Agents relating to the expression of CD61 on immune cells and uses thereof

Abstract

The invention relates to agents specific for a heterocomplex comprising CD61 and CD103, and methods and uses of such agents.

Description

[0001] AGENTS RELATING TO THE EXPRESSION OF CD61 ON IMMUNE CELLS AND USES THEREOF

[0002] Field of invention

[0003] The invention relates to integrins and agents specific for integrins, and methods and uses thereof, particularly in treating immune-related condition.

[0004] Background of the invention

[0005] Integrins are large, heterodimeric transmembrane glycoproteins often involved in facilitating adhesion between cells, and with the extracellular matrix. They require the pairing between an a and P subunit to exit the endoplasmic reticulum and reach the cell surface to become functionally active. The pairing and functions of integrins are undergoing active research. The expression and functional implications of integrins on cellular immunity in human diseases, including cancer, requires further investigation.

[0006] It is an object of the invention to provide a further or improved approach for targeting integrins that would provide clinical benefits, particularly in treating immune- related conditions.

[0007] Summary of the invention

[0008] In determining the mechanisms of protective anti-tumour immunity, the inventors identified the transient expression of CD61 on tumor-infiltrating lymphocytes (TILs) and its unconventional pairing with CD 103 at the immune synapse, which elevates T cell receptor signalling, improves anti-tumour cytotoxicity and mitigates tumour growth. The inventors found that the CD61+CD103+immune cells, e.g. CD61+CD103+TILs, exhibit enhanced effector functions and phenotype, while showing limited cellular exhaustion. Hence, the inventors identified a particularly effective population of immune cells, i.e. CD61+CD103+immune cells, that can be identified and targeted, e.g. by using an agent specific for the heterocomplex comprising CD61 and CD 103. Given that CD61+CD103+immune cells are found not only in tumor environments, but also in any inflammatory settings, the ability to identity and / or target CD61+CD103+immune cells e.g. CD61+CD103+TILs, provides broad utilities in clinical settings, e.g. in therapy, diagnosis and prognosis, as described herein. In particular, therapeutics targeting CD61+CD 103+ immune cells, e.g. CD61+CD 103+ TILs, in tumor environments, in which CD61 and CD 103 colocalise is advantageous in that the beneficial effects would be focused on the particularly effective population of immune cell (e.g. those that exhibit enhanced effector functions and phenotype, whilst showing limited cellular exhaustion), with minimal off-target toxicity. Similarly, therapeutics targeting CD61+CD 103+ immune cells in inflammatory settings, e.g. skin, joint, gut inflammatory disease, in which CD61 and CD 103 colocalise is advantageous in that the beneficial effects would be focused on the particularly effective population of immune cells (e.g. those that exhibit enhanced effector functions and phenotype, whilst showing limited cellular exhaustion), with minimal off-target effects.

[0009] In particular, the inventors demonstrated that CD61 expression on cancer-specific T cells co-localised and paired, transiently, with CD 103 at the immune synapse (see Figures 2 and 10-12). Interestingly, CD61 expression on cancer- specific T cells did not co-localise and pair with its canonical integrin partners, CD41 and CD51 at the immune synapse (see Figure 10). The functional significance of this was elucidated by the observations that CD61 co-localised with the T cell receptor (see Figure 3), and inhibition of CD61 reduced T cell receptor signalling, which suggests that CD61 functionally enhances TCR signalling (see Figures 3 and 13). The inventors further demonstrated that CD61 expression increased T cell cytotoxicity (see Figure 4), and that CD61+T cells reduced tumour size in vivo using mouse models (see Figures 4 and 13). It was also shown that CD61 expression was elevated in T cells within tumour models (Figures 5 and 8). The observation of transient synaptic surface expression of CD61 and its direct modulation of TCR downstream signalling indicates that this integrin presents a good target for modulating the effectiveness of antigen- specific cytotoxic and effector responses, while limiting the cellular exhaustion of activated T cells and therapeutic side effects.

[0010] The inventors further demonstrated that CD103+CD8+T cells localise in tumordominant regions and their spatial association with CD61 suggest a role of this integrin axis in modulating T cell function within the tumor microenvironment (see Figures 17 and 20). The inventors further demonstrated that CD61+CD8+T cells are located near E- cadherin+ tumour cells (see Figure 19).

[0011] Accordingly, the invention provides an agent specific for a heterocomplex comprising CD61 and CD 103. The invention also provides an agent comprising (i) a moiety specific for CD61 and (ii) a moiety specific for a tumor antigen.

[0012] The invention also provides a heterocomplex or a fusion protein comprising CD61 and CD103.

[0013] The invention also provides a polynucleotide encoding the fusion protein of the invention.

[0014] The invention also provides a vector comprising the polynucleotide of the invention.

[0015] The invention also provides a population of immune cells modified to express CD61, CD 103 or a heterocomplex comprising CD61 and CD 103, optionally wherein the immune cells additionally express an antigen binding protein on the cell surface.

[0016] The invention also provides a pharmaceutical composition comprising the agent, or a population of immune cells of the invention, and a pharmaceutically acceptable carrier.

[0017] The invention also provides a method of treating an immune-related condition in a subject, comprising administering to the subject a therapeutically effective amount of the agent, or the population of immune cells of the invention, or the pharmaceutical composition of the invention.

[0018] The invention also provides use of the agent, or a population of immune cells of, or a pharmaceutical composition of the invention, in the manufacture of a medicament for treatment of an immune-related condition.

[0019] The invention also provides a method of predicting outcome of a therapy for an immune-related condition for a subject, comprising detecting the presence of CD61+CD 103+ immune cells in a sample from said subject using the agent of the invention.

[0020] The invention also provides a method of identifying or enriching a population of CD61+CD 103+ immune cells, comprising detecting the presence of CD61+CD 103+ immune cells using the agent of the invention.

[0021] The invention also provides a method of diagnosing an immune-related condition in a subject, comprising detecting the presence of CD61+CD 103+ immune cells using an agent of the invention. Brief description of the figures

[0022] Figure 1. CD61 is expressed in human CD103+CD8+T cells a, Network plot showing clustering of enriched proteins in cancer- specific CD103+T cell clones (Clones from 2 cancer patients, assay performed with 2 biological repeats). Highlighted circles and protein names indicate groups more likely to be associated with immune activities, such as epigenetic, TGF-1 -related, immune, integrins and metabolism. b, Heatmap showing selected proteins enriched on both CD103+NY-ESO-l-specific and SSX-2-specific T cell clones, but downregulated on both CD 103" T cell clones. Cytotoxic T cell (CTL) clones’ annotation: CD103+T cell clone (left) or CD 103’ T cell clone (right). Antigen (Ag) specificity: NY-ESO-1 -specific T cell clones (light grey), SSX-2-specific T cell clones (dark grey). Expression level by log2 fold-change (FC) values, with a gradient of light grey to dark grey. (Paired clones from 2 cancer patients, assay performed with 2 biological repeats), c, Graph showing the frequency of CD61+cells (out of total CD103+TILs) by flow cytometry (right Y-axis, indicated by bars), and the CD61+CD103+colocated TILs by IHC (left Y-axis, indicated by diamond dots), of each NSCLC patient. Ni IHC = 31 patients; flow cytometry = 19 patients, d-e, Percentage of CD61+CD103+and CD61'CD103+T cells of paired peripheral blood, para tumor tissue and tumour tissue by flow cytometry plots and line plot, n = 19 patients. *** p < 0.001, one-way ANOVA with Tukey’s multiple comparison test. f. Histogram plots showing CD61 expression on seven pairs of CD103+and CD 103" T cell lines, from 7 different cancer patients (four patients with NY-ESO-l-specific, and one patient each with SSX-2-specific, Tyrosinase-specific and Melan- A- specific).

[0023] Figure 2 CD61 transiently co-localises with CD103 during synapse formation a, Horizontal bar graph showing the average median fluorescence intensity (MFI) of CD61 on CD103+T cell lines, from 7 different cancer patients, following either activation by aCD3 / CD28, or by co-culture with antigenic cancer cells, or no activation, median ± SEM, denoted as *** p < 0.001, one-way ANOVA with Tukey’s multiple comparison test. The assay was performed with 3 biological replicates per experiment across three independent experiments, b, Representative synapse images of integrin b7, CD103, CD61 and merged, of a cancer- specific CD103+TCR-T cell at 10 minutes post synaptic formation. An enlarged box with an arrow showing zoom-in synaptic microclusters images with colocalisation of CD103 and CD61, in areas with reduced integrin b7. c, Representative dotplot showing CD 103 and CD61 co-localisation by Pearson’s correlation coefficient (PCC) at 5, 10, 15 minutes post synaptic formation, p = 0.9435, each dot represents one cell contact / microcluster or one T cell, d, Representative dot-plot showing CD 103 and integrin b7 negative co-localisation by Pearson’s correlation coefficient (PCC) at 5, 10 and 15 minutes post synaptic formation, p = 0.197, each dot represents the average PCC per synapse, e, Representative synapse images showing IRM (internal reflection microscopy denoting the area of synapse), integrin b7, CD49d and merged, of a cancer- specific CD103+TCR-T cell at 5 minutes post synaptic formation. An enlarged box with an arrow showing zoom-in images of co-localised integrin b7 and CD49d. f, Representative coimmunoprecipitation (Co-IP) immunoblot images of CD 103 and CD61-flag on anti-flag IP pulldown lysate and whole cell lysate, of CD103'CD61-flag+T cell line, CD103+CD61- flag" T cell line and CD103+CD61-flag+T cell line. Molecular weight (MW) of CD103: ~150kDa, of CD61-flag: ~100kDa. The assay was performed with 2 repeats, g, Volcano plot showing enriched proteins on CD103+CD61+T cell line lysate in comparison to CD103+CD6T T cell line lysate. The assay was performed with 3 biological replicates, single repeat, h, Flow cytometry plots of intracellular and surface staining of CD61-flag with CD103-HA following initial transduction of primary T cell line with CD 103 (left), followed by secondary transduction with CD61 (right). The assay was performed with 3 biological repeats per experiment, across three independent experiments, b-d. Each TIRFM was performed with a minimum of 50 cells per experiment across three independent experiments, c-f. Microscopic big scale bar = 5 mm, small scale bar = 1 mm, for b, e.

[0024] Figure 3. CD61 enhances TCR signalling. a, Representative dot-plot showing TCRab and CD61 co-localisation (left) and TCRab and CD103 co-localisation (right) by Pearson’s correlation coefficient (PCC) at 5, 10, 15 minutes post synaptic formation, p = 0.9435, each dot represents one cell contact / microcluster per one T cell, b, Average median fluorescence intensity (MFI) of either Zap70 (pY292) or PLCgl (pY783) on WT CD61+T cell clone (from patient 1) following treatment with either 25nM, 50nM or lOOnM CD61 siRNA, or no treatment, c. Average MFI of either Zap70 (pY292) or PLCgl (pY783) on either WT CD61+, CD61KO, or WT CD61+T cell clones (from patient 1). d, Bar graph showing the average MFI of Zap70 (pY292) on CD61+T cell lines, from 7 different cancer patients following treatment with either aCD61 (neutralising treatment, IgG isotype control treatment, or no treatment, e. Average MFI of Lek on WT CD61+, CD61KOor WT CD61+T cell clones (from patient 1), including on WT CD61+T cell clone treated with either 25nM, 50nM or lOOnM CD61 siRNA, or no treatment, f. Bar graph showing the average MFI of Lek on CD61+T cell lines, from 7 different cancer patients following treatment with either aCD61 neutralising treatment, IgG isotype control treatment, or no treatment, g, Representative histogram plot of phosphorylated Zap70 (pY292) on WT CD61+T cell clone (from patient 1) following activation with or without Lek inhibition when using A770041 (right), with Genistein as positive control of tyrosine kinases inhibition (left), h, Schematic diagram of CD61 modulation of Zap70 phosphorylation via Lek and Vavl activity, under no inhibition (left), with reduced Zap70 phosphorylation after CD61 knock-down middle) and Lek inhibition right), a. Each TIRFM was performed with a minimum of 50 cells per experiment across three independent experiments, b-g. The assays were performed with 3 biological replicates, for a total of 3 experimental replicates, a-f, median ± SEM, denoted as *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant, with either one-way ANOVA with Tukey’ s multiple comparison test.

[0025] Figure 4. CD61 improves T cell cytotoxicity and tumour control a, Bar plots of CD107a median fluorescence intensity (MFI) between NY-ESO-1 -specific WT CD61+, CD61slRNA“d, CD61KOand WT CD6F T cell clones (from patient 1) following activation with an antigenic cancer cell, b, Horizontal bar graph showing MFI of CD 107a on CD61+T cell lines, from 7 different cancer patients following either aCD61 neutralising antibody treatment, IgG isotype control treatment, or no treatment, c, Line plot showing percentage of antigenic cancer cell death, following cancer cell co-culture with NY-ESO-1 -specific WT CD61+, CD61slRNA’treated, CD61KOor WT CD6L T cell clones (from patient 1). d, Horizontal bar graph showing the percentage of antigenic cancer cell death, following cancer cell co-culture with CD61+T cell lines, from 7 different cancer patients, following either aCD61 neutralising antibody treatment, IgG isotype control treatment, or no treatment, e, Kinetic analysis of mice tumour volume after adoptive transfer of either WT CD61+or WT CD6TT cell clones (patient 1). The arrow shows timepoints of T cell injections, f-g, Dot plots of mice tumour volume after 2nd(Day 10) or 3rd(Day 16) T cell injection of either WT CD61+or WT CD61" T cell clones (from patient 1). h, Kaplan-Meier survival curves of skin cutaneous melanoma (SCM) patients left) and stage 1 lung cancer (LC) patients (right) using TCGA dataset. Patients groups: (i) patients with CD61+CD103+CD8+CD3+cells (Gl, light grey), or (ii) patients with CD6T CD103+CD8+CD3+cells (G2, dark grey). The starting number of SCM patients analysed: nm = 16 patients, no2 = 122 patients. Starting number of LC patients analysed: nm = 85 patients, no2 = 22 patients, a-d. Assay was performed with 3 replicates per experiment, a total of 3 experimental repeats, e-g. In vivo assay performed with n = 8-10 mice. a-g. median ± SEM, denoted as *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant, with either one-way ANOVA with Tukey’s multiple comparison tests (a-e), or two-tailed / -test with Wilcoxon adjustment (f-g).

[0026] Figure 5. CD61+TILs have enhanced anti-tumor effector phenotypes in NSCLC. a-b, Expression of cytolytic molecules (granulysin, granzyme M, granzyme B), degranulation marker CD107a, chemokines (CCL-5, XCL-2) and cytokines (TNFa, IFNg) between CD61+and CD61 ’ TILs, by representative flow cytometry plots of 1 patient (Patient 7) and dot plots of the average median fluorescence intensity (MFI) and frequency of 19 NSCLC patients, c, Dot plot showing percentage of CD61+TILs expressing granulysin and granzyme M following ex vivo aCD61 neutralising antibody treatment or no treatment ex vivo, on 19 NSCLC patients, d, Line plot on frequency of combinatorial effector signatures positive (IFNg+TNFa+CCL5+XCL2+granzyme M+granzyme B+granulysin+) cells between CD61+and CD61" TILs, on 19 NSCLC patients, e, The frequency of CD61+CD103+CD8+co-located cells (full-coloured triangle) or CD61" CD103+CD8+co-located cells (empty triangle) present within tumor body, clustering around tumor body, or further from islets, by IHC (proximity from tumor islets defined as per Methods section), f, Line plot on the frequency of cells with tumor-reactive combinatorial markers expression (CD39+CD103+) between CD61+and CD6T TILs. Statistical analysis conducted as median ± SEM, denoted as *** p < 0.001, * p < 0.05, ns = not significant, with either one-way ANOVA with Tukey’s multiple comparison test (c, e), or two-tailed / -test with Wilcoxon adjustment (d, f). n = 19 patients, each dot represents one patient, for b-e.

[0027] Figure 6. CD61+Trm TILs do not exhibit hallmarks of exhaustive phenotype a, The expression of PD-1, TIGIT and Tim-3 by representative flow cytometry plot of Patient 6 (top) and the frequency of PD-1+, Tim-3+and TIGIT+cells by box-whiskers plots (bottom) between CD61+TILs (full-coloured triangle) and CD61 ’ TILs (empty triangle), b- c, Dot plots on the frequency of triple co-expressed PD-l+Tim-3+TIGIT+cells and PD- 1+Tim-3'TIGI cells between CD61+TILs (full-coloured triangle) and CD6T TILs (empty triangle), d, Pie charts showing percentages cells in early differentiated stage (CD27+CD28+, light grey), intermediate differentiated stage (CD27+CD28‘, medium grey), and late differentiated stage (CD27'CD28‘, black) between CD61+TILs (top) and CD6 T TILs (bottom). Data represent average frequency of total 19 patients, e, Representative histogram plot (from one patient- Patient 7) of cells undergoing cellular divisions (denoted by number of CLSE peaks) between CD61+TILs (top) and CD6T TILs (bottom). Peaks at the dotted line represents cells that are not proliferating, f, Line plot on the frequency of proliferated cells between CD61+TILs (full-coloured triangle) and CD6T TILs (empty triangle).

[0028] Statistical analysis conducted as median ± SEM, denoted as *** p < 0.001, ** p < 0.01, ns = not significant, with two-tailed / -test with Wilcoxon adjustment (a-c, f). n = 19 patients, each dot represents one patient, for a-c, f.

[0029] Figure 7. Enriched proteins of cancer-specific CD103+CD8+T cells. a, Heatmap of 890 proteins by log2 fold-change values, on both CD103+and CD 103" cancer- specific T cell clones from 2 different cancer patients. Arrow denotes the 103 proteins enriched on the CD103+T cell clones. Assay is performed by 2 biological replicates, b, Pie chart on proteins classification by Gene Ontology NCBI annotation, on four major groups of cellular processes, metabolism, protein synthesis and trafficking and unknown. Proteins were also subdivided into specific biological roles. Numbers on the side of each legend represent number of proteins annotated to that particular subgroup. Number represents number of proteins annotated to the major group.

[0030] Figure 8. Gating strategy to identify CD61+TILs ex vivo.

[0031] Gating strategy illustrating identification of CD61+CD103+and CD61'CD103+TILs based on initial gating of: lymphocytes (FSC-A vs SSC-A), single cells (FSC-H vs FSC-A), Live CD3+cells (Live / Dead + dumping (CD56, CDl lc) vs CD3), CD41 and CD51 negative CD8+T cells (CD8 vs CD41 and CD51), Trmcells (CD103 vs CD69, CD45RO vs CD49a, CD62L vs CCR7, CD45RA vs CCR7). Gatings were performed based on reference cell population of total CD8+CD3+TILs. Subsequent analysis was then performed to analyse selected immunophenotype (refer to Fig. 5 and 6).

[0032] Figure 9. Summary of NSCLC patients’ clinical parameters and CD61 expression on TILs ex vivo. a, Pie charts on the summary clinical parameters of NSCLC patients, according to gender, age, tumor stage and NSCLC pathology type, used in this study, b, Representative IHC images of CD8, CD 103 and CD61 on three serial tumor resections, from one NSCLC patient. Arrow represents areas of co-localisation between CD8, CD 103 and CD61. Dotted lines indicate alignment of the serial tumor resections performed by Visiopharm. c, Plot showing frequency of CD61+cells (out of the total CD103+TILs) and the tumor stage of each patient. Each variety of dot represent individual cancer patient, n = 19 patients, p value = 0.0035, by Pearson correlation coefficient, d, Violin plots showing frequency of CD61+cells (out of total CD103+TILs), according to tumor pathology type, gender and age. n = 19 patients.

[0033] Figure 10. CD61 co-localises with CD103. a, Representative histogram showing CD61 expression on CD103+cancer- specific T cell clone (from patient 1), following activation either by aCD3 / CD28 or by NY-ESO-1+cancer cells activation, or no activation. Dark grey represents isotype control, light grey represents CD61 staining. The assay was performed with 3 biological replicates per experiment, a total of 3 repeats of experiments, b, Representative kinetic plot of CD61 expression by median fluorescence intensity (MFI) on cancer-specific CD103+T cell clone (from patient 1), following activation for either 0, 15 minutes, 30 minutes, 45 minutes, 1 hr, 1.25 hrs, 1.5 hrs, 1.75hrs, 2 hrs and 6-, 8-, 10- and 12-hours. The assay was performed with 3 biological replicates per experiment, a total of 3 repeats of experiments, c, Histograms showing the radially averaged MFI of antigen (denoted by HLA-A2NY-ESO-I), CD 103, CD61 and TCR plotted as relative to maximum MFI at 15 minutes, according to distance from the synapse centre at 5, 10, 15 minutes post synaptic formation. Data represent average MFI from 3 repeats of experiments, d, Dot-plots showing MFI of CD61, CD103 and TCRab at the point of synapse contacts, at 5, 10, 15 minutes post synaptic formation. Each dot represents one synaptic contact per cell. Each TIRFM was performed with a minimum of 50 cells per experiment across three independent experiments. *** p < 0.001, ns = not significant, one-way ANOVA with Tukey’s multiple comparison test, e, Flow cytometry plots showing CD61 expression against CD41, CD51 or CD 103 on CD103+cancer- specific T cell clone (from patient 5). f, Volcano plot showing enrichment of CD61 and CD 103 but downregulation of integrin b7 on the primary CD103+CD61+T cell line lysate compared to primary CD103+T cell line lysate, The assay was performed with 3 biological replicates.

[0034] Figure 11. CD61 interacts with CD103. a, Full immunoblot images for CD103 and CD61-flag on whole cell lysate (WCL) and pulldown lysates, of integrins-transduced primary T cell and U937 cells. Immunoblot of CD103 on pulldown lysate of transduced T cell (CD8 membrane 1), of transduced U937 (U937 membrane 1) (top), hnmunoblot of CD61-flag on pulldown lysate of transduced T cell (CD8 membrane 2), of transduced U937 (U937 membrane 2), on WCL of transduced T cell (CD8 membrane 4), of transduced U937 (U937 membrane 4) (middle). Immunoblot of CD103 and CD61-flag on WCL of transduced T cell (CD8 membrane 3), of transduced U937 (U937 membrane 3) (bottom). Molecular weight of CD61-flag: -100 kDa, of CD103: ~150kDa. U937 used as positive control of transduction, b, Schematic showing integrins cell surface rescue workflow. Primary T cells were activated overnight (Day 0) before primary transduction with CD103. Cells were stained for CD103-HA and CD61- flag or integrin b7-cMyc surface and intracellular expression on Day 6. Cells were reactivated overnight before secondary transduction with CD61-flag or integrin b7-cMyc. Staining was repeated on Day 12. Assay was performed with 2 experimental repeats.

[0035] Figure 12. Proteins and their associated pathways, that are enriched on cancerspecific CD61+T cells. Network plot showing enriched proteins and their associated pathways post in vitro T cells activation on the CD61+T cell. Assay was performed with 2 biological repeats.

[0036] Figure 13. Manipulation of CD61 affects T cell functions, a, Overlaid flow cytometry histograms showing downregulation of CD61 expression following serial CD61 siRNA treatment (+ 25nM, or 50nM, or lOOnM treatment) on WT CD61+T cell clone (of patient 1), and the abrogation of CD61 expression following CRISPR-Cas9 editing of the CD61 gene on WT CD61+T cell clones (top histogram). Note the CD61KOT clone derived from a starting population of 100% CD61 positive cells. WT CD61+T cell clones were transfected with non-targeting RNPs as control). CRISPR-Cas9-mediated CD61KOdemonstrates consistent CD61 abrogation across 4 passages of T cell expansion, b, Histogram plots showing phosphorylation level of Zap70 (pY292) on WT CD61+T cell clone, WT CD61+T clone treated with 25nM, 50nM or lOOnM siRNA, CD61KOT cell clone, WT CD61+T cell clone treated with anti-CD61 blocking antibody (PM6 / 13) and WT CD6T T cell clone (of patient 1). c, Histogram plots showing Zap70 (pY292) phosphorylation level on CD61+T cell lines, from 7 different cancer patients following either aCD61 neutralising antibody treatment, IgG isotype control treatment, or no treatment (four patients with NY-ESO-l-specific, and one patient each with SSX-2- specific, Tyrosinase-specific and Melan-A-specific). d. Schematic diagram of in vitro tumour growth assay, with NOD.SCID mice xenografted with NY-ESO-1+HCT116 tumour at day 0 before adoptive transferred with WT CD61+or CD61 ’ T clones (derived from patient 1) at day 2, day 8 and day 14. Tumor volume measurements were taken at intervals, at Day 7, 10, 13, 16 and 20 post xenografts. Figure 14. Effector immunophenotypes of NSCLC patients.

[0037] A set of heatmaps illustrating the MFI of IFNg, TNFg, granulysin, granzyme B, granzyme M, CD107a, CCL5 and XCL2, on CD61+CD103+and CD6rCD103+TILs, of each individual patient, n = 19 patients. Values represent MFI, with gradient. N = 19 patients.

[0038] Figure 15. Width lining of ‘clustering’ margin areas of E-Cadherinover'expressedtumor islets.

[0039] Dark grey areas showing positive E-Cadherinover'expressmgcells, with light grey staining denoting nuclear staining. Dashed lining denoting area margin of 1.5cm from the E- Cadherinover'expressingcells, determined using Visiopharm IHC APP algorithm (further detailed in Methods section).

[0040] Figure 16. TIRFM and super-resolution imaging of CD61 localisation with regards to membrane topography a. Imaging showing topological flatness of T cell contact zone to the bilayer, using wheat germ agglutinin (WGA) as membrane marker staining, with surface internal reflection contrast microscopy (denoted as IRM). Left and right images represent three independent experiments. Top panels: T cell clones synapses stained with WGA to label plasma membrane-associated glycans together with CD 103 and CD61 antibody staining. Scale bar:5 m. The black square indicates ROI zoomed in and displayed in further magnification at the bottom panels. Bottom panels: Further magnification of both IRM and WGA images demonstrating no increases in contact reflection (of IRM shades) and WGA fluorescence intensity (glycocalyx signal) indicative of no topological changes in the membrane, such as 3D protrusions being projected in 2D. Scale bar:500nm. White arrows represent areas of CD61 enrichment, where CD61 and CD 103 signals concentrate within the cell-bilayer interface at the synapse centre (white arrow, see also Figure 16c ii and iii), and poorly localise in microvilli (the non-interface area)). Scale bar:5pm. b. TIRFM imaging of CD103hlghCD61hlgh-sorted (top panel) and CD103lowCD61low-sorted (bottom panel) T cell clones stained for CD 103, NY-ESO-1 peptide loaded-HLA-A*02:01, IRM, CD61 and WGA staining. Higher levels of CD61 and CD 103 expression between the T cell clones, yet still showing co-localisation on points of contact. The central enrichment of antigenic peptide- HLA-A*02:01 is indicative of central clustering of TCR-bound NY-ESO-1 peptide loaded- HLA-A*02:01 molecules, where CD61 and CD103 are present in the TCR-pMHC microclusters (see also Figure 16a). Scale bar: 5pm. c. Maximum intensity projection of T cells interacting with antigen-, ICAM-1-, CD58- and E-Cadherin-coated SLB, imaged using Olympus SpinSR SoRam super-resolution microscopy. Plasma membrane glycans were labelled using WGA, and the integrins CD 103, and CD61 were labelled using fluorescence antibodies. CD61 and CD 103 signals concentrate within the cell-bilayer interface at the synapse centre (white arrow, see also ii and iii), and poorly localise in microvilli (the noninterface area). Scale bar: 5 pm. c-i. A zoom-in image showing a rare example of CD 103 presence at the tip of microvilli. Scale bar = 200nm. c-ii and iii. Zoom-in images showing two different orthogonal views demonstrating most of the CD 103 signal distributes in the cell-SLB interface, localising with CD61 in the cSMAC (ii) and synaptic cleft (iii). Images were acquired using an Olympic SpinSR SoRA super-resolution microscope at 60X 1.4NA (+3.2X) magnification. The assay was performed with 50,000 cells per well / replicate, for 3 biological replicates, for a total of 3 experimental repeats.

[0041] Figure 17. Sub-cellular spatial transcriptomic analysis revealed gene expression profile of different CD61+ CD8+ Trm clusters. Spatial transcriptomic analysis with 896,578 cells from 1,364 field-of- views (FOVs) across six lung cancer patients, a. Five CD8+ Trm cell clusters were identified. Among them, three clusters (CD8-1, CD8-4 and CD8-5) are CD61 positive T cell clusters, whereas cluster CD8-2 and CD8-3 are CD61 negative, b. ITGAE (CD103) expression on different T cell clusters. All three CD61+ CD8+ Trm clusters show high level expression of CD 103, indicating the association of these two genes.

[0042] Figure 18. Phenotypical and functional differences of three CD61+ CD8+ Trm clusters. Average immuno- score of immuno-responsive a. and inhibitory / exhaustive b. phenotypic features for 12 different T cell clusters. CD61+ cluster CD8-1 and CD8-5 show highest immuno-responsive scores with lower inhibitory / exhaustive immuno-score, whereas CD61+ cluster CD8-4 has a non-responsive / quiescent state, suggesting that cluster CD8-1 and CD8- 5 are highly tumor-responsive, c. Different functional properties of three CD61+ CD8+ Trm clusters. Consistent with Fig2b, Cluster CD8-1(CD61+A) and CD8-5 (CD61+C) are highly tumor-responsive, with CD8-1 having enhanced cytokine production, whereas CD8-5 has chemotactic-enrichment. Cells at cluster CD8-4 (CD61+B) again are non-responsive with quiescent state, possible early differentiated / or regulatory type, which requiring further analysis.

[0043] Figure 19. Close interaction of CD61+CD8 T cells with epithelia tumor cells. a. Distribution of CD61+ CD8 Trm clusters. In top 20 FOVs, the 3 clusters of CD61+ TILS shared similar or adjacent locations, b. cell count contribution across FOVs for each cluster. The most abundance CD61+ TILs (cluster CD8-1) is present in almost all area of the lung tumor tissue from patient 5748. These data suggested the abundance and widely distribution of CD61+ CD8+ T cells in the lung tumor, c. Nearest-neighborhood analysis shows that CD61+CD8 T cells are more proximate to epithelia tumor than any other cells, d. Spatial imaging shows CD61+CD8 T cells are clustered with lung epithelia tumor, e. Tumor epithelia that are proximal to CD61+TILs have enriched E-Cadherin expression, f. Validation of CD61+ TILs always nearby E-Cadherin+targets, by orthogonal method of synaptic microscopy.

[0044] Figure 20. CD103+CD8+T Cells Exhibit Preferential Enrichment in Tumor-Dominant Regions. Multiplex immunofluorescence (COMET) analysis was performed to assess the spatial distribution of CD103+tissue-resident CD8+T cells in pancreatic tumor tissues. Tumor regions were classified into tumor-dominant regions and stroma-dominant regions, a. Representative images of immunofluorescence staining showing the localization of CD8+T cells, CD103+cells, E-cadherin+epithelial structures, and nuclei. White arrows indicate CD8+T cells, dotted arrows indicate CD103+cells, and striped arrows indicate CD8+CD103+double-positive T cells. Scale bar: 100 pm. b. Quantification of CD8+T cells in the stromadominant and tumor-dominant regions, showing a higher density of CD8+T cells in the stroma-dominant regions, c. Proportional analysis of CD103+CD8+and CD103'CD8+T cells in each region, demonstrating a preferential localization of CD103+CD8+T cells in the tumor-dominant region. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t-test; *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant. Detailed description of the invention

[0045] Agents

[0046] The invention relates to identifying and / or targeting immune cells in which CD61 is transiently expressed and pairs with CD103. The immune cells may be tumor-infiltrating lymphocytes (TILs), T cells, NK cells, B cells, or NKT cells.

[0047] CD61, also known as integrin P3, is an integrin protein which is known to pair with its canonical integrin partners, integrin aV (CD51) and to integrin allb (CD41). The inventors surprisingly identified the upregulation of CD61 on human antigen- specific T cells (CD 103+ T cells; see Figure la). CD61 was previously thought to only be expressed by non-lymphocytic cells such as platelets, megakaryocytes and endothelial cells. While there is some evidence showing CD61 expression on murine T cells, the functional implications of CD61 on human antigen- specific T cells were unclear.

[0048] CD 103, also known as integrin aE, is an integrin protein which is known to pair with its canonical partner integrin P7. CD 103 is a key phenotypic marker of tumourinfiltrating T lymphocytes. In cancer, CD103+TrmTILs are known to be immunophenotypically diverse, ranging from terminally exhausted (layilin+), Trmprecursors (granzyme H+), and Trmtransitional-phase (XCL1+) cells. Certain CD103+TIL subtypes, such as the CD103+CD39+cells are tumor-reactive TILs, with clonal expansion observed in different cancer types. It was recently demonstrated that CD103+TGE-bl+cytotoxic T cells were efficient killers of antigenic cancer. Clinically, the enriched presence of CD103+T cells in cancer patients and pathogenically diseased patients has been associated with improved outcomes. However, the immune-associated proteins and the mechanisms that are utilised to promote effective cellular immune activities, responses and protection were poorly defined.

[0049] A useful agent with the invention may be an agent that is specific for the heterocomplex comprising CD61 and CD 103. The agent may independently act at any step associated with CD61 and CD 103 co-localisation, including modulating the binding kinetics, cell surface recycling, half-life of either or both of the integrins, or binding of the CD61 / CD 103 heterodimer with further components. The agent may modulate the functional effect of the association between CD61 and 103 in a positive way (i.e. an agonist) or a negative way (i.e. an antagonist).

[0050] The agonist may promote the functional effect of the association between CD61 and 103. For example, the agonist may promote the association between the integrins or stabilise the binding of the integrins.

[0051] The antagonist may reduce the functional effect of the association between CD61 and CD 103. The antagonist may at least partially reduce the association between CD61 and CD103, or may eliminate the association between CD61 and CD103. The antagonist may sterically block the association between CD61 and CD103. The antagonist may lead to partial or complete depletion of CD61 / CD 103 expressing cells.

[0052] The agent may not modulate the functional effect of the association between CD61 and CD103. The agent may not substantially modulate the functional effect of the association between CD61 and CD103.

[0053] The expression and localisation of CD61 and / or CD 103 may be determined by any appropriate techniques in the art. For example, expression and localisation be determined by imaging studies, such as immunohistochemistry or microscopy, e.g. total internal reflection fluorescent microscopy (TIRFM), co-immunoprecipitation, immunoblotting, interactomics analysis by LC-MS / MS, and flow cytometry.

[0054] Hence, the functional effect of the association of CD61 and CD 103 can be measured by determining any of the TCR signalling downstream activities. For example, the inventors showed that knock down or knock out of CD61 resulted in reduced T cell receptor (TCR) signalling via Lek and Zap70 phosphorylation, suggesting that the colocalisation of CD61 and CD 103 in an immune synapse formed between the T cell and a target cell elevates T cell receptor (TCR) signalling. Techniques appropriate for determining the activity of any of these components would be known by a person skilled in the art, e.g. as described in the Examples. For example, cytotoxicity assays, such as in vitro T cell CD107a assay or CFSE-based T cell cytotoxicity assay, proliferation assays, or kinetic assays, such as mouse model tumour growth kinetic assays.

[0055] The agent may bind directly to the heterocomplex comprising CD61 and CD 103. The agent may bind indirectly to the heterocomplex comprising CD61 and CD 103. The agent may bind in a reversible manner. The agent may bind in an irreversible manner.

[0056] Without being limited to theory, once the heterocomplex comprising CD61 and CD 103 is formed in vivo, there will be created new epitope(s), also referred to herein as neoepitope(s), which are not present in CD61 or CD 103 alone, i.e. before CD61 or CD 103 are present in the heterocomplex comprising CD61 and CD 103. Hence, an agent of the invention may bind to a neoepitope in the heterocomplex comprising CD61 and CD 103.

[0057] The agent may not bind to CD61 or CD 103 alone, i.e. before CD61 or CD 103 is present in the heterocomplex comprising CD61 and CD 103. Hence, the agent may bind to a neoepitope in the heterocomplex comprising CD61 and CD 103, and may not bind to CD61 or CD 103 alone.

[0058] The agent may be multi- specific, such as bi-specific. For example, the agent may comprise a first moiety that binds to CD61 and a second moiety that binds to CD 103. The agent may comprise a first moiety that binds to an epitope on CD61 and a second moiety that binds to neoepitope in the heterocomplex comprising CD61 and CD 103. The agent may comprise a first moiety that binds to an epitope on CD 103 and a second moiety that binds to neoepitope in the heterocomplex comprising CD61 and CD 103.

[0059] The agent may be a bi-specific agent, e.g. a bi-specific antibody, comprising an antigen binding domain of an antibody specific for CD61 and antigen binding domain of an antibody specific for CD61. The agent may be a bi-specific antibody comprising an antibody specific for CD61 and an antibody specific for CD61. The antibody specific for CD61 may be A-l l, anti-CD61 blocking antibody (PM6 / 13), etaracizumab, intetumumab, or rHIgM-22. The antibody specific for CD 103 may be Monoclonal Antibody to Inhibit CD103 for Inflammatory Disorders, anti-CD103 antibody clone EPR4166(2), or etrolizumab.

[0060] A useful agent with the invention may be an agent that is specific for CD61.

[0061] A useful agent with the invention may be an agent that is specific for CD61 and a tumor antigen.

[0062] The agent may bind to any epitope on CD61. The agent may bind CD61 and / or the tumor antigen in a reversible manner. The agent may bind CD61 and / or the tumor antigen in an irreversible manner. The tumor antigen may be NY-ESO-1, a MAGE family member (e.g. MAGE- A3, MAGE-A4, MAGE-B6, or MAGE-B18), BAME, SSX-2, or gplOO.

[0063] In the embodiment where the agent is an agonist, the agent may be linked to a moiety that is specific for a tumor antigen.

[0064] The agent may be linked to a therapeutic or prophylactic agent, such as an anticancer therapeutic, e.g. as described herein.

[0065] The agent may be linked to a label, for example, with a radio-label, a biotin-label, a chromophore-label, a fluorophore-label, or an enzyme-label.

[0066] The agent may be any molecule, either naturally occurring or synthetic. For example, the agent may be a small molecule (organic or inorganic), a protein, a nucleic acid, a polysaccharide, a lipid, or a fatty acid, a drug compound, or other compound.

[0067] The small molecule may be an organic molecule having a molecular weight of less than about 2500 daltons, e.g., less than 2000, less than 1000, or less than 500 daltons.

[0068] The nucleic acid may be an oligonucleotide, such as DNA, RNA, or a modified oligonucleotide. The oligonucleotide may be about up to Ikb in length, e.g. 10 to 500bp in length, e.g., about >10, >50, >100, >150, >200, >250, >300, >350, >400, >450 or >500 bp in length.

[0069] The protein may be from about 5 to about 25 amino acids in length, e.g., about 5, 10, 15, 20, or 25 amino acids in length. The protein may be an antibody, such as a nanobody, as described herein. The protein may be an affimer.

[0070] The term “antibody” referred to herein includes the various antibody formats disclosed herein, including those comprising various formats of heavy and / or light chains discussed herein.

[0071] The antibody may be selected from the group consisting of single chain antibodies, single chain variable fragments (scFvs), variable fragments (Fvs), fragment antigenbinding regions (Fabs), recombinant antibodies, monoclonal antibodies, fusion proteins comprising the antigen-binding domain of a native antibody or an aptamer, single-domain antibodies (sdAbs), also known as VHH antibodies, nanobodies (Camelid-derived singledomain antibodies), shark IgNAR-derived single-domain antibody fragments called VNAR, diabodies, triabodies, Anticalins, aptamers (DNA or RNA) and active components or fragments thereof. Further examples of possible antibody formats are disclosed in the review “The coming of Age of Engineered Multivalent Antibodies, Nunez-Prado et al Drug Discovery Today Vol 20 Number 5 Mar 2015, page 588-594, D. Holmes, Nature Rev Drug Disc Nov 2011:10; 798, Chan and Carter, Nature Reviews Immunology vol. 10, May 2010, 301.

[0072] The antibody may comprise a complete antibody having full length heavy and light chains, or an antigen-binding fragment thereof. For example, the microbe-targeting moiety may be a full-length antibody.

[0073] The antibody may be an antigen-binding fragment. An antigen-binding fragment of the invention binds to the same epitope of the parent antibody, i.e. the antibody from which the antigen-binding fragment is derived. An antigen-binding fragment of the invention typically retains the parts of the parent antibody that interact with the epitope. The antigen-binding fragment typically comprises the complementarity-determining regions (CDRs) that interact with the antigen, such as one, two, three, four, five or six CDRs. In some embodiments, the antigen-binding fragment further comprises the structural scaffold surrounding the CDRs of the parent antibody, such as the framework regions (e.g. FR1, 2, or 3) and / or the variable region domains of the heavy and / or light chains. Typically, the antigen-binding fragment retains the same or similar binding affinity to the antigen as the parent antibody. Antigen-binding fragments of antibodies include single chain antibodies (i.e. a full-length heavy chain and light chain); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH), scFv. The methods for creating and manufacturing antibody fragments are well known in the art (see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0074] The antibody may be multi- valent, e.g. bivalent, trivalent or tetravalent. The multivalent antibody may comprise multiple specificities e.g bispecific or may be monospecific, see for example WO 92 / 22853, WO05 / 113605, W02009 / 040562 and W02010 / 035012. A multi- specific antibody comprises at least two different variable domains, wherein each variable domain is capable of binding to a separate antigen or to a different epitope on the same antigen.

[0075] The constant region domains of an antibody, if present, may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. Typically, the constant regions are of human origin. In particular, human IgG (i.e. IgGl, IgG2, IgG3 or IgG4) constant region domains may be used. Typically, a human IgGl constant region.

[0076] The antibody may not comprise an Fc region., i.e. may not comprise CH2 and CH3 domains. However, constant domains such as CHI, CKappa / CLambda may be present.

[0077] The antibody may be an isolated antibody. An isolated antibody is an antibody which is substantially free of other antibodies having different antigenic specificities.

[0078] The antibody may be a monoclonal antibody (mAb). Monoclonal antibodies (mAbs) may be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example those disclosed in “Monoclonal Antibodies: a manual of techniques”(Zola H, 1987, CRC Press) and in “Monoclonal Hybridoma Antibodies: techniques and applications” (Hurrell JGR, 1982 CRC Press). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature, 352, 624-628 (1991) and Marks et al., JMol, Biol., 222(3):581-597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with an antigen of interest, for instance in form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen of interest. Monoclonal antibodies may also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rats, dogs, primates, etc.

[0079] The antibody may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human or humanised antibody. Typically, the antibody is a human antibody. Fully human antibodies are those antibodies in which the variable regions and the constant regions (where present) of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, but not necessarily from the same antibody.

[0080] The skilled person is readily able to determine the binding site (epitope) of an antibody using standard techniques. For example, techniques that may be used to determine antibody epitopes include hydrogen / deuterium exchange, X-ray crystallography and peptide display libraries (as described in the Examples). A combination of these techniques may be used to determine the epitope of an antibody.

[0081] The invention also provides a method of identifying an agent described herein. The method may comprise identifying the agent in an appropriate model, such as an in vitro, ex vivo or in vivo model. For example, the method may use a model of an immune-related condition, e.g. a tumor model or a model for a T-cell pathology for example skin, gut or joint or systemic inflammation. The model may comprise cells expressing CD61 and CD 103. The cells may express CD61 and CD 103 naturally or be engineered to overexpress CD61 and CD 103. The method may comprise screening a library of potential agents in the model. The method may comprise creating the library of potential agents. Suitable screening assays, e.g. high throughput screening assays, are known in the art. An example may be to use a phage-display antibody library to isolate human monoclonal antibodies (mAbs). The method may comprise determining the binding properties of the potential agents to the target, and / or determining the functional effects of the potential agents on the target. The method may further comprise reiterative directed agent synthesis to improve agent properties. The method may further comprise determining pharmacology, pharmacokinetics, safety and toxicity studies.

[0082] The target used in the method of identifying an agent described herein may be a heterocomplex comprising CD61 and CD 103, CD61 alone and / or CD 103 alone. Hence, the invention also provides the use of a heterocomplex comprising CD61 and CD 103 in a method of identifying an agent specific for CD61 and CD 103.

[0083] The target used in the method of identifying an agent described herein may be a fusion protein comprising CD61 and CD 103. Hence, the invention also provides the use of a fusion protein comprising CD61 and CD 103 in a method of identifying an agent specific for CD61 and CD103.

[0084] In embodiments for identifying an agent specify for the heterocomplex comprising CD61 and CD 103, the method may comprise eliminating agents specific for CD61 alone and CD 103 alone.

[0085] The invention also provides the use of a heterocomplex comprising CD61 and CD 103 as a target for immune-based therapy. Cells

[0086] Also provided herein is a cell that has been modified to express CD61 or CD 103, or both CD61 and CD 103.

[0087] The cell may express either (e.g. CD61) or both of these integrins constitutively. The cell may over-express either or both of these integrins. Any methods for constitutively expressing or over-expressing a protein may be used with the invention. For example, either (e.g. CD61) or both genes encoding the integrins may be driven by a constitutive promoter.

[0088] CD61 and CD 103 may be expressed as a fusion protein. Hence, the invention also provides a fusion protein comprising CD61 and CD 103. The invention also provides a polynucleotide encoding said fusion protein. The invention also provides a vector comprising said polynucleotide.

[0089] The cell described herein may be used in a method of identifying an agent described herein. The cell may be a mammalian cell, such as a human cell.

[0090] The cell expressing CD61 and / or CD 103 may be an immune cell. The modified immune cell may additionally express an antigen binding protein on the cell surface. The antigen binding protein binds to an antigen (e.g. in the context of a MHC complex or a HLA complex). Hence, the invention also provides a population of immune cells modified to express CD61, CD103, or a heterocomplex comprising CD61 and / or CD103. The immune cells may additionally express an antigen binding protein on the cell surface. The antigen binding protein may be specific for a tumor antigen. For example, the antigen binding protein may be a chimeric antigen receptor (CAR), a T cell receptor (TCR) or a modified T cell receptor. The immune cell may be a TIL, a T cell, a NK cell, a B cell, or a NKT cell. The immune cell may be a T cell. The T cell may be a CD8+ T cell, CD4+ T cell, regulatory T cell, effector T cell, tissue resident memory T cell, memory T cell, or gamma delta (y5) T cell. The antigen binding protein may be a protein comprising an antigen binding portion from an antibody or an antigen binding fragment thereof (e.g. a scFv, Fv, Fab, a Fab’, a F(ab’)2 fragment, sdAbs, a heavy chain variable domain (VH) or a nanobody (VHH or VNAR), such as a TCR mimetic. Such a modified immune cell may have therapeutic utility, as explained herein. Polynucleotides / vectors

[0091] The invention also provides one or more isolated polynucleotides (e.g. DNA) encoding an agent described herein, wherein the agent is a protein or a nucleic acid. The invention also provides one or more isolated polynucleotides (e.g. DNA) encoding a fusion protein comprising CD61 and CD 103. In one embodiment, the polynucleotide sequence is collectively present on more than one polynucleotide, but collectively together they are able to encode an agent described herein. For example, the polynucleotides may encode the heavy and / or light chain variable regions(s) of an antibody. The polynucleotides may encode the full heavy and / or light chain of an antibody described herein. Typically, one polynucleotide would encode each of the heavy and light chains.

[0092] Polynucleotides which encode an agent or a fusion protein described herein can be obtained by methods well known to those skilled in the art. For example, DNA sequences coding for part or all of the antibody heavy and light chains may be synthesised as desired from the corresponding amino acid sequences.

[0093] General methods by which the vectors may be constructed, transfection methods and culture methods are well known to those skilled in the art. In this respect, reference is made to “Current Protocols in Molecular Biology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0094] A polynucleotide described herein may be provided in the form of an expression cassette, which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the antibody or the fusion protein of the invention in vivo. Hence, the invention also provides one or more expression cassettes encoding the one or more polynucleotides that encoding an antibody or a fusion protein described herein. These expression cassettes, in turn, are typically provided within vectors (e.g. plasmids or recombinant viral vectors). Hence, in one embodiment, the invention provides a vector encoding an antibody or a fusion protein described herein. In another embodiment, the invention provides vectors which collectively encode an antibody described herein. The vectors may be cloning vectors or expression vectors. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide described herein.

[0095] The polynucleotides, expression cassettes or vectors described herein are introduced into a host cell, e.g. by transfection. Hence, the invention also provides a host cell comprising the one or more polynucleotides, expression cassettes or vectors of the invention. The polynucleotides, expression cassettes or vectors of the invention may be introduced transiently or permanently into the host cell, allowing expression of an antibody from the one or more polynucleotides, expression cassettes or vectors. Such host cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast, or prokaryotic cells, such as bacteria cells. Particular examples of cells include mammalian HEK293, such as HEK293F, HEK293T, HEK293S or HEK Expi293F, CHO, HeLa, NSO and COS cells, or any other cell line used herein, such as the ones used in the Examples. Preferably the cell line selected will be one which is not only stable, but also allows for mature glycosylation.

[0096] The invention also provides a method for the production of an antibody described herein, comprising culturing a host cell containing one or more vectors of the invention under conditions suitable for the expression of the antibody from the one or more polynucleotides of the invention, and isolating the antibody from said culture.

[0097] The invention also provides a method for the production of a fusion protein described herein, comprising culturing a host cell containing a vector of the invention under conditions suitable for the expression of the fusion protein from a polynucleotide of the invention, and isolating the fusion protein.

[0098] Pharmaceutical compositions

[0099] The invention provides a pharmaceutical composition comprising an agent or a population of immune cells as described herein. The composition may comprise a single agent or a single population of immune cells, or a combination (such as two, three or four) of the agents or populations of immune cells described herein. The pharmaceutical composition may also comprise a pharmaceutically acceptable carrier.

[0100] The composition of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0101] The agent or population of immune cells may be comprised in a composition that comprises a physiologically acceptable carrier or diluent.

[0102] Suitable pharmaceutically acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers include water, buffered water and saline.

[0103] Other suitable pharmaceutically acceptable carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. In addition, if desired, the pharmaceutical compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, and / or pH buffering agents.

[0104] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage.

[0105] Pharmaceutical compositions may comprise additional therapeutic or prophylactic agents as described herein.

[0106] The pharmaceutical composition may be administered orally, subcutaneously, intravenously, intradermally, intramuscularly or intranasally.

[0107] The pharmaceutical composition may be formulated as a tablet, capsule, powder, liquid, emulsion, suspension, a suppository or enema solution. In one embodiment, the pharmaceutical compositions may be formulated as a tablet or capsule. The pharmaceutical compositions may be formulated for slow- or delayed-release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

[0108] Also within the scope of the invention are kits comprising the agent, population of immune cells, or pharmaceutical composition described herein and instructions for use. The kit may further contain one or more additional reagents, such as an additional therapeutic or prophylactic agent as discussed herein. Methods and uses

[0109] The invention relates to the use of an agent, a population of immune cells, or a pharmaceutical composition described herein in a clinical setting, e.g. in therapy, diagnosis and prognosis.

[0110] For example, described herein is use of an agent, a population of immune cells, or a pharmaceutical composition described herein in a method of treatment of the human or animal body by therapy, e.g. for use as a medicament.

[0111] For instance, also provided is a method of treating an immune-related condition in a subject, the method comprising administering to the subject an effective amount of an agent, a population of immune cells, or pharmaceutical composition described herein. Hence, the invention also provides an agent, a population of immune cells, or a pharmaceutical composition described herein for use in a method of the immune-related condition. The invention also provides the use of an agent, a population of immune cells, or a pharmaceutical composition described herein for the manufacture of a medicament for the treatment of an immune-related condition. The invention also provides an agent, a population of immune cells, or a pharmaceutical composition of the invention for the treatment of an immune-related condition.

[0112] The immune-related condition may be cancer. The cancer may be any cancer, such as cancer in the lung, skin, head and neck, prostate, breast, ovarian, liver, endometrial, bladder, gall bladder, pancreas, colon, kidney, stomach, esophagus, cervix, nervous system, or soft tissues. The cancer may be melanoma, adenocarcinoma, carcinoma, or sarcoma. The cancer may be a solid cancer. The cancer may be lung cancer. The cancer may be skin cutaneous melanoma (SCM). Treatment of these immune-related conditions may benefit from enhancing the cytotoxicity and anti-tumor responses in the body. An agent described herein targets a particularly effective population of immune cells, i.e. CD61+CD103+immune cells, which exhibits enhanced effector functions and phenotype, whilst showing limited cellular exhaustion, and significant antitumour activity. Hence, augmentation of these immune cells by an agent that promotes the functional effect of the association of CD61 and CD 103 (i.e. an agonist) leads to increased cytotoxicity and antitumor responses of these TILs with minimal toxic side effects. An agent described herein may target a particularly effective population of immune cells, e.g. CD61+immune cells, to promote cytotoxicity and inflammatory responses of these immune cells. For example, the invention may provide a method of promoting cytotoxicity and inflammatory responses, comprising contacting immune cells with an agent specific for CD61 for targeting CD61+immune cells.

[0113] The immune-related condition may be associated with overreacted immune cells. The immune-related condition associated with overreacted immune cells may be an inflammatory disease, autoimmune disease, transplantation, allergy, or viral infection (e.g. acute viral infection). Treatment of these immune-related conditions may benefit from dampening the inflammatory responses in the body. An agent described herein targets a niche immune cell population which exhibits enhanced effector functions and phenotype, whilst showing limited cellular exhaustion. Hence, dampening these immune cells by an agent that reduces the functional effect of the association of CD61 and CD 103 (i.e. an antagonist) leads to reduced cytotoxicity and inflammatory responses of these immune cells with minimal off target effects. An agent described herein may target a particularly effective population of immune cells, i.e. CD61+immune cells to reduce cytotoxicity and inflammatory responses of these immune cells with minimal off target effects.

[0114] The therapeutic uses and methods may comprise administering a therapeutically effective amount of the agent, the population of immune cells, or the pharmaceutical composition.

[0115] Also provided is a method of formulating a composition for treating an immune- related condition, wherein said method comprises mixing an agent or a population of immune cells described herein with an acceptable carrier to prepare said composition.

[0116] The subject may have been previously treated for the cancer, such as using adoptive cell therapy.

[0117] The methods and uses of the invention may comprise identifying or enriching a population of CD61+CD 103+ immune cells, comprising detecting the presence of CD61+CD 103+ immune cells using an agent described herein.

[0118] In embodiments where the agent of the invention acts as an agonist, the methods and uses of the invention may comprise a method of enhancing the functional effect of the association between CD61 and CD103. The methods and uses of the invention may comprise augmenting CD61+CD 103+ immune cells activity in the subject.

[0119] In embodiments where the agent of the invention acts as an antagonist, the methods and uses of the invention may comprise a method of reducing the functional effect of the association between CD61 and CD103. The methods and uses of the invention may comprise reducing CD61+CD103+ immune cells activity in the subject.

[0120] The methods and uses described herein may comprise identifying or enriching a population of CD61+ immune cells, comprising detecting the presence of CD61+ immune cells using an agent described herein. The therapeutic methods and uses described herein may comprise inhibiting the disease state (e.g. the cancer), for example by arresting its development and / or causing regression of the disease state until a desired end point is reached. The therapeutic methods and uses of the invention may comprise achieving a partial response, a full response by the cancer. The therapeutic methods and uses of the invention may achieve remission of the cancer. The therapeutic methods and uses described herein may delay the growth of the cancer, arrest the growth of the cancer and / or reverse the growth of the cancer. The therapeutic methods and uses of the invention may reduce the size of the cancer by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or by 100%.

[0121] Typically, the therapeutic methods and uses are for a human subject in need thereof. However, non-humans animals such as non-human mammals are also contemplated. The non-human mammals may be rats, rabbits, sheep, pigs, cows, cats or dogs.

[0122] The agent, population of immune cells, or pharmaceutical composition described herein may be administered as a single dose. The agent or the pharmaceutical composition may be administered in a multiple dose regimen. For example, the initial dose may be followed by administration of a second or plurality of subsequent doses. The second and subsequent doses may be separated by an appropriate time. For example, the doses between doses may be administered once about every week, once about every 2 weeks, once about every 3 weeks, once about every four weeks, or once about every month.

[0123] The agent, population of immune cells, or pharmaceutical composition may be administered intravenously. Also provided is a method of performing adoptive cell therapy in a subject, the method comprising administering to the subject an effective amount of an agent described herein. Hence, the invention also provides an agent described herein for use in combination with adoptive cell therapy. The invention also provides the use of an agent for the manufacture of a medicament for adoptive cell therapy.

[0124] For example, the agent or the pharmaceutical composition may be administered with one or more further therapy, such as one or more further therapeutic agents.

[0125] The further therapy may be CAR-T cell therapy, TCR-T cell therapy, immunotherapy, anti-cancer therapy, chemotherapy, radiotherapy and / or surgery. The further therapeutic agent may be an anti-cancer agent. The further therapeutic agent may be an additional immune effector cell.

[0126] The further therapeutic agent may be a CAR-T cell or a TCR-T cell. Preferably, the further therapeutic agent is a TCR-T cell. The CAR-T cells express CARs. The CAR-T cells identifies cancer cell proteins expressed on the cancer cell surface. The TCR-T cells identify tumour- specific MHC-bound cancer antigen. The cancer antigen may be extracellular or intracellular. The TCR-T cell may comprise natural TCR or modified TCR. Without wishing to be bound by theory, an agent that promotes the functional effect of the association of CD61 and CD 103 augments the cytotoxicity and immune responses induced by CAR-T cells or TCR-T cells by augmenting a particularly effective population of immune cells, i.e. CD61+CD103+immune cells, which exhibits enhanced effector functions and phenotype, whilst showing limited cellular exhaustion.

[0127] The further therapy may be an immunotherapy, such as a checkpoint inhibitor that inhibits a checkpoint protein selected from PD-1, CTLA-4, PD-L1, PD-L2, B7-H3, B7-H4, BTLA, HVEM, TIM-3, GAL9, LAG-3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or a combination thereof. The further therapy may be nivolumab, pembrolizumab, atezolizumab, BMS-936559, MEDI4736, MPDL33280A or MSB0010718C, ipilimumab and tremelimumab, MEDI6469, PF-05082566 or a combination thereof.

[0128] The further therapy may be an anti-cancer therapy, such as an anti-cancer therapeutic selected from an alkylating agent (such as mechlorethamine, cyclophosphamide, chlorambucil, ifosfamidecysplatin, or platinum-containing alkylating agents such as cisplatin, carboplatin and oxaliplain), and anti-metabolite (such as a purine or pyrimidine analogue or an anti-folate agent, such as azathioprine and mercaptopurine), an anthracycline (such as daunorubicin, doxorubicin, epirubicin idarubicin, valrubicin, mitoxantrone or anthracycline analog), a plant alkaloid (such as a vinca alkaloid or a taxane, such as vincristine, vinblastine, vinorelbine, vindesine, paclitaxel or doestaxel), a topoisomerase inhibitor (such as a type I or type II topoisomerase inhibitor), a podophyllotoxin (such as etoposide or teniposide), a tyrosine kinase inhibitor (such as imatinib mesylate, nilotinib or dasatinib), an adenosine receptor inhibitor (such as A2aR inhibitors, SCH58261, CPI-444, SYN115, ZM241385, FSPTP or A2BR inhibitors such as PSB-1115), or adenosine receptor agonists (such as CCPA, IB- MECA and CI-IB-MECA) or a combination thereof. Combined administration of the agent or the pharmaceutical composition with the additional therapeutic agent may be achieved in a number of different ways. All the components may be administered together in a single composition. Each component may be administered separately as part of a combined therapy. For example, the agent or the pharmaceutical composition of the invention may be administered before, after or concurrently with the further therapeutic agent.

[0129] The biological activity and / or therapeutic efficacy of the administered agent or the pharmaceutical composition may be measured by known methods. For example, the method may comprise imaging, such as magnetic resonance imaging.

[0130] The invention also provides a method of delivering a therapeutic agent to a CD61+CD103+immune cell in a subject, the method comprising administering an agent described herein. The therapeutic agent may be an anti-cancer therapeutic or antiinflammatory, e.g. as described herein.

[0131] The method and uses of the invention also relate to a method of treating cancer, comprising administering to a subject a population of immune cells (such as T cells) that express: (i) a heterocomplex comprising CD61 and CD 103; and (ii) an antigen binding protein on the cell surface. The antigen binding protein may be a chimeric antigen receptor (CAR), a T cell receptor (TCR) or a modified T cell receptor.

[0132] The method and uses of the invention also relate to a method of treating an immune-related condition, comprising: (i) modifying patient T cells to overexpress CD61 and / or CD103; and

[0133] (ii) administering said modified immune cells to the patient in need thereof, optionally wherein the TCR is modified.

[0134] The invention provides a method of predicting outcome of a therapy for an immune-related condition for a subject, by determining the presence of CD61+CD 103+ immune cells that exhibit enhanced effector functions and phenotype, while showing limited cellular exhaustion, such as CD61+CD 103+ TILs or CD61+ TILs, in a sample from said subject. Hence, the invention also provides the use of CD61 / CD 103 as predictor of immune outcome including after checkpoint inhibition.

[0135] The method may comprise determining the level of CD61+CD 103+ immune cells in a first sample from said subject, and comparing the level of CD61+CD 103+ immune cells in a first sample from said subject, wherein a difference between the level of CD61+CD 103+ immune cells in the first sample compared to the second sample is an indication of whether or not the subject will respond to therapy. The first sample may be a test sample. The second sample may be a control sample, e.g. a standardised control level of CD61+CD 103+ immune cells in a healthy subject. The second sample may be a sample previously obtained from the subject, e.g. pre-treatment, or at an earlier time point during the course of therapy.

[0136] The level of CD61+CD 103+ immune cells may be determined by an agent described herein. Typically, the agent may not modulate the functional effect of the association between CD61 and CD 103, although agents that modulate the functional effect of the association between CD61 and CD 103 function may also be used. The agent may be labelled, as described herein.

[0137] The sample may be a blood sample, such as a serum sample.

[0138] The therapy may be an anti-cancer therapy, such as an immune checkpoint inhibitor therapy (e.g. as described herein). In this embodiment, an increase in the level of CD61+CD 103+ immune cells, e.g. CD61+CD 103+ TILs, particularly at tumor sites, during the course of the therapy may be an indicator of favourable prognosis. On the other hand, a decrease or a lack of increase in the level of CD61+CD 103+ immune cells, e.g. CD61+CD 103+ TILs, particularly at tumor sites, during the course of the therapy may be an indicator of poor prognosis. The therapy may be a therapy for a disease associated with overactive immune cells. The therapy may be an anti-inflammatory, e.g. as described herein. In this embodiment, a decrease in the level of CD61+CD 103+ immune cells during the course of the therapy may be an indicator of favourable prognosis. On the other hand, an increase or a lack of decrease in the level of CD61+CD 103+ immune cells during the course of the therapy may be an indicator of poor prognosis.

[0139] An inflammatory useful with the invention may be methotrexate, infliximab, cyclophosphamide, azathioprine, cyclosporin A, sulfasalazine, hydroxychloroquine, leflunomide, etanercept, or tumor necrosis factor-alpha (TNFa) or other cytokine blockers or antagonists.

[0140] The therapeutic methods and uses may comprise, prior to treatment with an agent, a population of immune cells, or a pharmaceutical composition described herein, determining whether the cancer expresses an antigen specifically targeted by an agent of the invention.

[0141] The invention also provides an agent, a population of immune cells, or a pharmaceutical composition described herein for use in a method of preventing immune- related conditions, comprising identifying a subject susceptible to an immune-related condition according to the methods of the invention, and administering an agent, a population of immune cells, or a pharmaceutical composition to the subject.

[0142] Also provided herein is a method of diagnosing an immune-related condition in a subject, comprising detecting the presence of CD61+CD 103+ immune cells using an agent described herein.

[0143] The methods and uses may comprise including a step of comparing levels of CD61+ and / or CD 103+ immune cells in a subject's sample to a reference. The reference may be (i) a threshold value, (ii) the corresponding levels of CD61+ and / or CD 103+ immune cells in a sample from a positive control, and / or (iii) the corresponding levels of CD61+ and / or CD 103+ immune cells in a sample from a negative control. The comparison provides a diagnostic indicator of whether the subject is susceptible to an immune-related condition or has an immune-related condition. As would be within the understanding of a person skilled in the art, whether the levels of CD61+ and / or CD 103+ immune cells is increased or decreased would depend on the reference used and the nature of the immune-related condition. For example, in a subject having cancer, the levels of CD61+ and / or CD 103+ immune cells in the blood or tissue would be at a higher level than the level in a negative control sample (non-cancer sample), and at a similar level as in a positive control sample (cancer sample).

[0144] Typically, the invention involves comparing levels of CD61+ and / or CD 103+ immune cells against a threshold value, and the optimal threshold value may be determined by training classifier algorithm to distinguish between "case" and "control" samples as explained above.

[0145] The invention also provides diagnostic devices and kits for detecting the presence of CD61+ and / or CD 103+ immune cells of the invention.

[0146] The invention also provides a diagnostic device for use in providing a diagnostic indicator of a subject susceptible to or having an immune-relate condition, wherein the device permits determination of the levels of CD61+ and / or CD 103+ on immune cells in a sample. The invention also provides a diagnostic device for use in discriminating an immune-related condition characterised by levels of CD61+ and / or CD 103+ immune cells, wherein the device permits determination of the levels CD61+ and / or CD 103+ on immune cells in a sample.

[0147] The immune cells may be T cells, NK cells, B cells, or NKT cells. The T cells may be TILs, CD8+ T cells, CD4+ T cells, regulatory T cells, effector T cells, tissue resident memory T cells, memory T cells, or gamma delta (y5) T cells.

[0148] The presence and / or level of CD61+CD 103+ immune cells may be determined by an agent described herein using standard techniques in the art, such as western blot analysis, radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, solution phase assay, solution phase assay, electrochemiluminescence immunoassay (ECLIA), ELISA assay or FACS.

[0149] Other

[0150] It is to be understood that different applications of the disclosed agents, cells, or pharmaceutical compositions of the invention may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0151] In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “an agent” includes two or more “agents”.

[0152] Furthermore, when referring to “>x” herein, this means equal to or greater than x. When referred to “<x” herein, this means less than or equal to x.

[0153] Unless otherwise provided, factors described herein are human homologs.

[0154] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0155] The following examples illustrate the invention.

[0156] Examples

[0157] Introduction

[0158] Integrins are large, heterodimeric transmembrane glycoproteins that facilitate adhesion between cells, and with the extracellular matrix1. They require the pairing between an a and b subunit to exit the endoplasmic reticulum and reach the cell surface to become functionally active2'8. The pairing and functions of integrin b3 (1TGB3, CD61) with its known cis integrin partners, integrin aV (ITGAV, CD51) and integrin allb 1TGA2B, CD41) have been well-documented on non-lymphocytic cells such as megakaryocytes, platelets and macrophages, as well as on endothelial cells5,9'13. While there are pieces of evidence of CD61 pairing with CD41 or CD51 on murine T cells14,15, the expression and functional implications of CD61 on antigen-specific T cell immunity in human diseases, including cancer, remains unclear.

[0159] On the other hand, it is well established that integrin aE (1TGAE, CD 103) pairs with integrin b7 (ITGB7) on murine and human immune cells. CD 103 is considered a key phenotypic marker of Tim cells in a variety of tissues including tumours. In cancer, CD103+Trmtumour-infiltrating T lymphocytes (TILs) are known to be immunophenotypically diverse, ranging from terminally exhausted (layilin+), Trm precursors (granzyme H+), and Trm transitional-phase (XCL1+) cells16. Certain CD103+TIL subtypes, such as the CD103+CD39+cells are tumor-reactive TILs, with clonal expansion observed in different cancer types17'19. Our recent study further demonstrated CD103+TGF-bl+cytotoxic T cells were efficient killers of antigenic cancer20.

[0160] Clinically, the enriched presence of CD103+T cells in cancer patients and pathogenically diseased patients has been associated with improved outcomes21'25. This positive clinical attribute makes CD103+T cells an important target for immunotherapy strategies. However, the immune-associated proteins and the mechanisms that are utilised to promote effective cellular immune activities, responses and protection remain poorly defined. Therefore, this study evaluates the mechanistic contributors of protective immunity on human antigen- specific cytotoxic CD8+T cells, using cancer as a disease model.

[0161] CD61 is expressed on CD103+CD8+T cell clones and Trm TILs

[0162] The presence and enrichment of CD103+TILs is considered a good indicator of desirable clinical prognosis and outcome17 19,21'23. To investigate how these T cells can promote protective immunity, HLA-A*02:01 -restricted CD103+and CD103' CD8+T cell clones were previously generated, from two separate patients20. The paired CD103+and CD 103" T cell clones isolated from a gastric cancer patient are characterised by the same TCR: TRAV8-6 TRAJ30, TRBV6-1 TRBJ2-7, recognising the SSX-2 tumour antigen. In contrast, the second pair of T cell clones were isolated from a melanoma patient with a distinct TCR: TRAV12-2 TRAJ31, TRBV12-4 TRBJ1-2, recognising the tumour antigen, NY-ESO-1.

[0163] As part of a discovery approach to identify CD 103 immune-related proteins, the proteomic profiles were first compared between the cancer- specific CD103+and CD 103" T cell clones from these two cancer patients. The differential expression analysis revealed 103 proteins enriched on the two CD103+T cell clones, compared to their respective paired CD103' T cell clone (Figure 7a, Table 1). Among these proteins, 70.8% (or 92 proteins) are associated with cellular processes, 10% (13 proteins) with metabolism and 16.9% (22 proteins) with protein synthesis and trafficking (Figure 7b). Several of these protein subgroups were identified to be linked to immune activities, including immune effectors / cytokines, integrins, metabolic -related, TGF-l-related and epigenetic-related groups (Figure la).

[0164] Due to being described as an integrin of non-immune cells such as platelets, endothelial cells and megakaryocytes in humans5 11 13, the enrichment of CD61 (Figure lb) in the CD103+clones were unexpected. To validate the in vitro proteomics approach, the existence of CD61+T cells within the total CD8+TILs in a larger cohort of patients’ ex vivo was next evaluated. Dual immunohistochemistry (IHC) and flow cytometric CD61 protein analyses was performed on a cohort of non-small cell lung cancer (NSCLC) patients’ samples obtained from the Oxford Radcliffe Biobank (Table 2, Figure 8, Figure 9a).

[0165] As evidenced by the dual IHC and flow cytometry analyses, the presence of CD61+T cells in the CD103+TILs population was confirmed, with variable frequencies across the NSCLC cancer patients, ranging from 2% to 77% (Figure 1c, Figure 9b). The variability in the frequency of the TILs subpopulation correlated with the NSCLC tumour stages (Figure 9c), but not with other clinical parameters evaluated such as NSCLC pathology type (including between adenocarcinoma and squamous cell carcinoma), gender and age (Figure 9d).

[0166] Importantly, regardless of the frequency variability between patients, the CD61+TILs subset was significantly enriched in the lung tumour tissue of these patients compared to the paired para tumor tissue and peripheral blood (Figure Id-e).

[0167] To further confirm the existence of CD61 on human antigen-specific CD8+T cells, CD61 expression was evaluated on seven pairs of cancer-specific CD103+and CD 103" T cell lines, from 7 different cancer patients. Indeed, positive CD61 expression on the seven CD103+T cell lines was identified, compared to their paired CD 103" T cell lines (Figure If).

[0168] CD61 transiently co-localises with CD103 during synapse formation

[0169] To evaluate the kinetic expression of CD61 on human T cells, the expression following different activation regimes was next measured. The cancer- specific CD103+T cell lines, sourced from seven different cancer patients demonstrated positive surface expression of CD61 following activation either by agonistic aCD3 / aCD28 antibody or following co-culture with antigenic cancer cells (Figure 2a, Figure 10a). In contrast, resting T cells showed undetectable levels of CD61 expression, which was consistently observed across all seven cancer patients (Figure 2a). Upregulation of CD61 peaked within the first 2 hours post T cell activation, before gradually decreasing over time (Figure 10b), suggesting that CD61 expression on human T cells is transient.

[0170] Considering the transient upregulation of CD61 on the T cells, its potential involvement in the temporal scales of cell-to-cell contacts was evaluated. Total internal reflection fluorescence microscopy (TIRFM) was used to analyse the recruitment and distribution of CD61 within the synaptic contacts formed between the NY-ESO-l-specific CD61+T cell line and supported lipid bilayers (SLBs)-containing physiological densities of antigenic pMHC, ICAM-1, CD58 and E-cadherin (SLB protein densities are as informed in the Methods section). A time-dependent increase of CD61, as well as of CD 103, was identified at the points of contact between the T cell membrane and the bilayer, within the central Supramolecular Activation Cluster (cSMAC) region, defined by the area of accumulation of antigen and TCRab (Figure lOc-d).

[0171] Interestingly, the CD61 co-localised with CD 103 at the points of contact (Figure 2b). The co-localisations’ Pearson’s correlation coefficient (PCC) between CD61 and CD103 was consistently positive throughout the 15 minutes of contact (Figure 2c). In contrast, integrin b7, the known canonical integrin partner of CD 103, was located in the outermost synaptic compartment, also known as the distal Supramolecular Activation Cluster (dSMAC) (Figure 2b). Evidently, the PCC for CD 103 and integrin b7 was consistently below zero, indicating they were inversely correlated at the synapse (Figure 2d). The dSMAC localisation of integrin b7 is likely due to its interaction with its alternative cis-integrin partner, the integrin a4 1TGA4, CD49d), rather than with CD103. Co-localisation between CD49d and integrin b7 was observed at the dSMAC microclusters (Figure 2e), which may reflect an early CD49d-integrin b7 pairing and positioning, leading to segregation of integrin b7 from CD 103.

[0172] With the central clustering of CD61 at the cSMAC, it was next evaluated whether CD61’s conventional cis- interacting integrin partners, CD41 and CD51, were relied upon for CD61 integrin heterodimerisation on the T cell’s surface. Unexpectedly, both CD41 and CD51 were not expressed on the cell surface of the NY-ESO-l-specific CD61+T cell line (Figure lOe). These data suggest that the absence of CD41 and CD51 on these T cells enables the unconventional pairing with CD 103, as per Figure 2b.

[0173] To further investigate the possible interaction between CD61 and CD 103 on human T cells, co-immunoprecipitation analysis was performed on CD61 pulldown lysates of primary T cells over-expressing both these integrins. CD61 was found to co- immunoprecipitate with CD103 when pulled down from the lysate of CD61-flag+CD103+T cell line, but not on the lysate controls: CD61-flag'CD103+T cell line lysate and the CD61-flag+CD103' T cell line lysate (Figure 2f and Figure I la). Additionally, this CD61:CD103 interaction is also reproducible on the monocytic cell line U937 overexpressing both these integrins (Figure I la), reaffirming the interaction of these molecules in human cells.

[0174] To further strengthen this observation, an analysis of the overall proteins that may co-immunoprecipitated with the CD61-flag protein was carried out. Apart from proteins that are commonly known to associate with integrin heterodimer complex formation, the presence of co -precipitated CD103 on the CD61-flag+CD103+T cell line was confirmed (Figure 2g, Figure 12), further demonstrating the existence of a CD61:CD103 integrins complex. Additionally, no enrichment of integrin b7 was detected on the CD61- flag+CD103+T cells (Figure lOf), suggesting the exclusion of integrin b7 from the CD61:CD103 complex.

[0175] As it is widely recognised that an interaction between an a and b integrin subunit is required for the cell surface expression of integrins, it was evaluated whether CD61 secondary transduction can promote CD 103 cell surface expression (Figure 11b). Following the primary transduction of CD103-HA on primary T cells, surface expression of the CD 103 was not observed (Figure 2h). However, a secondary transduction of CD61 on the same CD103+T cell line was able to rescue surface expression of CD 103 on the T cells (Figure 2h). Taken altogether, our findings using the multi-faceted approaches above have demonstrated the interaction between CD61 and CD 103 on human T cells.

[0176] CD61 enhances TCR signalling

[0177] The possible role of CD61 on T cell signalosome was next evaluated. In parallel to CD61 and CD 103 co-localisation at the cSMAC, o-localisation between CD61 with TCRab, as well as between CD 103 and TCRab was further observed (Figure 3a), suggesting that CD61 may modulate TCR signalling activity.

[0178] To assess the functional significance of CD61 towards proximal TCR signalling, CD61 knock-down T cells were generated using siRNA, and CD61 CRISPR knock-out (KO) T cells were generated, from the wild-type (WT) CD61+T cell clone of cancer patient 1 (Figure 13a) before functional evaluation. Interestingly, Zap70 and PLCgl phosphorylation levels were gradually decreased following serial CD61 siRNA treatments (Figure 3b and Figure 13b). Additionally, the CD61KOT cell clone demonstrated impaired phosphorylation of both these proteins, to levels comparable with those seen on WT CD6F T cell clone (Figure 3c and Figure 13b). To further verify the importance of CD61 in regulating Zap70 phosphorylation, the Zap70 (pY292) expression on WT CD61+T cell lines from 7 different cancer patients was next evaluated. Indeed, phosphorylated Zap70 level was significantly impaired following treatment with aCD61 neutralising antibody (Figure 3d and Figure 13c).

[0179] Given that a cytosolic adaptor protein, the kinase Lek, is known to maintain and directly sustain the phosphorylation of Zap70 at the TCR complex’s cytoplasmic domains, the potential protein linker between CD61 and Zap70 was next evaluated. WT CD61+T cell clone exhibited the highest Lek expression in comparison to the siRNA-treated and CD61KOT cell clones (Figure 3e). In agreement, treatment with aCD61 neutralising antibody led to a significant downregulation of Lek expression on WT CD61+T cell lines of 7 different cancer patients (Figure 3f), suggesting that CD61 is a modulator of Lck- dependent Zap70 phosphorylation.

[0180] To further confirm the involvement of Lek in the CD61-Zap70 phosphorylation axis, WT CD61+T cell clone was treated with either the Lck-specific inhibitor A770041 or the broader tyrosine kinase inhibitor amino genistein as a positive control. As expected, a significant impairment of the Zap70 phosphorylation on the WT CD61+T cell clone following the inhibition of Lek was observed (Figure 3g). Taken altogether, these observations suggest that CD61 may enhance TCR proximal signalling, including Zap70 phosphorylation in an Lck-dependent manner (Figure 3h). CD61 improves T cell cytotoxicity and tumour control

[0181] To determine the importance of CD61 on T cells’ anti-tumor immunity, in vitro T cell degranulation and cytotoxicity analyses was performed. The cancer- specific WT CD61+T cell clone was found to have significantly elevated expression of the cytolytic degranulation marker, CD 107a, compared to the T cells treated with CD61 siRNA, CD61KO, and the WT CD6T T cell clones (Figure 4a). Additionally, neutralising CD61 with blocking antibody also limited the degranulation capacity of the CD61+T cell, consistently observed across the CD61+T cell lines from 7 different cancer patients (Figure 4b).

[0182] Consistent with the increased degranulation activity, a higher frequency of cancer cell death induced by the WT CD61+T cell clone was observed across multiple time points, with CD61 siRNA-treated and CD61KOT cell clones exhibiting impaired T cell cytotoxicity (Figure 4c). To validate the importance of CD61 towards T cell cytotoxicity, WT CD61+T cell lines from 7 different cancer patients were treated with anti-CD61 neutralising antibody prior to the co-culture with antigenic cancer cells. It was confirmed that neutralising anti-CD61 antibody consistently limited the cytotoxic responses of these T cell lines (Figure 4d).

[0183] The in vivo physiological relevance of CD61+T cells was further assessed by evaluating the growth of xenografted antigenic tumours in NOD.SCID mice following adoptive transfer of either WT CD61+or CD6F T cell clones over time (Figure 12d). Tumor growth was significantly mitigated in mice injected with the CD61+T cells compared to the CD6F T cells (Figure 4e). Importantly, the differences in tumour sizes were readily observed after the second adoptive transfer of T cells, with more substantial differences seen following the third T cell injection (Figure 4f-g).

[0184] Since these in vitro and in vivo findings demonstrated the immune contribution of CD61+cancer- specific T cells towards tumour control, the possible clinical relevance of the CD61+TILs on patients’ overall survival (OS) probability, in a lung cancer (LC) patient cohort as well as a skin cutaneous melanoma (SCM) patient cohort from the TCGA database was explored17. The CD61hlCD103+CD8+CD3+SCM patients exhibited better OS prognosis compared to the CD6110CD103+CD8+CD3+patients (Figure 4h). In validating this survival pattern, the CD61hlCD103+CD8+CD3+LC patients were further found to also have improved OS prognosis compared to the CD6110CD103+CD8+CD3+patient (Figure 4h).

[0185] CD61+TILs exhibit enhanced anti-tumor effector phenotypes in NSCLC

[0186] To further dissect the relevant clinical immunophenotype of the CD61+T cells that may contribute towards enhanced cancer immunity and improved survival in cancer patients, multicolour flow cytometric profiling of tumors from 19 NSCLC patients was performed (Table 2). The CD61+and CD61 ’ TrmTILs were first stratified according to the well-established tissue-resident memory TILs phenotype of CD103+CD69+CD49a+CD45RO+(CD62L'CCR7'CD45RA') CD8+TILs (Figure 8).

[0187] Clinical immunophenotypic analyses showed significant upregulation of key anti-tumor effector cytokines, chemokines and cytolytic molecules (including granzyme M, granulysin, granzyme B, CD107a, CCL5, XCL2, TNFa, IFNg) on the CD61+TILs, compared to the CD61" TILs (Figure 5a-b and Figure 14). Additionally, it was confirmed that in particular, granulysin and granzyme M expression on CD61+TILs were dependent on CD61 activity, as treatment using an anti-CD61 blocking antibody demonstrated impaired expression of both cytokines (Figure 5c).

[0188] The CD61+TILs have significantly enriched combinatorial immune effector signatures compared to the CD61'TILs (Figure 5d). As upregulated immune effector signatures on TILs are highly indicative of tumor-responsiveness, it was hypothesised that CD61+TILs could be more infiltrative of the NSCLC tumor bodies. Using in-situ IHC approach, it was confirmed that the CD61+TILs (identified by CD61+CD103+CD8+colocalised cells) were a significantly present at higher frequency within tumor islets compared to the CD61' TILs (identified by CD61'CD103+CD8+co-localised cells) (Figure 5e).

[0189] Previous studies on TILs have established tumor-reactive TILs as marked by dual positive CD103+CD39+expression17'19. In the analysis of our NSCLC patient cohort, significant enrichment of these combinatorial markers was found on the CD61+TILs compared to the CD6T TILs (Figure 5f), therefore, suggesting that the TILs subset is likely immune reactive within the tumor microenvironment.

[0190] CD61+Trm TILs do not exhibit hallmarks of exhaustive phenotype

[0191] The tumor microenvironment is known to be immunosuppressive and this is well- established to contribute to chronic T cell exhaustion. The hallmarks of T cell exhaustion include (i) reduced antigen sensitivity, (ii) regression of effector responses, (iii) terminal stage of differentiation, and most importantly, (iv) co-expression of multiple immune inhibitory receptors20.

[0192] As shown in Figure 5, the CD61+TILs do not have regression of effector responses but instead exhibited enhanced immune effector phenotype. Therefore, the other hallmarks of cancer T cell exhaustion that may be exhibited by the TILs subset was next evaluated, namely the co-expression of multiple inhibitory receptors such as Tim-3, PD-1 and TIGIT, which have been previously shown by the inventors to be the most prominent inhibitory receptors co-expressed on total CD8+TILs, in a variety of cancers including NSCLC. Interestingly, the CD61+TILs exhibited enriched PD-1 expression, but reduced expression of Tim-3 and TIGIT, when compared to the CD61" TILs (Figure 6a). This observation was confirmed by the limited frequency of combinatorial expression of PD-l+Tim-3+TIGIT+by CD61+TILs, compared to the CD61 ’ TILs (Figure 6b). Instead, the CD61+TILs were highly enriched for the PD-1+Tim-3'TIGIT" population (Figure 6c). As PD-1 is also well- established to be a marker of activation on T cells, the current finding suggests that the PD- 1+Tim-3'TIGIT" Trm TILs population could be less exhaustive, and therefore more active and responsive in lung cancers.

[0193] Consistent with its more active and responsive nature, it was found that CD61+TILs are not at the terminal stage of differentiation (another hallmark of T cell exhaustion). Instead, they are enriched for early-differentiated cells (CD27+CD28+) (Figure 6d). In contrast, the CD61 ’ TILs were predominantly late stage differentiated.

[0194] Being at the earlier stage of maturation, it was further demonstrated that the CD61+TILs were capable of undergoing more cellular divisions compared to the CD61" TILs, with an increased frequency of proliferating cells (Figure 6e-f). Taken altogether, these findings highlighted CD61+TILs as proactive, tumor- responsive T cells exhibiting enhanced anti-tumor effector and cytotoxic immune responses, but lack the expression of multiple immunocheckpoints receptors (key hallmark of TILs exhaustion). These positive attributes therefore likely contribute towards the T cells capacity to mitigate tumor growth and improve survival.

[0195] CD61+ CD8+ Trm Clusters Exhibit Distinct Transcriptional Profiles

[0196] To define the transcriptional landscape of CD61+CD8+tissue-resident memory (Trm) cells in lung cancer, single-cell spatial transcriptomic analysis was performed on 896,578 cells from 1,364 fields-of-view (FOVs) across six patients. This analysis identified five distinct CD8+Trm clusters, among which three clusters (CD8-1, CD8-4, and CD8-5) expressed CD61, while CD8-2 and CD8-3 were CD61-negative (Figure 17a). Analysis of ITGAE (CD103) expression in these clusters demonstrated that all three CD61+CD8+Trm clusters exhibited high CD 103 expression (Figure 17b). This suggests an association between CD61 and CD 103 expression at the transcriptional level. These findings indicate that CD61+CD8+Trm cells represent a distinct subset of tumor-infiltrating lymphocytes (TILs) with tissue-residency properties.

[0197] Phenotypic and Functional Heterogeneity of CD61+ CD8+ Trm Clusters

[0198] To determine the functional implications of CD61 expression in CD8+Trm cells, the immuno-responsive and inhibitory / exhaustion-related phenotypic features across 12 different T cell clusters. Clusters CD8-1 and CD8-5 exhibited high immuno-activation scores and low inhibitory / exhaustive immuno-score, indicative of strong tumor-reactivity, whereas CD8-4 displayed a quiescent or non-responsive state, suggesting a potential differentiation or regulatory phenotype (Figure 18a, 18b). To validate the differential functional roles of CD61+Trm cells, the expression of key immune effector molecules was assessed. Further analysis revealed that CD8-1 (CD61+A) demonstrated elevated cytokine production, whereas CD8-5 (CD61+C) exhibited increased expression of chemotactic markers, consistent with a more exhausted but potentially migratory phenotype. In contrast, CD8-4 (CD61+B) remained phenotypically inert, suggesting a regulatory or transitional state that requires further investigation (Figure 18c). These findings confirm that CD61+Trm cells are functionally heterogeneous, with subsets exhibiting either a highly activated, cytotoxic phenotype or a more quiescent profile. The presence of functionally diverse CD61+Trm cells within tumors suggests potential roles in tumor control or immune evasion.

[0199] CD61+CD8+Trm Cells Are Preferentially Located Near E-cadherin+Tumor Cells

[0200] To explore the spatial distribution of CD61+CD8+Trm cells, the localization across the top 20 FOVs in lung tumor tissues was analysed. CD8-1, CD8-4, and CD8-5 clusters were frequently observed, with CD8-1 being the most abundant and widely distributed across the lung tumor in Patient 5748 (Figure 19a, 19b). Nearest-neighbor analysis demonstrated that CD61+CD8+T cells were significantly closer to epithelial tumor cells than any other cells (Figure 3c). Spatial imaging further confirmed that CD61+CD8+T cells preferentially clustered within lung epithelial tumor regions, supporting a role of CD61 expression in tumor-immune interactions (Figure 19d). Additionally, epithelial tumor cells in close proximity to CD61+TILs exhibited increased CDH1 (E-cadherin) expression, suggesting that CD61+CD8+Trm cells preferentially localize near epithelial tumor cells, with an association to E-cadherin-expression (Figure 19e). This observation was further validated using synaptic microscopy, which confirmed the consistent localization of CD61+TILs near E-cadherin+epithelial targets (Figure 19f). E-Cadherin, a cell adhesion molecule, is a well-established ligand for CD10326,27. These findings indicate that CD61+CD8+Trm cells are not only abundant in lung tumors but also preferentially associate with epithelial tumor cells, suggesting a role in tumor- immune interactions that may influence anti-tumor responses.

[0201] CD103+CD8+T Cells Exhibit Preferential Enrichment in Tumor-Dominant Regions

[0202] Using the COMET multiplex immunofluorescence technique per manufacturer's workflow instructions', the tumor tissue was segmented into two distinct regions: the tumor-dominant region and the stroma-dominant region, to analyze the spatial distribution of CD103+tissue-resident CD8+T cells (Trm). The results demonstrated that although CD8+T cells were more enriched in the stroma-dominant region, the majority were CD 103" CD8+T cells, whereas CD103+CD8+T cells were predominantly localized in the tumor-dominant region (Figure 20). Discussion

[0203] This study demonstrated an unexpected CD61 expression on human cytotoxic CD8+T cells in a cancer disease model. With the absence of its cis integrins partners CD41 and CD51, CD61 can co-localise and pair transiently with CD 103 at the cell-to-cell contacts. On the contrary, integrin b7 was clearly segregated apart from the CD 103, towards the distal synaptic space. The discovery using in vitro proteomics model was validated and confirmed using multi-faceted approaches with larger cohort of in situ, in vitro and ex vivo patients’ samples. As for its mechanistic functions on human cells, CD61 co-localisation with the TCR augmented proximal TCR signalling activities and contributed towards elevating anti-tumor cytotoxicity was demonstrated. This in turn allowed for a better control of tumor growth, evidenced in the immunocompromised mice model used in this study. Clinically, the presence of CD61+TILs were associated with improved T cell anti-tumor efficacy, mediated through enhanced anti-tumor effector functions and phenotypes whilst mitigating hallmarks of cellular exhaustion.

[0204] Integrins are well-established to only become functionally active following cellular activation and the pairing between an a and b subunit2'8. CD 103 was previously known to exclusively paired with integrin b7 in mediating cellular adhesion, primarily between intraepithelial CD8+T cells and the E-Cadherin-expressing epithelial and endothelial cells3. Additionally, the CD 103: integrin b7 pair functions similarly to another known integrin pair found on T cells, the integrin aL:b2 heterodimer, which promotes synapse assembly and stability, particularly when these integrin pairs are present at the dSMAC and peripheral SMAC (pSMAC). In contrast to these studies, minimal co-clustering between CD 103 and integrin b7 at the pSMAC and dSMAC was shown. Instead, CD 103 colocalised together with CD61 at the cSMAC. The distinct segregation between CD 103 and integrin b7, and the pairing between CD 103 and CD61 on these human cell models suggest that CD 103 is likely a promiscuous and dynamic integrin, that is not restricted to single integrin partner as previously thought.

[0205] The pairing and functions of CD61 with its canonical cis integrin partners, CD41 and CD51, have been well-documented on non-lymphocytic cells, such as megakaryocytes, platelets and macrophages, as well as on structural cells such as endothelial cells5,9'13. While there is evidence of CD61 expression on murine T cells14,15, the expression and functional implications of CD61 on antigen-specific immunity in human diseases, including cancer, were not known. Most remarkably, it was observed that CD61 expression on human cancer-specific CD8+T cells can occur in the absence of CD41 and CD51. Utilising synapse microscopy, the differential rearrangement of CD61 and integrin b7 in relation to CD 103 was demonstrated. The CD61 was enriched in its colocalisation with CD 103 in the synaptic microclusters, as well as evidenced by the enrichment of both proteins in the Co-IP lysates and flow cytometry approaches. Significant to the field, the study uncovered the first example of CD61 pairing with an I- domain-containing integrin a subunit, CD 103.

[0206] CD61 upregulation was only observed on the CD103+T cells, but not on the CD103" T cells. The link between CD61 and CD103 co-expression is likely due to their upregulation by TGF-bl. Not only has the recent study shown that the CD103+T cell clones can specifically express TGF-bl20, other studies have also demonstrated TGF-bl as a well-established cytokine required for sustaining phenotypic expression of CD 103 on certain human and murine cells. In parallel, a recent study has now additionally shown that TGF-bl can also induce the upregulation of CD61 mRNA and protein expression in a dose- and time-dependent manner.

[0207] The enrichment of CD61 on the cSMAC alongside the TCR suggests that CD61 may be involved in co- stimulatory signals, by promoting T cell activation, signalling activities and effector functions. This is because the dynamic cSMAC is well-established as a centripetally enriched zone for the TCR and its associated signalling molecules such as inhibitory and co-stimulatory receptors, for inducing net signalling outcomes and cytolytic activity. In contrast, the pSMAC, where most integrins such as integrin aL:b2 are known to be usually clustered and maintained, is primarily responsible for the assembly and firm adhesion of the synaptic structure and cell-to-cell contacts. It is known that CD61 can signal via Fyn kinase, but whether this also occurs in T cells remains unclear. Evidently, this study showed that CD61 is involved in modulating TCR-dependent ZAP70 phosphorylation, importantly through the intermediary Lek protein, providing an example of the potential mechanism by which CD61 can operate in human immune cells.

[0208] The transient nature of CD61 expression implicates the high turnover rate of this protein and the dynamic pairing with an I-domain-containing a integrin subunit. This high turnover could either be associated with the need to regulate the effects of CD61 on immune activities, or more likely, is dependent on the stability of protein-protein interaction. A recent study on an unconventional integrin pairing between CD51 and CD29 has suggested that low intra-heterodimer integrin affinity can lead to better functional activity. For CD61, the potentially low affinity interactions with its partners, such as CD 103, are likely needed depending on different situations and cell types. For example, CD61 interaction with a non-integrin partner, the heparan sulfate proteoglycans, can prompt vesicular endocytosis, leading to internalisation and the loss of affinity interaction.

[0209] Evidently, this study has that the immune potency and functions of CD61 on human T cells is spatially-, temporally- and TCR activation-dependent. At least in this model, the TCR-pMHC interactions, is necessary to trigger the temporal recruitment of CD61, via CD 103, to the cell surface and the spatial reorganisation and function within the synapse. This fine spatial-temporal regulation of CD61 and its kinetic association with the TCR and its signalling may explain why CD61+TILs in NSCLC cancer patients exhibited elevated anti-tumor activities and proliferative immunophenotype, but limited cellular exhaustion phenotype. This may therefore contribute towards better tumor control.

[0210] The invention described herein relates to the modulation of immune cell function through the targeting of the CD61 / CD 103 complex. The findings indicate that the tumor microenvironment may influence the retention and spatial positioning of Trm cells, thus affecting their functional role in tumor immunity. The presence of CD103+CD8+ T cells in tumor-dominant regions and their spatial association with CD61 suggest a role of this integrin axis in modulating T cell function within the tumor microenvironment.

[0211] The association between CD61 and CD 103 appears to play a critical role in the functional properties of specific T cell populations, including their tissue residency, activation potential, and cytotoxic capacity. We hypothesise that disruption or enhancement of this complex modulates these advantageous effects, and co-localisation of CD 103 and CD61 (in tumor-dominant areas) might correlates with improved prognosis- thereby offering a novel strategy for immune modulation.

[0212] Additionally, it is also hypothesised that CD61 expression on T cells may also independently contribute to T cell function, even in the absence of CD103. These findings suggest that the spatial distribution of CD103+CD8+ T cells and CD61 are key determinants of the tumor-immune landscape, offering new insights for immune modulation strategies.

[0213] In conclusion, the inventors have found that: i) the transient expression of CD61 and its unconventional pairing with CD 103 at the immune synapse enhances T cell receptor signalling, improves anti-tumour cytotoxicity and mitigates tumour growth; and ii) clinically, CD61+ tumour infiltrating lymphocytes (TILs) exhibit enhanced effector functions and show limited cellular exhaustion. This potentiates a new target for immunebased cellular therapies, for cancer and / or for autoimmune disease.

[0214] Methods

[0215] Paired. NSCLC patients’ peripheral blood, paratumor and tumor tissues procurement and processing

[0216] Confirmed non-metastatic NSCLC patients were recruited from the John Radcliffe Hospital, Oxford, United Kingdom, between December 2020 and April 2021. Subjects included both females and males who were between 63 to 80 years old. The study was reviewed and approved by the Oxford Radcliffe Biobank (ORB) Tissue Access Committee to obtain pseudonymised tissue samples and associated clinical data from patients recruited under ORB (Ethical approval reference numbers: 09 / H0606 / 5+5 and 19 / SC / 0173). All procedures were performed according to the Declaration of Helsinki guidelines. Clinical parameters of individual patients are as described in Table 2, with summary in Figure 9. Prior to surgery, patient provided informed written consent for collection of paired peripheral blood, paratumor and tumor tissues. Sizes of tissue samples collected were kept consistent between each patient (tumor resection volume of no more than 90mm3and paratumor resection weight of maximum 0.1g). Samples were collected and stored in RPML1640 on ice and de-identified prior to tissue processing. Tumor samples were confirmed using immunohistology by the ORB. Paired peripheral blood, paratumor and tumor tissue samples were used for ex vivo multicolour flow cytometry (FC) analyses, while the paraffin-embedded tumor slides of the same patients were used for immunohistochemistry (IHC). In total, 31 patients were used for IHC analysis and 17 of the same patients were used for FC analysis.

[0217] For the ex vivo multicolour flow cytometry, the cell suspension used for the assay was isolated from tissue as previously described20. Briefly, tissues were cut into small pieces using a pair of scissor and forceps before enzymatically dissociated into cell suspensions in RPMI-1640 using human tumor dissociation kit (Miltenyi Biotech), following the protocol provided by the supplier. Following the enzymatic dissociation, cells were filtered through 100mm strainer to remove indigestible parts of the tissue, with dead cells or debris removed by centrifugation at 1,500 rpm for 10 minutes. Cells were then resuspended in RPMI-1640 supplemented with 10% FCS (Sigma Aldrich), 2 mM L- glutamine (Sigma Aldrich) and 1% v / v (500U / ml) penicillin / streptomycin (Sigma Aldrich). For peripheral blood, the PBMC were isolated using Ficoll-Hypaque gradient isolation, followed by centrifugation to pellet the PBMC and resuspended in the same complete media as above.

[0218] Patient-derived cancer-specific CD8+T cell clones

[0219] HLA-A*02:01 -restricted T cell receptor (TCR)-matched CD103+and CD103" NY- ESO-li57-i65-specific and SSX-24i-49-specific CD8+T cell clone pairs were generated from gastric cancer and melanoma patients, as previously described20. Additionally, HLA- A*02:01 -restricted NY-ESO-l-specific T cell lines were generated from patients anonymously identified as Patient 1-4, SSX2-specific T cell line identified from Patient 5, Tyrosinase-specific T cell line identified from Patient 6 and melan-A-specific T cell line identified from Patient 7- to further validate the data observed the T cell clones. The identification of patients with the specific T cell lines were as previously described20Briefly, mononuclear cells were stimulated with tumor-associated antigens of the following: lOmg / ml SSX-24i-49-specific KV9 peptide (KASEKIFYV, SEQ ID NO: 1) (Peprotech), NY-ESO-li57-i65-specific SC9 peptide (SLLMQITQC, SEQ ID NO: 2) (Peprotech), melan-A / MART-126-35*A27L-specific EV10 peptide (ELAGIGILTV, SEQ ID NO: 3) (Peprotech) or Tyrosinase369-377*N370Dvariant-specific YV9 peptide (YMDGTMSQV, SEQ ID NO: 4) (JPT) in RPMI-1640 media, supplemented with 10% v / v heat-inactivated human AB serum (National Blood Service), 2mM L-glutamine (Sigma Aldrich), 1% v / v (500U / ml) penicillin / streptomycin (Sigma Aldrich) (hereon termed as H10), recombinant human IL-2 (200U / ml) (Peprotech) and recombinant human IL- 15 (0.5ng / ml) (Peprotech) for 14 days at 37°C. After 14 days, antigen- specific T cells expressing or not expressing CD103 were sorted using PE-conjugated HLA-A*02:01 / cancer peptide tetramers, and cultured in vitro using the same media as above, for another 14 days. After, the purity of sorted populations was confirmed by tetramer staining, and confirmed to have >90% purity. Validated cancer- specific T cells were stored in several batches for future assays, maintained for approximately 2 months and passaged once after every thaw, tested for mycoplasma monthly and re-authenticated using the tetramer staining method prior to every assays. Each pair of T cell clones used in this study have the same T cell receptor, so that any immune-related functional differences observed are likely contributed by the differential proteins’ expression of each T cell clone. siRNA treatment of WT CD61+T cell clone

[0220] For knocking-down CD61, siRNA targeting CD61 transcript was purchased commercially from ThermoFisher Inc., catalogue number 4392420, assay IDs: s7580, s7581, s7582. Triple siRNA targeting CD61 treatment was performed according to Lipofectamine RNAiMax protocol (ThermoFisher). Briefly, the WT CD61+T cell clone (from patient 1) was firstly seeded into a 96 well round bottom plate at 2M / ml of H10 and recombinant human IE-2 (200U / ml) (Peprotech). Serial dilution of each siRNA was prepared in 100ml Opti-MEM (for final concentration of 25nM, 50nM and lOOnM for each siRNA), in parallel but separately, adding 3ml of the Lipofectamine RNAiMax reagent to 100ml Opti-MEM before merging in 1:1 ratio and incubated at room temperature for 20 minutes to form transfection complexes. Solution was then added drop wise onto cells solution 3-7 days prior to co-culture with target cancer cells, incubated at 37°C for specific time duration required for each T cell assays (T cell cytotoxicity and Phosflow as described below). Validation data is as shown on Figure 13a. Surface staining was performed 5 days post siRNA treatment and before each T cell functional assays, to ensure consistent CD61 downregulation on the T cells. CRISPR / Cas9-editing ofCD61 on cancer-specific CD6!+T cells

[0221] For knocking-out CD61, CRISPR-Cas9 approach was undertaken. The ablation of gene of interest, CD61, was achieved by transfection with Cas9-gRNA RNP complexes. Prior to transfection, the Day 8 post- feeder expanded WT CD61+T cell clone (from patient 1) was washed three times with 10-volumes of prewarmed Opti-MEM-I medium (ThermoFisher). Cells were resuspended to a final concentration of 3 x 107cells / mL. In parallel, RNP complexes were assembled in two steps. First, 200 pmol of Alt-R CRISPR- Cas9 tracrRNA (200 mM stock, Integrated DNA Technologies (IDT)) were mixed with 200 pmol of Alt-R CRISPR-Cas9 predesigned ITGB3 crRNA (200 mM stock, IDT), and incubated at 95°C for 5 minutes, with the resultant duplex guide RNA allowed to cool to room temperature. The duplex gRNAs were then mixed with 124 pmol of Alt-R S. pyogenes CRISPR-Cas9 Nuclease V3 (IDT) and incubated at 37 °C for 15 minutes. The resultant RNPs were allowed to cool to room temperature and then supplemented with 200 pmol of Alt-R Cas9 Electroporation Enhancer (200 mM stock, IDT). Cells were then mixed with the RNP solution and immediately transferred to a 2-mm cuvette (Bio-Rad), electroporated at 290 V for 2 ms using an ECM 830 Square Wave electroporator. Immediately after transfection, cells were cultured in a 37 °C with 5% CO2 incubator with prewarmed, RPMI 1640 supplemented with 100 U / ml recombinant human IL-2 for 6 days. After 6 days, the CD61 CRISPR KO T cells were sorted after 2 hours of activation with anti-CD3 / CD28 antibody (StemCell Technologies) at 37°C. While waiting a minimum of 5 days from CRISPR / Cas9 edition ensured full degradation of pre- synthesised and stored CD61, the pre-sort activation ensured adequate trafficking of remaining CD61 to the surface of the edited T-cell clones and enabled the selection of truly CD61 negative (CD61KO) single T cells from the initial pool of 100% positive wildtype CD61+ T cells (compare Fig.13a, top grey histogram (CRISPR / Cas9 input) to blue histogram representing T cell clones after four passages). Sorted T cells were cultured and expanded in vitro using serial dilution cloning and feeder cell-stimulation, for another 14 days. The purity of clones and lines was confirmed by CD8 and CD61 staining, and confirmed for purity >90%. Surface staining was performed regularly at the start of all functional assays to ensure consistent CD61 abrogation on the T cells (Fig.13a). For controlling CRISPR-Cas9 editions the inventors preferred flow cytometry-based measurement of CD61 in preactivated cells over PCR-based Alt-R CRISPR-Cas9 Control Kits (IDT) as these were bulk measurements not enabling the precise selection of single CD61KOcell clones. Validated cells were stored in several batches for future assays, passaged once after every thaw, and verified for abrogation of CD61 protein levels with flow cytometry at 14 days post T cell expansion (as per Figure 13a) prior to functional assays. Cells were tested for mycoplasma monthly.

[0222] Mice

[0223] Immunodeficient NOD SCID gamma (NSG) mice (strain NOD.Cq-Prkdc scid Il rgtmiwji / sd)were|->recilocally at the Department of BioMedical Services (BMS), University of Oxford. Details of experimental model and in vivo assay used is according to previous study28, and further described in the Methods Details section. All mice were housed in ventilated cages, maintained under specific pathogen-free conditions and used at 8-10 weeks of age. All mice experiments were performed in accordance with Animals (Scientific Procedures) Act 1986 and according to the University of Oxford Animal Welfare and Ethical Review Body (AWERB) guidelines, and operating under the UK Home Office license PBA43A2E4.

[0224] TCR-engineered NY-ESO-1 -specific TCR-T cells generation from primary CD8+T cells (TCR-T)

[0225] TCR DNA template design The NY-ESO-1 TCR sequence used in this is as described in previous paper20. DNA templates were designed in silico and synthesised by Gene Art (ThermoFisher Scientific). The plasmids were used directly as the repairing template. TCR construct for CRISPR-Cas9-mediated HDR repair is designed with the following structure: 5’ homologous arm, P2A, TCR-b, T2A, TCR-a, bGHpA tail, 3’ homologous arm. To facilitate TCR expression, the TCR sequence is codon optimised and sequence confirmed by Sanger sequencing. Both 5’ and 3’ homologous arm sequences were used as previously described29.

[0226] Primary human CD8+T cells isolation PBMCs were isolated from the peripheral blood of a healthy human donor using Ficoll-Hypaque gradient isolation as described above. Primary CD8+T cells were then isolated using the CD8+T cell isolation kit (Miltenyi Biotec.) before being activated in vitro with 25ml / ml ImmunoCult Human CD3 / CD28 T cell activator (StemCell Technologies) for two days.

[0227] Orthotopic TCR replacement in primary human CD8+T cells

[0228] NY-ESO-1 transgenic T cell was generated by using orthotopic TCR replacement system with modifications. Briefly, exogenous NY-ESO-1 TCR was inserted into the primary T cell trac gene locus, together with the blockage of trbc gene expression. CRISPR gDNA sequences used are: 5'-AGAGTCTCTCAGCTGGTACA-3' (SEQ ID NO: 5) for trac and 5'-GGAGAATGACGAGTGGACCC-3' (SEQ ID NO: 6) for trbc (targeting both trbcl and lrbc2). Two days post primary T cell activation, T cells were harvested and washed with PBS before resuspension in P3 Primary Cell Nucleofector Solution (Lonza). The CRISPR RNP complex was generated with sgRNA (Integrated DNA Technologies) and Alt-R S.p. Cas9 Nuclease V3 protein (Integrated DNA Technologies) by incubation at room temperature for 15-20 minutes. Cells were then electroporated with CRISPR RNPs in presence of DNA HDR repairing template using the 4D Nucleofector X unit (Lonza). After electroporation, cells were plated and incubated with pre- warmed allogenic feeders. After a week, cells were sorted using the NY-ESO-1 tetramer on the BD LSR Fusion (BD Biosciences). The sorted TCR-T cells were then expanded by allogenic feeders and confirmed for TCR antigen specificity using the tetramer in parallel to each time T cell assay was performed.

[0229] CD61, CD 103 and. integrin b7 ov erexpression system on primary CD8+T cells, NY-ESO- 1-specific TCR-T cells and U937 cells

[0230] Primary CD8+T cells and U937 cells over-expressing CD61, CD 103 and / or integrin b7 were generated using a lentiviral transduction strategy. Primary CD8+T cells were first isolated from healthy donor using CD8 T cell isolation kit, human (Miltenyi Biotec).

[0231] Generating integrins-tag plasmids

[0232] Briefly, LentiX cells were plated in 6-well plates at 650,000 cells per well in DMEM supplemented with 10% FCS, 2 mM L-glutamine and 1% v / v (500U / ml) penicillin / streptomycin and incubated for overnight at 37°C. Cells were next co-transfected with the packing plasmids pMD2G (0.26 mg / well; Addgene plasmid #12259) and psPAX2 (0.5 mg / well; Addgene, plasmid #12260), as well as the relevant lentiviral expression vector plasmid (at 0.75 mg / well; pHR backbone) in Opti-MEM and FuGENE HD transfection reagent. All the integrins were cloned into the pHR-SIN plasmid backbone (Addgene, plasmid 79121) with full length gene sequence of either integrin aE (for CD 103 protein), integrin b3 (for CD61 protein) or integrin b7 (with relevant tag). DNA fragments for the various genes were custom purchased from IDT and ThermoFisher. The full-length protein sequences were obtained from Uniprot. DNA sequence integrity and identity was confirmed for all plasmids by Sanger sequencing. To improve the lentiviral transfection efficacy and titre, ViralBoost Reagent (Alstem) were added to the LentiX cell culture media at the time of transfection. Lentiviruses were harvested 72 hours after transfection and dead cells or debris were removed by centrifugation at 3,500 rpm for 5 minutes. Lentiviruses were placed on ice following harvesting and prior to transduction. U937 transduction was performed as a positive control of transduction.

[0233] Generating integrins-tag-transduced cells

[0234] 0.5M freshly-isolated primary CD8+T cells were first activated overnight with 10 mg / well OKT3 (Biolegend) or lOml / well aCD3 / CD28 (StemCell Technologies). For transduction, IM WT U937 cells or 0.5M of overnight-activated primary CD8+T cells were cultured with 3ml of respective lentivirus in a T25 flask upright and incubated at 37°C for a minimum of 2 hours. 2mls of culture media was then added before further incubation for 5 days. Culture media was changed whenever the media colour turns yellow. After 5 days, cells were collected and stained to determine the transduction efficiency and surface expression of the corresponding proteins. Briefly, cells were first stained with Live / Dead Fixable Aqua Stain Kit (ThermoFisher) for 20 minutes at 4°C prior to staining with BV421 anti-CD103 (Biolegend), PE / Cy7 anti-CD61 (Biolegend) and FITC anti-integrin b7 (Biolegend) for 20 minutes at 4°C. Cells were then permeabilised with BD CytoFix / CytoPerm Solution for 20 minutes at 4°C prior to intracellular staining with AF647 anti-cMyc (Biolegend) and PE anti-FLAG (Biolegend) for 20 minutes at 4°C. Samples were then acquired on Attune Nxt flow cytometer (ThermoFisher) and analysed on FlowJo V.10 (BD Biosciences). Liquid chromatography-mass spectrometry (LC-MS / MS)

[0235] Samples preparation Paired CD103+and CD 103" cell clones were activated for 3 hours or 6 hours with 10ml aCD3 / CD28 (StemCell Technologies) at 37°C, with nonactivated T cells as a normalisation control. IM cells were used per treatment in order to extract sufficient amount / concentration of proteins. To retain proteins and prevent secretion of molecules outside the cells, 0.7mg / ml Monensin and Img / ml Brefeldin A (BD Biosciences) were added per sample. After T cell activation at the set timepoints, cells were washed with PBS thoroughly three times. Each cell pellet was then lysed with 1% NP40 cell lysis buffer (ThermoFisher), IX protease inhibitor cocktail (Sigma Aldrich) and ImM phenylmethylsulfonyl fluoride, PMSF (ThermoFisher) on ice for 1 hour. Subsequently, the cell solution was vortex in 10 minutes intervals during the ice incubation. Subsequantly, the cell solution was microcentrifuged at 13,000rpm for 10 minutes at 4°C. Supernatants containing proteins were transferred into new tubes and frozen on dry ice.

[0236] Samples digestion Samples were thawed and proteins were denatured in 8M urea for 30 minutes. Protein reduction was performed with lOmM tris(2- carboxyethyl(phosphine) (TCEP) for 30 minutes at room temperature before undergoing alkylation with 50mM iodoacetamide for another 30 minutes at room temperature in the dark. Samples were then diluted to 1.5mM urea with 50mM triethylammonium bicarbonate (TEAB) before the proteins were digested with 1.5mg trypsin and incubated overnight at 37°C. Overnight digested samples were cleaned on SOFA HRP C18 and evaporated to dryness using a vacuum centrifuge. Samples containing the dried peptides were then reconstituted in 5% dimethyl sulfoxide (DMSO) and 5% formic acid.

[0237] LC-MS / MS Samples were then analysed using Ultimate 3000 UHPEC (ThermoFisher Scientific) connected to an Orbitrap Fusion Eumos Tribrid (ThermoFisher Scientific). Briefly, peptides were loaded onto a trap column (PepMapC18; 300pm x 5mm, 5pm particle size, Thermo Fischer) and separated on a 50cm-long EasySpray column (ES803, Thermo Fischer) with a gradient of 2-35% acetonitrile in 5% dimethyl sulfoxide, 0.1% formic acid at 250 nE / min flow rate over 60 minutes. Eluted peptides were then analysed on an Orbitrap Fusion Eumos Tribrid platform (instrument control software v3.3). Data were acquired in data-dependent mode, with the advance peak detection (APD) enabled. Survey scans were acquired in the Orbitrap at 120 k resolution over a m / z range 400 -1500, AGC target of 4e5 and S-lens RF of 30. Fragment ion spectra (MS / MS) were obtained in the Ion trap (rapid scan mode) with a Quad isolation window of 1.6, 40% AGC target and a maximum injection time of 35ms, with HCD activation and 28% collision energy.

[0238] Proteomics analysis

[0239] Enriched proteins analysis The log2 fold change values of each protein at 3 and 6 hours were calculated by normalising the log2 values of each protein of these timepoints with the log2 values at the 0 hours timepoint. Subsequently, the TGF-bl fold-change values of the paired T cells was used as the threshold to exclude any proteins with lower foldchange than TGF-bl stimulated cells. The inventors used the TGF-bl fold-change values as the evaluating threshold because the inventors have previously identified TGF-bl as a protein exclusively expressed by the CD103+T cell clones, but not by the CD 103" T cell clones20. Therefore, it can be assumed that any proteins with values lower than that of TGF-bl is least likely to be expressed by the CD103+T cell clones. This initial step resulted in a filtered paired dataset of 890 proteins, from the original 3405 proteins recorded in the dataset. To assess the enriched proteins of the CD103+T cell clones, the inventors cross-referenced these proteins to the Gene Ontology NCBI annotation database, categorising the proteins according to their known biological activities. Primarily focusing on the proteins of cellular processes, the inventors utilised the STRING interactions database to stratify the proteins into their specific functional protein subgroups. Heatmap of selected interested proteins were carried out using R. All proteins known to be associated with TCR signalling were originally identified using the Reactome annotation database before being analysed in relation to the proteomics dataset to evaluate the proteins upregulated or downregulated on the T cells.

[0240] Combined network plot for 3 vs 0 hours and 6 vs 0 hours for the paired NY-ESO-1- specific T cell clones gene list were converted to Entrez IDs (org.Hs.eg.db version 3.11.4). Upregulated proteins list was used as input for over-representation analysis (clusterProlifer version 3.18.0, ReactomePA version 1.32.0) to find REACTOME pathways with enriched proteins (with p value cut off 0.01 and p adjusted value cut off 0.05). The resulting output was used to create a concept network plot.

[0241] Barplots Barplot were constructed for selected proteins of a pathway using log2 fold-change values for specific (proteins (ggplot2 version 3.3.2). Data deposition details is as mentioned in the ‘Data and Code Availability’ section below.

[0242] Multicolour flow cytometry TILs immunophenotyping and analysis

[0243] For each immunophenotyping staining, IM cells of paratumor tissue, tumor and peripheral blood were first stained with Live / Dead Fixable Aqua Stain Kit (ThermoFisher) for 20 minutes at 4°C. For surface staining, cells were washed and then stained with dumping markers: BV510 anti-CD56 (Biolegend) and BV510 anti-CDl lb (Biolegend), T cells markers: BUV805 anti-CD8 (BD Biosciences) and BV650 or APC / Cy7 anti-CD3 (BD Biosciences), integrins: BUV395 anti-CD103 (BD Biosciences), BV421 or AF647 anti-CD61 (BD Biosciences), PerCP / Cy5.5 or FITC anti-CD41 (Biolegend), APC anti- integrin b7 (Biolegend) and PE or FITC anti-CD51 (Biolegend), tissue-resident T cell markers: PerCP / Cy5.5 anti-CD45RO (Biolegend), PE or BUV496 anti-CD49a (BD Biosciences), PE / Cy7 or BV605 anti-CD69 (Biolegend), BUV486 anti-CD62L (BD Biosciences), PE / Cy7 anti-CCR7 (Biolegend) and BV786 anti-CD45RA (BVD Biosciences), tumor-reactive TILs marker: APC / Cy7 or BV785 anti-CD39 (BD Biosciences and Biolegend), T cell differentiation markers: PE / Cy7 anti-CD27 (BD Biosciences) and BUV496 anti-CD28 (BD Biosciences), inhibitory markers: BUV737 anti- PD-1 (BD Biosciences), BV421 or BB515 anti-Tim-3 (BD Biosciences) and PE anti- TIGIT (BD Biosciences) for another 20 minutes at 4°C. For intracellular cytokine staining, cells were T cells were treated with 0.7mg / ml Monensin and Img / ml Brefeldin A (BD Biosciences), washed and permeabilised with BD CytoFix / CytoPerm Solution for 20 minutes at 4°C, before stained with cytokines: APC / Cy7 anti-IFNg (Biolegend) and BV785 anti-TNFa (Biolegend), cytolytic molecules: PE anti-granulysin (Biolegend), AF488 anti-granzyme M (ThermoFisher), AF647 anti-granzyme B (BD Biosciences), chemokines: BUV737 anti-CCL5 (Biolegend) and FITC anti-XCL2 for another 20 minutes at 4°C. Following antibodies staining, cells were fixed with IX CellFix (BD Biosciences) and acquired on BD LSR Symphony (BD Biosciences) and analysed on FlowJo V.10 (BD Biosciences).

[0244] Immunohistochemistry analysis

[0245] Lung tumor resection slides were provided by the ORB, cut at 5mm thickness on supercharged frost slides. Adjacent slides of individual patients were separately stained for CD 103, CD8 and CD61 using antibodies against CD 103 (Leica, clone EP206), CD8 (Leica, clone 4B11) and CD61 (Abeam, clone VLPL2). Once stained, the slides were digitised and data analysed using the Visiopharm Integrator System (VIS) platform version 2020.09.0.8195. Image analysis protocols are implemented as Analysis Protocol Packages (APP) in VIS. Several APPs were designed to quantify slides stained with CD103, CD8 and integrin b3. Prior to the image analysis, it is important to outline the Region of Interest (ROI), a number of auxiliary APPs were designed to detect ROIs. The Tissuealign analysis module was used to align and subsequently analyse digitised serial slides.

[0246] Image alignment Tissuealign module was used to align 5 digitised serial slides. The alignment was performed both on a large scale, and on a finer detailed level, to get the best possible match of the 5 tissue slides.

[0247] Detecting ROIs The first auxiliary APP runs on the slide using threshold classification that identifies the tissue regions. The second auxiliary APP runs on the integrin b3 slide using DeepLabv3 network of the VIS Al module that identifies the integrin b3positive regions. The ROIs are then superimposed on the aligned CD8 slide to outline various regions for subsequent analysis limited to the inside of the specific regions. A CD8 APP was run on the ROIs outlined by the integrin b3 app to find co-located cells between integrin b3 and CD8. The inventors follow the same process to find collocated cells between integrin b3, CD8 and CD103.

[0248] Analysis HDAB-DAB colour deconvolution band is used to detect positively stained cells on the integrin b3, CD 103, CD8 slides. To enhance the stained cells, while suppressing the background variation, several pre-processing steps were included. The colour deconvolution bands were inputted into a threshold classifier. Thresholding classification method defines a threshold for a given feature, and assigns one class to all pixels with a feature value above or equal to that value, and another class for the rest. The classification rule is defined as: where T is the user-selected threshold (cut-off value), A and B are the labels / classes to which the pixel is assigned. As post-processing steps; a method for cell separation which is based on shape and size is used, cell areas that are too small are removed and finally applying unbiased counting frames to avoid the cells that are intersecting with neighbouring tile boundaries counted twice (or more).

[0249] Quantitative Output Variables and Calculations The output variables obtained from the APPs include, (i) total tissue area, (ii) total tumor islets areas by areas of overexpression of E-Cadherin. (iii) CD61, CD 103 and CD8 positive cell areas, and (iv) colocated positive cells area between CD61, CD103 and CD8. Firstly, to determine tumor islets, the inventors used E-Cadherin over-expression as tumor marker, because E- Cadherin is well-established to over-express on epithelial tumor cells. On a serial section, the inventors identified E-Cadherin positive staining area, in which E-Cadherin overexpression level analysis was performed using Visiopharm IHC Analysis Protocol Packages (APP) algorithm to separate tumor cells and normal epithelia. Cells present at “within’ areas were defined as cells located within the E-Cadherinover'expressedstained regions, cells present at “clustering’ represent cells located within 1.5cm (c< 1.5cm) from the E-Cadherinover'expressedregions, and cells present at “distal” represent cells located over 1.5cm (c>1.5cm) from the E-Cadherinover'expressedregions. The width denoting ‘clustering’ areas is as shown on Fig. 15, in which the APP algorithm is used to set the 'clustering' as the half average of the distance between one E-Cadherinover'expressedstained region with another.

[0250] Preparation of Glass-supported lipid bilayers

[0251] Acid- and plasma (5 min)-cleaned Coverslip Glass D 0.17 + / - 0.005 mm (Schott Nexterion, #1472315) was attached to Sticky-Slide VI04chambers (Ibidi, #80608) to assemble Six-well imaging chambers. To form glass- supported lipid bilayers (SLB), a liposome master mix containing 0.1875% vokvol of 0.4 mM CapBio, 12.5% vokvol 0.4 mM DGS-NTA(Ni) in a 0.4 mM DOPC matrix (to 100% vokvol) was incubated on the glass for 30 min at RT to allow spreading. To remove excess liposomes, assembled SLB were washed three times with HBS / HSA buffer and then blocked using a 5% BSA solution containing 5 pg / mL of Streptavidin either unconjugated or conjugated with Dylight 405 (ThermoFisher Scientific, #21831) and 100 pM NiSC for no longer than 20 min at RT. After three washes SLB were then incubated with the recombinant proteins to reconstitute an antigen-presenting cell membrane composed of 200 molec. / pm2of ICAM-1, 100 molec. / pm2of CD58, 100 molec. / pm2of E-Cadherin (SinoBiological), and 30 molec. / pm2of biotinylated antigenic HLA-A2 NY ESO-1 peptide complex (HLA-A*02:01 loaded with NY-ESO-li57-i65-specific SC9 peptide (SLLMQITQC, SEQ ID NO: 7) at effective 5 pg / mL. Most recombinant proteins were designed with a twelve histidine tag and were produced in-house using HEK293T cells as described elsewhere. Calibration of recombinant protein densities was performed on bead-supported lipid bilayers (BSLBs) as described elsewhere. E-Cadherin was calibrated with flow cytometry on cells and BSLBs using an in-house AF647-labelled anti-CD324 (E-Cadherin) (Biolegend). After 30 min at RT, SLB were washed three times and incubated with 0.75 x 106cells per well for either 5, 10 or 15 minutes at 37°C. After stimulation, cells were immediately fixed for 10 min with prewarmed 4% PFA in PBS containing 2 mM MgCh, washed three times with HBS / HSA buffer, and stained using BV421 anti-Integrin b7 (BD Biosciences), AF488 anti-CD103 (Abeam), AF647 anti-CD61 (integrin b3) (Biolegend) either at 1 pg / mL for 15 min at RT or 1:100 dilution at 4°C overnight. After four washes, cells were imaged immediately using Total Internal Reflection Fluorescence Microscopy (TIRFM).

[0252] Total internal reflection fluorescent microscopy (TIRFM)

[0253] Imaging of T cell clone immune synapses was performed on an Olympus 1X83 inverted microscope equipped with a 4-line (405 nm, 488 nm, 561 nm, and 640 nm laser) illumination system. The system was fitted with an Olympus UApON 150x 1.45 numerical aperture objective, and a Photometries Evolve delta EMCCD camera to provide Nyquist sampling. Quantification of fluorescence intensity was performed with Fiji / ImageJ (National Institute of Health). For co-localisation analyses, the inventors used the EzColicalisation plugin with a combination of manual and Costes’ method-assisted thresholding to identify relevant pixel values, as previously described30. For analysing the sectional distribution of each integrin within T-cell immune synapses, the inventors used custom-written Fiji / ImageJ macros to segment cells based on either the antigen (Ag) channel or the integrin b7 integrin channel. The inventors then performed radial averaging on all the channels from the segmented micrographs by rotating them 1° x 359 times before averaging all the rotated copies from each channel. The inventors then averaged all the radial averages from each channel before the inventors drew a diagonal line plot on the resulting micrographs to analyse the radial mean fluorescence intensity of the signal from each channel. When segmenting cells on the Ag, the radial averages were centred on the cSMAC. When segmenting on the integrin b7 channel, the radial averages were centred on the whole contact area, as this signal was mainly found in the periphery.

[0254] To ensure flatness in the topography of the contact zone between T cell membrane and the bilayer, the inventors used the bilayer system well-established in their previous studies31'34, where T cell membrane flatness was ensured using ICAM1:LFA-1 interactions on the substrate, restricting the formation of significant membrane protrusions.

[0255] Co-immunoprecipitation of CD61, CD 103 and. integrin b7

[0256] Integrins-transduced primary CD8 T cells and / or U937 cells were washed twice with ice cold PBS and lysed in Pierce IP Lysis Buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 5% glycerol containing PhosSTOP phosphatase inhibitors and cOmplete protease inhibitor cocktail). Whole cell lysates were incubated on ice for 10 minutes and centrifuged at 13,000g at 4°C for 5 minutes to remove dead cells or cell debris. Pre-cleared lysates were incubated with Anti-FLAG M2 magnetic beads or anti- cMyc antibody, at 4°C overnight. The proteins bound by anti-flag antibody were pulled down by the magnetic beads according to manufacturer’s protocol. Briefly, the samples were incubated with beads overnight, followed by three times of washes with PBS. Pulldown samples were eluted by competition using the 3X FLAG peptide. The elution was carried out in room temperature via the incubation of beads in O.lmg / mL 3X FLAG peptide for 30 minutes. Beads were then removed, and samples were subjected to immunoblotting analysis. Immunoblotting

[0257] Samples were loaded onto the 4%-15% gradient Criterion TGX precast gels (BioRad). Proteins were transferred onto the nitrocellulose membrane, and the membrane was then blocked in 5% skim milk in TBS containing 0.1% Tween 20 (TBST) for 1-2 h. After blocking, the membrane was incubated overnight at 4 °C with primary antibodies (purified anti-beta- actin antibody (Sigma-Aldrich), purified anti-CD103 antibody clone EPR4166(2) (Abeam), purified anti-cMyc antibody (Sigma Aldrich) or purified anti-FLAG M2 antibody (Sigma- Aldrich). The membranes were then incubated with relevant IRDye secondary antibodies (Li-COR) in TBST containing 5% skim milk after washing 3-4 times in TBST. Gel was imaged on Li-COR Odyssey.

[0258] CD61 interactomics analysis by LC-MS / MS

[0259] Eluted co-immunoprecipitated proteins were further processed for mass spectrometry analysis using S-trap (Protifi). Proteins were reduced with 10 mM DTT in H2O, followed by alkylation with 20 mM iodoacetamide in H2O in the dark. Samples were acidified by addition of 12% phosphoric acid (to a final concentration of -1.1%), diluted with 90% methanol in 100 mM triethylammonimum bicarbonate (TEAB, 640 ml of methanol mixture per 100ml of sample), and captured on S-TrapTM mini columns (ProtiFi). Columns were washed with 90% methanol in 100 mM TEAB followed by centrifugation at 4000g (400ml per column x3). Captured proteins were digested with trypsin (1:30 w / w) overnight at room temperature. Peptides were first eluted with 50mM TEAB (80ml, 4000g for 1 minute), followed by elution with 0.5% TFA in H2O (80ml, 4000g for 1 minute) and finally eluted with 50:50:0.5 acetonitrile:MilliQ:TFA mixture, and driedin a vacuum concentrator. Dried peptides were dissolved in Buffer A (98% MilliQ- H2O, 2% CH3CN and 0.1% TFA). 2.2% of the tryptic peptides were analysed by LC- MS / MS using a U3000 HPLC connected to an Orbitrap Ascend tribrid instrument (ThermoFisher). The tryptic were loaded onto a PepMacC18 trap column (300mm x 5mm, 5mm particle size, ThermoFisher) and separated on a 50cm EasySpray column (ES803, ThermoFisher) using a 60 minutes linear gradient from 2 to 35% acetonitrile, 0.1% formic acid and at 250nl / min flow rate. MS data were acquired in data-independent mode (DIA) with minor changes from previously described method35,36. Briefly, MSI scans were acquired in the Orbitrap over the mass range of 350-1650m / z, with a 45k resolution, maximum injection time of 91ms, an AGC set to 125% and a RF lens at 30%. MS2 scans were then collected using the tMSn scan function, with 40 variable width DIA scan windows at 30k orbitrap resolution, normalised AGC target of 1000%, maximum injection time set to auto and a 30% collision energy. Raw mass spectrometry files were label free quantified using DIA-NN (version 1.8) in library- free mode using the Uniprot proteome UP000005640 (2022) as FASTA file. Data were further processed in Perseus (version 1.6.2.3). Volcano plots were generated using the processed data and plotted using VOlcaNOseR. Data deposition details is as mentioned in the ‘Data and Code Availability’ section below.

[0260] Phosflow staining assay

[0261] T cells (clones or lines) were co-culture with cancer cells at an E:T ratio of 1:2 at 37 °C for 15 minutes, 30 minutes or 2 hours. Following T cell activation, cells were stained with Live / Dead Fixable Aqua Dead Cell Stain Kit (Thermo Fisher) before fixed with BD Cytofix Fixation Buffer for 10 minutes at 37 °C. Cells were then permeabilised using BD Phosflow Perm Buffer III for 30 minutes at 4°C before stained with FITC anti-ZAP70 (BD Biosciences), AF647 anti-ZAP70 (pY292) (BD Biosciences), AF488 anti-PCLgl (BD Biosciences), AF647 anti-PLCgl (pY783) (BD Biosciences), AF647 anti-Lck (BD Biosciences). Cells were then acquired immediately on Attune Nxt flow cytometer (ThermoFisher) and analysed on FlowJo V.10 (BD Biosciences). To assess contribution of CD61 towards TCR signalling proteins activities, T cells were treated with lOnM aminogenistein (tyrosine kinases inhibitor, Santa Cruz Biotechnology) and 7.5nM A770041 (Lek inhibitor, Sigma Aldrich) before the T cell activation and the Phosflow assay were performed. To evaluate the T cell cytotoxic efficacy, the CD61+T cell lines from seven cancer patients were treated with anti-CD61 (lOmg / ml, PM 6 / 13, Novus Biotechnology), in parallel with the T cell activation.

[0262] In vitro T cell CD 107 a staining assay

[0263] T cells (clones or lines) were co-cultured with cancer cells at an E:T ratio of 1:2 at 37°C for 4 hours, in presence of PE / Cy7 anti-CD107a (Biolegend) staining. Following T cellsactivation, cells were fixed and samples were then acquired on Attune Nxt flow cytometer (ThermoFisher) and analysed on FlowJo V.10 (BD Biosciences). To evaluate the T cell cytotoxic efficacy, the CD61+T cell lines from seven cancer patients were treated with anti-CD61 (lOmg / ml, PM6 / 13, Novus Biotechnology), in parallel with the T cell activation.

[0264] Carboxyflourescein diacetate succinimidyl ester (CFSE)-based T cell cytotoxicity assay

[0265] Cancer cells were initially stained with 0.5mg / ml CFSE (ThermoFisher) before coculture with T cells (clones or lines) at an E:T ratio of 1:2 at 37°C for either 2, 4, 6 and 8 hours. Cells were then stained with 7-AAD (BD Biosciences) and BV421 anti-E-Cadherin (Biolegend) and PE / Cy7 anti-CD8 (BD Biosciences) before acquiring on the Attune Nxt flow cytometer (ThermoFisher) and analysed on FlowJo V.10 (BD Biosciences). To evaluate the T cell cytotoxic efficacy, the CD61+T cell lines from seven cancer patients were treated with anti-CD61 (lOmg / ml, PM6 / 13, Novus Biotechnology), in parallel with the T cells activation.

[0266] Mice xenograft and tumor growth kinetics assay

[0267] Immunodeficient NSG mice (strain NOD.Cq-Prkdc scid n2rgtmlwjl / SzJ) were xenografted with NY-ESO-1+HCT116. A WT HCT116 is absent of the NY-ESO-1 antigen as previously described20, and therefore was transduced with lenti virus expressing the NYESO-1 protein linked to co-expression of eGFP via an internal ribosomal entry site (IRES) link, as previously described. Transduced cells were sorted based on eGFP positive expression and cultured for 3 passages in RIO before confirmation of NY-ESO-1 expression by eGFP through flow cytometry staining and used for in vivo experiment. IM NY-ESO-1+HCT116 cells in PBS solution were injected subcutaneously in 1:1 ratio with Matrigel Matrix solution (Corning) at Day 0. After 48 hours, mice were randomised into groups (n = 8-10), and IM T cells (WT CD61+or CD61- T cell clones (of patient 1)) in PBS solution were injected intravenously per mouse. Additional intravenous injections of T cells were carried out on Day 7 and 14 post tumor xenografts, with the same number of T cells. Measurements using digital callipers were taken on Day 4, 7, 10, 13, 16 and 20 post tumor xenografts. Tumor volume approximated according to the formula for ellipsoid volume (width / 2 x depth / 2 x length / 2 x p4 / 3). To keep the experiment in a blinded manner, the tumor measurements and decoding of the treatment group was performed by separate individuals.

[0268] Kaplan-Meier survival curve analysis

[0269] Clinical RNA expression datasets were downloaded from The Cancer Genome Atlas (TCGA) database using RTCGA (version 1.18.0) (Kosinski M, Biecek P (2021). RTCGA: The Cancer Genome Atlas Data Integration. R package version 1.24.0). Patient metadata for associated datasets were downloaded from cBioPortal. The datasets used were the skin cutaneous melanoma (TCGA, PanCancer Atlas), lung cancer (TCGA, PanCancer Atlas) and lung cancer (University of Cologne)17. The analysis of TCGA dataset utilises analytical pipeline that facilitates the identification of CD8A and CD3E -enriched samples (known to be enriched on T cells), which then were subjected to more granular analyses; though as TCGA dataset is not sigle cell resolution, the analyses performed may not necessarily of T cells. Using the surv_cutpoint function from the survminer R package, the inventors objectively determined the optimal cutpoint for CD8 expression using the following arguments: time=”Months.of.disease. specific. survival”, event=”Disease. specific. Survival. status”. This was categorised into a categorical variable using the surv_categorize function and samples with high CD8A and CD3E expression as categorised by above were classed as CD3+CD8+ samples. So, the CD3+CD8+ samples analysed would be from datapoints that show evidence of a high T cell proportion. The inventors then filtered out CD103+CD8+cells using 1TGAE (CD 103) gene marker. Lastly, patients were segregated based on high or low expression of 1TGB3 (CD61), specifically to identify two group of patients, having either CD61hlCD103+CD8+CD3+samples or CD6110CD103+CD8+CD3+samples. Optimal cut-points were calculated to distinguish between high or low expression of each of these four genes for each dataset (survival version 3.1-12; Themeau T (2021). A Package for Survival Analysis in R. R package version 3.2-13). Only patients that showed high expression of CD8A, CD3E and ITGAE were deemed as CD8A+CD3E+ITGAE+ and kept in the analysis. These patients were used to plot Kaplan-Meier survival curves between ITGB3+ and 1TGB3- patients (survminer version 0.4.9). For lung cancer datasets, the optimal cutpoints were calculated for stage I patients from each dataset separately, before being combined into a single survival plot. P values were determined by log-rank test.

[0270] Ex vivo T cells proliferation assay

[0271] IM cells of paratumor tissue or of tumor tissue were stained with 0.5mg / ml CFSE prior to activation with lOul aCD3 / CD28 (StemCell Technologies). The cells were incubated at 37 °C for 72 hours. After, the cells were stained with Live / Dead Fixable Aqua Cell Stain Kit (Thermo Fisher) for 20 minutes at 4°C before being stained with conjugated antibodies against BV650 anti-CD3 (BD Biosciences), BUV805 anti-CD8 (BD Biosciences), BUV395 anti-CD103 (BD Biosciences), BV421 anti-CD61 (BD Biosciences), PerCP / Cy5.5 anti-CD45RO (Biolegend), PE anti-CD49a (BD Biosciences and Biolegend) and PE / Cy7 anti-CD69 (Biolegend). Following antibodies staining, cells were fixed with IX CellFix (BD Biosciences) and acquired on BD LSR Symphony (BD Biosciences) and analysed on FlowJo V.10 (TreeStar Inc.). Cells were considered proliferative based on decrease in CFSE fluorescence, within the 1stdownward peaks of CFSE onwards.

[0272] Statistical analysis

[0273] All graph generation and statistical analyses were conducted using GraphPad Prism software (except for survival curve as generated as mentioned above). Unless stated otherwise, data are summarised as median ± SEM. The number of patients used for each ex vivo analyses is shown in the figure legends. Each of the in vitro functional assays were repeated 3 times minimum. Statistically significant differences between 2 groups were assessed using two-tailed paired t test, with Wilcoxon adjustments for non-parametrically distributed variables. For comparison between more than 2 paired of groups, one-way ANOVA with Tukey multiple comparison test was performed. Multilayer statistical analyses between more than 2 groups and between more than 2 treatments, was carried out using two-way ANOVA with Tukey multiple comparison test. Correlation analyses were performed using non-parametric Spearman rank correlation. Statistical significance was set as * P< 0.05, ** P< 0.01 and *** P < 0.001, and were two-tailed. Spatial trans criptomics slides processing and data collection via CosMx SMI

[0274] The dataset was generated using the CosMx SMI instrument according to the company’s protocol. Briefly, slides were baked overnight at 60°C to enhance tissue adherence, followed by deparaffinisation in xylene (two washes of 5 minutes each) and 100% ethanol (two washes of 2 minutes each). Slides was then briefly air-dried at 60°C for 5 minutes before proceeding with antigen retrieval. Target retrieval was carried out in preheated CosMx lx target Retrieval Solution at 100°C for 15 minutes using a pressure cooker, followed by enzymatic permeabilization with proteinase K (3pg / mL) at 40°C for 30 minutes to optimise probe accessibility. In situ hybridisation was conducted using the CosMx Universal Cell Characterisation RNA 960-gene panel, supplemented with a custom add-on probes targeting additional genes of interest including CD69, ITGB3 (CD61 , Zap70, ITGA1 (CD49a), CD 101. Hybridisation was performed overnight at 37°C in a humidified chamber, followed by two stringent washes (25 minutes each) in proprietary CosMx wash buffer and additional washes in 2x SSC buffer (2 washes of 2 minutes each). To enable single-cell resolution of segmentation process, immunofluorescence staining was performed using CosMx segmentation markers, DAPI (nuclear stain), Pan-CK (epithelial marker), CD45 (immune marker) and CD298 / p2m (cell membrane marker for tissue segmentation). Sections were incubated with immunofluorescence antibodies for 1 hour at room temperature, followed by PBS (3 washes for 5 minutes each). Processed slides were assembled into flow cells using the CosMx Flow Cell Assembly Tool and loaded onto the CosMx SMI instrument. High-resolution fluorescence imaging was performed, capturing RNA targets at subcellular resolution.

[0275] Spatial Imaging

[0276] To perform spatial imaging of individual patient tissue section, images were recalled from the spatial image slot from the spatial seurat object, or captured from AtoMx software. The seurat spatial function, ImageDimPlot, was carried out on specific metadata column containing information of specific cell clusters of interest needed for visualisation- utilising specific parameter of boundaries = “segmentation” to call polygon data instead of centroid data. Color annotation for each cell clusters are made to accommodate color-blind readers, with specific metadata columns created to associate specific cell clusters to unique color hex code.

[0277] To further visualise specific FOV of a tissue section, the parent seurat object was subsetted to specific FOV of interest using metadata column, fov, before carrying out the ImageDimPlot function. Within the daughter seurat object, the visualisation using the function utilise the metadata column of x_slide_mm and y_slide_mm, which calls values specific to that FOV region instead of whole slide values. Similar to subsetting specific FOV, a zoomed-in visualisation of certain area within specific FOV involves subsetting the daughter seurat object based on parameters of specified x_slide_mm and y_slide_mm range of values. Visualisation using ImageDimPlot will plot only the specific values within the specified parameter. Cell counts of each cell clusters was extracted from specific metadata column containing clusters information using the table() function. Individual transcript spatial imaging was carried out using Bruker’ s proprietary built-in analytical tool available within AtoMx software.

[0278] Spatial immunophenotype analysis

[0279] To analyse the spatial immunophenotype of each CD8+T cell clusters, 12 phenotypic features was created and the median immunoscore for each feature was calculated for each T cell clusters, including i) inflammatory cytokines feature (IFNG, TNF, 1L32, ILIA, 1L1B, IL6), ii) cytotoxicity feature (GNLY, GZMB, PRF1, GZMA, GZMK, FASLG). iii) cell cycle / proliferation feature GL2RA, MKI67, PCNA, TOP2A, CCND1), iv) mitochondrial metabolism feature (GLUD1 , PPARA, MYC, MTOR, PPARG), v) chemokines feature (CCL5, CCR5, CXCR4, XCR1, XCL1 / 2, CCL3 / L1 / L3, CCL4 / LI / L2). vi) cytoskeleton / motility feature (MMP1, QAP3, STMN1, ARHGD1B, RAC2), vii) costimulatoryreceptors feature (IGOS, TNFRSF9, CD28, CD40LG, TNFRSF4, CD2, KLRK1), viii) co-stimulatory signalling feature (ZAP70, FYB1, ITK, HMGN2, CALM1 ), ix) inhibitory receptors feature (CTLA4, HAVCR2, PDCD1, TIGIT, LAG3). x) inhibitory signalling feature (DUSP2, ZFP36), xi) immunosuppressive / regulatory feature (TGFB1, IL10, FOXP3). and xii) differentiation feature (HMGB2, CD44, TBX21, RUNX3, EOMES). Visualisation was carried out using the radarchart function. Each feature was also merged into two categories: i) immuno-responsive features which include inflammatory cytokines, cytotoxicity, cell cycle / proliferation, chemokines, mitochondrial metabolism, co-stimulatory receptors and signalling genes, and ii) immune exhausted features which include inhibitory receptors and signalling, immunosuppressive / regulatory and differentiated genes. Average sum of immunoscores were calculated and plot for each T cell clusters using conventional barplot function.

[0280] Nearest neighbors anlaysis

[0281] For nearest neighbors analyses, KNN approach was used. For nearest neighbors to CD61+CD8+TILs, k = 10 was used and calculated using the get.knnx function. Normalisation was carried out to create relative proximity and density proximity, to adjust and normalise KNN based on the frequency of each cell clusters. Clusters that are densely proximate to CD61+TILs were grouped into one category and compared with the remaining epithelia / tumor clusters for their E-Cadherin CDH1) gene expression. Conventional / -test analysis with Wilcoxon adjustment, was performed. For proximate cell immediately adjacent to the different CD61+TIL clusters, k = 1 was used and calculated as above. Following this, correlation analysis between apoptotic epithelia / tumor density and CD61+A cluster was calculated using apoptotic genes available in the 960-plex gene panel (CASP3, BAX, CDKN1A), extracting spatial coordinates of the apoptotic epithelial cells and CD61+A separately and performed Kernel density estimation for the different cell type, prior to performing cor.test analysis using the conventional spearmen correlation test.

[0282] Tables

[0283] Table 1, List of 103 enriched proteins on the CD103+T cell clones.

[0284] Table showing the log2 fold-change values on both CD103+and CD 103" T cell clones, at 6 versus 0 hours timepoint analysis.

[0285]

[0286] Table 2, Clinical parameters of each patient used in this study.

[0287] Table 2 shows the clinical parameters evaluated for this study (Age, Sex, Tumor stage, Nodal invasion, Tumor pathology type, Usage in study).

[0288] References

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Claims

Claims1. An agent specific for a heterocomplex comprising CD61 and CD 103.

2. An agent comprising (i) a moiety specific for CD61 and (ii) a moiety specific for a tumor antigen.

3. The agent of claim 1 or claim 2, wherein the agent is a small molecule, an antibody or antigen-binding fragment thereof (e.g. a nanobody, a scFv, or a Fab), or a nucleic acid (e.g. an oligonucleotide).

4. The agent of any one of claims 1 to 3, wherein the agent is an antagonist that reduces the functional effect of the association of CD61 and CD103.

5. The agent of any one of claims 1 to 3, wherein the agent is an agonist that promotes the functional effect of the association of CD61 and CD103.

6. The agent of claim 5, further comprising a moiety that is specific for a tumor antigen.

7. A heterocomplex or a fusion protein comprising CD61 and CD 103.

8. A polynucleotide encoding the fusion protein of claim 7.

9. A vector comprising the polynucleotide of claim 8.

10. A population of immune cells modified to express CD61, CD 103 or a heterocomplex comprising CD61 and CD 103, optionally wherein the immune cells additionally express an antigen binding protein on the cell surface.

11. The population of immune cells of claim 10, wherein the antigen binding protein is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a modified T cell receptor, or a TCR mimetic.

12. The population of immune cells of claim 10 or claim 11, wherein the immune cells are T cells.

13. A pharmaceutical composition comprising the agent of any one of claims 1 to 6, or a population of immune cells of any one of claims 10 to 12, and a pharmaceutically acceptable carrier.

14. The agent of any one of claims 1 to 6, a population of immune cells of any one of claims 10 to 12, or a pharmaceutical composition of claim 13, for use in a method of therapy practised on the human or animal body.

15. The agent of any one of claims 1 to 6, a population of immune cells of any one of claims 10 to 12, or a pharmaceutical composition of claim 13, for use in a method of treating an immune-related condition.

16. A method of treating an immune-related condition in a subject, comprising administering to the subject a therapeutically effective amount of the agent of any one of claims 1 to 6, the population of immune cells of any one of claims 10 to 12, or the pharmaceutical composition of claim 13.

17. Use of the agent of any one of claims 1 to 6, a population of immune cells of any one of claims 10 to 12, or a pharmaceutical composition of claim 13, in the manufacture of a medicament for treatment of an immune-related condition.

18. A method of predicting outcome of a therapy for an immune-related condition for a subject, comprising detecting the presence of CD61+CD 103+ immune cells in a sample from said subject using the agent of any one of claims 1 to 6.

19. The agent, population of immune cells or pharmaceutical composition for use according to claim 15, the method of claim 16 or claim 18, or the use of claim 17, wherein the immune-related condition is cancer, such as lung cancer or melanoma, and optionally wherein the agent is an agonist.

20. The agent, population of immune cells or pharmaceutical composition for use according to claim 15, the method of claim 16 or claim 18, or the use of claim 17, wherein the immune-related condition is associated with overreacted immune cells, and optionally wherein the agent is an antagonist.

21. The agent, population of immune cells or pharmaceutical composition for use, the method, or the use according to claim 17, wherein the immune-related condition associated with overreacted immune cells is an autoimmune disease, transplantation, allergy, or viral infection (e.g. acute viral infection).

22. The agent, population of immune cells or pharmaceutical composition for use, the method, or the use according to claim 19 or claim 20, wherein the method comprises administering a further therapeutic agent concurrently or separately to the agent, population of immune cells or pharmaceutical composition, optionally wherein the anticancer agent is a CAR-T cell or a TCR-T cell.

23. A method of identifying or enriching a population of CD61+CD 103+ immune cells, comprising detecting the presence of CD61+CD 103+ immune cells using the agent of any one of claims 1 to 6.

24. A method of diagnosing an immune-related condition in a subject, comprising detecting the presence of CD61+CD 103+ immune cells using an agent described herein.

25. The method of claim 23 or claim 24, wherein the immune cells are TILs, T cells, NK cells, B cells, or NKT cells, optionally wherein the T cells are CD8+ T cells, CD4+ Tcells, regulatory T cells, effector T cells, tissue resident memory T cells, memory T cells, or gamma delta (y5) T cells.

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