5%; TMBIM6 KO#2 = 98%). D. Same as Figure 6C with sgRNA (NT1) and IDO1 (IDO KO#1 = 96,7%; IDO KO#2 = 66,8%). E. Barplots depicting the Median Fluorescent Intensity (MFI) of B2m-APC (left graph), panHLA-A,B,C-FITC (right graph) or HLA- A*24-APC (bottom left) staining using RA cells expressing or not a control sgRNA (NT1) or KO for IDO1 or TMBIM6 (same cell as shown in Figures 6C-D). Cells were treated or not for 48 hrs with IFNy prior staining. Values represent the average of 3 independent staining ± SD. *p<0,05; **p<0,01 and ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test. F. A barplot depicting the percentage of activated TCRTMBIM6W>F 1q-ce||S asmeasured by flow cytometry using anti-CD137 antibody staining. The same cell populations as in Figure 5e were incubated with the indicated peptides (10nM) and then co-cultured for 16 hrs with TCRTMBIM6W>F 1q-ce||S at an^.q- ratio of 1 :2. Values represent the average of the 3 independent co-cultures ± SD. ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test. G. Graph representing the percentage of activated TCRTMBIM6W>F 1ce||S UpOn C0-culture with TMBIM6 KO#2 RA cells. Prior to co-culture, RA cells were treated for 48 hrs with IFNy and IDO1 inhibitor (1MT) and then loaded with the indicated concentrations of either TMBIM6w>For its corresponding synthetic peptide TMBIM6wr. TCRTMBIM6W>F^-transduced T cells were incubated overnight (16 hrs) with the peptide-loaded cells at an E:T ratio of 1 :2 and subsequently examined by flow cytometry for activation by staining with anti-CD137 antibody. Dots represent the average of a technical duplicate ± SD. H. A scheme representing the strategy employed for performing immunopeptidomics in RA IDO1 KO cells. 2 different RA KO clones were used and exposed or not for 48 hrs to IFNy combined or not with tryptophan depleted medium (-W / IFN) prior lysis, HI_A pulldown and mass-spec analysis. Below the scheme a heat map depicting the abundance of both WT peptides and W>F peptides identified by immunopeptidomics in this 2 IDO1 KO clones. I. A barplot depicting the relative viability of RA cells overexpressing or not TMBIM6m, TMBIMG^ or MARTI 26-35 after 16 hrs co-culture with TOR TMBIM6W>F 1or TCRMART1T cells at an E:T ratio of 2:1. Prior co-culture RA cells were treated or not with IFNy for 48 hrs. Viability was normalized to the control situation (normal medium and no TOR T cell). Values represent the average of 3 independent co-cultures ± SD. ***p < 0.001 by 2-way ANOVA, followed by Bonferroni post hoc test. J. A barplot representing the relative cell viability of RA expressing a control sgRNA (NT1) or knocked-out for IDO1 or TMBIM6. Prior to co-culture with TCR TMBIM6W>FT cells for 48 hrs at an E:T ratio of 2:1 , RA cells were treated for 48 hrs with IFNy and KYNase. After 48 hrs, cells were rinsed and fixed with 4% formaldehyde and then stained with 0,1% Crystal Violet. The intensity of crystal violet was measured by a plate reader (490nM). Every dot represents the average of the 3 independent co-cultures + / -SD. ** p<0,01 and ***p < 0 001 by 1-way ANOVA, followed by Sidak post hoc test.

[0088] Figure 7 shows Specific activation of TCR™BIM6W>F 1T cells by PC-3 cancer cells. Related to Figure 5. A. Sanger sequencing images of PC-3 cells expressing a control sgRNA (NT1) or either IDO1 or TMBIM6 KOs. The list of primers used for the PCR is indicated in Table 2. The sgRNA sequences are marked by the black line. A red star annotates the nucleotide where the errors start to accumulate. The samples were also submitted to TIDE analysis to evaluate the efficiency of the knock-out (TMBIM6 KO#1A = 60,7%; TMBIM6 KO#1 B = 48,1%; IDO KO#1A = 27,4%; IDO KO#1 B = 34,7%). B. Immunoblot analysis for pSTATI , WARS, IDO1 and Tubulin of PC-3 cells knock-out for IDO-1 or TMBIM6 and exposed or not for 72 hrs to IFNy. TMBIM6 knockout was demonstrated using DNA sequencing and TIDE analysis (Figure 7A). C. Barplots depicting the Median Fluorescent Intensity (MFI) of HI_A-A*24:02-APC (left graph), B2m-APC (right graph) or panHI_A-A,B,C-FITC (bottom left) staining using PC3 cells expressing or not a control sgRNA (NT1) or KO for IDO1 or TMBIM6 (same cell as shown in Figures 7A-7B). Cells were treated or not for 72 hrs with IFNy prior staining. Values represent the average of 3 independent staining ± SD. *p<0,05; **p<0,01 and ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test. D. A barplot depicting the percentage of activated TCRTMBIM6W>F 1-ce||Smeasured by flow cytometry using anti-CD137 antibody staining. Co-culture experiments of the cell lines presented in Figures 7A-7B with the TCRTMBIM6W>F 1-ce||S at an^. -ratj0 Of q were performed as indicated in Figure 5B. Values represent the average of the 3 independent co-cultures ± SD. ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test. E. A barplot depicting the relative viability of PC3 cells expressing a control sgRNA(NTI) or KOs for IDO1 or TMBIM6 treated or not for 48 hrs with IFNy and then co-culture or not for 16 hrs with TCRTMBIM6W>F 1q-ce||S at an^.q-ratj0 Of 2:1. Viability was normalized to the situation without TCR T cells. Values represent the average of 3 independent co-cultures ± SD. ***p < 0.001 by 2-way ANOVA, followed by Bonferroni post hoc test. F. A barplot depicting the relative cell viability of PC-3 upon co-culture with TCRTMBIM6W>F 1T cells at an E:T ratio of 2:1. Prior 48 hrs of co-culture, PC-3 cells were exposed to 72 hrs IFNy and KYNase. PC-3 expressing a control sgRNA (NT1) or either IDO1 or TMBIM6 KOs were used. A ratio of 1 :2 between cancer cells and TCR T cells was used for this experiment. Values represent the average of the 3 independent co-cultures ± SD. ***p < 0.001 by 1-way ANOVA, followed by Sidak post hoc test. G. A barplot depicting the percentage of activated TCR™BIM6W>F 1T cells measured by flow cytometry using anti-CD137 antibody staining. Prior 16 hrs of co-culture at an E:T ratio of 1 :2, PC-3 cells were exposed to 72 hrs IFNy and KYNase, combined or not with 1-methyl-tryptophan (1-MT) or Epacadostat. Values represent the average of the 3 independent co-cultures ± SD ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test. H. Immunoblot analysis for pSTATI , WARS, IDO1 and Tubulin of PC-3 cells knock-out for IDO-1 or TMBIM6 and exposed or not for 72 hrs to IFNy. TMBIM6 knockout was demonstrated using DNA sequencing and TIDE analysis (Figure 7A). I. A barplot depicting the percentage of activated TCRTMBIM6W>F 1T cells measured by flow cytometry using anti-CD137 antibody staining. Prior 16 hrs of co-culture at an E:T ratio of 1 :2, PC-3 cells were exposed to 72 hrs IFNy and KYNase, combined or not with 1-methyl-tryptophan (1-MT) or Epacadostat. Values represent the average of the 3 independent co-cultures ± SD. ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test. J. Immunoblot analysis for pSTATI , WARS, IDO1 and Tubulin of PC-3 cells exposed or not for 72 hrs to IFNy in combination with 1-methyl-tryptophan (1-MT) or Epacadostat. K. A barplot depicting the percentage of activated TCRTMBIM6W>F 1q-ce||Smeasured by flow cytometry using anti-CD137 antibody staining. Prior 16 hrs of co-culture at an E:T ratio of 1 :2, PC-3 cells were exposed to either 72 hrs of IFNy and KYNase or to doxycycline, at the indicated timepoints. Values represent the average of the 3 independent experiments ± SD. **p<0.01 and ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test.

[0089] Figure 8 shows Characterization of non-transformed cell lines. Related to Figure 9. A. A Barplot depicting the Median Fluorescent Intensity (MFI) of HI_A-A*24-APC staining using MCF10-A, hTERT-RPE1 and 293T cells where either HLA-A*02:01 or HLA-A*24:02 have been overexpressed using lentiviral constructs. Cells were treated or not for 48 hrs with IFNy in combination or not with tryptophan-depleted medium prior staining. Values represent the average of 3 independent staining ± SD. *p<0,05 and ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test. B. A barplot depicting the percentage of TCRTMBIM6W>F 1activated T cells measured by flow cytometry using CD- 137 staining. TCRTMBIM6W>F 1-ce||S were C0-cultured for 16 hrs, at an E:T ratio of 1 :2, with either MCF10-A, hTERT-RPE1 , or 293T cells ectopically expressing HLA-A*24:02. Prior co-culture cells were loaded with different concentrations of TMBIM6W>Fpeptide or its WT counterpart as indicated. Values represent the average of a technical duplicate ± SD. C. Top panel: Flow cytometry gating strategy for evaluating T cell activation and killing of RA cells and A*24:02+fibroblasts after 24 hrs of co-culture with TCRctrl(top row) or TCRTMBIM6W>F 1(bottom row) transduced T cells. Target cells were labelled with CFSE prior to co-culture with target cells to properly visualize and gate during flow analysis. Target cell subset are gated as FSC / SSC, singlets, Live / Dead Fixable Near-IRne9, CFSE+CD3ne9events. To allow for proper evaluation of the number of remaining target cells, fluorescent beads were added to each well (10,000) and acquisition stop was set to 3,400 (shown in upper left plot). Activation of TCR transduced CD8+T cells was measured by upregulation of CD137 as FSC / SSC, singlets, Live / Dead Fixable Near-IRne9, CFSEne9CD3+CD8+mTCRp+CD137+. Bottom panel: Flow cytometry gating strategy for evaluating T cell activation and killing of PBMCs after 24 hrs of co-culture with TCRctrlor TCR™BIM6W>F.I autologous transduced T cells. Transduced T cells were labeled with cell-trace violet prior to co-culture with target cells to visualize and gate during flow analysis properly. Target PBMCs are gated as FSC / SSC, singlets, Live / Dead Fixable Near- |Rne9CTVne9CD45+and further distinguished as B cells (CD20+) or T cells (CD3+). To allow for proper evaluation of the number of remaining target cells, fluorescent beads were added to each well (10,000), and the acquisition stop was set to 3,400 (shown in the upper left plot). Activation of TCR transduced CD8+T cells was measured by upregulation of CD137 as FSC / SSC, singlets, Live / Dead Fixable Near-IRne9CTV+CD3+mTCRp+CD8+CD137+. D. A barplot depicting the percentage of activated TCRctrlT cells upon co-culture of 16 hrs with RA cells, fibroblasts, or autologous PBMCs from three healthy donors. Prior to co-culture, RA cells, fibroblasts, and PBMCs were loaded with either the TCRctrl-specific peptide or left untreated. Activation is shown as % CD137+of total CD8+mTCRp+T cells. Dots represent two or three technical replicates and lines represent the mean. E. A barplot depicting the relative viability of RA cells (CFSE+CD3-), fibroblasts (CFSE+CD3-), and PBMCs (CTV’CD45+) after 24hrs of co-culture with TCRctrltransduced T cells from three healthy donors. Target cells were either peptide-loaded with the TCRctrl-specific peptide or left untreated for 16 hrs before being co-cultured with TCRctrlcells at an E:T ratio of 2:1 (RA cells and PBMCs) or 4:1 (fibroblasts). Viability was quantified by flow cytometry, and each dot represents a technical replicate and shows a number of viable target cells after co-culture with TCRctrl(black). Viability is normalized to the mean of untreated target cells + TCRctrl’ and samples and lines represent the mean of two or three technical replicates.

[0090] Figure 9 shows non-transformed cell lines are not able to activate TCR™BIM6>F 1T cells. A. A barplot depicting the percentage of activated TCRTMBIM6W>F 1-ce||Smeasured by flow cytometry using anti-CD137 antibody staining. 16 hrs co-culture experiments of 3 non-transformed cell lines overexpressing either HLA-A*02:01 or HLA-A*24:02 and treated for 48 hrs with IFN© combined or not with tryptophan depleted medium with the TCRTMBIM6W>F 1-ce||S at an^. -ratj0 Of q:2wereperformed. RA cancer cells were used as positive control for T cell activation. Values represent the average of the 3 independent co-cultures ± SD. ***p < 0.001 by 1-way ANOVA, followed by Sidak post hoc test. B. A barplot depicting the percentage of activated TCRctrlor TCR™BIM6W>F 1T cells from three healthy donors measured by flow cytometry using anti-CD137 antibody staining. Prior co-culture, RA cells and HI_A-A*24:02 fibroblasts were treated or not for 48 hrs with IFN©. Dots represent technical replicates and lines represent the mean. C. A barplot representing viable RA cells and HI_A-A*24:02+fibroblasts (CFSE+CD3_) after 24 hrs of co-culture with TCRctrlor TCRTMBIM6W>F 1transduced T cells from three healthy donors. RA cells and fibroblasts were pretreated + / - IFNy for 48 hrs before being co-cultured with TCR transduced T cells at an E:T ratio of 4:1 and viability was quantified by flow cytometry. Each dot represents a technical replicate and shows number of viable RA cells and fibroblasts after co-culture with TCRctrl(green) and TCRTMBIM6W>F 1. Viability is normalized to the mean of TCRctrltreated samples and lines represent the mean of two technical replicates. D. Same as b, but here, autologous HI_A-A*24:02 PBMCs were used instead of fibroblasts. E A barplot representing viable RA cells (CFSE+CD3_), PBMCs (CTVnegCD45+), B cells (CTVnegCD45+CD20+) and T cells (CTVnegCD45+CD20negCD3+) after 24 hrs of co-culture with TCRctrlor TCRTMBIM6W>F 1transduced T cells from three healthy donors. PBMCs from the three TCR transduced donors (autologous) and RA cells were pre-treated + / - IFNy for48hrs before being co-cultured with TCR transduced T cells at an E:T ratio of 2:1. Viability was quantified by flow cytometry, and each dot represents a technical replicate and shows the number of viable target cells after co-culture with TCRctrl(green) and TCRTMBIM6W>F 1. viability is normalized to the mean of TCRctrltreated samples and lines represent the mean of two or three technical replicates

[0091] Figure 10 shows TCR™BIM6W>F 1T cells are highly specific against TMBIM6W>Fpeptide as opposed to WT counterpart. Related to Figure 11. A. A barplot depicting the percentage of activated TCRTMBIM6W>F 1q-ce||Smeasured by flow cytometry using anti-CD137 antibody staining following co-culture with the different indicated cell lines. Cancer cells were deprived of tryptophan by 48 or 72 hrs IFNy combined or not to tryptophan depleted medium, and then subsequently subjected to T cell activation assays using co-culture with TCRTMBIM6W>F 1q-ce||S at an^.q-ratj0 Of 1 :2 as indicated in Figure 5B. Values represent the average of the 3 independent co-cultures ± SD. ***p < 0.001 by 1-way ANOVA, followed by Sidak post hoc test. B. A barplot depicting the percentage of activated TCRTMBIM6W>F 1q-ce||Smeasured by flow cytometry using anti-CD137 antibody staining following co-culture with the different indicated cell lines. Cancer cells were deprived of tryptophan by 48 hrs combined or not to tryptophan depleted medium prior co-culture with TCR™BIM6W>F.I q-ce||S at an^.q-ratj0 Of q 2 as indicated in Figure 5B. Please note that another T cell donor was used for this experiment as compared to Figure 11. Values represent the average of the 3 independent co-cultures ± SD. ***p < 0.001 by 1-way ANOVA, followed by Sidak post hoc test. C. A barplot depicting the Median Fluorescent Intensity (MFI) of HLA-A*24-APC staining of various cancer cell lines treated or not for with IFN. Values represent the average of 3 independent staining ± SD. ***p < 0.001 by 1-way ANOVA, followed by Sidak post hoc test. D. Graph representing the percentage of activated TCR™BIM6W>F 1-ce||S UpOn C0-culture with various cancer cells. Prior to co-culture, cells were treated or not with IFNy for 24 hrs and then loaded with the indicated concentrations of either TMBIM6w>For its corresponding WT synthetic peptide. TCR™BIM6W>F 1-transduced T cells were incubated overnight (16 hrs) with the peptide- loaded cells at an E:T ratio of 1 :2 and subsequently examined by flow cytometry for activation by staining with anti-CD137 antibody. Dots represent the average of two or three technical replicates ± SD.

[0092] Figure 11 shows a wide array of IFNy-induced HLA-A*24:02 expressing cell lines that activate TCR™BIM6W>F-1T cells A barplot depicting the percentage of activated CD137+TCRTMBIM6W>F 1T cells as measured by flow cytometry following co-culture with different cancer cell lines at an E:T ratio of 1 :2, as indicated. The various cell lines were deprived of tryptophan by either IFNy or IFNy added to tryptophan-depleted medium, and subsequently co-cultured with TCRTMBIM6W>F 1T cells, as schematically illustrated in Figure 5b. Cell lines annotated in red are HI_A-A*24:02 positive, those in black are HLA-A*24:02 negative, and those in light red are HLA- A*24:02 but have a mutation in B2M. CRC: Colorectal cancer; LUAD / LUSC: Lung adenocarcinoma / squamous cell carcinoma; BRCA; Breast cancer; SKCM: Skin cutaneous melanoma; PDAC: Pancreatic adenocarcinoma; HCC: hepatic cellular carcinoma; PC: Prostate cancer; GBM: Glioblastoma. Values represent the average of 3 independent co-cultures ± SD. ***p < 0.001 by 1-way ANOVA, followed by Sidak post hoc test

[0093] Figure 12 showsAdoptive T cell therapy of TCR™BIM6W>F 1T cells enhance TCRMART1cancer cell killing A. A model depicting the concept of sequential targeting of a tumor-associated antigen followed by neoepitope targeting by TCR T cells. A suboptimal amount of TCRMART1is used to initiate tryptophan shortage in the cancer cells, which is followed by TCR™BIM6W>F 1treatment. B. A line graph depicting the relative viability of genetically modified RA cells, as indicated, upon 16 hrs of co-culture with different ratios of TCRMART1T cells. RA cells overexpressing NYESO-1157- 165 (purple) were used as control for RA cells overexpressing MARTI26-35 (yellow). Every dot represents the average cell viability of three independent co-cultures ± SD. ***p < 0.001 by 2- way ANOVA, followed by Bonferroni post hoc test. C. A barplot depicting the percentage of activated CD137+TCR™BIM6W>F 1-ce||Smeasured by flow cytometry upon 16 hrs co-culture with RA cells at an E:T ratio of 1 :2 overexpressing either MARTI26-35 or NYESO-1157-165 peptides. Prior to co-culture, RA cells were exposed for 48 hrs to various amounts of TCRMART1T cells in combination or not with IDO1 inhibitor (1-Methyl-tryptophan, 1 MT) and / or IFNy. Values represent the average of 3 independent co-cultures ± SD. ***p < 0.001 by 2-way ANOVA, followed by Bonferroni post hoc test. D. A barplot depicting relative cell viability of RA cells overexpressing either MARTI26-35 or NYESO-1157-165 peptides upon 16 hrs co-culture with TCR™BIM6W>F 1at anE:T ratio of 2:1. Prior to co-culture, RA cells were exposed for 48 hrs to various amounts of TCRMART1T cells in combination or not with IDO1 inhibitor (1-Methyl-tryptophan, 1MT). Values represent the average of 3 independent co-cultures ± SD. ***p < 0.001 by 2-way ANOVA, followed by Bonferroni post hoc test.

[0094] Figure 13 showsOptimization of the dual hitting TCR T cell adoptive strategy. Related to Figure 12. A. A barplot depicting the percentage of activated TCRMART1T cells measured by flow cytometry using anti-CD137 antibody staining following co-culture at an E:T ratio of 1 :2 with RA cells overexpressing TMBIM6wr, TMBIMO™^, NYESO-1157-165 or MARTI26-35 epitopes. Cancer cells were co-cultured with TCRMART1T cells for 16 hrs. Values represent the average of the 3 independent co-cultures ± SD. ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test. B. A barplot depicting the percentage of activated TCRTMBIM6W>F 1-ce||Smeasured by flow cytometry using anti-CD137 antibody staining. Prior to 16 hrs of co-culture, RA cells were exposed for 48 hrs to medium supplemented with IFNy combined or not with IDO1 inhibitors 1-methyl- tryptophan (1-MT) or Epacadostat. Values represent the average of the 3 independent co-cultures ± SD ***p < 0 001 by 1-way ANOVA, followed by Bonferroni post hoc test. C. A barplot depicting the relative cell viability of RA cells upon co-culture with TCRTMBIM6W>F 1orTCRMARTI -ce||S at anE:T ratio of 2:1. Prior to 16 hrs of co-culture, RA cells were exposed for 48 hrs to medium supplemented with IFNy with or without (1MT or Epacadostat). Viability was normalized to the situation without adding T cells. Values represent the average of the 3 independent co-cultures ± SD ***p < 0.001 by 2-way ANOVA, followed by Bonferroni post hoc test. D. A graph depicting the relative cell viability of RA overexpressing either NYESO-1157-165 or MARTI26-35 epitope upon co-culture with TCRMART1T cells. During the 48 hrs of co-culture, RA cells were exposed or not to IDO1 inhibitor (1MT). Viability was normalized to the situation without adding T cells. Values represent the average of the 3 independent co-cultures ± SD. ***p < 0.001 by 2-way ANOVA, followed by Bonferroni post hoc test. E. Graphs depicting the Median Fluorescent Intensity (MFI) of B2m-APC (left graph) and panHLA-A,B,C-FITC (right graph) of RA cells treated or not for 48 hrs with IFNy combined or not with IDO1 inhibitors (1 MT and Epacadostat). Values represent the average of 3 independent staining ± SD. *p < 0.05; **p < 0.01 and ***p < 0.001 by 1-way ANOVA, followed by Bonferroni post hoc test.

[0095] Figure 14 shows the rationale beyond the preclinical relevance of combining T cells targeting a classic neoantigen (MARTI) followed by T cells targeting the identified W>F peptide (TMBIM6). The graph represents the probability of survival of immunodeficient mice (n=8 per group) where 1 x 106PC-3 cells were injected into their fat pad. PC-3 were engineered to overexpress HLA- A*02:01 and either NYESO-1 or MART-1. When the tumour reached 150mm3, the mice were randomly distributed into the experimental groups. Then, the mice received either PBS (100uL) or 15 x 106of DMF-5 T cells by tail vein injection. In the meantime for 3 consecutive days the mice were treated intraperitoneally with IL-2 (1 OO.OOOU / inj.). 96 hrs after the T-cell transfer, mice received either PBS (100uL) or 15 x 106of TCRTMBIM6W>F 1q-ce||S taj|vejninjection. In the meantime for 3 consecutive days the mice were treated intraperitoneally with IL-2 (1 OO.OOOU / inj.). The tumours were measured 3 times a week and the end-point was reached when the tumour reached 1500mm3or when the mice showed a dramatic loss of weight according to the guidelines of the NKI animal facility. The mice expressing MARTI -PC3 tumours treated with both DMF5 and TCRTMBIM6W>F 1T cells (pink curve) show a median survival of 65 days as compared to mice treated with DMF5 T cells only (53 days, purple curve).

[0096] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0097] Various aspects of the invention are described in further detail below.

[0098] Detailed Description

[0099] The invention provides nucleic acid molecules that encode a binding protein that comprises T cell receptor components that specifically bind to a peptide comprising an amino acid sequence of an antigen or epitope of a Wto F substitutant peptide derived from TMBIM6. For example, the binding protein may specifically bind to a peptide according to SEQ ID NO: 58.

[0100] In some examples, the nucleic acid molecules may be part of a composition of isolated nucleic acids encoding the binding protein. The nucleic acid molecules may be distinct nucleic acid molecules within the nucleic acid composition. The TOR components of the binding protein may therefore be encoded by two (or more) nucleic acid molecules (with distinct nucleotide sequences) which, together, encode all of the TCR components of the binding protein. In other words, some of the TCR components may be encoded by one nucleic acid sequence in the nucleic acid composition, and others may be encoded by another (distinct) nucleic acid sequence within the nucleic acid composition. Alternatively, the nucleic acid molecules may be part of a single nucleic acid sequence. The TCR components of the binding protein may therefore all be encoded by a single nucleic acid sequence (for example with a single open reading frame, or with multiple (e.g. 2 or more, three or more etc.) open reading frames).

[0101] Nucleic acid molecules described herein may form part of a larger nucleic acid sequence that encodes a larger component part of a functioning binding protein. For example, a nucleic acid sequence that encodes TCR a chain CDR3 or a TCR Va domain with the specified features described herein may be part of a larger nucleic acid sequence that encodes a functional TCR a chain (including the constant domain). As another example, a nucleic acid sequence that encodes a TCR p chain CDR3 or a TCR Vp domain with the specified features described herein may be part of a larger nucleic acid sequence that encodes a functional TCR p chain (including the constant domain). As a further example, both nucleic acid molecules (for example each encoding at least a TCR a chain CDR3 and TCR p chain CDR3 respectively) may be part of a larger nucleic acid sequence that encodes a combination of a functional TCR Va domain or TCR a chain (including the constant domain) and a functional TCR Vp domain or TCR p chain (including the constant domain). In some examples, the sequence encoding the functional TCR Va domain or TCR a chain is separated from the sequence encoding the functional TCR p domain or TCR p chain by a linker sequence that enables coordinate expression of two proteins or polypeptides in the same nucleic acid sequence. More details on this are provided below.

[0102] The nucleic acid molecules described herein may alternatively encode a small component of a T cell receptor e.g. TCR a chain CDR3, TCR p chain CDR3, a TCR Va domain, or a TCR Vp domain only. The nucleic acid molecules may be considered as “building blocks” that provide essential components for peptide binding specificity. The nucleic acid molecules described herein may be incorporated into a distinct nucleic acid sequence (e.g. a vector) that encodes the other elements of a functional binding protein such as a TCR, such that when the nucleic acid sequence described herein is incorporated, a new nucleic acid sequence is generated that encodes e.g. a TCR a chain and / or a TCR p chain that specifically binds to a antigen derived from a W to F substitutant of TMBIM6 (TMBIM6W>F). The nucleic acid molecules described herein therefore have utility as essential components that confer binding specificity for a TMBIM6W>Fantigen, and thus can be used to generate a larger nucleic acid sequence encoding a binding protein with the required antigen binding activity and specificity.

[0103] The nucleic acid molecules described herein may be codon optimised for expression in a host cell, for example they may be codon optimised for expression in a human cell, such as a cell of the immune system, a inducible pluripotent stem cell (iPSC), a hematopoietic stem cell, a T cell, a primary T cell, a T cell line, a NK cell, or a natural killer T cell (Scholten et al, Clin. Immunol. 119: 135, 2006). The T cell may be a CD4+ or a CD8+ T cell. Codon optimisation is a well-known method in the art for maximizing expression of a nucleic acid sequence in a particular host cell. In some examples, one or more cysteine residues may also be introduced into the encoded TCR alpha and beta chain components (e.g. to reduce the risk of mispairing with endogenous TCR chains). In one example, the nucleic acid molecules described herein are codon optimised for expression in a suitable host cell, and / or are modified to introduce codons encoding one or more cysteine amino acids (e.g. into the constant domain of the encoded TCR a chain and / or the encoded TCR p chain) to reduce the risk of mispairing with endogenous TCR chains.

[0104] In some examples, a TCR constant domain is modified to enhance pairing of desired TCR chains. For example, enhanced pairing between a heterologous TCR a chain and a heterologous TCR p chain due to a modification may result in the preferential assembly of a TCR comprising two heterologous chains over an undesired mispairing of a heterologous TCR chain with an endogenous TCR chain (see, e.g., Covers et al, Trends Mol. Med. 16(2):11 (2010)). Exemplary modifications to enhance pairing of heterologous TCR chains include the introduction of complementary cysteine residues in each of the heterologous TCR a chain and p chain.

[0105] A binding protein that is encoded by the nucleic acid molecules described herein is specific for an antigen or epitope derived from a W to F substitutant derived from TMBIM6 (TMBIM6W>F) and comprises TMBIM6W>Fspecific-TCR components. However, the encoded binding protein is not limited to being a TCR. Other appropriate binding proteins that comprise the specified TMBIMG^ antigen specific -TCR components are also encompassed. For example, the encoded binding protein may comprise a TCR, an antigen binding fragment of a TCR, or a chimeric antigen receptor (CAR). TCRs, antigen binding fragments thereof and CARs are well defined in the art. A non-limiting example of an antigen binding fragment of a TCR is a single chain TCR (scTCR) or a chimeric dimer composed of the antigen binding fragments of the TCR a and TCR chain linked to transmembrane and intracellular domains of a dimeric complex so that the complex is a chimeric dimer TCR (cdTCR).

[0106] "Chimeric antigen receptor" (CAR) refers to a fusion protein that is engineered to contain two or more naturally-occurring amino acid sequences linked together in a way that does not occur naturally or does not occur naturally in a host cell, which fusion protein can function as a receptor when present on a surface of a cell. CARs described herein include an extracellular portion comprising an antigen binding domain (i.e., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as an scFv derived from an antibody or TCR specific for a cancer antigen, or an antigen binding domain derived or obtained from a killer immunoreceptor from an NK cell) linked to a transmembrane domain and one or more intracellular signalling domains (optionally containing co-stimulatory domain(s)) (see, e.g., Sadelain et al, Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016), and Stone et al, Cancer Immunol. Immunother., 63(11): 1163 (2014)).

[0107] A T cell receptor (TCR) is a molecule found on the surface of T cells (T lymphocytes) that is responsible for recognising a peptide that is bound to (presented by) a major histocompatibility complex (MHC) molecule on a target cell. The invention is directed to nucleic acid molecules and compositions thereof that encode binding proteins comprising TCR components that interact with a particular peptide in the context of the appropriate serotype of MHC. For example, for a TM Bl M6W>Fantigen the serotype may be HI_A-A. For example, for a TM Bl M6W>Fantigen the serotype may be HLA-C. In some examples, the serotype may be HI_A-A*24. In some examples, the serotype may be HI_A-A*24:02. In some examples, the serotype may be HLA-C*02. For example, in the context of HLA-A*24:02 (in other words, the encoded binding protein is capable of specifically binding to a TMBIMe^ antigen: HI_A-A*24:02 complex). For example, in the context of HI_A-C*02:02 (in other words, the encoded binding protein is capable of specifically binding to a TMBIM6W>Fantigen: HLA-C*02:02 complex). Peptides that are presented by HI_A- A*24:02 to TCRs may be described as being “HLA-A*24:02 restricted”. Peptides that are presented by HI_A-C*02:02 to TCRs may be described as being “HLA-C*02:02 restricted”. The TCR is composed of two different polypeptide chains. In humans, 95% of TCRs consist of an alpha (a) chain and a beta (P) chain (encoded by TRA and TRB respectively). When the TCR engages with peptide in the context of HLA (e.g. in the context of HI_A-A*24:02 or HI_A-C*02:02), the T cell is activated through signal transduction. The alpha and beta chains of the TCR are highly variable in sequence. Each chain is composed of two extracellular domains, a variable domain (V) and a constant domain (C). The constant domain is proximal to the T cell membrane followed by a transmembrane region and a short cytoplasmic tail while the variable domain binds to the peptide / HI_A-A complex. The variable domain of each chain has three hypervariable regions (also called complementarity determining regions (CDRs)). Accordingly, the TCR alpha variable domain (referred to herein as a TCR Va domain, TCR V alpha domain, Va domain or V alpha domain, alpha variable domain etc) comprises a CDR1 , a CDR2 and CDR3 region. Similarly, the TCR beta variable domain (referred to herein as a TCR Vp domain, TCR V beta domain, Vp domain or V beta domain, beta variable domain etc) also comprises a (different) CDR1 , CDR2, and CDR3 region. In each of the alpha and beta variable domains it is CDR3 that is mainly responsible for recognizing the peptide being presented by the HLA molecules.

[0108] As will be clear to a person of skill in the art, the phrase “TCR a chain variable domain” refers to the variable (V) domain (extracellular domain) of a TCR alpha chain, and thus includes three hypervariable regions (CDR1 , CDR2 and the specified CDR3), as well as the intervening sequences, but does not include the constant (C) domain of the alpha chain, which does not form part of the variable domain.

[0109] As will be clear to a person of skill in the art, the phrase “TCR p chain variable domain” refers to the variable (V) domain (extracellular domain) of a TCR beta chain, and thus includes three hypervariable regions (CDR1 , CDR2 and the specified CDR3), as well as the intervening sequences, but does not include the constant (C) domain of the beta chain, which does not form part of the variable domain.

[0110] Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single- chain TCRs containing variable regions of TCRs attached by a flexible linker, TCR chains linked by an engineered disulfide bond, single TCR variable domains, single peptide-MHC- specific TCRs, multi-specific TCRs (including bispecific TCRs), TCR fusions, TCRs comprising co-stimulatory regions, human TCRs, humanized TCRs, chimeric TCRs, recombinantly produced TCRs, and synthetic TCRs. In some examples, the TCR is a full-length TCR comprising a full-length a chain and a full-length p chain. In some examples, the TCR is a soluble TCR lacking transmembrane and / or cytoplasmic region(s). In some examples, the TCR is a single-chain TCR (scTCR) comprising Va and p linked by a peptide linker, such as a scTCR having a structure as described in PCT Publication No: WO 2003 / 020763, WO 2004 / 033685, or WO 2011 / 044186.

[0111] As used herein, the term "full-length TCR" refers to a TCR comprising a dimer of a first and a second polypeptide chain, each of which comprises a TCR variable region and a TCR constant region comprising a TCR transmembrane region and a TCR cytoplasmic region. In some examples, the full-length TCR comprises one or two unmodified TCR chains, e.g., unmodified a, P, y, or 5 TCR chains. In some examples, the full-length TCR comprises one or two altered TCR chains, such as chimeric TCR chains and / or TCR chains comprising one or more amino acid substitutions, insertions, or deletions relative to an unmodified TCR chain. In some examples, the full-length TCR comprises a mature, full-length TCR a chain and a mature, full-length TCR chain. In some examples, the full-length TCR comprises a mature, full-length TCR y chain and a mature, full-length TCR 5 chain.

[0112] In Some examples, an antigen-binding fragment of a TCR comprises a single chain TCR (scTCR), which comprises both the TCR Va and TCR Vp domains, but only a single TCR constant domain. In other examples, an antigen-binding fragment of a TCR comprises a chimeric TCR dimer in which the antigen binding fragment is linked to an alternative transmembrane and intracellular signalling domain (where the alternative transmembrane and intracellular signalling domain are not naturally found in TCRs). In further examples, an antigen-binding fragment of a TCR or a chimeric antigen receptor is chimeric (e.g., comprises amino acid residues or motifs from more than one donor or species), humanized (e.g., comprises residues from a non-human organism that are altered or substituted so as to reduce the risk of immunogenicity in a human), or human. Methods for producing engineered TCRs are described in, for example, Bowerman et al, Mol. Immunol, 5(15):3000 (2009), PCT Publication No WO2017210586A1 , . Methods for making CARs are well known in the art and are described, for example, in U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191 ; U.S. Patent Publication No. 2010 / 065818; U.S. Patent No. 8,822,647; PCT Publication No. WO 2014 / 031687; U.S. Patent No. 7,514,537; and Brentjens et al, 2007, Clin. Cancer Res. 73:5426.

[0113] Components of the TCR a chain variable (Va) domain

[0114] Provided herein is a nucleic acid molecule or compositions thereof that encodes a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a TMBIMe^ antigen as described herein. In some examples, the CDR3 of the TCR a chain comprises a sequence having at least 75% sequence identity to SEQ ID NO: 1.

[0115] In some examples, the CDR3 of the TCR a chain comprises a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 1. In some examples, the CDR3 of the TCR a chain consists of an amino acid sequence according to SEQ ID NO: 1.

[0116] As would be clear to a person of skill in the art, variants of the amino acid sequence shown in SEQ ID NO:1 may also be functional (i.e. retain their ability to confer specific binding to a TM Bl M6W>Fantigen (e.g. the peptide shown in SEQ ID NO:58) when the CDR3 is part of TCR Va domain). Such functional variants are therefore encompassed herein. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:1). In other words, appropriate (functional) Va domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO:1 by one or several (e.g. two etc) amino acids.

[0117] In some example, the CDR3 of the TCR a chain may be encoded by a nucleic acid sequence having at least at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 3. In some example, the CDR3 of the TCR a chain may be encoded by a nucleic acid sequence according to SEQ ID NO: 3. In some example, the CDR3 of the TCR a chain may be encoded by a genetically degenerate sequence of SEQ ID NO: 3 (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). For example, SEQ ID NO: 3 is codon optimised for humans. In some examples, the CDR3 of the TCR a chain may be encoded by a non-codon optimised sequence. Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of any one of SEQ ID NO:1. The term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one, two or more amino acids any one of SEQ ID NO: 1 , or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the CDR3.

[0118] Non-functional variants are amino acid sequence variants of any one of SEQ ID NO: 1 that do not specifically bind to a TMBIM6W>Fantigen (e.g. the peptide shown in SEQ ID NO:58). Nonfunctional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 1 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and nonfunctional variants are well known to a person of ordinary skill in the art.

[0119] In some examples, the CDR3 of the TCR a chain is comprised within a Va domain. For example, in one example there is provided a TCR Va domain comprising a CDR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 1. In one example there is provided a TCR Va domain comprising a CDR3 comprising an amino acid sequence according to SEQ ID NO: 1. In one example there is provided a TCR Va domain comprising a CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 1.

[0120] The encoded TCR Va domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence according to SEQ ID NO: 4, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to the TMBIM6W>Fantigen (e.g. the peptide shown in SEQ ID NO:58)). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 4. The term “variant” also encompasses homologues and fragments. In some examples functional variants are as defined above in respect of functional variants of CDR3 of the TCR a chain. For example, the CDR1 of the TCR a chain may comprise or consist of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 4.

[0121] In some examples, wherein the CDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 1 as described above, the CDR1 of the TOR Va domain may comprise a CDR1 amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 4. In some examples, wherein the CDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 1 as described above, the CDR1 of the TCR Va domain may consist of a CDR1 amino acid sequence according to SEQ ID NO: 4. In some example, the CDR1 of the TCR a chain may be encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 6. In some example, the CDR1 of the TCR a chain may be encoded by a nucleic acid sequence according to SEQ ID NO: 6. In some example, the CDR1 of the TCR a chain may be encoded by a genetically degenerate sequence of SEQ ID NO: 6 (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). For example, SEQ ID NO: 6 is codon optimised for humans. In some examples, the CDR1 of the TCR a chain may be encoded by a non-codon optimised sequence.

[0122] The encoded TCR Va domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 comprising an amino acid sequence according to SEQ ID NO: 7, or a functional variant thereof as described above. Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 7. The term “variant” also encompasses homologues and fragments. In some examples, functional variants of CDR2 refers to variants that retain the ability to specifically bind to HLA-A*24:02 and / or HLA-C*02:02. For example, the CDR2 of the TCR a chain may comprise or consist of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 7.

[0123] In some examples, wherein the CDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 1 as described above, the CDR2 of the TCR Va domain may comprise a CDR2 amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 7. In some examples, wherein the CDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 1 as described above, the CDR2 of the TCR Va domain may consist of a CDR2 amino acid sequence according to SEQ ID NO: 7. In some example, the CDR2 of the TCR a chain may be encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 9. In some example, the CDR2 of the TCR a chain may be encoded by a nucleic acid sequence according to SEQ ID NO: 9. In some example, the CDR2 of the TCR a chain may be encoded by a genetically degenerate sequence of SEQ ID NO: 9 (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). For example, SEQ ID NO: 9 is codon optimised for humans. In some examples, the CDR2 of the TOR a chain may be encoded by a non-codon optimised sequence..

[0124] The encoded TOR Va domain may therefore comprise the CDRs mentioned in detail above (or functional variants thereof), with appropriate intervening sequences between the CDRs.

[0125] For example, TCR Va domain may comprise a CDR3 comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , a CDR1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 4, and a CDR2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 7.

[0126] The encoded TCR Va domain may comprise an amino acid sequence of SEQ ID NO: 10, or a functional variant thereof (i.e. wherein the variant TCR Va domain retains the ability to specifically bind to a TM Bl antigen (e.g. the peptide shown in SEQ ID NO:58) when part of a binding protein described herein). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of any one of SEQ ID NO: 10. The term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of any one of SEQ ID NO: 10, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein. In one example, the encoded TCR Va domain may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 10, whilst retaining the ability to specifically bind to a TM Bl M6W>Fantigen (e.g. the peptide shown in SEQ ID NO:58). In other words, a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 10 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO: 10 may all be in regions of the TCR Va domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ I D NO: 1 , SEQ I D NO: 4 and / or SEQ I D NO: 7 and still have 25% (or less) sequence variability compared to SEQ ID NO: 10). In other words, the sequence of the CDRs of SEQ ID NOs: 1 , 4 and 7 may be retained whilst the rest of the sequence is varied.

[0127] As another example, the encoded TCR Va domain may comprise an amino acid sequence of SEQ ID NO: 10, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions.

[0128] In examples where the TCR Va domain has the amino acid sequence of SEQ ID NO: 10, the TCR Va domain may be encoded by the nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:12. In some example, the TCR Va domain may be encoded by a genetically degenerate sequence of SEQ ID NO: 12 (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). For example, SEQ ID NO: 12 is codon optimised for humans. In some examples, the TOR Va domain may be encoded by a non-codon optimised sequence.

[0129] In some examples, the TOR Va domain may include a leader sequence or leader peptide. A “leader peptide” refers to a peptide having a length of about 5-30 amino acids that is present at the N-terminus of newly synthesized proteins that form part of a secretory pathway. In some examples, the leader peptide forms part of the transmembrane domain of a protein.

[0130] In one example, the encoded TCR Va domain leader peptide may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 19. In one example, the encoded TCR Va domain leader peptide may have an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 19. In one example, the encoded TCR Va domain leader peptide may be encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 20. In one example, the encoded TCR Va domain leader peptide may be encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 20. Other Va leader sequence, such as Va25, can also be used.

[0131] As such, in one example, the encoded TCR Va domain may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 13, whilst retaining the ability to specifically bind to a TM Bl M6W>Fantigen (e.g. the peptide shown in SEQ ID NO:58). In other words, a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 13 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO: 13 may all be in regions of the TCR Va domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 1 , SEQ ID NO: 4 and / or SEQ ID NO: 7 and still have 25% (or less) sequence variability compared to SEQ ID NO: 13). In other words, the sequence of the CDRs of SEQ ID NO: 1 , 4 and 7 may be retained whilst the rest of the sequence is varied.

[0132] As another example, the encoded TCR Va domain may comprise an amino acid sequence of SEQ ID NO: 13, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions.

[0133] In examples where the TCR Va domain has the amino acid sequence of SEQ ID NO:13, the TCR Va domain may be encoded by the nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15. In some example, the TCR Va domain may be encoded by a genetically degenerate sequence of SEQ ID NO: 15 (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). For example, SEQ ID NO: 15 is codon optimised for humans. In some examples, the TOR Va domain may be encoded by a non-codon optimised sequence.. For the avoidance of doubt, the nucleic acid sequence encoding the TOR Va domain may also encode a TOR a chain constant domain. An example of a suitable constant domain is encoded in the MP71-TCR-flex retroviral vector. However, the invention is not limited to this specific constant domain, and encompasses any appropriate TCR a chain constant domain. The constant domain may be murine derived, human derived or humanised. Methods for identifying or generating appropriate constant domains are well known to a person of skill in the art and are well within their routine capabilities.

[0134] By way of example only, the constant domain may be encoded by or derived from a vector, such as a lentiviral, retroviral or plasmid vector but also adenovirus, adeno-associated virus, vaccinia virus, canary poxvirus or herpes virus vectors in which murine or human constant domains are pre-cloned. Recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors as published by R Monjezi, et al., 2017). Moreover, naked (synthetic) DNA / RNA can also be used to introduce the TCR. As an example, a pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes as described in LV Coren et al., BioTechniques 2015 may be used to provide an appropriate constant domain. Alternatively, single stranded or double stranded DNA or RNA can be inserted by homologous directed repair into the TCR locus (see Roth et a / 2018 Nature vol 559; page 405). As a further option, non - homologous end joining is possible.

[0135] For example, the TCR a constant domain may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0136] In examples where the TCR a constant domain has the amino acid sequence of SEQ ID NO: 16, the TCR a constant domain may be encoded by the nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18.

[0137] Examples of specific TCR a chain amino acid sequences that include a TCR Va domain described herein with an appropriate constant domain are shown in SEQ ID NOs: 21 (without leader sequence) and 24 (with leader sequence). Appropriate functional variants as described herein of SEQ ID NOs: 21 and 24 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 21 or 24. In some examples, the TCR a chain comprises or consists of the amino acid sequence according to SEQ ID NO: 21 or 24.

[0138] In examples where the TCR a chain has the amino acid sequence of SEQ ID NO:21 , the TCR a constant domain may be encoded by the nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23.

[0139] In examples where the TOR a chain has the amino acid sequence of SEQ ID NO:24, the TOR a constant domain may be encoded by the nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26.

[0140] Components of the TCR 8 chain variable (VB) domain

[0141] Provided herein is a nucleic acid molecule or compositions thereof that encodes a polypeptide comprising a CDR3 of a TCR B chain polypeptide that specifically binds to a TM Bl M6W>Fantigen as described herein. In some examples, the CDR3 of the TCR B chain comprises a sequence having at least 75% sequence identity to SEQ ID NO: 27.

[0142] In some examples, the CDR3 of the TCR B chain comprises a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 27. In some examples, the CDR3 of the TCR B chain consists of an amino acid sequence according to SEQ ID NO: 27.

[0143] As would be clear to a person of skill in the art, variants of the amino acid sequence shown in SEQ ID NO: 27 may also be functional (i.e. retain their ability to confer specific binding to a TM Bl M6W>Fantigen (e.g. the peptide shown in SEQ ID NO:58) when the CDR3 is part of TCR VB domain). Such functional variants are therefore encompassed herein. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 27). In other words, appropriate (functional) VB domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO: 27 by one or several (e.g. two etc) amino acids.

[0144] In some example, the CDR3 of the TCR B chain may be encoded by a nucleic acid sequence having at least at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 29. In some example, the CDR3 of the TCR B chain may be encoded by a nucleic acid sequence according to SEQ ID NO: 29. In some example, the CDR3 of the TCR B chain may be encoded by a genetically degenerate sequence of SEQ ID NO: 29 (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). For example, SEQ ID NO: 29 is codon optimised for humans. In some examples, the CDR3 of the TCR B chain may be encoded by a non-codon optimised sequence.

[0145] Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of any one of SEQ ID NO: 27. The term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one, two or more amino acids any one of SEQ ID NO: 27, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the CDR3.

[0146] Non-functional variants are amino acid sequence variants of any one of SEQ ID NO: 27 that do not specifically bind to a TM Bl M6W>Fantigen (e.g. the peptide shown in SEQ ID NO:58). Nonfunctional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 27 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and nonfunctional variants are well known to a person of ordinary skill in the art.

[0147] In some examples, the CDR3 of the TCR p chain is comprised with a Vp domain. For example, in one example there is provided a TCR Vp domain comprising a CDR3 comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 27. In one example there is provided a TCR VP domain comprising a CDR3 comprising an amino acid sequence according to SEQ ID NO: 27. In one example there is provided a TCR p domain comprising a CDR3 comprising an amino acid sequence consisting of SEQ ID NO: 27.

[0148] The encoded TCR p domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence according to SEQ ID NO: 30, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to the TMBIM6W>Fantigen (e.g. the peptide shown in SEQ ID NO:58)). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 30. The term “variant” also encompasses homologues and fragments. In some examples functional variants are as defined above in respect of functional variants of CDR3 of the TCR p chain. For example, the CDR1 of the TCR p chain may comprise or consist of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 30.

[0149] In some examples, wherein the CDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 27 as described above, the CDR1 of the TCR p domain may comprise a CDR1 amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 30. In some examples, wherein the CDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 27 as described above, the CDR1 of the TCR p domain may consist of a CDR1 amino acid sequence according to SEQ ID NO: 30. In some example, the CDR1 of the TCR p chain may be encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 32. In some example, the CDR1 of the TCR p chain may be encoded by a nucleic acid sequence according to SEQ ID NO: 32. In some example, the CDR1 of the TCR p chain may be encoded by a genetically degenerate sequence of SEQ ID NO: 32 (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). For example, SEQ ID NO: 32 is codon optimised for humans. In some examples, the CDR1 of the TCR p chain may be encoded by a non-codon optimised sequence.

[0150] The encoded TCR Vp domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 comprising an amino acid sequence according to any one of SEQ ID NOs: 33, or a functional variant thereof as described above. Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of any of SEQ ID NOs: 33. The term “variant” also encompasses homologues and fragments. In some examples, functional variants of CDR2 refers to variants that retain the ability to specifically bind to HLA-A*24:02 and / or HLA-C*02:02. For example, the CDR2 of the TCR p chain may comprise or consist of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 33.

[0151] In some examples, wherein the CDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 27 as described above, the CDR2 of the TCR Vp domain may comprise a CDR2 amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 33. In some examples, wherein the CDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 27 as described above, the CDR2 of the TCR Vp domain may consist of a CDR1 amino acid sequence according to SEQ ID NO: 33. In some example, the CDR2 of the TCR p chain may be encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 35. In some example, the CDR2 of the TCR p chain may be encoded by a nucleic acid sequence according to SEQ ID NO: 35. In some example, the CDR2 of the TCR p chain may be encoded by a genetically degenerate sequence of SEQ ID NO: 35 (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). For example, SEQ ID NO: 35 is codon optimised for humans. In some examples, the CDR2 of the TCR p chain may be encoded by a non-codon optimised sequence.

[0152] The encoded TCR Vp domain may therefore comprise the CDRs mentioned in detail above (or functional variants thereof), with appropriate intervening sequences between the CDRs.

[0153] For example, TCR Vp domain may comprise a CDR3 comprising or consisting of an amino acid sequence according to SEQ ID NO: 27, a CDR1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 30, and a CDR2 comprising or consisting of an amino acid sequence according to SEQ ID NO: 33.

[0154] The encoded TCR Vp domain may comprise an amino acid sequence of SEQ ID NO:36, or a functional variant thereof (i.e. wherein the variant TCR Vp domain retains the ability to specifically bind to a TM Bl antigen (e.g. the peptide shown in SEQ ID NO:58) when part of a binding protein described herein). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of any one of SEQ ID NO:36. The term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of any one of SEQ ID NO:36, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein. In one example, the encoded TCR Vp domain may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 36, whilst retaining the ability to specifically bind to a TM Bl M6W>Fantigen (e.g. the peptide shown in SEQ ID NO:58). In other words, a functional TCR Vp domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:36 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO:36 may all be in regions of the TCR Vp domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 27, SEQ ID NO: 30 and / or SEQ ID NO: 33). In other words, the sequence of the CDRs of SEQ ID NOs: 27, 30 and 33 may be retained whilst the rest of the sequence is varied.

[0155] As another example, the encoded TCR Vp domain may comprise an amino acid sequence having at the amino acid sequence of SEQ ID NO: 36, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions).

[0156] In examples where the TCR Vp domain has the amino acid sequence of SEQ ID NO:36, the TCR VP domain may be encoded by the nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:38. In some example, the TCR p domain may be encoded by a genetically degenerate sequence of SEQ ID NO: 38 (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). For example, SEQ ID NO: 38 is codon optimised for humans. In some examples, the TCR p domain may be encoded by a non-codon optimised sequence. In some examples, the TCR p domain may include a leader sequence or leader peptide. A “leader peptide” refers to a peptide having a length of about 5-30 amino acids that is present at the N-terminus of newly synthesized proteins that form part of a secretory pathway. In some examples, the leader peptide forms part of the transmembrane domain of a protein.

[0157] In one example, the encoded TCR p domain leader peptide may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 42. In one example, the encoded TCR p domain leader peptide may have an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 42. In one example, the encoded TCR Vp domain leader peptide may be encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 43. In one example, the encoded TCR p domain leader peptide may be encoded by a nucleic acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 43.

[0158] As such, in one example, the encoded TCR Vp domain may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 44, whilst retaining the ability to specifically bind to a TM Bl M6W>Fantigen (e.g. the peptide shown in SEQ ID NO:58). In other words, a functional TCR Vp domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:44 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO:44 may all be in regions of the TCR Vp domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 27, SEQ ID NO: 30 and / or SEQ ID NO: 33). In other words, the sequence of the CDRs of SEQ ID NOs: 27, 30 and 33 may be retained whilst the rest of the sequence is varied.

[0159] As another example, the encoded TCR Vp domain may comprise an amino acid sequence of SEQ ID NO: 44, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions.

[0160] In examples where the TCR Vp domain has the amino acid sequence of SEQ ID NO:44, the TCR VP domain may be encoded by the nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46. In some example, the TCR p domain may be encoded by a genetically degenerate sequence of SEQ ID NO: 46 (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). For example, SEQ ID NO: 46 is codon optimised for humans. In some examples, the TCR p domain may be encoded by a non-codon optimised sequence. For the avoidance of doubt, the nucleic acid sequence encoding the TCR p domain may also encode a TCR p chain constant domain. An example of a suitable constant domain is encoded in the MP71-TCR-flex retroviral vector. However, the invention is not limited to this specific constant domain, and encompasses any appropriate TCR p chain constant domain. The constant domain may be murine derived, human derived or humanised. Methods for identifying or generating appropriate constant domains are well known to a person of skill in the art and are well within their routine capabilities.

[0161] By way of example only, the constant domain may be encoded by or derived from a vector, such as a lentiviral, retroviral or plasmid vector but also adenovirus, adeno-associated virus, vaccinia virus, canary poxvirus or herpes virus vectors in which murine or human constant domains are pre-cloned. Recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors as published by R Monjezi, et al., 2017). Moreover, naked (synthetic) DNA / RNA can also be used to introduce the TCR. As an example, a pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes as described in LV Coren et al., BioTechniques 2015 may be used to provide an appropriate constant domain. Alternatively, single stranded or double stranded DNA or RNA can be inserted by homologous directed repair into the TCR locus (see Roth et al 2018 Nature vol 559; page 405). As a further option, non - homologous end joining is possible.

[0162] For example, the TCR p constant domain may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 39.

[0163] In examples where the TCR constant domain has the amino acid sequence of SEQ ID NO:39, the TCR p constant domain may be encoded by the nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41.

[0164] Examples of specific TCR p chain amino acid sequences that include a TCR V p domain described herein with an appropriate constant domain are shown in SEQ ID NOs: 47 (without leader sequence) and 50 (with leader sequence). Appropriate functional variants as described herein of SEQ ID NOs: 47 and 50 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 47 or 50. In some examples, the TCR p chain comprises or consists of the amino acid sequence according to SEQ ID NO: 47 or 50.

[0165] In examples where the TCR p chain has the amino acid sequence of SEQ ID NO:47, the TCR p constant domain may be encoded by the nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 49.

[0166] In examples where the TCR a chain has the amino acid sequence of SEQ ID NQ:50, the TCR a constant domain may be encoded by the nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52.

[0167] In some examples, a nucleic acid composition described herein encodes a TM Bl M6W>Fantigenspecific binding protein having a TCR Va domain with a CDR3 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 1 ; and a TCR Vp domain with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:27. In addition, the TMBIM6W>Fantigen may comprise or consist of the sequence shown in SEQ ID NO: 58. Furthermore, the TCR Va domain may be part of a TCR a chain having a constant domain and the TCR p domain may be part of a TCR p chain having a constant domain. In some examples, the CDR3 of the Va domain may be encoded by a nucleic acid sequence comprising the sequence of any one of SEQ ID NOs: 3; and the CDR3 of the Vp domain may be encoded by a nucleic acid sequence comprising the sequence of any one of SEQ ID NO: 29.

[0168] In this particular example, the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of SEQ ID NOs: 10 or 13; and the p domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of SEQ ID NOs: 36 or 44. In such cases, the Va domain may be encoded by a nucleic acid sequence comprising the sequence of any one of SEQ ID NOs: 12 or 15; and the Vp domain may be encoded by a nucleic acid sequence comprising the sequence of any one of SEQ I D NOs: 38 or 46.

[0169] In one example, the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NOs: 21 or 24; and the Vp domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NOs: 47 or 50. In one example, the Va domain comprises the amino acid sequence of SEQ ID NO: 21 or 24 and the Vp domain comprises the amino acid sequence of SEQ ID NO: 47 or 50. In such cases, the Va domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 23 or 26; and the Vp domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 49 or 52.

[0170] For the avoidance of doubt, this particular example encompasses components of TCR clone TCRTMBIM6W>F 1exemplified herein. The different components of TCR™BIM6W>F 1anc|thejrrespective SEQ ID NOs are summarised in Table 1 below.

[0171] As stated in more detail elsewhere herein, the nucleic acid molecules, TMBIM6 variant peptide binding proteins, or compositions thereof described herein encode both a TCR Va domain and a TCR Vp domain, which form the binding protein that is capable of specifically binding to the TMBIM6W>Fantigen. In examples where the TCR Va domain and the TCR p domain are encoded by the same nucleic acid sequence, the TCR Va domain and TCR Vp domain may be joined together via a linker, e.g. a linker that enables expression of two proteins or polypeptides from the same vector. By way of example, a linker comprising a porcine teschovirus-1 2A (P2A) sequence may be used, such as 2A sequences from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A) or Thosea asigna virus (T2A) as published by A.L. Szymczak et al., Nature Biotechnology 22, 589 - 594 (2004) or 2A-like sequences. 2A and 2A-like sequences are linkers that are cleavable once the nucleic acid molecule has been transcribed and translated. Another example of a linker is an internal ribosomal entry sites (IRES) which enables translation of two proteins or polypeptides from the same transcript. Any other appropriate linker may also be used. As a further example, the nucleic acid sequence encoding the TCR Va domain and nucleic acid sequence encoding the TCR Vp domain may be cloned into a vector with dual internal promoters (see e.g. S Jones et al., Human Gene Ther 2009). The identification of appropriate linkers and vectors that enable expression of both the TCR Va domain and the TCR Vp domain is well within the routine capabilities of a person of skill in the art.

[0172] In some examples, the linker comprises an amino acid sequence that can induce ribosomal skipping. For example, a 2A peptide. Examples of 2A peptides include T2A, P2A, E2A, and F2A. In some examples, the linker comprises P2A amino acid sequence. In some examples, the linker comprises an amino acid sequence derived from a porcine teschovirus. For example, the linker comprises or consists of a porcine teschovirus derived P2A sequence. In some examples, the linker comprises or consists of a sequence according to SEQ ID NO: 53.

[0173] Additional appropriate polypeptide domains may also be encoded by the nucleic acid sequences that encode the TOR Va domain and / or the TOR Vp domain. By way of example only, the nucleic acid sequence may comprise a membrane targeting sequence that provides for transport of the encoded polypeptide to the cell surface membrane of the modified cell. Other appropriate additional domains are well known and are described, for example, in WO2016 / 071758.

[0174] In one example, the nucleic acid molecules, TMBIM6 variant peptide binding proteins, or compositions thereof described herein may encode a soluble TOR. For example, the nucleic acid molecules, TMBIM6 variant peptide binding proteins, or compositions thereof may encode the variable domain of the TCR alpha and beta chains respectively together with an immune- modulator molecule such as a CD3 agonist (e.g. an anti-CD3 scFv). The CD3 antigen is present on mature human T cells, thymocytes and a subset of natural killer cells. It is associated with the TCR and is involved in signal transduction of the TCR. Antibodies specific for the human CD3 antigen are well known. One such antibody is the murine monoclonal antibody OKT3, which is the first monoclonal antibody approved by the FDA. Other antibodies specific for CD3 have also been reported (see e.g. W02004 / 106380; U.S. Patent Application Publication No. 2004 / 0202657; U.S. Pat. No. 6,750,325). Immune mobilising mTCR Against Cancer (ImmTAC; Immunocore Limited, Milton Partk, Abington, Oxon, United Kingdom) are bifunctional proteins that combine affinity monoclonal T cell receptor (mTCR) targeting with a therapeutic mechanism of action (i.e. , an anti-CD3 scFv). In another example, a soluble TCR of the invention may be combined with a radioisotope or a toxic drug. Appropriate radioisotopes and / or toxic drugs are well known in the art and are readily identifiable by a person of ordinary skill in the art.

[0175] In one example, the nucleic acid molecules, TMBIM6 variant peptide binding proteins, or compositions thereof may encode a chimeric single chain TCR wherein the TCR alpha chain variable domain is linked to the TCR beta chain variable domain and a constant domain which is e.g. fused to the CD3 zeta signalling domain. In this example, the linker is non-cleavable. In an alternative embodiment, the nucleic acid composition may encode a chimeric two chain TCR in which the TCR alpha chain variable domain and the TCR beta chain variable domain are each linked to a CD3 zeta signalling domain or other transmembrane and intracellular domains. Methods for preparing such single chain TCRs and two chain TCRs are well known in the art; see for example RA Willemsen et al, Gene Therapy 2000.

[0176] The invention also provides isolated nucleic acid sequences that encode a peptide of the invention (and corresponding vectors). All general statements herein relating to nucleic acid sequences and vectors apply equally. A person of skill in the art would readily identify suitable nucleic acid sequences and vectors on the basis of the peptide sequences provided herein. As such, there is also provided a binding protein encoded by the nucleic acids described herein. For example, a TMBIM6 variant peptide binding protein having a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:1 and a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 27. In some examples, the TCR Va domain comprises a CDR3 amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 and the TCR Vp domain comprises a CDR3 amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 27. In some examples, the TCR Va domain comprises a CDR3 amino acid sequence consisting of SEQ ID NO:1 and the TCR Vp domain comprises a CDR3 amino acid sequence consisting of SEQ ID NO: 27.

[0177] In some examples, the TCR Va domain comprises an amino acid sequence having at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10 or 13.

[0178] In some examples, the TCR Va domain further comprises a TCR a chain constant region. For example, a TCR a chain constant region comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to or consists of SEQ ID NO: 16.

[0179] In some examples, the TCR Vp domain further comprises a TCR p chain constant region. For example, a TCR p chain constant region comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to or consists of SEQ ID NO: 39.

[0180] In some examples, TCR Vp domain has an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 36 or 44.

[0181] In some examples, the TCR Va domain comprises a CDR1 having an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to or consisting of SEQ ID NO: 4. In some examples, the TCR Va domain comprises a CDR2 has an amino acid sequence of having an amino acid sequence having at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to or consisting of SEQ ID NO: 7.

[0182] In some examples, the TCR Vp domain comprises a CDR1 having an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to or consisting of SEQ ID NO: 30. In some examples, the TCR Vp domain comprises a CDR2 having an amino acid sequence having at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to or consisting of SEQ ID NO: 33.

[0183] In some examples, the TCR Vp domain comprises a leader peptide positioned at the N-terminal. For example, the leader sequence comprise an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to or consisting of SEQ ID NO: 42. Other suitable leader sequences will be known. In some examples, the TCR Va domain comprises a leader peptide positioned at the N-terminal. For example, the leader sequence comprise an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to or consisting of SEQ ID NO: 19. Other suitable leader sequences will be known.

[0184] As such, in some examples, there is provided a TMBIM6 variant peptide binding protein comprising TCR a chain comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 21 or 24.

[0185] As such, in some examples, there is provided a TMBIM6 variant peptide binding protein comprising a TCR p chain comprising or consisting of an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 47 or 50.

[0186] As such, in some examples, there is provided a TMBIM6 variant peptide binding protein comprising TCR a chain comprising or consisting of an amino acid sequence having at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 21 and a TCR chain comprising or consisting of an amino acid sequence having at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 47. As such, in some examples, there is provided a TMBIM6 variant peptide binding protein comprising TCR a chain comprising or consisting of an amino acid sequence having at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 24 and a TCR p chain comprising or consisting of an amino acid sequence having at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 50. In some examples the TMBIM6 variant peptide binding protein comprises or consists of a T cell receptor (TCR) or an antigen binding fragment thereof comprising or consisting of an amino acid sequence having at least 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 55.

[0187] In some examples the TMBIM6 variant peptide binding protein comprises a TCR, an antigen binding fragment of a TCR, or a chimeric antigen receptor (CAR) as described herein. In some examples the TMBIM6 variant peptide comprises a single chain TCR (scTCR) or a chimeric TCR dimer in which the antigen binding fragment of the TCR is linked to an alternative transmembrane and intracellular signalling domain.

[0188] For example, provided herein is a TCR encoded by the nucleic acids described herein. For example, a TCR comprising an amino acid sequence according to any one or more of SEQ ID NOs: 1 , 4, 7, 10, 13, 21 , 24, 27, 30, 33, 36, 44, 47, 50, or 55. In some examples, there is provided a TCR comprising or consisting of an amino acid sequence according to SEQ ID NO: 55. The binding proteins or TCRs may be for use as medicaments as described herein.

[0189] Also provided are isolated nucleic acid molecules. For example, one or more isolated nucleic acid molecules comprising or consisting of a sequence according to any one or more of SEQ ID NOs: 3, 6, 9, 12, 15, 23, 26, 29, 32, 35, 38, 46, 49, 52, or 57. The isolated nucleic acid molecules may be for use as medicaments as described herein.

[0190] Antigen

[0191] The binding proteins described herein may also be expressed as part of a transgene construct that encodes additional accessory proteins, such as a safety switch protein, a tag, a selection marker, a CD8 co-receptor p-chain, a-chain or both, or any combination thereof.

[0192] The TMBIM6W>Fantigen that is specifically bound by the binding proteins described herein comprises the amino acid sequence shown in SEQ ID NO:58. The antigen may be an antigenic fragment (i.e. a portion) of the sequence shown in SEQ ID NO:58, it may consist of the sequence of SEQ ID NO:58 or it may comprise (i.e. include within a longer sequence) the sequence of SEQ ID NO:58. The TMBIMe^ antigen is capable of being presented by at least HLA-A*24:02 and / or HI_A-C*02:02. In some examples, TMBIMe^ antigen is capable of being presented by at least HI_A-A*24 family members and / or HI_A-C*02:02. The encoded binding protein may therefore be capable of specifically binding to a TMBIMe^ antigen:HLA-A*24:02 and / or HI_A-C*02:02 complex, wherein the TMBIM6W>Fantigen is an antigenic fragment of the sequence shown in SEQ ID NO: 58, or wherein the TMBIM6W>Fantigen comprises or consists of the amino acid sequence shown in SEQ ID NO: 58.

[0193] The TMBIM6W>Fantigen may be comprised within or derived from a TM Bl M6 protein that has undergone a tryptophan (W) to phenylalanine (F) substitution. Tryptophan (W) to phenylalanine (F) substitution may be induced in a subject by creating a tryptophan shortage in a cell. Therefore, in some examples, the level of antigen and antigen complex in a subject may be increased by decreasing a level of tryptophan in cells of the subject. For example, in cancer cells of a subject. In some examples, tryptophan (W) to phenylalanine (F) substitution and ergo production of the TMBIM6W>Fantigen described herein or TMBIM6W>Fantigen complex described herein may be induced by a disease, for example, a caner. In some examples, the subject may suffer from a disease such as cancer but tryptophan (W) to phenylalanine (F) substitution and ergo production of the TMBIM6W>Fantigen or TMBIM6W>Fantigen complex described herein may be induced by use of other or additional compounds (such as the additional therapeutics described herein).

[0194] Vectors and Modified Cells

[0195] Also provided herein are vectors or vector systems which include a nucleic acid molecule as described herein. The vector or vector system may have one or more vectors. The binding protein components that are encoded by the nucleic acid molecules described herein may be encoded by one or more nucleic acid sequences, for example in a nucleic acid composition. In examples where all of the binding protein components are encoded by a single nucleic acid molecule, the nucleic acid molecule may be present within a single vector (and thus the vector system described herein may comprise one vector). In examples where the binding protein components are encoded by two or more nucleic acid molecules (wherein the plurality of nucleic acid molecules, together, encode all of the components of the binding protein) these two or more nucleic acid molecules may be present within one vector (e.g. in different open reading frames of the vector), or may be distributed over two or more vectors. In this example, the vector system will comprise a plurality of distinct vectors (i.e. vectors with different nucleotide sequences).

[0196] Any appropriate vector can be used. By way of example only, the vector may be a plasmid, a cosmid, or a viral vector, such as a retroviral vector or a lentiviral vector. Adenovirus, adeno- associated virus, vaccinia virus, canary poxvirus, herpes virus, minicircle vectors and naked (synthetic) DNA / RNA may also be used (for details on minicircle vectors, see for example non- viral Sleeping Beauty transposition from minicircle vectors as published by R Monjezi et al., Leukemia 2017). Alternatively, single stranded or double stranded DNA or RNA can be used to transfect lymphocytes with a TCR of interest (see Roth et al 2018 Nature vol 559; page 405).

[0197] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been operably linked. The vector can be capable of autonomous replication or it can integrate into a host DNA. The vector may include restriction enzyme sites for insertion of recombinant DNA and may include one or more selectable markers or suicide genes. The vector can be a nucleic acid molecule in the form of a plasmid, a bacteriophage or a cosmid. Preferably the vector is suitable for expression in a cell (i.e. the vector is an “expression vector”). Preferably, the vector is suitable for expression in a human T cell such as a CD8+T cell or CD4+T cell, or stem cell, iPS cell, or NK cell. In certain aspects, the vector is a viral vector, such as a retroviral vector, a lentiviral vector or an adeno-associated vector. In some example, the vector is selected from the group consisting of an adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or synthetic RNA.

[0198] Preferably the (expression) vector is capable of propagation in a host cell and is stably transmitted to future generations.

[0199] The vector may comprise regulatory sequences. "Regulatory sequences" as used herein, refers to, DNA or RNA elements that are capable of controlling gene expression. Examples of expression control sequences include promoters, enhancers, silencers, TATA- boxes, internal ribosomal entry sites (IRES), attachment sites for transcription factors, transcriptional terminators, polyadenylation sites etc. Optionally, the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. Regulatory sequences include those which direct constitutive expression, as well as tissue-specific regulatory and / or inducible sequences.

[0200] In some examples, the vector comprises the nucleic acid sequence of interest operably linked to a promoter. "Promoter", as used herein, refers to the nucleotide sequences in DNA to which RNA polymerase binds to start transcription. The promoter may be inducible or constitutively expressed. Alternatively, the promoter is under the control of a repressor or stimulatory protein. The promoter may be one that is not naturally found in the host cell (e.g. it may be an exogenous promoter). The skilled person in the art is well aware of appropriate promoters for use in the expression of target proteins, wherein the selected promoter will depend on the host cell.

[0201] The vector may comprise a transcriptional terminator. “Transcriptional terminator” as used herein, refers to a DNA element, which terminates the function of RNA polymerases responsible for transcribing DNA into RNA. Preferred transcriptional terminators are characterized by a run of T residues preceded by a GC rich dyad symmetrical region.

[0202] The vector may comprise a translational control element. “Translational control element”, as used herein, refers to DNA or RNA elements that control the translation of mRNA. Preferred translational control elements are ribosome binding sites. Preferably, the translational control element is from a homologous system as the promoter, for example a promoter and its associated ribozyme binding site. Preferred ribosome binding sites are known, and will depend on the chosen host cell.

[0203] The vector may comprise restriction enzyme recognition sites.

[0204] The vector may comprise a selectable marker. "Selectable marker" as used herein, refers to proteins that, when expressed in a host cell, confer a phenotype onto the cell which allows a selection of the cell expressing said selectable marker gene. Generally this may be a protein that confers a new beneficial property onto the host cell (e.g. antibiotic resistance) or a protein that is expressed on the cell surface and thus accessible for antibody binding. Appropriate selectable markers are well known in the art.

[0205] In some examples, the vector may comprise a suicide gene. “Suicide gene” as used herein, refers to proteins that induce death of the modified cell upon treatment with specific drugs. By way of example, suicide can be induced of cells modified by the herpes simplex virus thymidine kinase gene upon treatment with specific nucleoside analogs including ganciclovir, cells modified by human CD20 upon treatment with anti-CD20 monoclonal antibody and cells modified with inducible Caspase9 (iCasp9) upon treatment with AP1903 (reviewed by BS Jones, LS Lamb, F Goldman, A Di Stasi; Improving the safety of cell therapy products by suicide gene transfer. Front Pharmacol. (2014) 5:254). Appropriate suicide genes are well known in the art.

[0206] Preferably the vector comprises those genetic elements which are necessary for expression of the binding proteins described herein by a host cell. The elements required for transcription and translation in the host cell include a promoter, a coding region for the protein(s) of interest, and a transcriptional terminator.

[0207] A person of skill in the art will be well aware of the molecular techniques available for the preparation of (expression) vectors and how the (expression) vectors may be transduced or transfected into an appropriate host cell (thereby generating a modified cell described further below). The (expression) vector system described herein can be introduced into cells by conventional techniques such as transformation, transfection or transduction. “Transformation”, “transfection” and “transduction” refer generally to techniques for introducing foreign (exogenous) nucleic acid molecule into a host cell, and therefore encompass methods such as electroporation, microinjection, gene gun delivery, transduction with retroviral, lentiviral or adeno-associated vectors, lipofection, superfection etc. The specific method used typically depends on both the type of vector and the cell. Appropriate methods for introducing nucleic acid molecules and vectors into host cells such as human cells are well known in the art; see for example Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y; Ausubel et al (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY; Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Luchansky et al (1988) Mol. Microbiol. 2, 637-646. Further conventional methods that are suitable for preparing expression vectors and introducing them into appropriate host cells are described in detail in WO2016 / 071758 for example.

[0208] In some examples, the host cell is contacted with the vector system (e.g. viral vector) in vitro, ex vivo, and in some examples, the host cell is contacted with the vector system (e.g. viral vector) in vivo.

[0209] The term "host cell" includes any cell into which the nucleic acid molecule or vector described herein may be introduced. Once a nucleic acid molecule or vector system has been introduced into the cell, it may be referred to as a “modified cell” herein. Once the nucleic acid molecule or vector is introduced into the host cell, the resultant modified cell should be capable of expressing the encoded binding protein (and e.g. correctly localising the encoded binding protein for its intended function e.g. transporting the encoded binding protein to the cell surface).

[0210] In some examples, the nucleic acid molecules or vectors may be introduced using CRISPR technology. Insertion of the nucleic acid molecules at the endogenous TCR locus by engineering with CRISPR / Cas9 and homologous directed repair (HDR) or non-homologous end joining (NHEJ) is therefore encompassed. Other conventional methods such as transfection, transduction or transformation of cells may also be used.

[0211] The term “modified cell” refers to a genetically altered (e.g. recombinant) cell. The modified cell includes at least one exogenous nucleic acid molecule (i.e. a nucleic acid sequence that is not naturally found in the host cell). In the context of the invention, the exogenous molecule comprises at least one of the T cell receptor component parts described herein.

[0212] The term “modified cell” refers to the particular subject cell and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0213] The host cell (and thus the modified cell) is typically a eukaryotic cell, and particularly a human cell (e.g. a T cell such as a CD8+T cell or a CD4+T cell, or a mixture thereof, or a hematopoietic stem cell, an iPSC, or gamma-delta T cell, or NK cell). The host cell (and thus the modified cell) may be an autologous or allogeneic cell (e.g. such as a CD8+T cell or a CD4+T cell, or a mixture thereof, or a hematopoietic stem cell, an iPSC, or gamma-delta T cell, or NK cell). “Allogeneic cell” refers to a cell derived from a different individual to the individual to which it is later administered. In other words, the host cell (and thus the modified cell) may be an isolated cell from a distinct individual compared to the subject to be treated. “Autologous cell” refers to a cell derived from the individual to which it is later administered. In other words, the host cell (and thus the modified cell) may be an isolated cell from the subject that is to be treated.

[0214] In the context of the methods of treatment described herein, the host cell (and thus the modified cell) may be for administration to an HLA-A*24:02 positive human subject. In view of this, the host cell (and thus the modified cell) may be HLA-A*24:02 positive but be TMBIM6W>Fnegative (i.e. modified cells can either be HI_A-A*24:02 positive or negative).

[0215] In the context of the methods of treatment described herein, the host cell (and thus the modified cell) may be for administration to an HLA-C*02:02 positive human subject. In view of this, the host cell (and thus the modified cell) may be HLA-C*02:02 positive but be TMBIMG^ negative (i.e. modified cells can either be HI_A-C*02:02 positive or negative).

[0216] The host cell (and thus the modified cell) may be any cell that is able to confer anti-tumour immunity after TCR gene transfer. Non limiting examples of appropriate cells include autologous or allogeneic a CD8 T cell, a CD4 T cell, Natural Killer (NK) cells, NKT cells, gamma-delta T cells, inducible pluripotent stem cells (iPSCs), hematopoietic stem cells or other progenitor cells and any other autologous or allogeneic cell or cell line (NK-92 for example or T cell lines) that is able to confer anti-tumor immunity after TCR gene transfer.

[0217] Advantageously, the modified cell is capable of expressing the binding protein encoded by the nucleic acid molecules or vectors described herein (i.e. the TCR component parts) such that the modified cell provides an immunotherapy that specifically targets cells that express TMBIMG^, and thus can be used to treat or prevent diseases or conditions. In some example, the subject a HI_A-A*24:02 positive human subject. In some example, the subject a HLA-C*02:02 positive human subject.

[0218] Pharmaceutical compositions

[0219] The nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems) or modified cells as described herein may be provided as part of a pharmaceutical composition. Advantageously, such compositions may be administered to a human subject in need thereof.

[0220] A pharmaceutical composition may comprise a nucleic acid molecule, TMBIM6 variant peptide binding proteins, or compositions thereof, vectors (or vector systems) or modified cells as described herein along with a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

[0221] Compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds. As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected nucleic acid molecules, compositions thereof, vectors or modified cells as described herein without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0222] Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. a nucleic acid molecule, vector, or modified cell as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

[0223] Adjuvants are pharmacological and / or immunological agents that modify the effect of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.

[0224] Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

[0225] Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.

[0226] In some examples, the pharmaceutical composition includes IFNy. T cells activated by cancerspecific antigens (neoantigens) secrete interferon-gamma (IFNy), which induces IDO1 in cancer cells. IDO1 catabolizes tryptophan to metabolites along the kynurenine pathway, generating intracellular tryptophan shortage. Therefore, without being bound by theory, IFNy may induce tryptophan to phenylalanine (W>F) substitutions and therefore increase a level of target epitopes in a target cell. For example, increase a level of an epitope comprising or consisting of an amino acid sequence according to SEQ ID NO: 58. In some examples, the pharmaceutical composition may include about 100 ll / rnl to about 500 ll / rnl of IFNy. In some examples, the pharmaceutical composition may include about 250 ll / rnl.

[0227] In some examples, the pharmaceutical composition may include one or more activators of IFNy. Activators of IFNy is used to refer to any compound that may increase or induce production of IFNy in a subject or target cell. For example, one or more compounds that lead to an increase in a level of IFNy in a target cell. For example, the expression of IFNy may be induced by IL-12, IL- 15, IL-18, and type I IFN. In some examples, the additional therapeutic is IFNy (which herein is referred to as an activator of IFNy).

[0228] In some examples, the activator of IFNy may be nucleic acid or peptide based therapeutic that increases levels of IFNy. For example, a nucleic acid, such as an mRNA or DNA coding an mRNA that encodes IFNy or a compound that indirectly leads to an increase in IFNy. In some examples, the additional therapeutic is an antibody fusion protein or nucleic acid encoding an antibody fusion protein comprising IFNy. For example, see Di Nitto C, Gilardoni E, Mock J, et al. An Engineered IFNy-Antibody Fusion Protein with Improved Tumor-Homing Properties. Pharmaceutics. 2023;15(2):377. Published 2023 Jan 22. doi:10.3390 / pharmaceutics15020377. In some examples, the antibody fusion protein targets a cancer antigen and therefore is able to provide targeted delivery of IFNy or activators thereof to a cancer or tumor. For example, cancer antigens include PSMA, alpha-fetoprotein, prostate-specific antigen, carcinoembryonic antigen, carbohydrate antigen 19-9, cancer antigen 15-3, cancer antigen 125, BCR, ABL, CD19, CD20, CD30, CD33, CD52, CTLA-4, EGFR, EpCAM, HER2, PAP, PD-1 , VEGF, MARTI , gp100, MAGE- A3, CEA, MAGE-A4, MAGE-A10, WT-1 and VEGF-R2.

[0229] In some examples, the additional therapeutic is an immunofiliment comprising IFNy or an activator of IFNy. Immunofiliments are synthetic filamentous polymers decorated with a variety of immunomodulating molecules to mimic natural antigen presenting cells. Immunofilaments are designed to specifically activate and expand immune cells such as antigen specific cytotoxic T cells, CAR-T cells, or NK cells.

[0230] For examples of immunofiliments see Weiss, Lea, et al. "Direct in vivo activation of T cells with nanosized immunofilaments inhibits tumor growth and metastasis." Acs Nano 17.13 (2023): 12101-12117, Weiss, Lea, et al. "Immunofilaments Provide a Nanoscale Platform for In Vivo T Cell Expansion and Cancer Immunotherapy." bioRxiv (2022): 2022-10, and Gerrits, Lotte, et al. "Semi-Flexible Immunobrushes Facilitate Effective and Selective Expansion of Antigen-Specific T Cells." Advanced Functional Materials 34.14 (2024): 2307606. In some examples, the pharmaceutical composition includes Kynureninase (KYNase). Kynureninase or L-Kynurenine hydrolase (KYNU) (EC 3.7.1.3) is a PLP dependent enzyme that catalyses the cleavage of kynurenine (Kyn) into anthranilic acid (Ant). Upregulation of tryptophan catabolism by IDO1 and / or tryptophan 2,3-dioxygenase (TDO) leads immune suppression in tumors. These enzymes catalyze the oxidation of tryptophan to N-formyl L-kynurenine, which is rapidly converted by formamidases to Kyn. Elevated concentrations of Kyn and higher plasma Kyn / Trp ratios are frequently observed in advanced stage cancer patients and correlate with poor prognoses. In some examples, KYNase may reduce suppression of T Cell activity. In some examples, the pharmaceutical composition may include a KYNase at a concentration of 3x102to 10x102mg / ml. In some examples, the pharmaceutical composition may include a KYNase at a concentration of about 7.2 mg / ml.

[0231] Kit of Parts In one aspect, there is provided a kit of parts that includes a nucleic acid composition, a vector system, a modified cell or pharmaceutical compositions as described herein and one or more of IFNy, activators of IFNy, a KYNase and / or one or more further therapeutics. For example, the kit of parts may include one or more of a further T-cell receptor; a modified cell comprising a further T-cell receptor or comprising a nucleic acid encoding the further T-cell receptor; an immune checkpoint inhibitor; and / or a chimeric antigen receptor cell therapeutic.

[0232] For example, the kit may include a nucleic acid composition, TMBIM6 variant peptide binding protein, a vector system, a modified cell or pharmaceutical compositions as described herein and a KYNase.

[0233] For example, the kit may include a nucleic acid composition, TMBIM6 variant peptide binding protein, a vector system, a modified cell or pharmaceutical compositions as described herein and IFNy.

[0234] For example, the kit may include a nucleic acid composition, TMBIM6 variant peptide binding protein, a vector system, a modified cell or pharmaceutical compositions as described herein and one or more activators of IFNy.

[0235] For example, the kit may include a nucleic acid composition, TMBIM6 variant peptide binding protein, a vector system, a modified cell or pharmaceutical compositions as described herein and an additional therapeutic.

[0236] The kit of parts may include one or more of a buffer, a diluent, a carrier or excipient. In some examples, the kit of parts includes a set of instructions for using the kit of parts. In some examples, the component parts of the kit are provided in one or more containers such as vials or tubes.

[0237] In some examples, the activator of IFNy may be nucleic acid or peptide based therapeutic that increases levels of IFNy. For example, a nucleic acid, such as an mRNA or DNA coding an mRNA that encodes IFNy or a compound that indirectly leads to an increase in IFNy. In some examples, the additional therapeutic is an antibody fusion protein or nucleic acid encoding an antibody fusion protein comprising IFNy. For example, see Di Nitto C, Gilardoni E, Mock J, et al. An Engineered IFNy-Antibody Fusion Protein with Improved Tumor-Homing Properties. Pharmaceutics. 2023;15(2):377. Published 2023 Jan 22. doi:10.3390 / pharmaceutics15020377. In some examples, the antibody fusion protein targets a cancer antigen and therefore is able to provide targeted delivery of IFNy or activators thereof to a cancer or tumor. For example, cancer antigens include PSMA, alpha-fetoprotein, prostate-specific antigen, carcinoembryonic antigen, carbohydrate antigen 19-9, cancer antigen 15-3, cancer antigen 125, BCR, ABL, CD19, CD20, CD30, CD33, CD52, CTLA-4, EGFR, EpCAM, HER2, PAP, PD-1 , VEGF, MARTI , gp100, MAGE- A3, CEA, MAGE-A4, MAGE-A10, WT-1 and VEGF-R2.

[0238] In some examples, the additional therapeutic is an immunofiliment comprising IFNy or activator of IFNy. Immunofiliments are synthetic filamentous polymers decorated with a variety of immunomodulating molecules to mimic natural antigen presenting cells. Immunofilaments are designed to specifically activate and expand immune cells such as antigen specific cytotoxic T cells, CAR-T cells, or NK cells.

[0239] For examples of immunofiliments see Weiss, Lea, et al. "Direct in vivo activation of T cells with nanosized immunofilaments inhibits tumor growth and metastasis." Acs Nano 17.13 (2023): 12101-12117, Weiss, Lea, et al. "Immunofilaments Provide a Nanoscale Platform for In Vivo T Cell Expansion and Cancer Immunotherapy." bioRxiv (2022): 2022-10, and Gerrits, Lotte, et al. "Semi-Flexible Immunobrushes Facilitate Effective and Selective Expansion of Antigen-Specific T Cells." Advanced Functional Materials 34.14 (2024): 2307606.

[0240] Treatment of a subject

[0241] The nucleic acid molecules, TMBIM6 variant peptide binding proteins and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein may be for use as medicament or in methods of treating disease in a subject in need thereof.

[0242] In one example, the nucleic acid molecules, TMBIM6 variant peptide binding proteins and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein may be for use in inducing or enhancing an immune response (e.g. a cell mediated response) or used in methods of in inducing or enhancing an immune response in a subject.

[0243] The term “induced or enhanced immune response” refers to an increase in the immune response (e.g. a cell mediated immune response such as a T cell mediated immune response) of the subject during or after treatment compared to their immune response prior to treatment. An “induced or enhanced” immune response therefore encompasses any measurable increase in the immune response that is directly or indirectly targeted to the disease or condition being treated (or prevented).

[0244] In another example, the pharmaceutical composition may be for use in stimulating a cell mediated immune response. In such an example, the target cell population or tissue may be a TMBIM6W>Fexpressing target cell population or tissue. For example, it may be a cancer or tumour cell or tissue that expresses TMBIMe^.

[0245] In another example, the pharmaceutical composition may be for use in stimulating a cell mediated immune response to a target cell population or tissue in an HLA-A*24:02 positive human subject. In another example, the pharmaceutical composition may be for use in stimulating a cell mediated immune response to a target cell population or tissue in an HLA-C*02:02 positive human subject. In such an example, the target cell population or tissue may be a TMBIMe^ expressing target cell population or tissue. Typically, it is a TMBIM6W>Fexpressing malignant target cell population or tissue. For example, it may be a target cell population or tissue comprising a TMBIMe^ expressing tumour or cancer.

[0246] The pharmaceutical composition may also be for use in providing anti-tumour immunity to an HLA-A*24:02 positive human subject. The pharmaceutical composition may also be for use in providing anti-tumour immunity to an HLA- C*02:02 positive human subject. In some examples, the nucleic acid molecules, TMBIM6 variant peptide binding proteins and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein may not be HLA restricted and so for use in any subject.

[0247] Advantageously, the nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein may be formulated for use in T cell receptor (TCR) gene transfer, an approach that is rapid, reliable and capable of generating large quantities of T cells with specificity for the peptide (e.g. SEQ ID NO:58), regardless of the patient’s pre-existing immune repertoire. Using TCR gene transfer, modified autologous cells suitable for infusion may be generated within a few days.

[0248] In some examples, the nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein may be for use in methods of adoptive immunotherapy such as adoptive T cell therapy. As used herein, the term “adoptive immunotherapy” or “adoptive cell therapy” (ACT) refers to a process whereby autologous or allogeneic cells of various hematopoietic lineages (e.g., lymphocytes or T-cells) are transferred to a patient or subject to treat disease. The term “adoptive T-cell therapy” refers to a process whereby autologous or allogeneic T-cells are transferred to a patient or subject to treat disease.

[0249] Adoptive T cell therapy has been used to treat hyperproliferative diseases, such as cancers, by providing an antigen-specific immune response. One method involves the use of genetically modified T cells that express an antigen-specific protein having an extracellular domain that binds to an antigen. Recombinant T cell receptors have been used to provide specificity to T cells. In other methods, heterologous T cell receptors, specific for a particular antigen, have been expressed in T cells to provide an antigen-specific immune response. Methods of adoptive T cell therapy are well known in the art, see for example WO2016 / 071758.

[0250] In another example, the nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein may be for use in treating an HLA-A*24 positive human subject having a disease or condition associated with an elevated level of TMBIM6W>F. In another example, the nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein may be for use in treating an HLA-C*02 positive human subject having a disease or condition associated with an elevated level of TMBIM6W>F. Typically, the disease or condition associated with an elevated level of TMBIM6W>Fmay be a hyperproliferative disease or condition such as cancer. In some examples, the nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein may be for use in treating an HLA-A*24:02 and / or HLA-C*02:02 positive human subject having a disease or condition associated with an elevated level of TM Bl M6W>Fantigen.

[0251] In some examples, the subject to be treated suffers from or is suspected of suffering from cancer. For example, the subject may have or be suspected of having a cancer which expresses a TM Bl M6W>Fantigen. “Cancer” refers a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may result in the formation of malignant tumours or cells that invade neighbouring tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0252] In some examples, the cancer is glioblastoma, prostate cancer, pancreatic cancer, non-small cell lung carcinoma, melanoma, breast cancer, gastric cancer; a head and / or neck cancer, a cancer related to viral infection or colorectal cancer. In some examples, the cancer is glioblastoma. In some examples, the cancer is prostate cancer. In some examples, the cancer is pancreatic cancer. In some examples, the cancer is non-small cell lung carcinoma. In some examples, the cancer is melanoma. In some examples, the cancer is breast cancer. In some examples, the cancer is gastric cancer. In some examples, the cancer is a head and / or neck cancer. In some examples, the cancer is a cancer related to viral infection. In some examples, the cancer is colorectal cancer.

[0253] “Glioblastoma” refers to a tumour of the brain and / or spinal cord, originating from cell populations in the brain such as glial cells, astrocytes, oligodendrocytes, neural stem cells, or cells of an existing astrocytoma.

[0254] “Prostate cancer” refers to any cancer that originates in the prostate. Prostate cancer is classified as an adenocarcinoma, or glandular cancer, that begins when normal semen-secreting prostate gland cells mutate into cancer cells.

[0255] "Gastric cancer" refers to malignant tumours occurring in the stomach, including gastric adenocarcinoma occurring in the gastric mucosal epithelium and malignant lymphoma, myosarcoma, and stromal tumours occurring in the submucosa, but is not limited thereto.

[0256] “Pancreatic cancer” refers to “locally advanced pancreatic cancer” and “metastatic pancreatic cancer.” “Locally advanced pancreatic cancer” refers to tumours that arise in pancreatic exocrine or neuroendocrine tissue, but distant metastases are absent. In contrast, “metastatic pancreatic cancer” refers to cancer spreading from the site from which it originates in the pancreas to involve another part of the body, for example, the liver.

[0257] “Non-small cell lung carcinoma” or “non-small cell lung cancer” refers to any type of epithelial lung cancer other than small cell lung cancer (SCLC). The most common types of non-small cell lung cancer are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma.

[0258] “Melanoma” refers to a condition characterized by the growth of a tumour arising from the melanocytic system of the skin and other organs. Most melanocytes occur in the skin, but are also found in the meninges, digestive tract, lymph nodes and eyes. “Breast cancer” refers to any malignancy of the breast tissue, including, for example, carcinomas and sarcomas.

[0259] “Head and / or neck” cancer refers to other malignancies, except brain cancer, located in the head and neck region. In some examples, head and / or neck cancer may be oral cancer, nasopharyngeal carcinoma, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, sinus cancer, salivary gland cancer or the like. Head and neck cancer commonly occurs in the oral cavity, nasal cavity, throat, sinus, salivary gland, larynx and the like.

[0260] “Colorectal cancer” refers to any cancer of the large bowel, which includes the colon (the large intestine from the cecum to the rectum) and the rectum.

[0261] “Cancer related to viral infection” refers to any cancer that may be caused or the result of a viral infection. Some examples of pathogenic viruses causing infections that may be related to or cause cancers include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1 , HAV-6, HSV-II , and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

[0262] In some examples, the subject has or is suspected of having cancer and is undergoing treatment with one or more additional therapeutics as described herein. In some examples, the subject has or is suspected of having cancer and has been treated with a therapeutic or agent that induces production of the TMBIM6W>Fantigen as described herein. For example, leads to or induces tryptophan deficiency in the cells of the subject. For example, in cancer cells of a subject.

[0263] In some examples, the nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein may be administered in combination with one or more additional therapeutics. For example, the additional therapeutic may be administered, prior to, subsequently to and / or concurrently with the nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein.

[0264] In particular, the one or more additional therapeutics may increase a level of a TMBIM6 variant peptide, wherein the variant comprises a W to F substitution in the subject. For example, the TMBIM6 variant peptide may comprise an amino acid sequence according to SEQ ID NO: 58.

[0265] In some examples, the additional therapeutic may be a compound that increases the level of IFNy in the subject. For example, the additional therapeutic may be an activator of IFNy. In some examples, the additional therapeutic is IFNy (which herein is referred to as an activator of IFNy). Without being bound by theory, IFNy, may induce IDO1 in the target cells (i.e. cancer cells). IDO1 catabolizes tryptophan and may therefore generate an intracellular tryptophan shortage. This may in turn increase levels of Wto F substitutions and increase levels of TMBIM6 variant peptide as described herein. Activators of IFNy (e.g. compounds that increase IFNy) may include one or more of IL-12, IL-15, IL-18, and type I IFN.

[0266] In some examples, the activator of IFNy may be nucleic acid or peptide based therapeutic that increases levels of IFNy. For example, a nucleic acid, such as an mRNA or DNA coding an mRNA that encodes IFNy or a compound that indirectly leads to an increase in IFNy. In some examples, the additional therapeutic is an antibody fusion protein or nucleic acid encoding an antibody fusion protein comprising IFNy. For example, see Di Nitto C, Gilardoni E, Mock J, et al. An Engineered IFNy-Antibody Fusion Protein with Improved Tumor-Homing Properties. Pharmaceutics. 2023;15(2):377. Published 2023 Jan 22. doi:10.3390 / pharmaceutics15020377. In some examples, the antibody fusion protein targets a cancer antigen and therefore is able to provide targeted delivery of IFNy or activators thereof to a cancer or tumor. For example, cancer antigens include PSMA, alpha-fetoprotein, prostate-specific antigen, carcinoembryonic antigen, carbohydrate antigen 19-9, cancer antigen 15-3, cancer antigen 125, BCR, ABL, CD19, CD20, CD30, CD33, CD52, CTLA-4, EGFR, EpCAM, HER2, PAP, PD-1 , VEGF, MARTI , gp100, MAGE- A3, CEA, MAGE-A4, MAGE-A10, WT-1 and VEGF-R2.

[0267] In some examples, the additional therapeutic is an immunofiliment comprising IFNy or a compound that indirectly leads to an increase in IFNy. Immunofiliments are synthetic filamentous polymers decorated with a variety of immunomodulating molecules to mimic natural antigen presenting cells. Immunofilaments are designed to specifically activate and expand immune cells such as antigen specific cytotoxic T cells, CAR-T cells, or NK cells.

[0268] For examples of immunofiliments see Weiss, Lea, et al. "Direct in vivo activation of T cells with nanosized immunofilaments inhibits tumor growth and metastasis." Acs Nano 17.13 (2023): 12101-12117, Weiss, Lea, et al. "Immunofilaments Provide a Nanoscale Platform for In Vivo T Cell Expansion and Cancer Immunotherapy." bioRxiv (2022): 2022-10, and Gerrits, Lotte, et al. "Semi-Flexible Immunobrushes Facilitate Effective and Selective Expansion of Antigen-Specific T Cells." Advanced Functional Materials 34.14 (2024): 2307606. In some examples, the additional therapeutic may be a further T-cell receptor. For example, the additional therapeutic may be a nucleic acid molecule, compositions thereof or vector encoding a binding protein or TCR different to those described above (i.e. capable of TMBIM6 variant peptide comprises or consists of and amino acid sequence according to SEQ ID NO 58) (e.g. a second T cell receptor). The additional therapeutic may be a nucleic acid molecule, compositions thereof or vector encoding a further binding protein or TCR that is comprised within a modified cell as described herein. For example, the modified cells described herein may include a nucleic acid molecule, compositions thereof or vector encoding a binding protein or TCR as described above (i.e. capable of TMBIM6 variant peptide comprises or consists of and amino acid sequence according to SEQ ID NO 58) and a second acid molecule, compositions thereof or vector encoding a second TCR. In some examples, the additional therapeutic may be a further modified cell comprising a further T-cell receptor or comprising a nucleic acid, compositions thereof or vector encoding the further T-cell receptor.

[0269] The further TCR may be any TCR that may increase the level of TMBIMG^ in a subject. The further TCR may lead to an intracellular tryptophan shortage in a subject. Thereby increasing the level of W to F substitution and thereby increasing the level of TMBIMG^ in a subject.

[0270] Examples of antigens that may be targeted by the further TCR include, but are not limited, NY- ESO-1 , HPV16-E6, HPV16-E7, HBV, MCPyV, TP53, KRAS G12D, MARTI , gp100, MAGE-A3, CEA, MAGE-A4, MAGE-A10, and / or WT-1. For example, the further TCR may be a DMF5 TCR. In some examples, the further TCR or further modified cell (e.g. T cell) is a TCR or modified cell that targets a tumour-associated antigen.

[0271] For example, the further TCR or further modified cell targets MARTI , gp100, MAGE-A3, CEA, MAGE-A4, MAGE-A10, and / or WT-1. In some examples, the further TCR or further modified cell targets MART 1 .

[0272] In some examples, the subject has been previously or is undergoing treatment with adoptive T cell therapy. For example, using one or more further TCRs as described herein. For example, the subject has undergone or is undergoing treatment with one or more further TCRs or modified cells and is then treated using a nucleic acid molecules and compositions thereof, TMBIM6 variant peptide binding proteins, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein.

[0273] Therefore, in one aspect, there is provided a method of treating a subject using nucleic acid molecules or compositions thereof, vectors (or vector systems), modified cells as described herein wherein the subject has been treated with or is being treated with a further TCR or further modified cell as described herein. For example, the subject has been treated with or is being treated with a TCR or modified cell that targets MARTI . In one example, there is provided nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein for use in treating a subject wherein the subject has been treated with or is being treated with a further TCR or further modified cell as described herein. For example, the subject has been treated with or is being treated with a TCR or modified cell that targets MART 1.

[0274] In some examples, the additional therapeutic is a immune checkpoint inhibitor. As used herein, an “immune checkpoint inhibitor” means an agent that inhibits proteins or peptides (e.g. immune checkpoint proteins) which are blocking the immune system, e.g., from attacking cancer cells. In some examples, the immune checkpoint protein blocking the immune system prevents the production and / or activation of T cells. An immune checkpoint inhibitor can be an antibody or antigen-binding fragment thereof, a protein, a peptide, a small molecule, or combination thereof. Typically, the inhibitor interacts directly to a target immune checkpoint protein (or its ligand, where appropriate) and thereby disrupts its function / biological activity. For example, it may bind directly to a target immune checkpoint protein (or its ligand, where appropriate). In one example, direct binding to a target immune checkpoint protein (or its ligand, where appropriate) inhibits, prevents or reduces the formation of protein complexes which are needed for immune checkpoint protein function / biological activity.

[0275] A review describing immune checkpoint pathways and the blockade of such pathways with immune checkpoint inhibitor compounds is provided by Pardoll in Nature Reviews Cancer (April, 2012), pages 252-264. Immune check point inhibitor compounds display anti-tumor activity by blocking one or more of the endogenous immune checkpoint pathways that downregulate an antitumor immune response. The inhibition or blockade of an immune checkpoint pathway typically involves inhibiting a checkpoint receptor and ligand interaction with an immune checkpoint inhibitor compound to reduce or eliminate the signal and resulting diminishment of the anti-tumor response.

[0276] The immune checkpoint inhibitor compound may inhibit the signalling interaction between an immune checkpoint receptor and the corresponding ligand of the immune checkpoint receptor. The immune checkpoint inhibitor compound can act by blocking activation of the immune checkpoint pathway by inhibition (antagonism) of an immune checkpoint receptor (some examples of receptors include CTLA-4, PD-1 , and NKG2A) or by inhibition of a ligand of an immune checkpoint receptor (some examples of ligands include PD-L1 and PD-L2). In such examples, the effect of the immune checkpoint inhibitor compound is to reduce or eliminate down regulation of certain aspects of the immune system anti-tumor response in the tumor microenvironment.

[0277] In some examples, the immune checkpoint inhibitor inhibits the CTLA-4 pathway or the PD- L1 / PD1 pathway (examples thereof are provided in, e.g., WO 2016 / 062722).

[0278] In some examples, the immune checkpoint inhibitor is an anti-CTLA-4 antibody or derivative or antigen-binding fragment thereof. Examples of anti-CTLA-4 antibodies and derivatives and fragments thereof are described in, e.g., US 6,682,736; US 7,109,003; US 7,123,281 ; US 7,411 ,057; US 7,807,797; US 7,824,679; US 8,143,379; US 8,491 ,895, and US 2007 / 0243184. In some examples, the anti-CTLA-4 antibody is tremelimumab or ipilimumab.

[0279] In some examples, the immune checkpoint inhibitor is an anti-PD-L1 antibody or derivative or antigen-binding fragment thereof. In some examples, the anti-PD-L1 antibody or derivative or antigen-binding fragment thereof selectively binds a PD-L1 protein or fragment thereof. Examples of anti-PD-L1 antibodies and derivatives and fragments thereof are described in, e.g., WO 01 / 14556, WO 2007 / 005874, WO 2009 / 089149, WO 2011 / 066389, WO 2012 / 145493; US 8,217,149, US 8,779,108; US 2012 / 0039906, US 2013 / 0034559, US 2014 / 0044738, and US 2014 / 0356353. In some embodiments, the anti-PD-L1 antibody is MEDI4736 (durvalumab), MDPL3280A, 2.7A4, AMP-814, MDX-1105, atezolizumab (MPDL3280A), or BMS-936559.

[0280] In some examples, the immune checkpoint inhibitor is an anti-PD-1 antibody or derivative or antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody selectively binds a PD-1 protein or fragment thereof. In some embodiments, the anti-PD1 antibody is nivolumab, pembrolizumab, or pidilizumab.

[0281] In some examples, the immune checkpoint inhibitor is an anti-NKG2A compound, such as an anti-NKG2A antibody. Examples of anti-NKG2A antibodies and derivatives and fragments thereof are described in WO 2016 / 041947, the content of which is hereby incorporated by reference in its entirety including, but not limited to, the sequence listings.

[0282] In some examples, the immune checkpoint inhibitor compound is a small organic molecule (molecular weight less than 1000 daltons), a peptide, a polypeptide, a protein, an antibody, an antibody fragment, or an antibody derivative. In some embodiments, the immune checkpoint inhibitor compound is an antibody. In some embodiments, the antibody is a monoclonal antibody, specifically a human or a humanized monoclonal antibody.

[0283] Methods for the preparation and use of immune checkpoint antibodies are described in the following illustrative publications. The preparation and therapeutic uses of anti-CTLA-4 antibodies are described in U.S. Patent Nos. 7229628 (Allison), 7311910 (Linsley), and 8017144 (Korman). The preparation and therapeutic uses of anti-PD-1 antibodies are described in U.S. Patent No. 8008449 (Korman) and U.S. Patent Application No. 2011 / 0271358 (Freeman). The preparation and therapeutic uses of anti-PD-L1 antibodies are described in U.S. Patent No. 7943743 (Korman). The preparation and therapeutic uses of anti-TIM-3 antibodies are described in U.S. Patent Nos. 8101176 (Kuchroo) and 8552156 (Tagayanagi). The preparation and therapeutic uses of anti-LAG-3 antibodies are described in U.S. Patent Application No. 2011 / 0150892 (Thudium) and International Publication Number W02014 / 008218 (Lonberg). The preparation and therapeutic uses of anti-KIR antibodies are described in U.S. Patent No. 8119775 (Moretta). The preparation of antibodies that block BTI_A regulated inhibitory pathways (anti-BTLA antibodies) are described in U.S. Patent No. 8563694 (Mataraza).

[0284] Immune checkpoint inhibitors that may be administered to a subject include but are not limited to an anti-PD-1 antibody, anti-PD-L1 antibody, anti-l_AG-3 antibody, anti-TIGIT antibody, anti- KLRB1 antibody, anti-LILRB2 antibody, anti-LILRB4 antibody, anti-LILRB2 and LILRB4 antibody and / or anti-TIM-3 antibody. Examples of immune checkpoint inhibitors include atezolizumab, ipimilumab, pembrolizumab, lambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK), pidilizumab (CT-011 , CURETECH LTD) and tislelizumab. Exemplary anti-PD-L1 antibodies include MDX-1105 (MEDAREX), MEDI4736 (MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB). Other examples include LILRB2 and LILRB4 antibodies described in US20190194327A1.

[0285] The inhibitor need not be an antibody, but can be a small molecule or other agent or compound. If the inhibitor is an antibody it may be a polyclonal, monoclonal, fragment, single chain, or other antibody variant construct. Inhibitors may target any immune checkpoint protein known in the art, including but not limited to, CTLA-4, PDL1 , PDL2, PD1 , B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1 , CHK2, A2aR, and the B-7 family of ligands. Combinations of inhibitors for a single target immune checkpoint or different inhibitors for different immune checkpoints may be used. In particular the immune checkpoint therapy may be an inhibitor of one or more of CD274 (PD-L1), PDCD1 LG2 (PD-L2), TIGIT, HAVCR2 (TIM-3), LAG-3, KLRB1 , LILRB2 and / or LILRB4.

[0286] Therefore, in one aspect, there is provided a method of treating a subject using nucleic acid molecules or compositions thereof, vectors (or vector systems), modified cells as described herein wherein the subject has been treated with or is being treated with a checkpoint inhibitor. In one example, there is provided nucleic acid molecules and compositions thereof, TMBIM6 variant peptide binding protein, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein for use in treating a subject wherein the subject has been treated with or is being treated with a checkpoint inhibitor.

[0287] For example, the immune checkpoint inhibitor may be selected from one or more of Nivolumab, Atezolizumab, avelumab, durvalumab , and ipilimumab.

[0288] In some examples, the additional therapeutic is a chimeric antigen receptor therapeutic. Chimeric antigen receptor therapeutics typically include chimeric antigen receptor cells, which may be chimeric antigen receptor T cells, chimeric antigen receptor NK cells, and the like. The term "chimeric antigen receptor" (CAR), as used herein, refers to a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide from which the extracellular domain is derived, and at least one intracellular domain. The "chimeric antigen receptor (CAR)" is sometimes called a "chimeric receptor", a "T-body", or a "chimeric immune receptor (CIR) " The "extracellular domain capable of binding to an antigen" means any oligopeptide or polypeptide that can bind to a certain antigen. The "intracellular domain" or "intracellular signalling domain" means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell. In certain embodiments, the intracellular domain may comprise, alternatively consist essentially of, or yet further comprise one or more costimulatory signalling domains in addition to the primary signalling domain. The "transmembrane domain" means any oligopeptide or polypeptide known to span the cell membrane and that can function to link the extracellular and signalling domains. A chimeric antigen receptor may optionally comprise a "hinge domain" which serves as a linker between the extracellular and transmembrane domains. Examples of CAR therapeutics Abecma®, Breyanzi ®, Kymriah ®, Tecartus ®, Yescarta ®, and Carvykti ®. Other examples of CAR therapeutics can be found in, for example, WO2019220109A1 , US11034750B2, W02013123061A1 , US20130287748A1 ,

[0289] WO2014055668A1 , WO2014138704A1 , WO2015075468A1 , and WO2017216561A1 .

[0290] Therefore, in one aspect, there is provided a method of treating a subject using nucleic acid molecules or compositions thereof, vectors (or vector systems), modified cells as described herein wherein the subject has been treated with or is being treated with a chimeric antigen receptor therapy. In one example, there is provided nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein for use in treating a subject wherein the subject has been treated with or is being treated with a chimeric antigen receptor therapy.

[0291] In some examples, the subject to be treated has been identified as having a disease or condition and the subject includes cells that express a peptide that comprises or consists of SEQ ID NO: 58. In some examples, a sample has been taken from the subject and the presence of a peptide comprising or consisting of SEQ ID NO: 58 has been detected in the sample. In some examples, the subject has cancer. In some examples, the presence of a peptide that comprises or consists of SEQ ID NO: 58 is indicative that the subject has cancer.

[0292] As used herein, the terms “treat”, “treating” and "treatment" are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a condition, disorder or symptom (i.e. in this case a haematological malignancy). Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom. “Treatment” therefore encompasses a reduction, slowing or inhibition of the amount or concentration of malignant cells, for example as measured in a sample obtained from the subject, of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to the amount or concentration of malignant cells before treatment.

[0293] As used here in the term “subject” refers to an individual, e.g., a human, having or at risk of having a specified condition, disorder or symptom. The subject may be a patient i.e. a subject in need of treatment in accordance with the invention. The subject may have received treatment for the condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment in accordance with the present invention.

[0294] The compositions described herein can be administered to the subject by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be by infusion or by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, percutaneous administration.

[0295] The compositions described herein may be in any form suitable for the above modes of administration. For example, compositions comprising modified cells may in any form suitable for infusion. As further examples, suitable forms for parenteral injection (including, subcutaneous, intramuscular, intravascular or infusion) include a sterile solution, suspension or emulsion; suitable forms for topical administration include an ointment or cream; and suitable forms for rectal administration include a suppository. Alternatively, the route of administration may be by direct injection into the target area, or by regional delivery or by local delivery. The identification of suitable dosages of the compositions of the invention is well within the routine capabilities of a person of skill in the art.

[0296] The nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein are for administration in an effective amount. An “effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response. The effective amount to be used will depend, for example, upon the therapeutic (or non-therapeutic) objectives, the route of administration, and the condition of the patient / subject. For example, the suitable dosage of the composition of the invention for a given patient / subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein for example severity and type of haematological malignancy, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. The dosages and schedules may be varied according to the particular condition, disorder or symptom the overall condition of the patient / subject. Effective dosages may be determined by either in vitro or in vivo methods.

[0297] The the nucleic acid molecules, TMBIM6 variant peptide binding proteins, and compositions thereof, vectors (or vector systems), modified cells or pharmaceutical compositions as described herein are advantageously presented in unit dosage form.

[0298] Methods of generating binding proteins (e.g. TCRs)

[0299] A method of generating a binding protein that is capable of specifically binding to a peptide containing a TMBIMG^ antigen and does not bind to a peptide that does not contain the TMBIM6W>Fantigen is also provided, comprising contacting a nucleic acid composition (or vector system) described herein with a cell under conditions in which the nucleic acid composition is incorporated and expressed by the cell.

[0300] In the context of the binding proteins described herein, the TMBIM6W>Fantigen comprises or consists of the seguence of SEQ ID NO:58, or a functional fragment or variant thereof.

[0301] The method may be carried out on the (host) cell ex vivo or in vitro. Alternatively, the method may be performed in vivo, wherein the nucleic acid composition (or vector system) is administered to the subject and is contacted with the cell in vivo, under conditions in which the nucleic acid seguence is incorporated and expressed by the cell to generate the binding protein. In one example, the method is not a method of treatment of the human or animal body.

[0302] Appropriate in vivo, in vitro and ex vivo methods for contacting a nucleic acid seguence (or vector systems) with a cell under conditions in which the nucleic acid seguence (or vector) is incorporated and expressed by the cell are well known, as described elsewhere herein.

[0303] As stated elsewhere herein, the binding protein comprise a TCR, an antigen binding fragment of a TCR, or a chimeric antigen receptor (CAR). Further details are provided elsewhere herein.

[0304] As such, in one aspect there is provided a TCR molecule encoded by one more of the nucleic acid seguences as described herein. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0305] Aspects of the invention are demonstrated by the following non-limiting examples.

[0306] EXAMPLES EXAMPLE 1 Materials and Methods Cell culture and reagents

[0307] Excepted for MD55A-3, all cancer cell lines originated from the American Tissue Culture Collection (ATCC) and grown in the recommended culture media. In detail, 888-Mel, Colo-320, MDA-MB-231 , HepG2, HEK293T, HT-29, hTERT RPE-1 , MIA Paca-2, PC-9, SW-480, WiDrwere cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO), supplemented with 10% fetal bovine serum (Sigma) and 100 U / ml penicillin-streptomycin (GIBCO). BT-20, D10, DU-145, DLD- 1 , HCT-15, NCI-H1299, NCI-H187, NCI-H226, MDA-MB-435S, PC-3 and RA were maintained in Roswell Park Memorial Institute 1640 Medium (RPM1 1640, GIBCO) supplemented with 10% fetal bovine serum (Sigma) and 100 U / ml penicillin-streptomycin (GIBCO). SUM159PT were maintained in DMEM / F12 medium supplemented with 10% FBS, insulin (5 pg ml-1, Sigma), hydrocortisone (1 pg ml-1, Sigma) and 100 U / ml penicillin-streptomycin (GIBCO). MD55A-3 was derived from metastatic melanoma tumor resections12and were maintained in Roswell Park Memorial Institute 1640 Medium (RPMI 1640, GIBCO) supplemented with heat-inactivated 10% fetal bovine serum (Sigma), 25 mM HEPES (GIBCO) and 100 U / ml penicillin-streptomycin (GIBCO). MCF-10A were cultured in DMEM / F-12, HEPES medium (Thermo-Fisher scientific) supplemented with 5% horse serum (Thermo-Fisher scientific), EGF (10 ng / ml; Millipore), insulin (10 pg / ml; Sigma), and hydrocortisone (500 ng / ml; Sigma). All cell lines were maintained in a humidified atmosphere containing 5% CO2 at 37 °C, tested regularly and were found negative for mycoplasma contamination (EZ-PCR mycoplasma kit; Biological Industries). Tryptophan-free DMEM / F-12 medium was purchased from US Biologicals and IFNy (PeproTech) was used at 250 U / ml for 48 hrs or 72 hrs as indicated. IDO inhibitors; 1- methyl-L-tryptophan (Sigma) was dissolved in 0.1 M NaOH at a 20 mM concentration adjusted to pH 7.5, filter-sterilized and used at a final concentration of 300 pM for 48 or 72 hrs; Epacadostat (Selleckchem) was diluted in DMSO and used for 48 or 72 hrs at 200nM. Polyethylenimine (PEI, Polysciences) was dissolved in water at a concentration of 1 mg / ml, after which it was filter- sterilized, aliquoted and stored at -20 °C. Doxycycline was used at a final concentration of 1 pg / ml for 24, 48 or 72 hrs (see Figure 7K).

[0308] T cells culture’. Untransduced CD8+ T cells, TCRMART1and TCRTMBIM6W>F 1Transduced T cells were either maintained in TexMACS medium (Miltenyi) supplemented with 5ng / mL of interleukine- 7 (IL-7) and of interleukine-15 (IL-15) (Immunotools, resuspended in UltraPure Water) or were maintained in RPMI 1640 medium supplemented with 10% human serum (One lambda), penicillin-streptomycin, and with 150 U / ml IL-2 (Proleukin, Novartis).

[0309] Thawing TCR transduced T cell’. T cells were allowed to thaw at 37°C and then resuspended in 10 mL of IMDM medium (GIBCO) supplemented with 20% FBS and 0.1mg / mL DNAse (Stem Cell Technologies). After 15 min incubation at 37°C, T cells were centrifuged 1500 rpm 5min and them cultivated at a concentration of 1 x 106cells per mL.

[0310] Lentiviral production and transduction

[0311] For lentivirus production: 3,5 x 106HEK 293T cells were seeded per 100 mm dish or 5 x 105per 6 wells plate, one day prior transfection. For each transfection, 10 pg of the pLenti vector of interest or of pLentiV2 for CRISPR-Cas9 sgRNA, 5 pg of pMDL RRE, 3.5 pg pVSV-G AND 2.5 pg of pRSV-REV plasmids were mixed in 500 pl of serum-free DMEM. For transfection in a 6 well plate, a ratio 1 :6 was applied. Next, 500 pl of serum-free DMEM (100pL for the 6 wells plate) containing 2 pl of a 1 mg / ml PEI solution per pg of plasmid DNA was added. The entire mix was vortexed and left for 20 min at room temperature after which it was added to the HEK 293T cells to be transfected. The next day, the medium was replaced and the lentivirus-containing supernatants were collected 48 and 72 hrs post transfection, and snap frozen in liquid nitrogen. Target cells were transduced by supplementation of the lentiviral supernatant with 8 pg / ml polybrene (Sigma). One day after transduction, the transduced cells were selected by addition of 5-10 pg / ml blasticidin (Invivogen) or 2 pg / mL puromycin (Bio-connect) to the medium.

[0312] Amino Acid mass spectrometry

[0313] Cancer cells were treated for 48 hrs with IFNy (as described above). Then, 100 ocL of the supernatant was collected. 100 ocL of sample was mixed with 100 ocL of internal standard mix (10 ocM l-Trp-d5 and 1 ocM l-Kyn-d4 in water) and 30 ocl of trifluoro acetic acid. After centrifugation, 2 ocl of the supernatant was subjected to LC-MS / MS consisting of an UltiMate 3000 Autosampler and HPLC pump (Thermo Scientific, Waltham, MA, USA) and API4000 MS / MS (Sciex, Framingham, MA, USA). Separation was performed on a Symmetry C18 column (2.1 x150 mm, particle size 3.5 pM, Waters, Milford, MA, USA). Mobile phase A (0.1% formic acid in water) and B (methanol) was used in a 5 min gradient from 20 to 95%B maintained for 3 min followed by reequilibration at 20%B. MRMs for acquisition were: l-Trp (205.1 / 187.9 and 205.1 / 146.3), l-Kyn (209.3 / 192.1 and 209.3 / 146.1), l-Trp-d5 (210.3 / 192.0 and 210.0 / 192.0) and l-Kyn-d4 (213.2 / 196.1 and 213.2 / 150.2).

[0314] Immunopeptidomics

[0315] Immunoprecipitation of HLA-peptides

[0316] For every cancer cell line presented in this paper, 108cells per replicate were used. Cells were seeded accordingly and treated or not with either IFNy (250 lU / mL) or a combination of tryptophan depleted medium and IFNy for 48 hrs. In order to improve tryptophan depletion by endogenous IDO1 , PC-3, Mia-Paca-2, SUM159PT and D10 were treated for 72 hrs with IFNy. After IFNy exposure, cells were washed with PBS and harvest by trypsinization. Cells were centrifuged (5min 1500rpm) and then washed with PBS. The dry pellet was snap frozen in liquid nitrogen. Then, cell pellet was lysed as described in previous study1840. W6 / 32 antibody cross linked to protein-A sepharose 4B beads was used for the immunoaffinity purification. Please note that for RA cells, immunopeptidomics have been reanalyzed from a previous study18.

[0317] Mass-Spectrometry

[0318] After vacuum concentration of the IP eluates in a Speedvac, peptides were analyzed by LC- MS / MS on an Orbitrap Exploris 480 Mass spectrometer, connected to either an Easy nLC1200 LC system (Thermo Scientific) or an Evosep One LC system (Evosep Biotechnology, Odense, Denmark). Prior to LC separation with the Evosep One, peptides were reconstituted in 0.1% formic acid and 50% of the sample was loaded on Evotip Pure™ (Evosep) tips. Peptides were then eluted and separated using the pre-programmed “Extended Method” (88 min gradient) on an EV1137 (Evosep) column with an EV1086 (Evosep) emitter. Nanospray was achieved using the Easy-Spray NG Ion Source (Thermo Scientific) with a liquid junction set-up at 1.9 kV.

[0319] Prior to LC separation with the nLC1200, peptides were reconstituted in 2% formic acid, after which 50% of the sample was directly loaded onto the analytical column (ReproSil-Pur 120 C18- AQ, 2.4pm, 75 pm x 500 mm column, packed in-house in fritted Empty Self Pack NanoLC column tubes with integrated emitter tip (CoAnn Technologies LLC, WA, United States). Peptides were eluted in a 110-minutes gradient containing a linear increase from 6% to 30% solvent B (solvent A was 0.1% formic acid / water and solvent B was 0.1 % formic acid / 80% acetonitrile) followed by washout at 90% solvent B. Nanospray was achieved using the Nanospray FlexTM Ion source (Thermo Scientific) with a liquid junction set-up at 2.0 kV.

[0320] On the Exploris 480, data-dependent acquisition was performed as follows. Full scan MS was acquired at resolution 60,000 with MS1 mass range 350-1700 m / z, normalised AGC target was set to 100% and maximum injection time was 50 ms. Dynamic exclusion was set to 10 sec. and MS2 spectra were acquired at 15,000 resolution. The top 10 precursors per cycle were HCD fragmented when their charge states were 2-4, whereas the top 5 precursors per cycle were subjected to HCD fragmentation if they were singly charged. MS2 isolation window was 1.1 m / z, the normalized collision energy was 30, the normalized AGC target was set to 50% and the maximum injection time was 100 ms.

[0321] Bioinformatics analysis of immunopeptidomics

[0322] (A) Database preparation: UniProt database UP000005640 was downloaded. All instances of canonical “Tryptophans” were identified, and a sequence of 10 amino acids upstream and downstream (-10 Upstream- W- +10 Downstream) was extracted and all tryptophans were substituted to Phenylalanine. This sequence collection was concatenated to the Uniprot database

[0323] (B) Search: Fragpipe41v20.0 with its native nonspecific- HI_A workflow (preset parameters) was used to identify immunopeptides. Manifest files listed all RAW files with corresponding experiment ID. Each replicate was scanned independently. The files were scanned in DDA mode

[0324] (C) Motif Analysis: Motif scanning and enrichment analysis was done using NetMHCspan61. Motif construction was done using MixMHCp6465with top 4 detected motifs using all identified peptides.

[0325] (D) Benign Tissue Analysis: The raw files for multiple samples (as available) in 25 different benign tissues (of top interest) were downloaded and analyzed in the same manner as described above.

[0326] Code availability

[0327] The scripts used in this study are available with additional details at https: / / github.com / apataskar / substitutants_manuscript.

[0328] Public dataset

[0329] The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http: / / proteomecentral.proteomexchange.org) via the PRIDE partner repository66with the dataset identifier PXD041893 (Reviewer account details: Username: reviewer_pxd041893@ebi.ac.uk Password: JxWpQlft).

[0330] Priming of W>F neoepitope reactive naive CD8 T cells

[0331] Peripheral blood mononuclear cells (PBMCs) from HI_A-typed healthy blood donors were obtained from the blood bank of Oslo University Hospital (ethical approval # 2018 / 879). Isolation of mononuclear cells was performed by density-gradient centrifugation (Axis-Shield). The study was approved by the Regional Ethics Committee (REC) and informed consent was obtained from healthy donors in accordance with the declaration of Helsinki and institutional guidelines (REC 2018 / 2006 and 2018 / 879). Priming and isolation of T cells reactive to W>F neoepitopes was performed as previously described12 18. After 10 days in culture, the W>F neoepitope co-cultures were screened by flow cytometry for the presence of W>F neoepitope reactive CD8 T cells by combinatorial pMHC staining12. Positive CD8 T cells were identified by Boolean gating as live CD8+ T cells staining positive for two pMHC multimer fluorochromes with the same W>F neoepitope (FlowJo (TreeStar) v10.6.2 software). Reactive CD8 T cells were subsequently single cell sorted into V-bottom 96-well plates for TCR sequencing (FACS Aria II).

[0332] TCR sequencing and cloning

[0333] Paired TCR-a and TCR-p sequences from neoepitope reactive single cells were obtained by three nested PCRs with multiplexed primers covering all TCR-a and TCR-p V genes according to the published protocol67with minor modifications68. In brief, cDNA synthesis was performed and the first PCR reaction in two separate steps. Primer sequences and cycling conditions for all three PCR reactions are provided in the original protocol67. The single cell TCR library was then sequenced using paired-end 300 bp Illumina MiSeq sequencing. MiXCR script was used to analyze sequencing data and reconstruction of full-length TCR chains was performed with the ImmuneScape VDJassembler web-based tool. Output was manually verified for each TCR in the IMGT database. Variable TCR-a and TCR-p fragments of identified TCRs were codon-optimized, synthesized and cloned into pMP71 retroviral vector by Genscript.

[0334] TCR transduction

[0335] Two different protocols for T cells transduction have been used in this study

[0336] 1. The TCR™BIM6W>F.Iwastransduced into healthy donor T cells as previously described46. In brief, PBMCs from healthy donors were isolated and cultured for three days in 12-well tissue culture plates coated with anti-CD3 (OKT3, eBioscience) and anti-CD28 (CD28.6, eBioscience) antibodies. PBMCs were plated at 2 x 106per well and the T-cell medium (TexMACS) was supplemented with 5ng / ml IL-7 and IL-15 (PeproTech). Retroviral supernatant was generated using Phoenix-AMPHO cells (4 x 106cells in 10cm petri dishes) plated 24 hrs before transfection with y-retroviral vector DNA mixed with X- tremeGENE9 DNA Transfection reagent (Roche Diagnostics) and Opti-MEM. After 24 hrs, the medium was refreshed and cells were incubated at 32°C and 5% CO2 for 24 hrs. The following day, antibody-stimulated PBMCs were harvested and resuspended in TexMACS medium supplemented with 5ng / ml IL-7 and IL-15 mixed 1 :1 with retroviral supernatant. PBMCs were plated in Retronectin (20pg / ml, Takara) pre-coated non-tissue-culture- treated 6-well plates and spinoculated at 900g for 60min before being cultured for 24 hrs. To ensure high transduction efficiency, a second spinoculation was performed the following day with fresh retroviral supernatant. Transduction efficiency was determined 5 days after final spinoculation by staining with anti-mouse TCR-p chain antibody and / or pMHC multimer and analysed by flow cytometry (see Figures 4A-B).

[0337] 2. Retrovirus was produced by transfecting FLY-RD18 packaging cells with the corresponding TCR-plasmid (TMBIM6W>F 1or TCRMART1clone DMF5) using Xtremegene 9 transfection reagent (Roche)69. In parallel, CD8 T cells were isolated from healthy donor PBMCs (Sanquin Blood Bank) using the CD8 T Cell Isolation Kit (Miltenyi Biotec). Isolated cell fractions were stimulated with CD3 / CD28 Dynabeads (Life Technologies) in T cell medium with 100 ll / rnl IL-2. After 48 hrs, retroviral supernatants were collected and used to infect prestimulated CD8+ T cells by spinoculation (2,000 g for 90 min) in Retronectin (Takara)-coated plates. Transduction efficiency was measured 72 hrs later by staining with an anti-mouse TCRp (Miltenyi) antibody and using flow cytometry (Fortessa, BD sciences). TCRMART1Transduced T cells were then selected with 2.5 pg / ml puromycin (Bioconnect) for 48 hrs whereas TCRTMBIM6W>F 1were ce||-sorted upon mTCR (Miltenyi) staining (BD FACSAria™ Fusion Flow Cytometer). After selection, T cells received fresh medium and IL-2 every 3-4 days. After 12-14 days of culture, transduced T cells were expanded using Rapid Expansion Protocol if necessary.

[0338] Rapid Expansion Protocol (REP)

[0339] TCRMART1and TCRTMBIM6W>F 1Transduced T cells were expanded using the rapid expansion protocol (REP) as previously described70. Briefly, TCR transduced T cells were cultured with 30 ng / ml anti-CD3 antibody (clone OKT-3; eBioscience) and 3000 U / ml of IL-2 in a 1 :1 mixture of RPMI 1640 and AIM-V medium (Gibco) supplemented with 5% human serum (One Lambda), in the presence of irradiated (40 Gy) allogeneic PBMCs (200:1 feeder / T cell ratio). After 7 days of culture, the medium was refreshed with a mixture of 1 :1 RPMI 1640 and AIM-V medium (Gibco) supplemented with 5% human serum and 3000 U / ml IL-2 . The purity of the resultant culture was checked by FACS looking at the % of CD8+ and TCRp double positive T cells. After REP completed, T cells were subsequently used directly for T-cell co-culture assays or cryopreserved in liquid nitrogen.

[0340] Recipients were seeded in a 6 cm plate the day before peptide loading. On the day of peptide loading, cells were washed with PBS and then cultured overnight with RPMI 10% FBS + P / S and with the indicated peptide (TMBIMO^ and TMBIM6wr). The synthetic peptides were used at various concentration (e.g. see Figure 10D). The peptide were ordered at GenScript Biotech with a purity >70%. In case of IFNy stimulation combined with peptide loading, cells were treated with IFNy 24 hrs prior peptide loading (Figure 10D).

[0341] T-cell activation

[0342] Cancer cells were treated or not with IFNy or a combination of tryptophan depleted medium and I FNy for 48 or 72 hrs as indicated in the figures. If cells were treated with I FNy, Kynureninase was also added to the medium to circumvent the inhibitory effect of kynurenine (7.2x102mg / mL purified PEG-HIS-mpKynureninase49and 2 mM pyridoxal 50-phosphate hydrate (Sigma)). Then, cancer cells were rinsed with PBS and harvest using PBS-EDTA. Cells were counted and 100,000 cancer cells were co-cultured in RPMI medium for 16 hrs in a 96 U-shaped well plates with 50,000 untransduced T cells, TCRTMBIM6W>F 1or TCRMART1Transduced T cell. In case cancer cells have been exposed to IFNy, KYNase was maintained during the co-culture. As a positive control for T cell activation, TCR T cell were treated with ionomycin (Sigma, 1 pg / mL) and Phorbol 12-myristate 13-acetate (pma, sigma aidrich, 20ng / mL). Then, the co-culture was transferred to a 96 V-shaped bottom plate and centrifuged at 4°C (4min, 1500rpm). Cell pellet was resuspended in PBS-0.5% BSA and centrifuged at 4°C (4min, 1500rpm). Then, cells were incubated with a 50pL mastermix of TCRmp-PE (Miltenyi, 1 :100), CD8-vioblue (Miltenyi, 1 :100), CD137-APC (Miltenyi, 1 :100) and with 1 :1000 Live-Dead cells Near-IR (thermofisher) in PBS-BSA 0.1% on ice in the dark for 30min. Then cells were rinsed twice with PBS-BSA 0.1%. Samples were analyzed on a BD LSR Fortessa (BD Biosciences). Data were analyzed using FlowJo V10 software (FlowJo).

[0343] Flow cytometry-based assay for evaluating TCRTMBIM6W>F 1safety with A*24:02+ fibroblasts PBMCs from three healthy HLA-A*24:02+ donors were transduced to stably express either the TCRTMBIM6W>F 1or an HIV-specific HLA-A*24:02-restricted control TCR (TCRctrl)71. Transduced T cells were maintained in TexMACS medium supplemented with pen / strep and 5ng / ml IL-7 and IL- 15 (PeproTech). Before co-culture, RA cells and Fibroblasts were cultured in T75-flasks in the presence or absence of IFNy for 48 hrs. Peptide loaded RA cells and fibroblasts were used as positive control for the TCRctrlspecific peptide (RYPLTFGWCF)71. To facilitate identification by flow cytometry, RA cells and fibroblasts were harvested and labelled with 2pM Carboxyfluorescein succinimidyl ester (CFSE, Life technologies) before being plated into 96-well flat-bottom plates at 50,000 cells / well (RA cells) or 25,000 cells / well (fibroblasts). Target cells were then co-cultured with either TCRTMBIM6W>F 1orTCRctrl-ce||Sfor24 hrs at an E:T ratio of 4:1. Co-culture medium was supplemented with Kynureninase and pyridoxal 50-phosphate hydrate (Sigma) as described above. Following 24 hrs of co-culture, cells were harvested, washed, and stained with a mix of anti-human antibodies against CD3 (PerCP-Cy5.5-hCD3, Biolegend), CD8 (hCD8-BV785, Biolegend), CD4 (hCD4-BV711 , Biolegend), CD137 (CD137-AF647, Biolegend) and of antimouse TCRp (mTCRp-PE, Biolegend) and Live / Dead NIR (Thermofisher) for 20 minutes in the dark at RT. Before flow cytometry analysis, cells were washed and re-suspended in 200pl FACS buffer containing 10,000 CountBright Absolute Counting Beads (ThermoFisher) and an equal number of bead events (3,400) were recorded from every well. The gating strategy is shown in Figure 8C.

[0344] Killing assay co-culture

[0345] Cancer cells were seeded in a 6cm plate. The next day, cells were treated or not with either IFNy or Tryptophan depleted medium and IFNy for 48 hrs (or 72 hrs when mentioned in figure legend). If IFNy was provided to the cells, 7.2x102mg / mL purified PEG-HIS-mpKynureninase49and 2mM pyridoxal 50-phosphate hydrate (Sigma) were also added for the course of the treatment18. Also in case IDO1 inhibitors were used, 1-Methyl-tryptophan (1MT) or Epacadostat were added at the same time as IFNy but remove during the course of T cell co-culture. After the time of treatment, cells were rinsed with PBS and collected using PBS-EDTA (Versene, ThermoFisher). Then, cells were counted and cells were seeded in a 96 wells plate or either 24 wells or 12 wells plate depending on the chosen readout. In the meantime, TCRTMBIM6W>F 1orTCRMART1Transduced T cells were collected, counted and resuspended in RPMI 10% FBS. Then co-culture between cancer cells and untransduced, TCRTMBIM6W>F 1orTCRMARTI -ce||were sef.UpUSing different ratio with the ratio 1 :2 as the one used in figures when not specified. After 16 hrs of co-culture, medium was refreshed with RPMI containing 10% FBS and 10pL of Resazurin was added in every wells (Serva, Heidelberg, Germany). After 2 hrs of incubation, fluorescence intensity was measured using a Tecan plate reader.

[0346] If co-culture lasted for 48 hrs, cells were refreshed, rinsed with PBS and fixed for 30 min at RT with 4% formaldehyde. Then cells were stained using Crystal Violet (0.1%) for 1 hr at RT in the dark. Then wells were thoroughly washed with water and dried overnight. To quantify killing efficiency, wells were unstained using a 10% acetic acid solution and measured at 590nM using a TECAN.

[0347] HI A-stainina

[0348] Cells were seeded in a 6 wells plate and then treated or not for 48 hrs with IFNy combined or not with tryptophan depleted medium. Then, cells were rinsed with PBS and detached using PBS- EDTA. Cells were transferred into 96 V-shape wells plate and centrifuged at 4°C (1500rpm, 4 min). Cell pellet was resuspended in PBS-0.5% BSA and centrifuged at 4°C (4 min, 1500rpm). Then, cells were incubated with a 50 pL mastermix of antibodies against B2m (1 :500; APC antihuman p2-microglobulin, Biolegend), panHLA-A,B,C (1 :100; FITC Mouse Anti-Human HI_A-ABC, BD Biosciences), HLA-A*24 (1 :200; Anti-HLA-A24 (Human) mAb, Sanbio-MBL) and Live / Dead NIR (1 :1000; thermofisher) for 30 min at 4°C in the dark. If needed, cells were washed twice with PBS-0.1% BSA before being incubated with the corresponding secondary antibody (1 :500; F(ab')2-Goat anti-Mouse IgG (H+L) Secondary Antibody, APC, eBioscience™, thermofisher). Then cells were rinsed twice with PBS-BSA 0.1 % and resuspended in 100 pL of PBS-BSA 0.1%. Samples were analyzed on a BD LSR Fortessa (BD Biosciences). Data were analyzed using FlowJo V10 software (FlowJo).

[0349] Western blot

[0350] Straight lysates from cells were made in 6 wells by addition of 200pL of 1x Laemmli buffer without 2-mercaptoethanol and bromophenol blue. Samples were boiled and protein content was assessed by performing BCA protein quantification (Thermofisher). Then, 2-mercaptoethanol and bromophenol blue were added and the same amount of protein per samples (30 pg) was loaded and run on SDS-PAGE gels and blotted on 22pm pore size nitrocellulose membranes (Santa Cruz). Then, membranes were stained overnight in PBS supplemented with 1 % BSA and 0.1% Tween with the appropriate antibodies: phospho-GCN2 (Thr899, 1 :1000, E1V9M Cell Signaling), GCN2 (1 :1000, #3302S Cell Signaling), phospho-STAT1 (Tyr701 , 1 :2000 58D6 Cell Signaling), WARS (1 :1000, 3A12 Bio-Connect), IDO1 (1 :2000, #86630S Cell Signaling), Tubulin (1 :10000, yl1 / 2 SC-53029 Santa Cruz).

[0351] Subsequent staining were performed with the appropriate LI-COR secondary antibodies at 1 :10000 (IRDye 800CWanti-Rabbit IgG, IRDye 800CWGoat anti-Rat IgG, IRDye 800CW Donkey anti-Mouse IgG, IRDye 680RD Donkey Anti-Mouse IgG, IRDye 680RD Donkey Anti-Rabbit IgG; Li-COR). Visualization was performed by use of an Odyssey infrared scanning device (Li-COR). Generation of CRISPR-Cas9 sgRNA

[0352] For CRISPR-Cas9 cloning, pLenti-CRISPR-V2 plasmid was digested using BsmBI (R0734L, NEB) and FastAP (EF0654, ThermoFisher Scientific) enzymes. Then the digested vector was purified using Gel purified kit (Promega). In the meantime, oligonucleotides against IDO1 or TMBIM6 (Table 2) were annealed and phosphorylated using T4 PNK. The digested vector and the annealed product were ligated using T4 DNA Ligase (ThermoFisher). Finally, the reaction product was used to transform DH5a-bacteria (ThermoFisher). All resulting plasmids were sequence verified by Sanger sequencing (Macrogen).

[0353] DNA extraction and Tide analysis

[0354] 200,000 cells were harvested from a 6 wells plate and lysed overnight at 55°C in 500 pL solution of Tris 100mM pH8, EDTA 5mM, SDS 0.2% and 200mM NaCI containing 2uL of proteinase K (20mg / mL stock solution from Sigma). Then, tubes centrifuged at 14.000rpm for 10 min at 4°C. 200 pL of the supernatant was transferred to a new tube and supplemented with 200 pL of isopropanol. Samples were then centrifuged at 14000rpm for 10 min at 4°C. Then the DNA pellet was washed twice using 70% Ethanol. Last, the pellets were resuspended in 100 pL of UltraPure™ DNase / RNase-Free Distilled Water (Invitrogen). Then, PCR were performed using Phusion Polymerase (thermofisher) following manufacturer’s instruction. Primers used for amplifying IDO1 and TMBIM6 are listed in Table 2. Cells expressing a control sgRNA (sgNT1) were used as a control for TIDE analysis. The PCR product was purified from agarose gel (Qiagen) and sent for Sanger sequencing (Macrogen). The analysis was done via tide.nki.nl following the guideline instructions.

[0355] Generation of reporter plasmids mVenus and mRFP were amplified by PCR using the primers listed in Table 2. The resulting PCR product was cloned into pLenti-blast vector by restriction-ligation cloning into the Xbal and Notl sites. TMBIM6m, TMBIM6W>F, MART126-35 and NYESO-I 157-165 were fused to fluorescent reporters using ultramers primers (IDT).

[0356] Plasmid expressing HLA-A*02:01 or HLA-A*24:02 were obtained from J.Olweus lab. HLA- A*02:01 and HLA-A*24:02 were amplified by PCR using the primers listed in Table 2. The resulting PCR product was then cloned into pLenti-blast vector by restriction-ligation cloning into the Xbal and Notl sites. All resulting plasmids were sequence verified by Sanger sequencing (Macrogen).

[0357] Statistics

[0358] One way-ANOVA or Two-ANOVA followed by Bonferroni or Sidak post hoc test was used for all statistics analysis used in the paper as mentioned in the legends of every figures. Prism 7 software was used for all statistical analyses and for data visualization. Statistical details about n number and p value are reported in Figure legends Results

[0359] Identification of shared inducible W>F substitutant neoepitopes

[0360] To test the possibility of using W>F substitutant epitopes as targets for adoptive T cell therapy, the inventors first set out to identify commonly expressed W>F substitutant neoepitopes in cancer cells depleted from tryptophan. Efforts were focused on cell lines expressing HLA-A*24:02, mainly because of the enrichment of phenylalanine residues at the second and last positions of the consensus motif for 9-mer bound peptides (NetMHCpan4.1 https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ), in addition to its high representation worldwide, and in the Asian population in particular3738(Figure 1A and 2A). For the identification of neoepitopes, cells were treated either with IFNy alone or combined with tryptophan depletion in case IDO-1-induced tryptophan depletion was not optimal, and subjected the various cell lines to immunopeptidomics, a mass spectrometry approach to identify cell surface exposed immuno-epitopes3940(Figures 1 B and 2B). A target-decoy database was created containing concatenated wild-type (WT) and tryptophan-substitutant database and scanned H LAMS with Fragpipe41(see methods). In total, 17 cell lines were examined, of which 13 expressed HLA-A*24 (11 expressed HLA-A*24:02 and 2 expressed HLA-A*24:03), 3 control cell lines that did not, and one (DLI145) where HLA-A*24:02 has been ectopically expressed (DU145HLA) (Figure 2C). Peptide signature retrieval using MixMHCp2.1 validated HLA-A*24:02 expression in all the assigned cell lines, including its appearance in DU145HLA(Figure 3A). This experiment uncovered a total of 469 W>F neoepitopes in the treated samples of which 13 were commonly detected in at least 5 cell lines (Figures 1C and 3B). As expected, all the commonly detected neoepitopes are also predicted to bind HLA-A*24:02 by NetMHCpan4.1 (Figure 1C). In contrast, no W>F substitutant neoepitopes were detected in DLD1 , a cell line that is positive for HLA-A*24:02 but defective in peptide loading due to mutations in the Beta-2-Microglobulin (B2M) gene, a key component of HLA presentation42(Depmap portal; Figures 1C and 3B). Moreover, only 1 of the 13 HLA-A*24:02 W>F substitutant neoepitopes was detectable in control untreated conditions, and none of those peptides were detected in cell lines expressing other HLA alleles whereas 10 appeared in the DU 145HLAcells - confirming their inducibility and HLA-A*24:02 specificity (Figures 1C and 3B). In contrast, 11 out of 13 WT-matched W>F neoepitopes were reproducibly detected in at least five of the HLA-A*24:02-presenting cell lines (Figure 3C;). Only two W>F-corresponding wild-type peptides (including the one corresponding to the W>F neoepitope group that was also detected in untreated cells) were not detected in any cell population (Figure 3C). Finally, none of the 13 identified common W>F neoepitopes was detected in an immunopeptidome collection dataset of 25 benign tissues, confirming their treatment-specific induction and potential to be bona fide cancer targets43(Figure 1C; https: / / www.iedb.org / home_v3.php). Thus, by immunopeptidomics, we identified 18 common HLA-A*24:02 W>F neoepitopes.

[0361] Identification and characterization of TCR T cells To identify a TCR targeting the TMBIM6W>Fneoepitope, TMBIMG^ peptide was synthesized for priming naive CD8+T cells isolated from healthy HLA-A*24:02+donors4647. Monocyte-derived dendritic cells (Autologous MoDC), isolated from peripheral blood mononuclear cells (PBMCs), were pulsed with the TMBIMG^ peptide and co-cultured with autologous naive CD8+T cells. After co-culture, combinatorial peptide-MHC multimer staining followed by flow cytometry analyses showed T cell reactivity towards TMBIMG^ peptide18. Subsequently, the TMBIMG^ reactive CD8 T cell population was single-cell sorted for TCR identification by RNA sequencing. Four pairs of TCR alpha / beta sequences were identified that were transduced into healthy donor PBMCs (data not shown). Transduction efficiency was determined either by mTCRp or by tetramer staining (Figures 4A-B). One TCR in particular, TCRTMBIM6W>F 1, demonstrated high sensitivity and specificity for TMBIM6W>Fas compared with the corresponding WT peptide TMBIM6WT(EC50™BIM6W>F=14,3nM and EC50™BIM6m=144nM; Figures 5A and 4C). These results demonstrate the identification of a TCR with the potential to target a W>F substitutant neoepitope in a specific manner.

[0362] Next, the capacity and specificity of TCRTMBIM6W>F 1to target endogenously expressed TMBIMG^ peptide was characterized following IFNy-induced tryptophan depletion. CD8 T cells, derived from healthy donor PBMC, were transduced with a retrovirus vector encoding TCR™BIM6W>F 1. Them, pre-treated RA glioblastoma cells were pre-treated for 48 hrs with either mock, IFNy, or a combination of IFNy and tryptophan depletion, medium was refreshed, RA cells were mixed with TCRTMBIM6W>F 1T cells for 16 hrs, when T-cell reactivity was determined by measuring the expression of the activation marker CD137 using flow cytometry48(Figure 5B). In these T-cell reactivity assays, we added Kynureninase (KYNase) to IFNy treatment to negate kynurenine’s suppressive effect on T cell activity49(Figure 6A). Figure 5C shows that while mock-treated cells did not activate T cells, high T cell reactivity of -40% was observed when cells were pretreated with either IFNy or IFNy with tryptophan depletion. Tryptophan depletion alone did not activate T cells by the generated W>F substitutants18, presumably due to a blockade in peptide processing and presentation. To control for background T cell activity and ensure reproducibility, the experiment was repeated with CD8+ T cells isolated from a new donor and the same effect was observed, while untransduced T cells showed no reactivity towards IFNy-treated RA cells (Figure 6B).

[0363] TMBIM6w>F-targeting TCR T cells activation is dependent on IDO1 and TMBIM6 expression

[0364] As IFNy boosts HI_A expression and peptide processing (Figure 3B)50’51, increased T-cell activation can alternatively be explained by an enhanced presentation of the TMBIM6mepitope. To distinguish TCRTMBIM6W>F 1reactivity towards TMBIM6W>Ffrom background TMBIM6wr, monoclonal RA cells were generated with IDO1 or TMBIM6 gene knockouts (KOs) from two independent single guide RNAs (sgRNAs) per gene. The KOs were confirmed by TIDE (Tracking of Indels by Decomposition) and immunoblot analyses (http: / / tide.nki.nl / ; Figures 5D and 6C-6D). Furthermore, immunoblot analysis for STAT1 phosphorylation (Tyrosine 701) confirmed intact activation of the IFNy pathway in all clones (Figure 5D). Immunoblot analysis for phosphorylated Threonine 899 of GCN2 (a marker for amino acid shortage52) confirmed enhanced phosphorylation following IFNy treatment in the parental and the two TMBIM6KOcell lines, and its expected lack of induction in the two IDO1KOclones (Figure 5D). As predicted, activation assays of TCR™BIM6W>F.I Tce||Sco-cultured with either TMBIM6KOor IDO1KOclones revealed a marked reduction compared with parental RA cells (from 40% to <10%, Figure 5E). HI_A-dependent variability in the clones was excluded by staining the cells with anti-HLA-A*24, anti-pan-HI_A and anti-B2m antibodies (Figure 6E). Finally, to control for the ability of the KO clones to present peptides, the clones were loaded them with a low concentration of the TMBIMG^ peptide prior to co-culture with TCRTMBIM6W>F 1-ce||Sjn absenceOf IFNy treatment. Figure 6F shows that all clones used in this study had the capacity to activate TCRTMBIM6W>F 1-ce||S even at very |OWconcentrations of the TMBIM6W>Fpeptide (10nM). Moreover, the incubation of TMBIMG^, but not control TMBIM6mpeptide, with TMBIM6KORA cells treated with IFNy, confirmed the specificity of TMBIMG™^ peptide recognition by TCRTMBIM6W>F 1q-ce||S(pjgure6G). The expression of TMBIM6mand TMBIM6W>Fwas confirmed in RA-IDO1KOcells that were treated with IFNy by immunopeptidomics (Figure 6H). High levels of TMBIM6wrpeptide that are further increased by IFNy treatment (Figure 5F) was observed. In contrast, TMBIM6W>Fpeptide was not detected in the cells even if they were treated with IFNy, unless IFNy was combined with tryptophan-depleted medium (Figure 5F), indicating the generation of this substitutant peptide by tryptophan shortage. Similar behaviour was observed for all W>F neoepitopes detected in RA cells (Figure 6H).

[0365] High specificity of TCRTMBIM6W>FT cells towards TMBIM6w>Fas opposed to TMBIM6mpeptide Then, the ability of TCRTMBIM6W>F 1T cells to specifically recognize target cells was investigated. Initially, either TMBIM6W>F, TMBIM6m, or MARTI 26-35 peptide amino acid sequences fused to fluorescence markers in RA cells was overexpressed and co-cultured them with TCRTMBIM6W>F 1T cells for 16 hrs (Figure 2G). Figure 5h shows a strong T cell activation upon TMBIMe^ peptide presentation that was comparable to MARTI peptide recognition by TCRMART1. Thus, TCRTMBIM6W>F 1arms T cells with a specific recognition activity towards RA cells engineered to express TMBIMe^ as opposed to the WT counterpart.

[0366] Encouraged by these results, TCRTMBIM6W>F 1T cell killing of RA cells that either ectopically express TMBIM6W>F, TMBIM6m, or were induced to express endogenous TMBIMG^ neoepitope following IFNy treatment was assessed. These experiments were controlled with the IDO1KOclones that showed an intact IFNy pathway induction and full capacity to present peptides but lacked the ability to induce intracellular tryptophan depletion and thus to generate W>F substitutants following IFNy treatment (Figures 5D-2E and 6H). RA cells were treated with either mock or IFNy for 48 hrs, replaced the medium, and co-cultured the cells with TCRTMBIM6W>F 1T cells for either 16 or 48 hrs, and cell viability was assessed (Figure 5G). First, Figure 5i shows that the ectopic expression of TMBIMG^ strongly and specifically sensitizes RA cells to TCR- TMBIM6W>F, but not TCRMART1-mediated T cell killing. TCRMART1-mediated T cell killing using overexpression of MARTI 26-35 epitope was controlled for (Figures 51 and 61). Then, Figure 5j shows that successive IFNy treatment of control RA cells transduced with a non-targeting sgRNA vector (NT1) followed by co-culture with TCRTMBIM6W>F 1-ce||S rec|ucec|ce|| viability to about 50%. In comparison, co-culture of mock-treated RA cells with TCRTMBIM6W>F 1- not(-!ave any significant impact on cell viability. Moreover, the additional effect of TCR™BIM6W>F 1T cells to IFNy treatment was dependent on the endogenous expression of either IDO1 or TMBIM6 (Figures 5J and 6J). Finally, similar results were obtained with the prostate cancer PC-3 cell line. Briefly, it was observed that IFNy-treatment-dependent activation and tumor-cell killing of TCR™BIM6W>F 1q- cells were both TMBIM6 and IDO1-dependent, as observed by genetic disruption of TMBIM6 and genetic and chemical inhibition of IDO1 (Figures 7A-7I). Moreover, doxycycline-mediated induction of IDO1 by itself was sufficient to activate TCRTMBIM6W>F 1q-ce||S a|beit to a lower level than IFNy, likely due to the lack of IFNy-mediated acceleration of peptide processing and presentation53(Figures 7K-7J). Thus, the identification of a TCR that mediates the recognition and killing of cancer cells induced to express a common neoepitope endogenously is demonstrated here.

[0367] Cancer-specific recognition of TCRTMBIM6>F 1T cells

[0368] Next, the degree to which TCRTMBIM6W>F 1q-ce||Sspecifically target malignant cells was assessed. Initially, three non-transformed cell lines (MCF-10A, RPE-1 , and 293T cells) that expressed comparable levels of TMBIM6 transcripts (Depmap portal) were examined. Cells were transduced with HI_A-A*24:02 and treated with either IFNy or tryptophan depletion conditions (Figure 8A). Interestingly, in TCRTMBIM6W>F 1q-ce|| activation assays, no activity was detected compared with RA cells treated with IFNy combined or not with tryptophan depleted-medium (Figure 9A). Loading these non-transformed cells with TMBIM6W>Fpeptide, but not TMBIM6wr, allowed specific activation of TCRTMBIM6W>F 1q-ce||Sindicating that the levels of expressed HLA-A*24:02 were sufficient to allow T cell activation if the epitope was present (Figure 8B). To further explore the potential for targeting of non-malignant, TCRTMBIM6W>F 1q-ce|| activation assays were performed with fibroblasts derived from three HLA-A*24:02 donors, using three different donors of CD8 T cells. No T cell activation and no T cell-mediated killing was observed in fibroblasts treated with IFNy as compared to control RA cells (Figures 9B-9C and 8C). Similar results were obtained using PBMCs, B cells, and T cells (Figures 9D-9E, 8C). To control for the capability of T cells from these donors to efficiently kill target cells, cells were transduced with TCRHIVand loaded RA, fibroblasts and PBMCs with the corresponding target HIV peptide. As expected, efficient killing was observed in all cases (Figures 8D-E). Altogether, these data demonstrate an exquisite specificity of TCRTMBIM6W>F 1T cells towards IFNy-treated cancer cells.

[0369] Broad activation of TCR™BIM6W>F 1T cells by tryptophan-depleted HLA-A*24:02+ cancer cells The broad expression of induced common neoepitopes, such as TMBIMG^, suggests the potential to elicit anti-cancer reactivities across tissues and tumor types, which is restricted only by the HLA type, host gene expression, and the ability of tumor cells to present epitopes. The identification of the TCRTMBIM6W>F 1provided a sensitive tool to test this potential. To this end, a panel of 16 cell lines originating from a variety of tissues with a shared expression of HLA-A*24:02 and TMBIM6, as determined by mRNA expression (depmap.org; Figure 2C;) were collected. As controls, representative cell lines that either express other HLA types than HLA-A*24:02 (MDA- MB-231 , D10, MD55A-3, Figure 2C), and two cell lines (HCT15 and DLD1) that express HLA- A*24:02 but cannot present peptides due to B2M gene mutations (Figure 3B) were used. In a few cases where IFNy insufficiently induced IDO1 to deprive cells of tryptophan, prolonged IFNy exposure to 72 hrs (instead of 48 hrs) or combined IFNy and tryptophan deprived medium were used to boost the generation of W>F substitutants (Figures 2B and 10A). Figure 11 shows that 8 out of the 14 HLA-A*24:02-expressing cell lines, representing 7 out of the 8 cell lines where TMBIM6w>Fwas detectable by immunopeptidomics, significantly activated TCRTMBIM6W>F 1-ce||Swhen treated with IFNy alone or combined with tryptophan-deprived media. As expected, none of the control cell lines activated TCRTMBIM6W>F 1-ce||Ssignificantly following such treatments, indicating the specific activity of TCRTMBIM6W>F 1T cells (Figure 11). Interestingly, TMBIM6w>Fand TMBIM6wrpeptides were not detected in the immunopeptidomics of three of six of the HLA- A*24:02-expressing cell lines that did not activate the TCR™BIM6W>F 1q-ce||S(cOlo320, HepG2, and NCI-H1299; Figure 30). As these cell lines expressed TMBIM6 mRNA at high levels (Colo320: 7.4; HepG2: 7,6; NCI-H1299: 8.1 , log2(TPM+1) depmap.org), and presented HLA- A*24:02 signature in the immunopeptidomics analysis (Figure 3A), a likely explanation for the lack of immunoreactivity is inefficient TMBIM6 peptide processing for presentation. Finally, IFNy- mediated tryptophan-depletion of cell lines expressing HLA-A*24:03, such as HT-29 or WiDr, where the TMBIMe^ peptide was not detected by immunopeptidomics, could still activate TCRTMB|M6W>F 1T cells (Figure 10B). Altogether, the results demonstrate that TCRTMBIM6W>F 1confers broad T cell reactivity towards an inducible TM Bl neoepitope, provided that the cells can process and present this neoepitope.

[0370] Interestingly, it was noted that variable T cell reactivity following IFNy treatment in the various cell lines (Figures 11 and 10B). To assess whether this is due to differential expression of HLA- A*24:02, HLA-A*24 was quantified by flow cytometry analysis in representative cell lines (Figure 10C) but observed no correlation (Figures 11 and 10B). For example, both WiDr and SUM159PT cell lines have more HLA-A*24 than RA cells, but showed a lower ability to stimulate TCRTMBIM6W>F 1T cells using two different CD8 T cell donors (Figures 11 and 10B). Furthermore, peptide loading experiments confirmed this finding (Figure 10D; e.g., SUM159PT has lower reactivity than PC-3) and also confirmed the specific reactivity to the TMBIMe^ epitope compared with its TMBIM6mcounterpart. Adoptive T cell therapy of TCRTMBIM6W>F 1T cells enhance TCRMART1cancer cell killing

[0371] Finally, the relevance of TCR™BIM6W>F 1T cells in a proposed clinical setting was assessed. IFNy treatment of patients with solid cancer types is not generally applied due to differential effects in the tumor microenvironment and upon systemic exposure54. Instead, local secretion of IFNy in the tumor microenvironment can be achieved by anti-tumor antigen-targeting TCRs, such as TCRMART1 26. Therefore, whether exposure of cancer cells to TCR™BIM6W>F 1-ce||S canjmprovethe efficacy of TCRMART1T cells in a combined immunotherapy approach was tested (Figure 12A). A setting of TCRMART1treatment that activates the T-cells but results in suboptimal killing of cancer cells expressing MARTI , but not the NYESO-1157-165 epitope was first identified, to mimic inefficient effect of these T cells when injected at a low number into patients. It was first shown that MARTI -specific T cell activation of TCRMART1by RA cells expressing MARTI26-35, but not NYESO-1157-165 or TMBIM6W>Fepitopes (Figure 13A). Based on data shown in Figure 12b, the ratio of -1 :10 TCRMART1T cells / cancer cells was selected for further experiments, resulting in moderate killing in the chosen experimental setting. Next, T-cell activation against RA cells ectopically expressing either MARTI 26-35 or control NYESO-1157-165 epitopes was examined for this purpose (RA-MART1 and RA-NYESO1 , respectively). These cells were pre-exposed to TCRMART1for 48 hrs, washed, TCRTMBIM6W>F 1-ce||S were app| jec| for 16 hrs, and T cell activation and T cell-mediated killing was assessed. Figure 12C shows T-cell activation towards MART1- expressing cells already at 1 :30 TCRMART1T cells / cancer cells that was as powerful as IFNy treatment alone. TCRMART1T cells / cancer cells with 1 -Methyl tryptophan (1-MT) were incubated with an IDO1 inhibitor, to examine the dependency on tryptophan depletion-mediated TMBIM6W>Fepitope production. Figure 12C shows that IDO1 blockade prevented TCRTMBIM6W>F 1activation, similar to the effect seen with IFNy treatment (Figure 13B). Similar effects were observed when cancer cell viability was assessed. Efficient and specific TCRTMBIM6W>F 1q-ce|| kj|| jngwasobserved towards RA-MART1 , but not RA-NYESO1 cells in an IDO1-dependent manner (Figures 12D and 13C). Finally, it was confirmed that the 1MT IDO1 inhibitor neither affected MARTI -specific TCRMART1T cell killing of RA cells expressing MARTI nor peptide presentation by RA cells (Figures 13D-13E).

[0372] Discussion

[0373] Taken together, the experiments above provide evidence that W>F neoepitopes are attractive targets for adoptive T-cell therapy. W>F neoepitopes are treatment inducible, broadly expressed, have potentially strong immunogenicity, and show high tumor-specificity - provoking T-cell reactivity that is stronger than that of their wild-type counterparts. This is in sharp contrast to neoantigens driven by somatic cancer mutations that are rarely broadly expressed and generally weakly immunogenic due to counter-selection by the tumor microenvironment. While on one abundant neoepitope was focused on, the identification of a dozen common neoepitopes binding to HLA-A*24:02 suggests that combined TCR T cell therapy, where two or more TCRs are pooled to target one tumor, may be feasible to increase anti-tumor activity, overcome tumor heterogeneity, and induce synergistic anti-tumor T-cell responses in the patient. Beyond HLA- A*24:02, also other well-distributed HLA alleles, such as HI_A- A*26:01 , B*35:01 , B*44:02, and C*04:01 , favor peptides with phenylalanine in their sequence. Tumor types expressing these HLA alleles can also be amenable to adoptive T cell therapy using W>F neoepitopes. Finally, evidence is provided for a new adoptive T cell therapy approach where the treatment with TCR T cells targeting a tumor-associated antigen induces W>F neoepitopes that are subsequently targeted by TCR™BIM6W>F 1T cells.

[0374] It is shown that TCRTMBIM6W>F 1-ce||Scooperate with TCRMART1T cells in target cell killing in an IDO1-dependent manner. Directing efforts towards targeting other HLA-A*02:01 cancer- associated antigens, such as MAGE-A3, MAGE-A4, or MAGE-A1055'57, or exploring cancer- associated antigens binding to other HLA types, such as MAGE-A3 and WT-15859, should enhance opportunities to establish a cancer-specific tryptophan-depleted environment favorable for efficient killing by TCRTMBIM6W>F 1T cells.

[0375] It has already been demonstrated that tumors infiltrated by T-cells harbor more W>F proteins18. Thus, further investigation to characterize the presence of W>F neoepitopes in tumor biopsies may indicate patients who could benefit from TCR^ therapy due to the intrinsic characteristics of the tumor microenvironment. Additionally, given that classical immunotherapy, such as PD-1 targeting, recruits T cells within tumors, combining PD-1 blockade therapy with TCR^ therapy could present an intriguing alternative60.

[0376] EXAMPLE 2

[0377] Cancer cells exposed to IFN gamma activated a metabolic pathway leading to tryptophan shortage. As cancer cells experienced tryptophan-shortage, they started producing aberrant proteins because of the mis-incorporation of phenylalanine instead of tryptophan generating neopeptides that can be recognized by T cells. Since IFNg treatment, in a clinical setting, is difficult to achieve, an alternative strategy could be the targeting of cancer-associated antigens by dedicated T-cells such as DMF5 recognizing MARTI antigen. By doing so, activated T cells will locally secrete IFNg forcing cancer cells to produce aberrant peptides and therefore being primed for second wave of T-cells injection towards W>F epitopes. This approach has been validated in vitro above which shows clinical application whereby T cells recognizing W>F neoepitope can enhance cancer cell killing by cancer associated T cells.

[0378] The aim of this experiment is to perform immunopeptidomics after T-cells injection at different time points. The idea is to find the optimal window where after T-cells exposure, cancer cells start to express W>F neoepitope due to local IFNg release. There is expected to be an enrichment of W>F peptides only in the tumor expressing MARTI antigen and treated with DMF5 T cells. As in vitro this strategy takes around 48h to 72h there is a need to assess different time points to find the best setting because T-cells need to travel to the tumors, to become activated and to start secreting IFNg.

[0379] The goal of this experiment is to find the optimal setting for inducing a new class of neoepitopes at the surface of the cancer cells. This class of antigens is the result of aberrant translation due to amino-acid shortage. By treating mice with specific T cells, once activated, the latest will locally secrete IFN gamma that is known to induce tryptophan shortage in the cancer cells therefore leading to production of new immunological targets. The aim is to define the best time between T-cells injection and aberrant peptides production.

[0380] Experimental design:

[0381] Cancer cells (PC-3) that will express the MART 1 associated antigen or not will be used and will they will be treated with either PBS or with DMF5 T cells recognizing MARTI . The cancer cells will be injected in the mammary fat pad of NSG female mice. 1x10A6 cells PC-3 cells will be injected as this setting has already been used in vivo. After 3 to 4 weeks, when the tumors will be around 150 to 200mm3, 10x10A6 DMF5 T cells or PBS will be injected by the tail vein of the animals. In the meantime, IL-2 (100.000U / injection) will be injected for 3 consecutive days.

[0382] Then, the tumors will be harvested at 72h, 96h or 120h after T-cells injection. The tumors will be snap frozen and we will perform immunopeptidomics to unveil the immunopeptidome of the tumors.

[0383] After the harvest of the tumors will be lysed, and immunopeptidomics wil be carried out to catch MHC-class I epitopes with the goal of identifying W>F neoepitopes.

[0384] 3 mice per time points and per conditions will be used. PBS versus DMF5 T cells will be compared at every time point for both cell lines and tumor treated with DMF5 T cells for both group will also be compared. W>F peptides are expected to only be present in PC3-MART1 treated with DMF5 T cells. Anova or T-test will be performed depending on which groups will be compared. Mass- spectrometry will be used to compare the peptide intensity obtained.

[0385] Table 2

[0386] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0387] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0388] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0389] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0390] Example 3

[0391] Materials and Methods

[0392] All mouse experiments were approved by the Netherlands Cancer Institute Animal Experimental Committee, and performed under the approval AVD30100202011584 WP 24.1.11064, 11065 and 11066. 1 x 106PC-3 cells overexpressing HLA-A*02:01 and either MARTI26-35 or NYESO-1157-165 were injected into mammary gland #4 of 6-8 week old NOD-SCID I L2R-null (jax) (NSG) mice. Tumor size was measured by caliper measurement and tumor volumes were calculated by the formula V = 1 / 2(LW2 ), where L corresponds to the length and W to the width of the tumor. After tumor volumes reached 150 mm 3, mice (n = 9 per group) were either treated by tail vein injection of PBS (control group) or received 15 x 106TCR MARTI T cells in a total volume of 100mL of PBS. After 96hrs, the mice were treated with PBS (control group) or received 15 x 106TCR TMBIM6W>F.I -]-ce||Sjn a tota|v0|ume Of i00mL of PBS. The size of the tumor was measured 3 times a week and the endpoint was defined as a volume of 1500mm3or when a dramatic loss of weight was observed as defined by the NKI animal facility guidelines. Results

[0393] Mice were inoculated with PC3 prostate cancer cells (which had HLA 24:02 and express TMBIM6- W>F neoepitope following IFNg). The cells were also engineered to ectopically express HLA-A* 02:01 and either the MART 1 epitope or NYESO-1 as a negative control. When the tumors reached 150mm in volume, they were treated with TCR-T cells against MART 1 \9DMF5) and 3 days later with a TCR T cells against TMBIM6 - Bi1 (TCR according to SEQ ID NO: 55). As expected, the mice treated with PBS (control) or with DMF5 T cells but that did not express the MART 1 epitope (NYESO-1 group) quickly reached the experimental endpoint with an average median survival of 28 days. The targeting of MARTI epitope by DMF5 T cells increased the median survival to 53 days. Furthermore, the addition of TCR ™BIM6W>F 1T cells prolonged the median survival to 65 days suggesting that the combination of a classical neoantigen targeting (MARTI) to a specific W>F peptide can improve the killing of the tumours and therefore increase the survival of the mice (Figure 14).

[0394] Sequences

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[0412] References

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Claims

Claims1. An isolated nucleic acid composition that encodes a TMBIM6 variant peptide binding protein capable of binding to a peptide: HLA complex, wherein the peptide comprises a TMBIM6 variant peptide comprising SEQ ID NO: 58, the TMBIM6 variant peptide binding protein having a TCR a chain variable (Va) domain and a TCR p chain variable (VP) domain, the composition comprising:(a) an isolated nucleic acid molecule that encodes a TCR Va domain having 90% sequence identity to SEQ ID NO: 10 and comprises a CDR3 amino acid sequence consisting of SEQ ID NO:1; a CDR1 amino acid sequence consisting of SEQ ID NO: 4; and a CDR2 amino acid sequence consisting of SEQ ID NO: 7; and(b) an isolated nucleic acid molecule that encodes a TCR Vp domain having 90% sequence identity to SEQ ID NO: 36 and comprises: a CDR3 amino acid sequence consisting of SEQ ID NO: 27; a CDR1 amino acid sequence consisting of SEQ ID NO: 30; and a CDR2 amino acid sequence consisting of SEQ ID NO: 33.

2. A TMBIM6 variant peptide binding protein capable of binding to a peptide:HLA complex, wherein the peptide comprises a TMBIM6 variant peptide comprising SEQ ID NO: 58, wherein the TMBIM6 variant peptide binding protein comprises: a) a TCR Va domain having 90% sequence identity to SEQ ID NO: 10 and comprises; a CDR3 amino acid sequence consisting of SEQ ID NO:1; a CDR1 amino acid sequence consisting of SEQ ID NO: 4; and a CDR2 amino acid sequence consisting of SEQ ID NO: 7; and b) a TCR Vp domain having 90% sequence identity to SEQ ID NO: 36 and comprises: a CDR3 amino acid sequence consisting of SEQ ID NO: 27; a CDR1 amino acid sequence consisting of SEQ ID NO: 30; and a CDR2 amino acid sequence consisting of SEQ ID NO:

333. The isolated nucleic acid composition of claim 1 , wherein the CDR3 of (a) is encoded by a nucleic acid sequence according to SEQ ID NO 3.

4. The isolated nucleic acid composition of any of claims 1 and 3, wherein the CDR3 of (b) is encoded by the nucleic acid sequence according SEQ ID NO: 29.

5. The isolated nucleic acid composition of any of claims 1 , 3 or 4 or the TMBIM6 variant peptide binding protein of claim 2, wherein (a) further comprises a TCR a chain constant region.

6. The isolated nucleic acid composition of any of claims 1 and 3 to 5, wherein the TCR Va domain is encoded by the nucleic acid sequence according to SEQ ID NO: 12.

7. The isolated nucleic acid composition of any of claims 1 and 3 to 6, or the TMBIM6 variant peptide binding protein of claim 2 or 5, wherein (b) further comprises a TCR p chain constant region.

8. The isolated nucleic acid composition of any of claims 1 , 3 and 4 to 7, wherein the Vp domain is encoded by the nucleic acid sequence according to SEQ ID NO: 38.

9. The isolated nucleic acid composition of any of claims 1, 3 and 4 to 8, or the TMBIM6 variant peptide binding protein of any of claims 2, 5 and 7, wherein the TCR Va domain has at least 90% sequence identity to SEQ ID NO: 13.

10. The isolated nucleic acid composition of any of claims 1, 3 and 4 to 9, or the TMBIM6 variant peptide binding protein of any of claims 2, 5, 7 and 9, wherein the TCR p domain has at least 90% sequence identity to SEQ ID NO: 44.

11. The isolated nucleic acid composition of any of claims 1, 3 and 4 to 9, or the TMBIM6 variant peptide binding protein of any of claims 2, 5, 7, and 9, wherein the TCR Va domain and / or TCR Vp domain each comprise a leader peptide positioned at the N- terminal; optionally wherein the TCR Va domain leader peptide comprises an amino acid sequence according to SEQ ID NO: 19 and / or wherein the TCR Vp domain leader peptide comprises an amino acid sequence according SEQ ID NO: 42.

12. The isolated nucleic acid composition of any of claims 1, 3 and 4 to 11, or the TMBIM6 variant peptide binding protein of any of claims 2, 5, 7, 9, 10 and 11, wherein the nucleic acid molecule or molecules encode or the TMBIM6 variant peptide binding protein comprises: a) a TCR a chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 21 or 24; and / or b) a TCR p chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 47 or 50; or the isolated nucleic acid composition of any of claims 1, 3 and 4 to 11:I. wherein the TCR a chain is encoded by the nucleic acid sequence according to SEQ ID NO: 23 or 26; and / orII. wherein TCR p chain is encoded by the nucleic acid sequence according to SEQ ID NO: 49 or 52.

13. The isolated nucleic acid composition of any of claims 1 , 3 and 4 to 12, wherein the nucleic acid molecule or molecules encode an amino acid sequence according to SEQ ID NO: 55; or the TMBIM6 variant peptide binding protein of any of claims 2, 5, 7 and 9 to 12; wherein the TMBIM6 variant peptide binding protein comprises or consists of an amino acid sequence according to SEQ ID NO: 55.

14. The isolated nucleic acid composition of any of claims 1, 3 and 4 to 13, wherein the nucleic acid molecule or molecules comprises or consists of a sequence according to SEQ ID NO: 57.

15. The isolated nucleic acid of any of claims 1 , 3 and 4 to 14, wherein the nucleic acid molecule or molecules encodes a T cell receptor (TCR) or an antigen binding fragment thereof; or the TMBIM6 variant peptide binding protein of any of claims 2, 5, 7 and 9 to 13; wherein the TMBIM6 variant peptide binding protein comprises or consists of a T cell receptor (TCR).

16. The isolated nucleic acid composition of any of claims 1, 3 and 4 to 15 or the TMBIM6 variant peptide binding protein of any of claims 2, 5, 7, 9 to 13 and 15, wherein the encoded binding protein or TMBIM6 variant peptide binding protein comprises a TCR, an antigen binding fragment of a TCR, or a chimeric antigen receptor (CAR).

17. The isolated nucleic acid composition or TMBIM6 variant peptide binding protein of claim 16, wherein the antigen binding fragment of a TCR is a single chain TCR (scTCR) or a chimeric TCR dimer in which the antigen binding fragment of the TCR is linked to an alternative transmembrane and intracellular signalling domain.

18. A vector system comprising a nucleic acid composition according to claims 1 or 17.

19. The vector system of claim 18, wherein the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus,lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.

20. A modified cell transfected or transduced with a nucleic acid composition according to claims 1 , 3, 4 to 17, or a vector system according to claim 18 or 19.

21. The modified cell of claim 20, wherein the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line.

22. The modified cell of claim 20 or 21 , wherein the modified cell is a human cell.

23. The modified cell of any of claims 20 to 22, wherein the modified cell expresses the TMBIM6 variant peptide binding protein.

24. A pharmaceutical composition comprising a nucleic acid composition according to any of claims 1 , 3, and 4 to 17, a TMBIM6 variant peptide binding protein of any of claims 2, 5, 7, 9 to 13 and 15 to 17, a vector system according to claim 18 or 19, or a modified cell according to any of claims 20 to 23, and a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

25. The pharmaceutical composition according to claim 24, further comprising IFNy and / or activators of IFNy.

26. The pharmaceutical composition according to claim 24 or 25, further comprising a KYNase.

27. A nucleic acid composition according to claims 1, 3, and 4 to 17, a TMBIM6 variant peptide binding protein of any of claims 2, 5, 7, 9 to 13 and 15 to 17, a vector system according to claim 18 or 19, or a modified cell according to any of claims 20 to 23, or a pharmaceutical composition according to any one of claims 24 to 26 for use a medicament.

28. A method of treating or preventing cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of claims 24 to 26.

29. A pharmaceutical composition according to any one of claims 24 to 26 for use in treating or preventing cancer in a subject.

30. The method of claim 28 or pharmaceutical composition for use according to claim 29, wherein the cancer is associated with tryptophan depletion.

31. The method of claim 28 or 30 or pharmaceutical composition for use according to claim 29 or 30, wherein the cancer is associated with increased IDO1 activity and / or IFNy activity.

32. The method of claim 28 and 30 to 31 or pharmaceutical composition for use according to any one of claims 29 to 31, wherein the cancer is glioblastoma, prostate cancer, pancreatic cancer, non-small lung carcinoma cell, melanoma, breast cancer, gastric cancer; a head and / or neck cancer, a cancer related to viral infection or colorectal cancer.

33. The method of any of claims 28 and 30 to 32, wherein the method induces or enhances a cell mediated immune response in the subject.

34. The pharmaceutical composition for use according to any of claims 29 to 32, wherein the pharmaceutical composition is for use in inducing or enhancing a cell mediated immune response in the subject.

35. The method of claim 28 and 30 to 33 or pharmaceutical composition for use according to any one of claims 29 to 32 and 34, further comprising administering at least one additional therapeutic.

36. The method according to claim 35 or pharmaceutical composition for use according to claim 35, wherein the additional therapeutic is selected from: a) activators of IFNy; b) a further T-cell receptor; c) a modified cell comprising a further T-cell receptor or comprising a nucleic acid encoding the further T-cell receptor; d) an immune checkpoint inhibitor; e) nucleic acid based therapeutics comprising a nucleic acid sequence encoding one or more activators of IFNy;f) protein based therapeutics comprising an amino acid sequence encoding one or more activators of IFNy; g) immunofilaments comprising activators of IFNy; and / or h) a chimeric antigen receptor cell therapeutic.

37. The method according to claim 35 or 36 or pharmaceutical composition for use according to claim 35 or 36, wherein the additional therapeutic increases a level of a TMBIM6 variant peptide in the subject, wherein the variant comprises SEQ ID NO: 58.

38. The method according to any one of claims 35 to 37 or pharmaceutical composition for use according any one of claims 35 to 37, wherein the additional therapeutic is administered, prior to, subsequently to and / or concurrently with the pharmaceutical composition.

39. The method of claim 28 and 30 to 33 and 35 to 38 or pharmaceutical composition for use according to any one of claims 29 to 32 and 34 to 38, wherein the subject is HLA- A*24 positive or HI_A-C*02:02 positive; optionally HI_A-*24:02 positive or HLA-C*02:02 positive.

40. A pharmaceutical composition according to any of claims 24 to 26 for use in treating or preventing cancer in a human subject, wherein the subject has been identified as having a cancer by the presence of a peptide in a sample isolated from the subject, wherein the peptide comprises or consists of SEQ ID NO: 58.

41. Use of a pharmaceutical composition according to any one of claims 24 to 26 in the manufacture of a medicament for treating or preventing cancer.

42. A kit of parts comprising: a) a nucleic acid composition according to claims 1, 3, and 4 to 17, a TMBIM6 variant peptide binding protein of any of claims 2, 5, 7, 9 to 13 and 15 to 17, a vector system according to claim 18 or 19, or a modified cell according to any of claims 20 to 23, or a pharmaceutical composition according to any one of claims 24 to 26; and one or more of: b) a KYNase; c) activators of I FNy d) a further T-cell receptor; e) a modified cell comprising a further T-cell receptor or comprising a nucleic acid encoding the further T-cell receptor;f) an immune checkpoint inhibitor; g) nucleic acid therapeutics comprising a nucleic acid sequence encoding one or more activators of IFNy; h) protein based therapeutics comprising an amino acid sequence encoding one or more activators of IFNy; i) immunofilaments comprising activators of IFNy; and / or j) a chimeric antigen receptor cell therapeutic.

43. An isolated nucleic acid composition that encodes a T cell receptor (TCR), wherein the TCR comprises:(i) a TCR Va domain comprising, SEQ ID NO: 10 or 13; and(ii) a Vp domain comprising, SEQ ID NO: 36 or 44.

44. A method of generating a binding protein that is capable of specifically binding to a TMBIM6 variant peptide and does not bind to a peptide that does not contain the TMBIM6 variant peptide, comprising contacting a nucleic acid composition according to any of claims 1 , 3, and 4 to 17 with a cell under conditions in which the nucleic acid composition is incorporated and expressed by the cell, wherein TMBIM6 the variant peptide comprises SEQ ID NO: 58.

45. The method of claim 44, wherein the method is ex vivo.

46. An isolated nucleic acid sequence comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs 12, 15, 23, 26, 38, 46, 49, 52 or 57.

47. An isolated nucleic acid sequence comprising or consisting of the nucleotide sequence of SEQ ID NOs: 12, 15, 23, 26, 38, 46, 49, 52 or 57 for use in therapy.

48. A T cell receptor (TCR), wherein the TCR comprises:(i) a TCR Va domain having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 10 or 13; and (ii) a p domain having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 36 or 44; wherein the Va domain comprises: a CDR3 amino acid sequence consisting of SEQ ID NO:1; a CDR1 amino acid sequence consisting of SEQ ID NO: 4; and a CDR2 amino acid sequence consisting of SEQ ID NO: 7; and the Vp domain comprises: a CDR3 amino acid sequence consisting of SEQ ID NO: 27;a CDR1 amino acid sequence consisting of SEQ ID NO: 30; and a CDR2 amino acid sequence consisting of SEQ ID NO: 33; or(ii) an amino acid sequence comprising or consisting of SEQ ID NO: 55.

49. The T cell receptor (TCR) according to claim 48 for use in therapy.

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